FACULTEIT DIERGENEESKUNDE / FACULTY OF VETERINARY MEDICINE

PRODUCTION OF
ANIMAL PROTEINS BY
CELL SYSTEMS
DESK STUDY ON CULTURED MEAT
(“KWEEKVLEES”)

H.P. HAAGSMAN, K.J. HELLINGWERF, B.A.J. ROELEN

 October 2009
Faculty of Veterinary Medicine
http://www.uu.nl
Utrecht
Production of animal proteins by cell systems

Desk study on cultured meat (“kweekvlees”)

H.P. Haagsman
K.J. Hellingwerf
B.A.J. Roelen

1
Contents

04 Preface
05 Introduction
07 Cultured meat: a short history
09 Cell culture
09 Prokaryotic cells
10 Eukaryotic cells
11 Culture media
12 Chemically defined culture media
12 Origin of components of culture media
15 Skeletal muscle from stem cells
15 Stem cells
16 Embryonic stem cells
16 Adult stem cells
17 iPS cells
17 Bioengineering and bioreactors for tissue cultures
18 Co-culturing
18 Mechanical stimulation
18 Electrical stimulation
18 Scale-up of muscle bioreactor cultures
19 Bioreactors for suspension cultures
22 Ethical and societal issues
23 Challenges
23 Generation of stem cell lines from farm animal species
24 Safe media for the culture of stem cells
24 Safe differentiation media to produce muscle cells
25 Tissue engineering of muscle fibers
25 Industrial bioreactors
25 Food processing technology
25 Consumer preferences and adapted marketing strategies
29 Current developments of cultured meat research
29 The Dutch 'In vitro meat' project funded by SenterNovem
30 Stem cell biology
30 Tissue engineering
31 Culture media
33 Press interest
33 International research
36 Strengths and weaknesses of the current approach
36 Strengths
36 Weaknesses
37 Opportunities
38 Threats
39 Spin-off
42 Policy making
45 Summary
47 Nederlandse samenvatting
50 References
52 Appendices
52 Interviewed experts
53 Alternative (animal) cell systems for protein production
55 List of speakers of the first in vitro meat symposium
56 Participants in a European FP7 application (2009)
57 Publications about in vitro meat
58 About the authors

2
Boxes:

08 Need for protein sources other than meat

10 The most important reasons to produce cultured meat
14 Photosynthesis
20 Life cycle assessment of cultured meat production (C2C)
21 Technological challenges
23 Best of both worlds
28 Which factors determine success or failure of artificial meat?
28 What would an introduction campaign focus on?
29 Presumed qualities of cultured meat
32 Organization of research on cultured meat
35 Role of The Netherlands in the development of cultured meat technology
37 The most important criteria that have to be met in order develop
marketable products
39 The role of companies in the development of cultured meat
42 Potential spin-offs of cultured meat research
43 Investments in cultured meat research and technology

3
Preface

This report was commissioned by the Ministry of Agriculture, Nature and Food Quality as
part of a survey on meat alternatives. It presents the current state of research and
development of cultured meat (‘kweekvlees”), a completely new idea to produce edible
skeletal muscle (i.e. meat) by culturing and differentiating stem cells of farm animal
species to skeletal muscle cells. This hypothetical method of producing ‘meat’ has been
patented by the Dutchman Willem van Eelen in 1999, and research on its feasibility has
been conducted by as part of a SenterNovem research project between 2005-2009.
The current study is performed by three researchers of two Dutch universities who also
took part in the SenterNovem research project. Since these authors were involved in the
‘in vitro meat’ research project they had already acquired a vast amount of knowledge
about the scientific aspects of cultured meat. Also in the past few years they have build
up an extensive global network of those working on or interested in this subject. These
contacts range from colleague scientists to process technologists, food specialists, and
representatives of vegetarian organizations to psychologists and sociologists. The authors
have used these contacts and did an extensive literature research for this report.
The study is therefore broad; not only scientific aspects are taken into account but also
societal and economic factors. This was not an easy task since research and development
of cultured meat is still in its infancy.
In this report, it is first described why there is a current dire need for meat alternatives.
Subsequently, the theoretical background and short history of cultured meat are
described and the types of culture media that can be used or developed. This is followed
by a section on bioengineering and process technology. Ethical and societal issues are
discussed, as well as challenges, strengths and weaknesses of the current approach.
Besides literature studies on technological and societal aspects of cultured meat, the
opinions of many interviewed experts were also an important source of information. The
opinions of the experts can be read throughout the report in the various boxes. We would
like to thank all experts that have contributed to this report.

The authors

4
Introduction

The use of livestock for the production of food has always been an essential part of man’s
existence on earth, and its impact has until recent years been primarily positive, both
economically and socially. However, current production methods are rather demanding.
Their impact may be direct, by ruminant methane production for example, or indirect,
such as expansion of soybean production for feed in South America, replacing rain
forests. About 70% of the fresh water use, 35% of land use and 20% of the energy
consumption of mankind is directly or indirectly used for food production, of which a
considerable proportion is used for the production of meat. The total area of ice-free land
in use for grazing is about 26% of the earth’s total, and an additional 33% of arable land
is in use for feed crop production. In total, livestock is responsible for 70% of agricultural
land exploitation, and without change of policies this percentage will only increase in the
near future.

It has been estimated that the global population will increase from 6 billion people in
2000 to 9 billion people in the year 2050. This population increase will be accompanied
by a rise in annual greenhouse gas emissions from 11.2 to 19.7 gigatonne of carbon
dioxide, carbon equivalent. It is anticipated that in the same period annual global meat
production will rise from 228 to 465 million tonnes due to rising incomes, urbanization
and growing populations (FAO 2006. Livestock’s long shadow, environmental issues and
options, Rome.
http://www.virtualcentre.org/en/library/key_pub/longshad/A0701E00.pdf).
The amount of additional land available for the required increase in production capacity,
however, is limited. This has serious consequences at various levels and makes it a major
policy focus. It is now evident that we are experiencing a climate change, and that
anthropogenic influences seem to be (at least partly) responsible for this. This includes
the current level of meat production. Feed crop production demands high levels of
energy, which in itself, leads to increased CO2 emission. Livestock species, particularly
ruminants, are responsible for greenhouse gas emissions, including methane from
alimentary tract fermentation and nitrous oxide that may be emitted from decomposing
manure and fertilizer. Indeed ruminants constitute the biggest anthropogenic source of
methane emissions.
If the climate change with increasing global temperatures that we experience today will
continue at the current pace this will have enormous consequences for plant and animal
life, including the human population. A significant rise in the sea-water level is expected
due to melting glaciers and ice caps, combined with an increased frequency in extreme
weather events. The water use for livestock and accompanying feed crop production also
has a dramatic effect on the environment such as a decrease in the fresh water supply,
erosion and subsequent habitat and biodiversity loss. In order to limit temperature
increase to an acceptable level it has been calculated that in 2050 greenhouse gas
emissions need to be between 40 and 80% reduced compared with the levels of 2000.

Land use, including that for the livestock sector, has increased dramatically in the past
decades, leading to loss and fragmentation of habitats. As a result the total area of
habitats important for biodiversity such as rain forests and wetlands has decreased
dramatically. Since the total land area of planet Earth is finite, the land surface that can
be used for the livestock sector either for cropping or grazing is limited. The future
increase in agricultural production will therefore have to stem from intensified agriculture
on land already used, and/or from a more efficient conversion of plant material to edible
meat products.

An additional complication is that, particularly in developed countries, animal welfare has

become a societal issue and keeping animals for consumption is a matter of debate for a
significant part of the population.

5
Based on the above, several scenarios can be sketched for the future of meat production.
Continuation of ‘business as usual’ will lead to further environmental degradation and
destruction of habitats. Solutions are however within reach, many of which are at political
levels, such as subsidy programs for environmental services and regulation of land rights.
Solutions can also come from the scientific sector, however, although these will not be
immediate. These solutions will need investment in the form of time and money, and
possibly changes in consumer’s habits.

Scientific innovations can and should come from all sectors involved, such as transport,
machinery, fertilizers etc. An important contribution can be made via the generation of
meat alternatives. Many of these innovations will be improvements of already existing
concepts and products. Great leaps can be made by radically new concepts that require
‘out of the box’ thinking.

An example of such a
concept is to make
edible products from
skeletal muscle cells,
cultured from stem
cells, outside the
animal in a bioreactor.
Although this
technology is still at a
very early stage, and
requires many
fundamental problems
and questions to be
solved, this technique
holds great promise as
a solution to reduce
livestock’s impact on
the environment. A
schematic overview of
how this technology
could look like can be
seen in the figure left.

6
Cultured meat: a short history

The idea of culturing animal parts in vitro for human consumption is not new. In fact,
already in the 1920s it was Winston Churchill who predicted that within 50 years from
then, animal parts would be ‘cultured separately under a suitable medium’ (Churchill,
essay ‘Fifty Years Hence’ later published in ‘Thoughts and adventures’, 1932) 1. In 2002 a
study was published in which the possibilities of culturing animal muscle protein for long-
term space flights or habituation of space stations were explored. For this, muscle tissue
from the common goldfish (Carassius auratus) ranging
from 3-10 cm in length was cultured in Petri dishes. The
results from the experiments were rather promising and a
limited increase in cell mass was observed when the
muscle tissue was cultured with crude cell extracts. In
addition, the cultured explants were washed, dipped in
olive oil with spices, covered in breadcrumbs and fried. A
test-panel judged these processed explants and, although
actual tasting was not performed according to Food and
Drug Administration rules, agreed that the product was
acceptable as food 2. This promising study was not
continued because of a lack of further funding.

In the Netherlands, it was Willem van Eelen in the early

1950s who independently had the idea of using tissue
culture for the generation of meat products. Since at that
time the concept of stem cells and the in vitro culture of
cells still had to emerge, it took until 1999 before van
Eelen’s theoretical idea was patented. Van Eelen, as part of
the company VitroMeat BV, sought collaboration with
academic partners, and in 2004 a consortium consisting of the Faculty of Biomedical
Technology (Technical University Eindhoven), the Swammerdam Institute of Life Sciences
(University of Amsterdam) and the Faculty of Veterinary Medicine (Utrecht University)
together with an industrial partner, the meat concern Meester Stegeman (at that time
part of Sara Lee Foods Europe) was founded. This consortium was awarded a
SenterNovem research grant for 4 years to study the proof of principle of culturing
skeletal muscle cells from farm animal stem cells.

7
Experts’ opinions (1): Need for protein sources other than meat

• There is definitely a market for meat substitutes. Examples are legume-based and
mycoprotein-based meat substitutes. Pros: the production is efficient, and the technology is
already mature. Cons: allergies, meat texture difficult to replicate, aftertaste, psychological
resistance to “substitute”

• First of all, the demand for meat is at present increasing. It will not be possible to produce all
that meat in an environmental and animal friendly way. So there is a rather conventional meat
market for in vitro meat. Another smaller market comprises the vegetarians that do not eat
meat for ethical reasons.

• If with conventional meat we mean the production of meat via ‘factory farming than there
certainly is a market for alternative protein sources. These alternatives will include biological
production and extensive farming. These has as advantages that they will cause less animal
suffering, but as disadvantage that it still is a very inefficient way of protein production.
Additionally, ‘meat protein’ can be produced using plants, such as for instance Quorn, soy etc.
These have as advantages that they are animal friendly and sustainable. Such products are
however no solution for the craving for meat.

• On a society level there is definitely a need and a market for cultured meat, but there is no
need for a different new 'taste' or something like that. I think that in the future cultured meat
will be safer than conventional meat. The production will also be more sustainable, but I do
not think that it will have a wider range of applications. I also do not think that it can ever be
tastier than conventional meat.

• There is a need for other protein sources but plant proteins will suffice if we returm to the
consumption patterns of the 1960s-1970s.

• There is a need for protein sources other than meat but the development of cultured meat will
only be a success if there will be an impulse with public money. Animal proteins could also be
produced by transgenic plants.

• Other protein sources are available like fungi and plants (soy, peas) that have been used to
make a variety of good products that are not expensive. The disadvantage of these products is
the lack of a good texture and a taste that does not approach the original ‘benchmark’.

• Due to the non-sustainability of traditional meat production I think there is a huge market for
this. However, unless people change their habits dramatically I do not think other products
than something that looks like, and tastes like, ordinary meat will have a great success. If the
processing technology become very advanced one may think that the actual protein sources
can be from bacteria, algae, plants, yeast as well as well as from tissue culture. However,
given that a muscle tissue product was offered in the same price range as other products, I
would think that it would be preferred by the consumers.

• There is a need for protein sources other than conventional meat. Cultured meat may be the
preferred alternative because it is, unlike the other products, animal-derived and with respect
to composition most like meat as we know it.

8
Cell culture

Prokaryotic cells. Research into the use of unicellular (micro)-organisms as a source for
human consumption has gained momentum in parallel with the first oil crisis. In that
period, from the mid seventies onwards, a lot of research has been conducted to get to
the use of single-cell protein as animal feed or human food. In the same period there was
a growing awareness of the world-hunger problem, and this also stimulated research on
alternative sources of food.

The fact that the microbial biomass indeed is suited as a human food is clear for a long
time. For example, local African tribes have centuries-old traditions of eating prokaryotic
cells. Some of these tribes made the biomass of the cyanobacterium Spirulina a main
component of their daily meal. In addition, many fermented food products contain large
numbers of living bacteria, especially from the group of the lactic acid bacteria. However,
continued research on single cell proteins has made clear that a prokaryotic biomass (i.e.
bacterial cells) has a specific disadvantage when these cells are used as the main
component of the diet of a mammal 3 4. Because of the high nucleic acid content of this
particular type of biomass, and the uric acid produced from nucleic acids, the
consumption of this type of biomass increases the risk of kidney stone formation.
Nevertheless, prokaryotic biomass can, provided that the required precautions of
concerning application, etc. are taken, become very useful as a source of nutrients of a
culture medium for mammalian cells.

Research on single cell protein has provided a lot of information regarding the theoretical
conditions, as dictated by the laws of Nature, on the large-scale culturing of unicellular
organisms. In particular, knowledge has been gained about energy requirement (driven
by the question whether influx or efflux of heat is required), supplementation of oxygen,
optimization of the composition of the growth medium, and efficiency of the conversion
of catabolic substrates (i.e. nutrients) towards biomass 5. Regarding the latter it has
become clear that after optimization many cell types can be cultured with a conversion
efficiency of sugars to biomass of up to 50% (based on weight). This is, however, only
possible provided that the cells can be cultured under optimal conditions, that means
with saturating levels of oxygen. Without oxygen, metabolism can only take place by
fermentation, a form of catabolism that provides only about 10% of the yield of aerobic
metabolism.

This particular type of research on single cell protein has been almost completely ended
at the end of the seventies when, because of, among others, the oil crisis, the prices for
raw materials like methanol and petroleum for the production of proteins increased
dramatically. Other factors also contributed to the termination of these projects,
including the discovery of traceable amounts of petroleum in the protein products.
Because of this, the knowledge transfer to those working outside the microbiology field
has not been optimal.

9
Experts’ opinions (2): The most important reasons to produce cultured meat

• Potential impact on reducing cardiovascular disease and greenhouse gas emissions

• To discontinue the use of animals as bioreactors to grow food and other products

• Animal ethics. This can be “meat without suffering”. Also environmental.

• There are a number of advantages of replacing conventional meat with cultured meat: 1)
Prevention of animal suffering; 2) Prevention of food scarcity that can be expected with an
increasing world population; 3) Liberation of land for nature (including wild animals); 4)
Cultured meat will be more sustainable and better for the climate.

• For me the most important reason to produce in vitro meat would be consumer demand. More
and more people are interested in cultured meat, and it can be a very successful product.

• Sustainability of the meat supply to an increasing population society, that is gaining buying
power and demanding for more protein. Environmental concerns as even more fertile land
would be used to grow meat (cows).

• It could be sustainability but that has not been proven yet.

• Avoid the use of animals in factory farming.

• Allegedly the safety and sustainability of cultured meat production are the most important
reasons to produce cultured meat but I have my doubts.

• Personally, the major reasons are the environmental impact and the effect it will have on
animal welfare.

• Nutrition of a growing world population with reduced ecological impact.

Eukaryotic cells. At the end of the 1900s it was discovered that is possible to keep
tissue alive outside the animal from which it was derived for several days in a warmed
physiological salt solution. With this method, however, the cells will eventually become
necrotic and the tissue will disintegrate because of lack of a bloodstream and therefore
insufficient supply of oxygen and nutrients to the cells. Growth of such tissue is not
possible, and culturing is merely maintenance. Alternatively, single cells can be liberated
from tissues by enzymatic digestion, and these cells can be kept in culture as a
suspension or attached to a suitable surface such as that of flasks or dishes. Equivalent
to culturing of whole tissue, single cells derived from most specialized tissues have
largely lost the capacity to divide. Indeed, tissues of adult animals also have ceased to
grow; intrinsic cellular mechanisms take care of cellular senescence after differentiation.
In vitro culture of cells derived from healthy tissue therefore results in cells that stop
dividing after a few population doublings.

Cells derived from tumors have escaped the mechanism of limited cell division and are
therefore immortal; in other words they can divide indefinitely. Indeed, the first human
cells that have been cultured for a prolonged time were derived from a patient with an
cervical tumor in 1951, and since then these cells, referred to as HeLa cells, are still in
culture and have become the most widely used cell-type in biomedical research.
Alternatively, cells can actively be immortalized by deliberate genetic modification such
as through irradiation, chemical mutagenesis or targeted introduction of specific genes.
When such cells can be sub-cultured for a prolonged period of time they are referred to
as a cell line.

10
In warm-blooded animals, such as mammals, cells are kept at a temperature of around
37°C. For proper in vitro culturing of such cells it is important that the cells are kept at
the appropriate temperature and under an appropriate gas atmosphere. Cells are
therefore cultured in insulated incubators with a humidified atmosphere of 36-39°C and
5% CO2 in air (depending on the species from which the cells were derived). Nowadays
mammalian cells are routinely cultured in vitro for research purposes in complex liquid
growth media containing salts, glucose, amino acids and other nutrients. Since not only
mammalian cells, but also bacteria, fungi and yeast thrive well in these rich media it is
essential that all reagents and equipment used for culturing are made and kept sterile.
Cells are cultured in sterile disposable culture flasks or dishes, and passaging is done
with sterile disposable pipettes. To create a sterile environment when handling the cells
(e.g. for passaging)
these procedures are
performed in laminar
flow cabinets (also
called tissue culture
hoods), by which a
sterile environment is
created by air that is
drawn through a
filter, and thereby
sterilized, and flowing
towards the user
(crossflow) or from
top to bottom
(downflow).

Since cells of a cell

line continue to
proliferate, the
bottom of the tissue
culture flask or dish in
which they are
growing will
eventually become completely covered with cells. Most animal cells show the
phenomenon of ‘contact inhibition’, i.e. they cannot grow over each other. When the
surface is fully covered by a monolayer of cells, they will gradually loose viability and die.
To avoid this, cells are routinely passaged to new flasks or dishes before they have
reached the maximal surface density. For this passaging (or splitting), cells are exposed
to low concentrations of enzymes which will hydrolyze the extracellular matrix between
the cells (and the plate) so that a suspension of single cells is formed. Part of this
suspension is subsequently transferred to a new flask.

Culture media

The possibilities of culturing mammalian cells in vitro has evolved primarily because of
biomedical interest (particularly cancer research), much less so because of a more
fundamental cell biological interest. A much richer and more complex culture medium is
needed for culturing mammalian cells than the medium needed for culturing prokaryotic
cells. Dependent on the cell type, many specific growth factors, vitamins, lipids, amino
acids, etc., are needed to maintain the viability of the cells and allow them to replicate.
In addition, in contrast to prokaryotic cells, many mammalian cell types prefer to be
attached to a solid surface.

To encourage attachment mammalian cells are usually cultured in flat plastic (for
instance polyethylene) culture flasks that have a large surface/volume ratio. If needed

11
the flasks can be coated with proteins such as, for instance, laminin. More recently,
several mammalian cell lines have become available that can be cultured in suspension.
In comparison to bacteria these mammalian cells are extremely sensitive to mechanical
stress, for instance caused by the forces that occur by stirring. This poses severe
limitations to the type of bioreactors that can be used for such cells 6.

Historically, mammalian cells have been cultured in liquid media containing blood plasma
and serum, of which the exact content was a) unknown and b) variable. Particularly, the
addition of fetal calf serum, usually 5-20% of the final concentration, has been very
successful. Even today, the majority of mammalian cells is cultured in medium containing
a substantial percentage of fetal calf serum. Apparently, the fetal calf serum contains the
growth factors that are required for mammalian cell proliferation. A disadvantage of the
ample availability of fetal calf serum (commercially available) for biomedical researchers,
is the fact that it has delayed the development of alternative serum-free culture media 7.

Research on mammalian cell cultures that is aimed at the basic understanding of cell
physiology has been less successful because of the complex, largely unknown and highly
variable composition of fetal calf serum. Furthermore, research on the effects of
individual protein growth factors in cell culture media has been limited, because of the
high costs of purified growth factors.

Chemically defined culture media. In the past 10-15 years there has been a
considerable improvement in the development of growth media that support large scale
culture of mammalian cells. This has been largely made possible by the increased use
(and the added value) of particularly antibodies, growth factors, other recombinant
proteins, etc. Examples of mammalian cells that are cultured in media with bovine serum
include PER.C6 cells (a human cell line developed by Crucell) and Chinese hamster ovary
(CHO) cells 8. Importantly, several mammalian cell lines can now also be cultured in
suspension, without adherence of the cells to a solid surface. Using this suspension
method, culture systems can be used with a much better productivity per volume 9.

Culture media that contain fetal bovine serum are being described as 'complex' media, to
stress the fact that the media are composed of many different factors of which the exact
nature and concentration is unknown. This makes it practically impossible to accurately
define the chemical composition of such media. In the past few years it has however
become clear that it is also possible to compose growth media for mammalian cell using
combinations of limited numbers of purified chemical compounds. Since the exact
composition of these chemically defined media is known, these media can be produced
with the guarantee that they do not contain animal products. The risk of contaminating
mammalian cells with animal components that create a health hazard is thus eliminated.
Importantly, production of cells and cell-derived products is much better standardized
with these chemically defined media.

For the past 10 years, much research has been conducted on the growth of cells like the
CHO and PER.C6 cell lines in chemically defined media. The composition of various such
'chemically defined media' for specific cell type purposes has been published in the past
few years. The price of these media, however, is still so high that these media can only
be used on a commercial basis for the generation of products with a very high added
value (such as monoclonal antibodies or therapeutics).

Origin of components of culture media. For some products, for instance those used
in human therapies, it is extremely important that the culture medium does not contain
animal derivatives. In those cases it has been demonstrated that complex amino acid or
protein mixtures can - instead of being harvested from animals - also be derived from
plants. Most of the specific animal proteins essential for mammalian cell culture will not
be naturally occurring in plants. But by using recombinant-DNA technology it has become
possible to let plant cells produce such animal proteins 10. One or several genes that

12
encode for animal growth factors can be introduced into plant cells. The plant cells will
produce the proteins that can subsequently be isolated by fractionation. Using these
techniques it is nowadays possible to efficiently produce culture media which are
completely free of animal-derived products 11.

The elemental composition of all living cells, including bacteria, plants cells, and animal
cells is carbon (C), hydrogen (H), oxygen (O), nitrogen (N), sulfur (S) and phosphorus
(P) (in order of numerical contribution) and the minerals potassium (K) and magnesium
(Mg). Other minerals are also needed but only in minute amounts, and these are
sufficiently available in for instance normal tap water. The composition of living cells
dictates that all culture media have to contain these elements, and the cells have to be
able to extract them from the medium, preferentially in balanced proportions.

On the basis of our textbook knowledge of cellular physiology and more recently acquired
knowledge of the genome, it is possible to define and compose culture media for simple
cell types in which these elements are present as molecules in such a way that they can
almost quantitatively be transformed into cell material. Vice versa, it is possible to
analyze the efficiency of conversion of a specific compound (like glucose) into cellular
material, provided that the compound is present in sufficient amounts. For the latter type
of analyses a chemostat is used, while for less complex cell culture experiments batch
cultures are mostly used.

The most extreme example of a simple efficient culture medium is a medium that can be
used for growth of cyanobacteria. These types of bacteria can grow efficiently by only
using carbon dioxide (CO2), phosphate, nitrogen gas and rain water. By using energy
from sunlight they can produce cell material (i.e. grow) from these compounds. There
are also non-photosynthesizing cells that can use CO2 as a carbon source, but that
quality is relatively rare. Conversely there are various examples of chemotrophic
organisms for which one single carbon-containing compound suffices to synthesize all the
complex molecules necessary for the formation of new cells. Commonly these properties
are specific for bacteria, although certain lower eukaryotic cells, such as yeast, can also
exhibit this type of metabolism.

For the in vitro culture of cells from more complex organisms, such as mammalian cells,
the composition of the culture medium is much more critical and therefore more
demanding. Mammalian cells are dependent on the supply of specific molecules that are
normally produced elsewhere in the body (for instance growth factors) and on
compounds that are directly taken up from the food. In addition, these cells need to burn
or metabolize part of their nutrients to produce energy in the form of adenosine
triphosphate (ATP). Energy is required for cell maintenance but also for various synthesis
processes (also termed: anabolism).

When sugars (carbohydrates) represent the main component in nutrients, two alternative
metabolic routes can be exploited: aerobic catabolism and anaerobic fermentation, which
leads to lactic acid production. Both processes will take place via glycolytic degradation of
the sugar. The use of either the aerobic pathway or the anaerobic pathway greatly affects
the energy yield per gram of sugar but much is still unknown about how these catabolic
pathways are regulated (see below).

The elementary building blocks needed by mammalian cells as a carbon source can be
divided into three classes: sugars, fatty acids and amino acids. Each of these classes is
made up of numerous representatives. Mammalian cells can synthesize most of these
compounds from one class to another, except for a group of essential amino acids that
have to be taken up via the bloodstream, or, in case of in vitro culture, via the culture
medium. This implicates that an almost unlimited variability in culture media is possible.
Historically, the sugar glucose has been the most important source of carbon in tissue
culture media. A possible disadvantage of using this sugar as carbon source is that it will

13
steer the catabolic process of the cells for the generation of metabolic energy towards
fermentation, which is a rather inefficient process. This can be somewhat compensated
by the use of an alternative monosaccharide that is easier to catabolize, for instance
because the monosaccharide is less efficiently taken up by the cells. Examples of such
alternative monosaccharides are galactose and fructose. Alternatively, the supply of
carbon and nitrogen can be combined using specific amino acids. Specific fatty acids can
also be used.

If it is not essential that the culture medium is chemically defined, it can be decided to
provide most of the nutrients for cell growth via a complex mixture of 'undefined'
components. Such mixtures can be derived from for instance hydrolysates of yeast cells
(i.e. yeast extract). Alternatively, hydrolysates from plant cells can be used (see above).
A disadvantage of these complex media is that it is much more difficult to determine
beforehand whether all necessary elements are present at the correct balanced ratios.
The possibility exist therefore that large amounts of non-metabolized compounds remain
present in the medium after culturing of the mammalian cells as a waste.

Despite this disadvantage, it is to be expected that the use of complex mixtures of

components, such as extracts from plant cells, in combination with partly purified growth
factors is the most straightforward method to develop culture media that can be used for
the generation of cultured meat on a large scale. The required growth factors can be
synthesized by the same plant cells from which the extracts are derived. This will be
extremely cost-effective, and can significantly reduce the price of culture media. As an
extra advantage, this production of culture medium offers the possibility of creating a 2-
step process for the generation of in vitro meat, relatively similar to the in vivo situation:
plant cells grow and photosynthesize using light, and these same cells can produce the
main ingredients for the culture medium used for the culture of the mammalian cells (see
also box on photosynthesis).

Photosynthesis

In photosynthesis, plants and cyanobacteria use carbon dioxide, water and minerals, to produce
biomass. The most efficient photosynthesizers are the photosynthetic microorganisms:
cyanobacteria and (green) algae. This is due to the absence of non-productive parts in the latter
organisms, like stems and roots, to their more complete surface coverage and because of their
lower maintenance energy requirement. Overall this leads to an approximately 10-fold higher
photosynthetic biomass yield for the microorganisms as compared to plants (10 versus 100 tonnes
per hectare per year under optimal conditions).

If this photosynthetic biomass would be hydrolysed for subsequent use as a food supply for muscle
cells, and one would assume a conversion yield into muscle cell biomass of 0.25 (g/g), for a
production facility of 10 tonnes of meat product annually, a surface area of 80 by 80 meters of an
algal mass culture would be required to provide the main medium ingredient. This calculation
shows that it should be feasible to integrate the algal mass culturing facility and the muscle cell
production facility into one operational unit.

14
Skeletal muscle from stem cells

Although meat can be referred to as

edible animal tissue, with meat we
commonly mean the flesh part of farm
animals, in other words the skeletal
muscle tissue of these animals. Skeletal
muscle is composed of bundles of muscle
fibers. When muscle tissue is formed,
single muscle cells (myoblasts) fuse with
each other and form multinucleated
myotubes, which assemble to form
muscle fibers. In vivo, skeletal muscle
tissue specific types of stem cells
(satellite cells), that reside in the existing
muscle, can become activated in
response to specific local factors that are
generated for instance in case of trauma.

With tissue engineering it is attempted to

mimic neo-organogenesis outside the
animal (ex vivo). For medical purposes,
tissue engineering of muscle tissue from
human cells holds promise for the
treatment of various diseases such as
muscular dystrophy and spinal muscular
atrophy. Additionally, engineered muscle
Traditional meat market
tissue can be used for surgical
reconstruction that may be needed after
traumatic injury or tumor ablation. The scientific and technological know-how for the
engineering of skeletal muscle tissue for regeneration purposes is in essence identical to
the knowledge needed for the in vitro production of skeletal muscle tissue from farm
animals for consumption purposes. In order to be effective the latter purpose requires
much larger numbers of cells that with current technologies only can be achieved with
bioreactors. These bioreactors need to be developed. In addition, a change in consumer’s
mind set might be needed. Nevertheless, many alternative methods are available to grow
muscle cells. For example muscle-derived stem cells can be grown on the surface of
micro-carriers suspended in growth medium and proliferate almost indefinitely (> 100
doublings). Such systems may also be used for the large-scale production of muscle
cells, which then could be processed to a meat-related product, after differentiation of
these cells into myoblasts. One could even envisage an edible nature of the micro-
carriers. This approach will allow the use of much simpler bioreactors than in the
approach to produce tissue cells. These simpler bioreactors will briefly be addressed
below.

Stem cells. Production of tissue in vitro necessitates the use of large quantities of cells,
but differentiated cells exhibit a limited proliferative capacity. In contrast, cells exist that
maintain or regain the capacity to self-renew, which means that these cells continue to
proliferate. Stem cells are unique in their capacity to remain in a rather undifferentiated
state for a substantial amount of population doublings while retaining the ability to
differentiate into at least one specific cell type 12. Stem cells have a tremendous potential
for human medicine as these cells may be used to repair damaged or diseased tissues in
our body 13. Indeed, it has been hypothesized to amplify stem cells and subsequently
introduce these into patients, such as is currently performed in bone marrow
transplantations. Alternatively, stem cells can be used for so-called tissue engineering
techniques by which complete tissues or organs are constructed outside the body (in
vitro). Non-stem cell based tissue engineering already has a diversity of applications

15
ranging from formation of cardiac valve substitutes, construction of cartilage or
construction of a urinary bladder 14. The list of tissues that can potentially be engineered
with stem cells is even more extensive and includes blood vessels, bone, cartilage, skin,
liver, cardiac muscle and skeletal muscle.

Different types of stem cells have been identified and cultured in vitro. The classification
of stem cells is largely dependent on the tissues or cell population from which they were
derived. Stem cells derived from pre-implantation (blastocyst stage) embryos are known
as embryonic stem cells, whereas stem cells derived form postnatal tissue are generally
called adult stem cells. The variety in the types of adult stem cells is obviously much
larger than the variety of embryonic stem cells.

Embryonic stem cells. The fertilized egg is a totipotent (from the Latin ‘totus’ meaning
entire) cell that, in mammals, will give rise to all the structures of the conceptus, both
embryonic (the fetus) and extra-embryonic (the yolk sac and umbilical cord). This cell is
not a stem cell since it cannot self-replicate without differentiation. As the embryo
develops by rapid cleavage divisions, simultaneously the first differentiation process
takes place by which lineages are being segregated: the trophectoderm that gives rise to
extra-embryonic structures and the inner cell mass that will give rise to the developing
fetus. The cells of the inner cell mass of several animal species can be taken from the
embryo and grown in the laboratory. When these cells are cultured under appropriate
conditions the cells will not differentiate as they would do in the conceptus but will
duplicate while maintaining the capacity to differentiate into all cells of the fetus. These
cells are called pluripotent (from the Latin ‘plures’ meaning many) embryonic stem (ES)
cells. There is an understandable academic interest in embryonic stem cells. Firstly, these
cells can provide information about early differentiation processes since they can
recapitulate the sequence of processes that take place in embryos after implantation into
the uterus which still are poorly understood. Secondly, when differentiated to specific
tissue types the cells can be used for screening of drugs or toxic compounds. Thirdly, it
has been proposed that, after differentiation, these cells can be used in human medicine
for cell replacement. The first embryonic stem cell lines were derived from mouse
blastocysts in 1981 15 16 but from then it took another 14 years before well-characterized
embryonic stem cells were derived from another species (rhesus monkey) 17. Human ES
cells were first derived in 1998 from surplus embryos that had been generated in fertility
clinics 18. Remarkably, well-characterized embryonic stem cell lines from other animal
species, including farm animals, have not been described, although many attempts have
been made to generate such cell lines 19, 20. Only recently the establishment of true
embryonic stem cell lines from rat embryos has been described, but derivation of these
cell lines required the addition of various inhibitors to the cells 21 22.

Adult stem cells. Adult stem cells comprise a more heterogeneous and in certain
aspects less well understood population of cells. The bodies of animals (and humans)
contain different groups of cells that sustain a certain level of self-renewal and it is
generally thought that these cells are necessary for regeneration and repair of tissues in
which for instance the cells have a short life span or when the tissue is damaged by
disease or trauma. To what extent these cells are true stem cells, i.e. capable of
unlimited self-renewal, or that this would be a more transient capacity, so that they
would eventually become a differentiated cell type, is in many cases not completely clear.
Similar to embryonic stem cells it has been hypothesized that pluripotent adult stem cells
do not exist in vivo but arise during in vitro culture 23.

Independent of their possible in vivo occurrence, adult stem cells or progenitor cells can
be excellent sources for the generation of cultured meat. In contrast to embryonic stem
cells, adult progenitor cells have been derived from farm animal species such as pig 24 25
26
and cattle 27, 28; these cells have, at least to a certain extent, the capacity to
differentiate into skeletal muscle cells. A disadvantage of adult stem cells could be their
limited differentiation potential, meaning that these cells can only differentiate into a

16
limited number of cell types. This is a serious drawback if stem cells are to be used for
biomedical purposes, but for the generation of cultured meat this would hardly be a
disadvantage since cells do not need to differentiate to other cells than myoblasts. For
cultured meat, it is however important that the cells have a minimal self-renewal
capacity since most adult stem cell types cannot be cultured in vitro indefinitely.

iPS cells. Differentiated cells can also be reprogrammed into an embryonic-like state by
introducing four (or less) genetic factors 29. These cells, called induced pluripotent stem
(iPS) cells, behave exactly like embryonic stem cells in that they self-renew with
conservation of their truly pluripotent character if cultured under the right conditions.
With this technique both mouse and human cells have been reprogrammed 30, 31. This
new technology has sparked the attention of many biomedical researchers, most
importantly because it opens up a possibility of creating human, even patient-derived,
pluripotent cells without the ethical difficulties that accompany pluripotent cells derived
from human embryos.

For the production of cultured meat, bovine or porcine iPS cells could be useful cells, as
these cells can also differentiate into muscle tissue. There are, however, some difficulties
in this approach. For the first iPS cells that were derived, the DNA that codes for four
transcription factors was delivered to the cells by retroviral infection. As a result, the viral
DNA integrated into the genome of the targeted cells at (multiple) random locations may
lead to uncontrolled behavior of the cells, and makes them non-suitable for large scale
production of food. For the production of an edible product, cells that have been infected
with a retrovirus also cannot be used, because of potential safety hazards. Very recently
however, human iPS cells have been generated that were made free of vector and
transgene sequences and it is anticipated that this technology will advance rapidly32.

The iPS technology proceeds rapidly and many labs are working on these cells, and
indeed the first iPS cells from farm animal species have recently been described. In 2009
two articles were published that describe the generation of porcine iPS cells 33 34.
Although the use of these cells for the generation of an edible product is debatable, it is
clear that these results are very promising.

Bioengineering and bioreactors for tissue cultures

Adult skeletal muscle tissue is characterized by elongated, multinucleated cells with a

highly organized network of cytoskeletal proteins. Skeletal muscle cells can reach
considerable lengths and adult cells have a large myonuclear domain. Culturing skeletal

17
muscle cells in vitro, however, results in cells with a relatively high nuclear density when
compared to the in vivo situation. Also, the characteristic highly organized architecture of
the cytoskeleton often is lacking under in vitro circumstances. This indicates that the cells
are immature, which makes them at that moment an inefficient protein source. Such
immature myotubes are referred to as primary myotubes. After fusion of the myoblasts,
primary myotubes need a secondary differentiation step in order to mature. Only then,
they will produce sufficient cell mass to be an efficient protein source.
It would be ideal to refrain from the addition of growth stimulating factors such as
hormones in the production of cultured meat although the use of growth factors
(produced in plants or lactic acid bacteria) may increase the efficiency of myotube
formation (see below).

In order to produce sufficient biomass in vitro, the primary myotubes need to be directed
towards secondary differentiation. The approach may be threefold:

Co-culturing. Commonly, cell cultures are expanded and differentiated in monoculture,

which creates a well-controlled environment without the interaction of different cell
types. The in vivo situation is distinctly different from this. In skeletal muscle, nerve
cells, cells forming blood vessels and fibroblasts that form the basal membrane are
ubiquitously present. It has been shown that in vitro, the presence of fibroblasts
improves the efficiency of myotube formation. Accruing evidence suggests that the basal
membrane is paramount in directing regeneration and controlled growth in many tissues,
including skeletal muscle. It regulates the activity of locally active growth factors such as
fibroblast growth factor (FGF), transforming growth factor beta (TFG-β) and hepatocyte
growth factor (HGF). Since the presence of extracellular matrix (ECM) gives meat its
texture and ‘bite’, co-culturing primary myotubes with fibroblasts may be a good way to
improve the maturation and to produce a meat-like texture at the same time. Co-
culturing has been shown to be very effective in the in vitro engineering of skeletal
muscle when the highly proliferative fibroblasts are seeded as a low percentage of the
total cell population.

Mechanical stimulation. In the body, regular activity of the skeletal muscle is a potent
stimulus for increase of muscle mass. Upon contraction, myotobes produce the IGF-1
splice variant mechano growth factor (MGF) which increases protein synthesis in the
myotubes. Furthermore, internal forces that occur inside the myotubes during contraction
are potent regulators of the cytoskeletal arrangement. In culture, it takes some time
before myotubes have matured enough to contract in such a way that they exert
significant intracellular forces. Stretch can be applied to the culture to yield a similar
effect on the immature myotubes. This procedure has the added advantage that the
myotubes will align in the direction of the applied stretch, which will make electrical
stimulation more effective, and it does stimulate the production of ECM proteins by the
fibroblasts in the culture.

Electrical stimulation. In vivo, nerve activity controls muscle contraction. Contraction

of developing myotubes upon electrical stimulation will increase the maturation of their
cytoskeletal structure. Moreover, contraction increases the total amount of the contractile
proteins actin and myosin. Since the contractile apparatus makes up by far the largest
part of the mature myotube, increased expression of those proteins is necessary to
achieve the myonuclear domain similar to that in the in vivo situation. If the electric
stimulus of the culture, and thus its contraction, is correctly tuned with the activity of the
stretch device, it will be possible to submit the cultured myotubes and surrounding ECM
to eccentric contractions. In vivo, such contractions have been shown to be a potent
stimulus for increase in muscle mass.

Scale-up of muscle culture bioreactors. If, in an experimental small-scale bioreactor,

the optimal myotube maturation protocol is established, the production of engineered
meat can be scaled up in specifically designed bioreactors. It is possible to culture cells

18
on flexible membranes. Mounting the culture over a post and applying a slight vacuum
under that post will suck down the membrane and stretch the cells in culture. The
amount of stretch must be tightly regulated as too much stress will damage the cells and
lead to cell death.

Electric stimulation can be applied by putting two electrodes into the culture medium, in
close proximity to the cells. Preferentially, such electrodes are of an inert material and
span a significant area. Carbon plate electrodes would suit these requirements. Polarity
should be changed during stimulation to avoid hydrolysis of the medium and separation
of positively and negatively charged ions. Stimulation over larger areas should be
avoided since it requires high voltages, which will increase the temperature of the culture
with detrimental effects to the cells, and an increased evaporation that will affect the
osmolarity of the medium.

Whereas the proliferation of stem cells may ultimately be possible in suspension cultures
in large fermentors, the production by mechanostimulation of mature secondary
myotubes, that contain high amounts of the nutritious proteins actin and myosin, may
always require a solid support. Since diffusion of gases, nutrients and metabolic products
is limited, the muscle tissue only grows in two dimensions. This necessitates the harvest
of the product as large sheets and subsequent processing into consumer products.
Alternatively, deformable micro-carrier beads of edible (non-animal) material may be
developed that enable production of secondary myotubes in suspension. Myotubes may
be used as an animal protein ingredient in a wide variety of products. Alternatively,
products with a meat-like appearance and texture can be made by using, for example,
cell printing techniques. Addition of fibroblasts (for firmness) and fat cells (for taste) to
the myotubes might result in meat completely of animal origin.

Bioreactors for suspension cultures. The possibility to grow cells in suspension

simplifies their proliferation considerably. The most simple and straightforward way is to
do this in a batch culture, well-stirred to facilitate oxygen transfer into the growth
medium. During the past few years a large variety of simple, often disposable, stirred-
tank type of reactors have been described, often for use at an industrial scale. Proper
reactor design can add a lot to the rate of oxygen transfer. For detailed studies of
physiology it is most ideal to grow cells in a chemostat: By continuous supply of fresh
medium and removal of cells plus medium, a constant, time-independent, environment
can be created which gives optimal opportunity to characterize metabolic processes. This
system also allows continuous supply of cells. Nevertheless, chemostats are challenging
devices for use of slow-growing mammalian cells. Generally, batch cultures and
chemostats do not result in very high cell densities. This parameter can be further
increased by running so-called fed-batch cultures, in which – after an initial phase of
exponential growth – the limiting nutrient is added in a steady supply. Accordingly, cell
densities of e.g. CHO-cells used for recombinant antibody production can increase up to
100 grams of cells per liter 35.

As explained above, the degree to which metabolism of the mammalian cells proceeds in
the presence of saturating amounts of oxygen determines the overall efficiency of growth
of the cells. For this reason it is important to be able to measure oxygen concentrations
in situ. The development of fluorescence lifetime probes has recently simplified these
measurements considerably, to the extent that this is a very straightforward
measurement in most large-scale culturing facilities.

With the use of bioreactors for suspension cells many scale-up problems in the
production of muscle-derived cells can be solved by the lessons learned in the single cell
protein producing industries (in for instance the BioProtein process operated by
Norferm). Therefore, is will be possible to operate reactors with a productivity of > kg
scale.

19
Life cycle assessment of cultured meat production (cradle to cradle)

Livestock production in total currently occupies about 30 % of the ice-free terrestrial surface of our
planet and amounts to 18 % of the global warming effect (FAO, 2006). The consumption of meat
has been predicted to double between 1999 and 2050, which will further increase its negative
impact on the environment. Cultured meat will be produced in a reactor by growing only muscle
cells/tissues, instead of growing the whole animals. Its development has started mainly from
attempts of producing space food for astronauts, but it could potentially offer many environmental,
heath, and animal welfare benefits in the future. To assess the overall environmental impact of
cultured meat production, it would be advisable to carry out a formal Life Cycle Assessment (LCA),
to estimate the energy-, water-, and land-use and the greenhouse gas (GHG) emissions. Here we
present some preliminary results that are based on an LCA study by Hanna Tuomisto (University of
Oxford, UK) and Joost Teixiera de Mattos (University of Amsterdam) that is still in progress.

The basic production unit considered is one ton (i.e. 1000 kg, of which 30% is dry matter en 20 %
protein) of cultured meat. With due scientific development this cultured meat product should be
producible from hydrolyzed algal biomass (for sugars and amino acids) and recombinant growth
factors produced via lactic acid bacteria. The most relevant input factors are the production of the
input materials and fuels, and production of the feedstock (presumably ~ 1.5 ton biomass with 50
% (w/w) protein), and the fermentation of muscle cells. The cost of nutrients (mainly: K+, Na+ and
inorganic phosphate are negligible, because of the low amounts required (~ 1 kg potassium and
0.1 kg phosphate).
Taking proper literature data one can estimate that 500 m2 algal mass culture will be required (for
cost estimate: see (Chisti, 2008)). The resulting extract will be fed into a 1000 l stainless steel
fermenter, which will have to run for two months (for costs: see Akiyama et al., 2003). It was
estimated that the average energy use is 45-60% lower; greenhouse gas emissions are 80-95%
lower; land use is 98% lower and water use is 90-98% lower. Only poultry production has a lower
energy use compared to cultured meat.

References

Akiyama, M., Tsuge, T., Doi, Y., 2003. Environmental life cycle comparison of
polyhydroxyalkanoates produced from renewable carbon resources by bacterial fermentation.
Polymer Degradation and Stability 80, 183-194.

Chisti, Y., 2008. Response to Reijnders: Do biofuels from microalgae beat biofuels from terrestrial
plants? Trends in Biotechnology 26, 351-352.

FAO, 2006. Livestock’s long shadow –environmental issues and options. Food and Agricultural
Organization of the United Nations, Rome, p. 390.

20
Experts’ opinions (3): Technological challenges

• I assume that the technology would involve standard mammalian cell culture techniques
(anchorage-dependent cells on microcarriers cultured to high density in large-scale
bioreactors). This can be challenging. In addition to the standard engineering challenges
(sufficient mass transfer, mixing, etc. while minimizing shear profiles) the costs of goods will
need to be extremely low to be competitive.

• There are many challenges for the production of in vitro meat, but I would say the most
important is the achievement of adult phenotype muscle in vitro, which has never been
reported in the scientific literature. This is important because it is adult phenotype muscle
that has the protein density, texture, and other important features that make meat an
important dietary component. Failing this, what you will have will be more like loose
connective tissue, not "meat" in any real sense of the word.

• There are quite a number of challenges that have to be overcome: (a) Generation of stem cell
lines from farm animal species; b) Proliferation of these stem cells without differentiation; c)
Efficient differentiation into muscle cells; d) Large-scale production of myofibers; e) Large-
scale production of cheap growth media.

• Here are some hurdles to be overcome. What are the best (stem) cells? What is the best
design of the bioreactor? And not least in the early phase (if the bioreactor can produce just
cells on some surfaces etc): What is the best way to process the product? In vitro meat
involves some basic biology but there are no fundamentally basic problems.

• The technological hurdles that need to be overcome are: (a) efficient differentiation of the
cells; (b) engineering of tissue that is larger than only a few cell layers; (c) processing of the
cells to something tasty and visually attractive

• To make a product with the required structure that is safe.

• I would suggest research is done to move the muscle cells into a suspension type culture; this
would greatly simplify the manufacturing process and eliminate potentially harmful agents.
Consideration should be made as to the culture vessels, capacity, disposable or permanent
stainless steel.

• Large-scale production, reduction of costs and producing the right texture (bite) are important
hurdles.

• The most critical steps are to be able to produce a serum free cell culture medium at a low
enough price, to be able to produce 3d muscle tissue either from stem cells, satellite cells or
mesenchymal cells having a phenotype very close to normal tissue, and then to be able to
scale up this process to an industrial level.

• An important challenge is to increase the mass of the tissue from a few cell layers with natural
diffusion to many cell layers that still are able to take up nutrients efficiently. Another hurdle is
consumer acceptance. The consumer may not like ‘high-tech’ food.

• In vitro meat may be made (a) through suspension culture of single cells or small cells clumps
that are not differentiated or (b) by tissue culture of differentiated cells. Single cell or cell
clump culture is an established technology that is used for the production of
BioPharmaceuticals with (very) high added value, such as monoclonal antibodies. The major
challenges for meat production based on singe cell technology are in my opinion: (a) The very
high cost of goods of the current processes (must be performed under sterile conditions to
prevent microbial contaminations, use of expensive culture media, etc.) (b) The lack of
resemblance to meat (texture, appearance, etc.). Tissue culture for the generation of muscle
tissue, potentially including blood vessels etc., may result in something more resembling
meat. The costs however may be even higher and the technology and especially the scale-up
still need significant further development.

21
Ethical and societal issues

The concept of cultured meat appeals to many people. Since the pioneering work of
Benjaminson and Mironov, and particularly since the start of the Dutch Vitro Meat
Consortium in 2005, many articles appeared in newspapers and magazines about
cultured meat. These articles, and also programs on radio and television, helped to start
a discussion about a future technology that is not even in its infancy. It may seem
somewhat premature to start a societal discussion at such an early stage. However, food
is a subject that evokes many emotions: it is, if we recall the turmoil associated with the
introduction of genetically modified foods, a good idea to educate citizens about all
aspects of producing animal proteins for human consumption by cultured cells.

Does food in general evoke strong feelings; meat is a commodity that sometimes is even
controversial. Indeed, it can be stated that controversy exists in the minds of people: the
different opinions about meat between citizens and consumers are striking. One may
argue that the disconnection between animals and meat that exists in consumer’s minds
would make it more difficult to lure people and convince them to eat cultured meat. On
the other hand, if a product is available that has all the characteristics of meat (like good
sensory properties), but is not produced from animals, citizens would probably buy it for
a variety of reasons. These could range from worries about the environment or animal
welfare to the fear of zoonotic diseases.

Unfortunately, no scientific studies have been conducted yet on society acceptance of

cultured meat production. It must be said, however, that the authors experienced a
positive attitude by the audience in national and international debates. Also web-surveys
and internet-discussions indicated that many people are in favor of the production of
‘victimless’ meat (see box). Other people, however, have expressed feelings of disgust
because they consider cultured meat as another step away from ‘natural’ food
production. In addition, the cultural background probably also plays an important role.
Interesting in this respect is that among the many requests for interviews none came
from journalists of Mediterranean countries. In contrast, dozens of requests came from
the Nordic countries (and many more from English- and German-speaking countries).
Social-cultural aspects of acceptance of cultured meat are probably important and should
be subjects of investigation. In addition, it will be interesting to compare the views of
people on current intensive farming with organic farming and the production of animal
proteins by cell systems. Replacement of (part of) the conventional meat production by
cultured meat would entail social, technological and economic changes. However, it is to
be expected that these transitions occur relatively slowly and farmers do not need to be
worried.

22
Best of both worlds

In their paper ‘Vegetarian Meat: Could Technology save Animals and Satisfy Meat Eaters’ Hopkins
and Dacey argue that cultured meat would be the best of both worlds: eat meat and not harm
animals. They specifically analyze the most important and most morally complex objections to
cultured meat and conclude:

“Cultured meat has the potential to make eating animals unnecessary, even while satisfying all the
nutritional and hedonic requirements of meat eaters. It also has the potential to greatly reduce
animal suffering. As such, the development of cultured meat would seem to have a moral claim on
us – whether moral vegetarians (for whom a greater opportunity exists to reduce animal suffering)
or conflicted meat eaters (for whom practice could now cohere with beliefs) or even for recreational
hunters (for whom ancillary arguments about providing food would fall by the wayside and require
defenses of getting pleasure from animal death per se). The development of cultured meat, then,
is not merely an interesting technological phenomenon, but something that we may be morally
required to support. In doing so, we recognize that morality is not something that must simply
respond to new technologies as they arrive, throwing us into confusion, but rather that morality
may champion and assist in the development of new technologies, as a step toward the production
of a world that in fact, and not merely in ideal, mirrors the moral vision we possess for it.”

Holmes, P.D. and Dacey, A., 2008. J. Agric. Environ. Ethics 21, 579-596.

Challenges

Cultured meat technology is still in its infancy. Important challenges for the production of
animal proteins by cell systems are:

1) Generation of suitable stem cell lines from farm animal species

2) Safe media for culturing of stem cells

3) Safe differentiation media to produce muscle cells

4) Tissue engineering of muscle fibers

5) Industrial bioreactors

6) Food processing technology

7) Customer preferences and adapted marketing strategies

1. Generation of suitable stem cell lines from farm animal species. For the
generation of cultured meat it is important that cells from farm animal species (like pig,
cattle or chicken) can be cultured in large quantities starting from a relatively small
number. Stem cells would fit this criterion. Embryonic stem cells have been cultured for
many generations. Since under normal conditions each cell will duplicate roughly every
day, if one would start with 10 cells within 2 months these 10 cells would have
proliferated to 1.2 E19 cells. If we assume that 0.5 gram of meat is approximately
equivalent to 116 million cells, this would amount to about 50,000,000 kg of meat
(assuming that these cells have also differentiated to skeletal muscle cells).

Culturing embryonic stem cells would be ideal for this purpose since these cells have an
(almost) infinite self-renewal capacity. In theory, one such cell line would be sufficient to
literally feed the world. Despite attempts from many research groups including our own
36
it has so far not been possible to culture cell lines with unlimited self-renewal potential

23
from pre-implantation embryos of farm animal species. Many attempts have been made,
however, to generate permanent cell lines from embryos of animals including hamster 37,
mink 38, rabbit 39, pig 40, cattle 41, sheep 42and goat 43. Until now, true embryonic stem
cell lines have only been generated from mouse, rhesus monkey, human and rat
embryos. It is to be anticipated that the social resistance to cultured meat obtained from
mouse, rat or rhesus monkey will be considerable and will not result in a marketable
product. The reasons why it has not been possible to culture embryonic cells from farm
animal species without differentiation of these cells, and thereby a loss of proliferative
capacity, is unknown. Apparently, the culture conditions as have been derived to keep
mouse and human embryonic cells undifferentiated are different from the conditions that
will be required for embryonic cells of farm animal species. Most likely, fundamental
research on the early development of embryos of these species can shed light on this
issue and thereby provide clues on how to maintain cells undifferentiated.

Another strategy would involve the use of adult stem cells from farm animal species.
Adult stem cells have been derived from, for instance, pig and cattle. A disadvantage of
these cells is that their in vitro proliferation capacity is not unlimited. However, it has
been demonstrated that these cell can proliferate in vitro for several months, and that
these cells also have the capacity to differentiate into skeletal muscle cells, albeit not
very efficiently. Nevertheless, for now, adult stem cells are the most promising cell type
for use in the production of cultured meat.

2. Safe media for culturing of stem cells. Mammalian adult and embryonic stem cells
are routinely cultured in a rich broth that contains salts, sugars, amino acids and other
supplements. For proper culturing, serum from fetal calves (aptly called fetal calf serum)
is added to the medium. Exactly which factors in the serum are important for proper cell
growth is unknown, but it is well-accepted that some batches of serum are better suited
for the culture of stem cells than others. Therefore, laboratories usually test different
batches of fetal calf serum for their suitability for stem cell culture. For the generation of
an animal-free protein product, the addition of fetal calf serum to the cells would not be
an option and it is therefore essential to develop a serum-free culture medium. Indeed
such media have already been generated and are available from various companies for
biomedical purposes; however, their price is incompatible with the generation of an
affordable edible product. A cell culture medium therefore has to be developed that
enables culturing of cells for an affordable price but that does not contain products of
animal origin.

Mouse and human embryonic stem cells are cultured on a feeder layer of embryonic
fibroblasts for the maintenance of stem cell characteristics, but recently culture media
and methods have been developed that do not require culturing on layers of feeder cells.
The medium that will be developed for the culturing of farm animal stem cells should also
be compatible with feeder free culture. Adult stem cells are less dependent on such a
layer of feeder cells for their proliferation.

3. Safe differentiation media to produce muscle cells. For stem cell culturing it is
important that these cells remain undifferentiated and maintain their capacity to
proliferate. For the production of cultured meat these cells have to be differentiated to
skeletal muscle cells. This differentiation process needs to be specific and efficient:
Specific in the sense that no other cell types must be formed and efficient in the sense
that the majority of the cells will differentiate into muscle cells. Differentiation of the cells
will have to be initiated with a specific (set of) growth factor(s). Currently, the most
efficient method to let (mouse) stem cells differentiate into skeletal muscle cells is to
culture them in a medium that contains 2% horse serum instead of 10 or 20% fetal calf
serum. For the generation of cultured meat, however, it is essential that the cells are
cultured and differentiated without animal products, so without horse serum. A
chemically defined culture medium therefore has to be developed that (efficiently)
enables the differentiation of stem cells to skeletal muscle cells.

24
4. Tissue engineering of muscle fibers. In the absence of blood flow that provides
oxygen and nutrients to the cells and removes metabolic end products, the possibility to
form a 3-dimensional structure of cells is restricted. The in vitro culturing of cells is
limited to only a few layers of cells, which would represent tissue of a thickness of less
than a millimeter at maximum because limitations in nutrient diffusion. This problem also
has to be addressed by those who pursue tissue engineering for biomedical purposes.
Culturing of cells on biological or synthetic scaffolds may provide a solution to this
problem. In this way the scaffold would provide shape and structure to the engineered
tissue. In the case of cultured meat, the scaffold should be either edible or
biodegradable. A more straightforward solution would be the processing of thin layers of
cells into a (meat-like) product.

5. Industrial bioreactors. Production of sufficient numbers of muscle cells for the

generation of edible products will require large-scale culturing. Since stem cells and
skeletal muscle cells require a solid surface for culturing (in contrast to, for instance,
blood cells that can be cultured in suspension) a large surface area is needed. Culturing
should be performed in large bioreactors containing many sheets of printed cells, cells
grown on scaffolds, or cells cultured on microspheres that can be kept in suspension.

In a model for mammalian muscle cells in a 3-D matrix, cells are supported and supplied
within the bioreactors such that the natural tissue builds ‘self-organizing constructs’,
where the 3D self-organization of tissues allows the provision of the nutrient supply,
aeration, waste removal etc. (one of the models from the in vitro meat economics study;
see supplementary materials). This subsequently allows cell, and consequently, tissue
growth. It was concluded from this study that several areas require further development:
(1) the mechanism for cell support and growth within the bioreactor; (2) the mechanism
for harvesting; (3) the need for pharmaceutical grade cleanliness and ability to sterilize;
(4) instrumentation and process control.

6. Food processing technology. Depending on the starting material (suspensions of

small myotubes, myofibers on scaffolds, microspheres, etc.) new technologies need to be
developed to make attractive products. It is expected that at first small pieces of cultured
muscle fiber will be produced that serve as raw materials for making a wide variety of
products (‘cultured meat inside’).

7. Consumer preferences and adapted marketing strategies. Why would a

consumer prefer cultured meat if meat from animals is available? If it is because of
sustainability or animal welfare issues, why not eat less meat and instead, more plant
proteins? Many questions can be asked and many factors determine consumer
preferences. Studies are required to determine the preferences and, consequently, the
marketing strategies. Interesting in this respect is the summary from a workshop on
cultured meat held on December 3rd, 2008 as part of an NWO application (see boxes).
This workshop was organized by scientists from Wageningen University (Dr. Cor vd
Weele and Dr. Hilde Tobi) and Utrecht University (Prof. dr. Henk Haagsman and Dr.
Bernard Roelen).

25
Participants of the valorisation workshop

Name Affiliation Field of expertise

Ing. C.J.G. Wever Ministry Agriculture, Nature & Food Quality
Food Quality (LNV)
Dr. J.C. Dagevos LEI (WageningenUR) Sociology of consumption
Dr. B.J. van ’t Hooft, Stegeman Meat industry
Drs. Ing. J.A.C. Peters Voedingscentrum Food quality & safety
Ir. C. van Dooren Voedingscentrum Food quality & sustainability
Mr. H.P. Voormolen Albron Catering & sustainability
V. Helder Vegetariërsbond Vegetarian concerns
Ir. C.P.G. Driessen Applied Pilosophy Group Technology & animal ethics
(WageningenUR)
K. Gruijters Katja Gruijters Food Design Food design

The workshop was moderated by Dr. Nicolien Wieringa of Groningen University. Several
important conclusions could be drawn from this workshop, in particular with respect to
consumer acceptance.
First of all it was acknowledged that various different consumer groups can be identified
in relation to cultured meat:
• Those that would readily eat cultured meat.
• Those that would accept cultured meat based on religious or fundamental principles
(for instance vegetarians).
• Those that do not immediately reject cultured meat, but will only accept when the
sustainability is demonstrated.
• Those that reject cultured meat.

During the workshop, three main areas of valorisation were recognised:

1) Sustainability
2) Societal embedding: reception and demand
3) Commercial production

Ad 1) Sustainability.
It was agreed between participants of the workshop that gains of sustainability are of
central importance for the success of a cultured meat product, and a basic requirement
for success. These gains in sustainability need to be calculated and indeed properly
demonstrated for credibility. It was also recognized that the sustainability of cultured
meat is not static but will be a topic of continuous development and improvement, most
likely in combination with other activities/discoveries.

Ad 2) Societal embedding: reception and demand.

On the one hand cultured meat is an alternative for 'traditional' meat from animals. For
many people meat is an important aspect of their daily food, because of the taste and
nutritional value and it is therefore important that an alternative should have a similar
taste and nutritional value. On the other hand it is important for a product to have its
own image and can be considered as something totally new and different. From this
perspective, it is not essential for the product to resemble and should in fact be clearly
distinctive from traditional meat.
From a perspective of social acceptance, the technological character of cultured meat can
have a negative value, and associations with Frankenstein, cloning, transgenesis and
unknown risks are close at hand. The name of the product can be of importance in this
aspect. In vitro meat and cultured meat are likely not to be names of consumer products
that will appeal to the average consumer. Marketing strategies designed by experts can
become very important in this respect. Transparency, that is a clear picture of the whole
production process could help in gaining public acceptance, whereby one can imagine
production in open agro-parks where in vitro meat cultures are being 'fed' with the

26
products of cultured algae. Technology would thus not increase but rather decrease the
distance between producers and consumers. It has to be mentioned that in the western
society, the distance between producers and consumers is already quite big, as one
thinks about (commercially successful) consumer products such as beer, soda drinks,
different types of candy, instant meals. This distance is therefore not necessarily
negative for commercial success, and could in fact also be beneficial (consumers simply
do not want to know). Proper consumer feedback and validation studies are therefore
essential for the introduction of a product like cultured meat.

Ad 3) Commercial production.
A requirement for commercial production is societal demand. Industry is clearly not
interested in a product that will not be profitable. On the other hand, there is a serious
commercial interest provided that technology has advanced more. In comparison with
animals, a product from a bioreactor could be attractive as it does not come with all the
vicissitudes of animals such as uneven growth, disease, consequences of stress, animal
killing etc.

During the workshop, marketing strategies for cultured meat were also discussed.
Because of the technology involved, it as suggested that the focus should be more on the
characteristics of the product rather than on the production process. Independent of
this, it was generally agreed that a broad acceptance is necessary for the product to
become commercially successful. The product should not only appeal to vegetarians or
an elite group of consumers. It was suggested that well known media cooks might be
called in for marketing help. Also, availability and price of traditional meat can be
important decision factors. The question arose whether a demand for cultured meat will
have to wait for a serious crisis in the availability of traditional meat. Finally, it is likely
that first products will not be end products but ingredients, which will most likely be less
offensive for many consumers.

Traditional product

27
Conclusions from the valorisation workshop (1)

Which factors determine success or failure of artificial meat?

Sustainability: Participants overwhelmingly emphasized sustainability as the central success

factor. They also stressed the need to quantify the gain in sustainability of cultured meat in
comparison with normal meat.

Name: The name of the product (in vitro meat; kweekvlees) was seen as a big risk factor. For
some, this had to do with Frankenstein-like associations, while others thought cultured meat
should not be presented as meat (see below).

Meat or no-meat? On the one hand, cultured meat is explicitly introduced as an alternative to the
problems of normal meat. Because people like meat, cultured meat should be as meat-like as
possible in order to be a real alternative. On the other hand: a new product needs a profile of its
own, otherwise it will not be able to compete. Some participants thought it should therefore not be
meat-like at all.

Process or product? On the one hand, food production as a process should be transparent, and
this is especially true for technologically produced food: “the mistakes of the GM debate should not
be repeated”. In order to expose big strengths of the product, such as sustainability, transparency
is also needed. On the other hand: embryonic stem cells, bioreactors and high tech production
trigger associations with cloning, Frankenstein etc. (Cultured meat therefore amounts to a “worst
case challenge for a marketing campaign”, as one participant of the valorization workshop put it).
Would it therefore be better to focus on the characteristics of the product rather than the
production process?

Place-independence. Cultured meat is not bound to soil or place, which opens up possibilities for
new places of production and for alternative land use. Given the fact that 80 % of agricultural soil
is directly or indirectly used for husbandry, this is not a trivial perspective.

Synergy. Cultured meat could be combined with other sustainable innovations. Algae cultures, for
example, might provide meat cultures with nutrients and energy.

Reliability. From a commercial perspective, animals are notoriously unreliable as a raw material,
due to illness, stress and uneven growth. Cultured meat is potentially a much more reliable
alternative.

Conclusions from the valorisation workshop (2)

What would an introduction campaign focus on?

Broad acceptance needed. In order to make a sustainability-difference, cultured meat should not
just appeal to vegetarians or other small elites; broad acceptance is needed. Wellknown media
cooks (etc.) might be called upon for marketing help.

Demand: crisis needed? At the moment, protein is not in short supply in the Western world. In
countries where protein supply is a problem, consumers might insist on “real” meat as soon as
increasing wealth allows this. The question arises whether substantial demand for cultured meat
will have to wait for a serious crisis in the availability of protein.

What kind of product? Participants thought it most likely that the (first) products will not be end
products, but ingredients. It is not easy to think of a campaign for a “mere” ingredient. Completely
new products gave more inspiration, even though their production may not be realistic, at least in
the short term.

28
Current developments in cultured meat research

The Dutch 'In vitro meat' project funded by SenterNovem. After having obtained a
patent an international patent on the 'industrial production of meat from in vitro cell
cultures' in 1999 (international application number PCT/NL98/00721), Willem van Eelen
sought collaboration with academia and industry to obtain funding in order to realize his
dream. In 2004, Utrecht University (Faculty of Veterinary Medicine, prof. dr. Henk
Haagsman and dr. Bernard Roelen), Eindhoven University of Technology (Department of
Biomedical Engineering, dr. Carlijn Bouten), University of Amsterdam (Swammerdam
Institute for Life Sciences, prof. dr. Klaas Hellingwerf), Meester Stegeman BV (at that
time part of Sara Lee foods, Peter Verstrate) and Vitro Meat BV (Willem van Eelen)
submitted a grant proposal to SenterNovem that was honored, resulting in the start of
the Dutch In Vitro Meat project in May 2005 that lasted until April 2009.
The project was subdivided into 3 different areas: a) stem cell biology, conducted at
Utrecht University; b) tissue engineering, conducted at Eindhoven Technical University;
and c) culture media, conducted at the University of Amsterdam.
To conduct this research, at Utrecht University 2 PhD students were appointed for a
period of 4 years, and 1 post-doctoral fellow for a period of 2 years. Also at Eindhoven
Technical University 2 PhD students were appointed for 4 years, and 1 post-doctoral
fellow for a 2 year period. At the University of Amsterdam, 1 technician was appointed on
this project for a period of 4 years.

Experts’ opinions (4): Presumed qualities of cultured meat

• There is no “objective” reason why in vitro meat should not have the same properties as
conventional meat. Safety and sustainability are two major issues that favor cultured meat.
• Production may be less safe because of risks of contamination.
• Cultured meat will be safer and more sustainable than conventional meat.
• Cultured meat may have a completely different risk profile than conventional meat. Much
attention should be paid to the safety of added substrates and other compounds of the culture
medium. So, less risks with respect to microbial contamination but more risk of contamination
of substrates.

29
Stem cell biology. To be able to generate sufficient skeletal muscle cells, these cells
have to be derived from stem cells. Stem cells have the capacity to self-renew, i.e. the
capacity to replicate without losing their characteristics. Also, for the generation of
cultured meat it is essential that the cultured stem cells can specialize into skeletal
muscle cells. Before the start of this project, no well-characterized stem cell lines of farm
animal species were available that fulfilled these criteria. At Utrecht University the focus
has therefore been on the generation of stem cell lines of pig origin, both of embryonic
and of adult origin. For the stem cells of embryonic origin, embryos of blastocyst stages
and inner cell masses of these embryos have been cultured at a variety of culture
conditions. Both in vitro produced and in vivo produced embryos have been used, but the
in vivo produced embryos gave far better results than the in vitro embryos, which is an
important finding. The main
difficulty of propagation of these
cells was to keep the cells
undifferentiated. Indeed the cells
could be cultured for few
passages, after which they had a
tendency to spontaneously
differentiate, primarily to a neural
lineage.
Cells have also been collected
from skeletal muscle of newborn
piglets. It is to be expected that
skeletal muscle tissue contains a
small fraction of stem cells, and
these cells can potentially be
used for propagation in vitro and
differentiation to skeletal muscle
cells. Indeed, cells were sorted
from the population of skeletal
muscle cells from newborn
piglets, and these cells have been
cultured in vitro for several
months, while maintaining the
capacity to specialize to skeletal
muscle tissue, and can therefore
be referred to as adult stem cells
(despite the fact that the original
cells were isolated form newborn
piglets they are still, confusingly,
referred to as adult stem cells). Cells are cultured in special incubators
The differentiation procedures
that are used until now are not very effective and other cells or differentiation conditions
need to be developed for the generation of large quantities of skeletal muscle cells.

Tissue engineering. The differentiation of naïve cells to skeletal muscle cells is rather
complicated and is dependent on many factors. By creating conditions that resemble the
environment, or niche, that stem cells undergo in vivo when stimulated to differentiate to
muscle cells, the group at Eindhoven Technical University has investigated which factors
are important for efficient differentiation. In particular, the influence of substrate
elasticity and protein coating has been investigated. A difficulty in these studies has
been lack of properly characterized stem cells of farm animal origin. For this reason,
stem cells of mouse origin were used. It is anticipated that results from these studies can
be directly used for studies with porcine cells.
When stem cells are freshly isolated from muscle tissue, their differentiation capacity has
been reported to be limited. One of the reasons for this limitation could be a lack of
pivotal niche factors, for instance substrate elasticity and extracellular matrix proteins.

30
The proliferative and differentiative response of primary (ie freshly isolated) mouse
muscle cells was investigated to different substrate stiffness and protein coatings in vitro.
It was discovered that the capacity to divide was primarily influenced by substrate
elasticity, with a substrate elasticity of 21 kPa, similar to the physiological elasticity of
skeletal muscle, being optimal. Differentiation capacity on the other hand was less
dependent on surface elasticity, but very much dependent on the type of surface protein,
with laminin and poly-D-lysine being the best stimuli for differentiation of myotubes.
In addition to these findings, researchers at Eindhoven Technical University also studied
the final maturation of skeletal muscle cells by electrical and mechanical stimulation. It
has been identified that, using the mouse C2C12 cell line as experimental model cells,
electrical stimulation leads to further maturation of differentiating myotubes.
Currently experiments are ongoing that analyze the influence of physical loads on cellular
differentiation. For this a computer-regulated bioreactor is used that uses vacuum
pressure to apply strains to cells (cyclic or static) that are cultured on flexible-bottom
culture plates. This can provide information on the behavior of muscle cells in a
mechanically-active 3 dimensional culture environment.

Culture media. At Amsterdam University studies have been conducted to optimize

culture media for in vitro cultured meat. Importantly, culture media to produce animal-
friendly meat should not contain animal derived factors such as fetal bovine serum, but
should contain enough nutrient including sugars and amino acids. Photosynthetic algae
can use energy from sunlight to produce nutrients for stem and muscle cell cultures.
Progress has so far mostly been on theoretical and small-scale experiments, and a
suitable culture medium is not yet available. A start was made with the production of
required growth factors by bacteria.

31
Experts’ opinions (5): Organization of research on cultured meat

• First and foremost, I would start with some focus groups to see what kind of reception this
conceptual product would receive in the market place. If there is a strong market, then
compile a team of large scale mammalian cell culture professionals.

• An ideal approach is one that attracts significantly more funding to accelerate research. It is
important to think strategically about which research milestones will attract further funding. I
believe the most important of these milestones is the production and human consumption of a
small amount of ground meat, in a form that resembles an existing product. Most of the public
and potential funders will remain skeptical about the technology until this milestone is
achieved. Even if it is a “$10,000 chicken nugget,” the case can be made that the costs will
decrease with improvements in the technology, but it must be shown that a product is
possible.

• Other analytic work can help support the case for cultured meat, including economic feasibility
analysis, environmental life cycle analysis, regulatory analysis, and consumer surveys. There
is opportunity for international collaboration, as there are many different proficiencies required
for success. Partners in East Asia should be especially valuable, as this technology could have
the greatest impact in that region. Other important things moving forward are transparency
about the research, in order to build trust with public and potential funders; and publication of
results in peer-reviewed literature, in order to build trust with scientists.

• I believe there should be a large effort to systematically investigate various animal stem cell
types, nutrition and bioreactor design.

• The most ideal approach would be when there is a collaboration between technological, cell
biological and ethical-social sciences.

• Most importantly, research on in vitro meat should be done in an international project,

perhaps even beyond EU only. Aside from technical aspects there should be research directed
on consumer acceptation.

• This project should be developed by a multidisciplinary group involving experts from different
areas going from cell culture to bioprocess engineering and polymer and food technologists.
An international consortium should also be created.

• First, public money is required. Stakeholders (consumers, producers, scientists) should act
together to start research on a large scale. It is pointless to only fund a small project.

• The market should decide if and how the technology must be developed. I am doubtful that
companies are interested at this point.

• The challenges involved are daunting and they cannot be met a small group of academics in
collaboration with a company or two. Like the thermonuclear fusion project, in vitro meat
technology development will need international concerted efforts from a whole range of
academic and engineering disciplines for many years before it becomes mature enough to be
taken over by commercial interests. An international body or society is needed to guide the
technology development and fuel it by attracting money from various sources.

• In my opinion, it is extremely important that there is sufficient attention for the societal
aspects of cultured meat, and that grant money is also used to study these aspects. For this
reason, I would plead for an interdisciplinary research centre.

• First step should be to develop the technology conceptually, including consumer acceptance.
Second step should be to mathematically model these concepts and to define under which
conditions they can become economically viable, so to define the development focus. Then
technology development programs could be initiated.

• First phase: make a good team consisting of cell biologists, microbiologists, engineers, etc.
Determine the best approach. Next, start a biotech company to test process technology. Sell
technology worldwide.

32
Scientific publications from the 'In vitro meat' project
o Roelen BA, Lopes SM. Of stem cells and gametes: similarities and differences. Curr
Med Chem, 2008; 15(13):1249-56.
o Wilschut KJ, Jaksani S, Van Den Dolder J, Haagsman HP, Roelen BA. Isolation and
characterization of porcine adult muscle-derived progenitor cells. J Cell Biochem,
2008; 105(5):1228-39.
o Boonen KJ, Post MJ. The muscle stem cell niche: regulation of satellite cells during
regeneration. Tissue Eng Part B Rev, 2008; 14(4):419-31.
o du Puy L, Chuva de Sousa Lopes S, Haagsman H, Roelen B. Differentiation of porcine
ICM cells into proliferating neural cells. Stem Cells Dev, 2009; doi:
10.1089/scd.2009.0075.
o Boonen KJ, Rosaria-Chak KY, Baaijens FP, van der Schaft DW, Post MJ. Essential
environmental cues from the satellite cell niche: optimizing proliferation and
differentiation. Am J Physiol Cell Physiol, 2009; 296(6):C1338-45.
o Du Puy L, Beqqali A, Monshouwer-Kloots J, Haagsman HP, Roelen BA, Passier R.
Cazip, a novel protein expressed in the developing heart and nervous system.
Accepted for publication.
o Wilschut KJ, Haagsman HP, Roelen BA. Extracellular matrix components direct
porcine muscle stem cell behaviour. Accepted for publication.
(publications recorded up to Oct 1, 2009; more manuscripts in preparation).

Press interest. Immediately from the start of the project the interest of the popular
press, both national and international, has been rather overwhelming. Indeed interviews
have been made and published in various media. In addition, particularly Henk
Haagsman and Bernard Roelen have been invited for lectures, seminars and workshops
on in vitro meat (both nationally and internationally) and have given a substantial
number of interviews for all types of media. This has helped the researchers to get a
feeling of the public opinion about in vitro meat. This information is crucially important
when future strategies are to be designed for the correct commercialization of a product
such as cultured meat.
Simultaneously, for public acceptance it has been and still is of importance to educate
future consumers correctly about cultured meat. Part of the media interest may come
from some form of sensationalism, but part of it is also driven by general interest and
sincere concern for the environment and animal welfare. A selection of news media that
interviewed the authors is indicated below.

National: Newspapers: NRC Handelsblad, Volkskrant, Algemeen Dagblad, Financieel

Dagblad, Spits, De Pers; magazines: Intermediair, Natuurwetenschap & Techniek, Vrij
Nederland; Radio: Radio 1 journaal, Vroege Vogels (VARA), Hoe? Zo! (TELEAC), Llink
FM; TV: Nieuwslicht (VARA), Noorderlicht (VPRO), RTV Utrecht, Dierenduel (VPRO).
International: Newspapers: De Standaard, De Morgen; Gazet van Antwerpen (Belgium),
Süddeutsche Zeitung; Frankfurter Allgemeine Zonntagszeitung, Westdeutsche Allgemeine
Zeitung (Germany), The Times; Daily record (UK), Globe and Mail (Canada), The New
York Times (USA).
International magazines: Der Spiegel, The Economist, Technology Review, Labor; Der
Standard (Germany); Scientific American (USA). Forskning & Framsteg (Sweden).
International TV: Nanovision (Germany); SVT (Sweden), Tagesthemen; W wie wissen,
Abenteuer Wissen (Germany), Kill it, cook it, eat it (UK), Future Food (Canada),
International Radio: (Belgium, Germany, Switzerland, Sweden, UK, USA).

International research. Although quite a lot of researchers have investigated, and still
are investigating, the generation of embryonic stem cells, it has not yet been possible to
establish an embryonic stem cell line from farm animal species. Most of these latter
studies emerged from a biomedical interest, not with the intention of using these cells for
food production. Nonetheless, there is a group of international researchers that
investigate cultured meat from different angles.

33
In April 2008, a three-day ‘in vitro meat’ symposium was organized at the Norwegian
Food Research Institute (Matforsk) in Aas, Norway and was hosted by the Norwegian
Institute of Life Sciences (UMB) and Matforsk. Cell biologists, tissue engineers, engineers,
entrepreneurs, NGO representatives and government officials from various European
countries but also from the US, Canada, Australia and New Zealand discussed the key
scientific challenges for the production of cultured meat. Issues that were discussed were
e.g. production of culture media, stem cell generation and culturing, and muscle tissue
engineering. Professor Stig Omholt (Chairman of the International In Vitro Meat
Consortium, Norwegian University of Life Sciences, Norway) presented at this meeting a
report made by eXmoor pharma concepts (UK) commissioned by him on behalf of the in
vitro meat meeting steering committee (members: Prof. Stig Omholt, Norway; Prof. Henk
Haagsman, Netherlands; Prof. José Teixeira, Portugal; Jason Matheny, USA, and Dr.
Bernard Roelen, Netherlands). The study presented in the report deals with the question
whether the production of in vitro meat can be financially viable as compared to the
factory gate prices for cheap meat products such as chicken. As it is currently not
possible to generate in vitro meat, several assumptions had to be made for this study:

1) Up front R&D en PR costs: Although these costs will be significant, they have been
ignored in the model. Instead, it is assumed that these costs will be met by
governments and charitable donations, but it is anticipated that these costs will be
substantial.

2) Capital costs: As these are unknown until the technology is available, the model used
factors from known technology.

3) Medium costs: Assumptions have been made based on medium that is currently
available for biomedical research in relatively small quantities. These costs have
been discounted to allow for larger volumes.

4) Financing costs: The model has assumed negligible business risks. Instead, it is
assumed that research has demonstrated the proof of principle of the technology,
and that the public opinion is positive (this will include acceptance by regulators that
grant licenses and acceptance by consumers).

5) It was concluded that the costs have to be incurred and the technology has to be
proven before manufacturers will invest in this technology. For the development of
culture media it was concluded that either on site production or a separate media
infrastructure would be necessary. Importantly, it was also concluded that the
following challenges will have to be met:

• Sufficient knowledge of the biology of the stem cell and its differentiation into muscle
cells.

• Tissue engineering on a very large scale.

• Maintenance of constant conditions around all individual cells in a large-scale reactor.

• Need of cell growth and differentiation and subsequent release from support without
damage upon harvesting.

• Need for on-site cleaning and sterilization systems in the large-scale reactors.

• Sophisticated instrumentation for measuring and controlling conditions within large-

scale reactors.

From the economic model it was concluded that a form of in vitro meat using mammalian
cells in a 3D matrix could be produced at around Euro 3500/tonne. For comparison, the

34
production of chicken meat equals about Euro 1800/tonne (unsubsidized) or Euro
1400/tonne (subsidized), and the costs for production of beef (current market price) is
around Euro 3550/tonne. Obviously, different capital cost and costs of the growth media
do affect the forecast of the breakeven price for in vitro meat, and these costs can be
reduced by for instance reducing the costs of the process plants, increasing the scale of
production or reducing the cost of media preparation.

Experts’ opinions (6): Role of The Netherlands in the development of cultured

meat technology

• The Netherlands seems to be taking a leading role in this research at this time. If
they stick with it they may emerge as the technology leaders in this area.

• The Dutch research effort into developing in vitro meat has the potential to
revolutionize agriculture, saves human lives, and significantly reduce climate change.
I sincerely hope that the Dutch government will expand its support of this research,
positioning The Netherlands as a global leader.

• I believe that in vitro meat would be important for a densely populated country like
The Netherlands with a large meat production. I hope the Netherlands will continue
as a leading country in the area.

• The Dutch government should stimulate the development of cultured meat, including
research on public reactions and acceptance.

• The Netherlands can be very important in the production of cultured meat as it is at

the moment one of the frontrunners. However, it all depends on the amount of
money invested.

• Positive, particularly when it leads to a reduction of millions of pigs in The

Netherlands.

• The Netherlands can and must be a leading country in the development of this
technology. The Dutch scientists have the required knowledge. It is important to act
swiftly.

• The Netherlands have played a pioneering role so far, and by proper funding of the
academic activity, the country will play a leading role for many years to come.

• I have high expectations of the Dutch research.

• I see culture meat development at the level of the academia. The Netherlands could
play a role but this will depend on the choices that are made. The development of
cultured meat is according to me not an essential investment for the Dutch
government in order to support innovation.

• I think this is a technology that will take 10 – 20 years to be established. As a global

leader in food and feed technology The Netherlands should at least assess the
feasibility and potential of this technology through mathematical modeling.

35
Strengths and weaknesses of the current approach

Strengths. Although the development of in vitro meat is still in its infancy, a substantial
group of international scientists is, indirectly, involved in this field (see first international
in vitro meat symposium: http://invitromeat.org/content/view/14/29/). The scientists
involved represent different disciplines, from cell biologists via process technologist to
ethicists. The multidisciplinary approach already from the start ensures product
development that fulfills all necessities.

The intense positive media interest that has, and still is, given to the Dutch in vitro meat
project (see supplementary materials) indicates that there is a definite interest for
(partial) replacement of traditional meat for cultured meat. The past decade has already
seen a change in consumer habits leading to development of organic and other eco-
labeled products, and tendencies towards vegetarianism and healthier diets. In this
respect, it is anticipated that indeed there is a healthy market for cultured meat. This is
further emphasized by the $1 million reward that has been offered by People for the
Ethical Treatment of Animals (PETA) to the first scientist who produces cultured meat and
brings it to market. However, there are some details attached to this contest. First it is
stated that the cultured meat should be of chicken origin and should be ready to sell to
the public by June 30, 2012. Further, the contestant must do both of the following: 1)
produce a cultured chicken-meat product that has a taste and texture indistinguishable
from real chicken flesh to non-meat-eaters and meat-eaters alike; 2) manufacture the
approved product in large enough quantities to be sold commercially; 3) successfully sell
it at a competitive price in at least 10 US states
(http://www.peta.org/feat_in_vitro_contest.asp). It seems unlikely that anybody can
meet these criteria by June 2012, but it does indicate the growing need for and interest
in meat alternatives.

Weaknesses. Currently, research on making an edible product from in vitro cultured

cells is basically only conducted in the Netherlands by the Dutch In Vitro Meat
Consortium: Hellingwerf/Teixeira de Mattos with 1 technician, University of Amsterdam;
Post with 2 PhD students, Technical University Eindhoven, Haagsman/Roelen with 2 PhD
students, Utrecht University; with feedback from In Vitro Meat BV (van Eelen) and
Stegeman (Verstrate). This research has been financed by a SenterNovem grant, started
in the spring of 2005, and ended March 31st, 2009. The relatively small basis of this
research consortium is of concern, and with the limited research that could be carried out
it will be difficult to secure future industrial support.

Limiting factors in the development of an edible product are the generation of the stem
cells that will be used, the characterization of these cells once generated and the optimal
conditions how to culture them. The cells are essential for the generation of cultured
meat, but in contrast to popular belief, correct bovine or porcine cells that can (a)
proliferate indefinitely and (b) differentiate efficiently to muscle cells, have not been
derived yet, nor are the culture conditions known to maintain such cells in a proliferative
state. The cells that were generated with financial support by SenterNovem do not fulfill
all the required criteria. Without the proper cells (the starting ingredients) it is difficult to
design culture media for proliferation and differentiation. Similarly, tissue engineering
research and the development of suitable bioreactors is limited if the proper cells are not
available. To circumvent these problems, researchers have used related cells as model
systems. The murine C2C12 myoblast cell line has been particularly useful in this
respect. This approach has provided interesting and practical results, but it is expected
that more concrete results should be available with cells of farm animal species before
companies are interested to invest.

36
Experts’ opinions (7): The most important criteria that have to be met in order
develop marketable products

• Cost competitiveness with a suitable product, and very careful attention to public relations and
advertising. Doing this wrong could be a disaster that makes GM foods appear to be "organic"
by comparison.

• Low cost, safe and nutritious. Texture and taste of ground meat should not be a problem to
duplicate, given what is already accepted by consumers.

• Products should be attractive to the senses. Additionally the products should have a proven
safety and the sustainability should be clearly demonstrated. There should also be a social
acceptance of technological food production. To my opinion, this should be achieved by a
transparent production process.

• Societal obstacles are a very important issue. Many people react with emotional aversion on
the subject of cultured meat. I will be very important to understand this aversion, and to
investigate how it can be maximized and more importantly how it can me minimized.

• There are quite a number of criteria that are very important for the success of a new product.
These are consumer acceptance, taste, continuity of production (it should be available all year
round), and it should be available at a competitive price.

• Cost and consumer acceptance. Ethical concerns might be a point to consider.

• To show the necessity to produce cultured meat and benefit for both consumer and
environment.

• The production of products with a good taste and ‘bite’,

• Comparable prices with ordinary meat, safe, tasty, healthy and having a resemblance with an
ordinary high-quality piece of meat.

• Products have to be generated that the consumer will accept and wishes to buy.

• The technology must have been developed to a large extent. Societal acceptance is very
important.

• Cost prize, equal or lower than real meat. Sustainability, significantly lower resource use per
kg produced meat. Quality, equal or better taste etc. Public acceptance, avoid “Frankenstein
food” image.

• Consumer acceptance, ‘transparent’ production process, not too expensive.

Opportunities. Man’s impact on the planet Earth has become alarmingly clear in the
past decades. Numerous reports have been produced with frightening scenarios of the
future and indeed without a change of policies there is a distinct possibility that our
planet will become inhabitable. Energy consumption and the livestock sector in particular
have been identified as the leading drivers of climate change, deforestation, pollution and
reduction of biodiversity. Simultaneously, the livestock sector is extremely important for
the agricultural economy and obviously for the human diet. Different solutions can, and
should be, developed to reduce the environmental impact of this sector, one of those
being the (partial) replacement of traditional meat with an edible product made from
animal proteins produced by cultured cells.

The possibility of making an edible meat product from cultured cells without the use of
animals may provide a change in agriculture and society at large. The infrastructure that
should accompany the implementation of such a technology is still lacking and has to be
developed. At this point in time the Netherlands is considered to be the leading country
with respect to the research on cultured meat. Although this notion may not be true, fact
is that Dutch scientists have a very good reputation if it comes to food and agricultural

37
sciences, environmental sciences, microbiology, stem cell biology, and tissue
engineering. Investment in research and development of this technology may create
opportunities for Dutch companies to become leaders in a new ‘green’ technology.

In addition, the spin-off know-how and technology in the field of tissue-engineering can
give Dutch universities and Dutch industry a significant scientific and technological
advantage in the medical application of tissue engineering (tissue regeneration).

Threats. One major threat in the development of cultured meat is the relatively slow
progress because of the limited number of scientists involved. Coverage by the media
has been beneficial for public awareness and initiating discussions about innovative ways
to produce animal proteins. On the other hand, media attention raised high expectations
by citizens and media alike. Many journalists expect us to show and eat a piece of lab-
grown meat and are surprised to hear that world-wide only four PhD students and one
technician work full-time on cultured meat. If research continues at the present pace and
progress remains slow, the present enthusiasm for the technology may dwindle. Most
people that were interviewed by us indicated that, at the present state of knowledge (and
the financial crisis), companies will not invest in cultured meat production. It is concluded
that public funding is required at this point in time to increase the research efforts with
respect to the production of animal proteins by cultured muscle cells. The major threat is
that, if that does not happen, potential investors and companies will never invest
because of a lack of tangible results. This threat may be thwarted by using the present
momentum of public enthusiasm to justify the investment of public seed funds.

Another threat may be the lack of consumer acceptance. However, from the start of the
project we have encouraged the involvement of representatives from consumer groups.
Also, there has been considerable media attention for the project, and through
radio/TV/newspapers/magazines the consumer has been (and will be) informed and
educated about cultured meat production. In general, the media attention has been
positive and supportive for an in vitro meat product. On the other hand, several media,
in the internet in particular on various webblogs, have been more skeptical and even
negative about the concept of cultured meat. ‘Frankenfood’ is a repetitively used word in
this respect. Consumer acceptance is of utmost importance; without it there may be a
product but no market. Knowledge of consumer's choices and factors that affect these
choices is therefore important. Consumer's motivation, habits and choices should
therefore be investigated. A proper feedback between those that study consumer's
profiles with those that study cell biology/culture media/tissue engineering is crucial.

A major threat for the implementation of the technology comprises the production costs.
Obviously the price of cultured meat should not differ too much from the price of
traditional meat. For several reasons it is assumed that the price of in vitro meat will be
similar to the price of regular meat. Firstly, the study by eXmoor pharma concepts
concluded that the forecast costs for cultured meat products will be around €3300/
tonne. This is in the same order as the unsubsidized price of chicken meat that is around
€1800/ tonne (see supplementary materials). Secondly, it has been predicted that the
price of regular meat will rise significantly, as the meat demand will only increase with
the growing world population while there is simply not enough feed for the animals. It is
therefore expected that the price of regular meat will only increase, whereas it is
predicted that the price of in vitro meat will decrease as the market becomes bigger.

38
Experts’ opinions (8): The role of companies in the development of cultured
meat

• Probably not until the risk has been mitigated substantially.

• Companies may invest but not until proof of principle is established by research funded by
governments or foundations. I think private commercial funding at this stage may be less
likely than public or foundation funding. Once proof of principle is established, it seems likely
that private commercial funding will play an important role.

• Not in this first phase. There is still much basic science to be done. I believe the first phase
has to be public.

• At the moment I do not think that companies will invest in research on in vitro meat. The
product is simply too immature yet, first a number of uncertainties need to be answered.

• I would strongly suggest a private-public association. However, I consider that public

institutions should have a key role in the launching of the project. In the second phase, private
companies should join the project.

• I favor development on a public-private basis.

• At first companies will not invest in cultured meat. It is important to show that the technology
is feasible.

• Companies will not invest before the technology is mature to allow very moderate R&D
investment costs and before they see that governments will through legislation make a market
for this. The FAO has already articulated favorable opinions about the concept, and they sent a
high-ranking representative to the first international workshop on in vitro meat production
held in Norway in 2008. They will probably continue to support this vision.

• It will take some time before this technology will become profitable, until this time public
funding is preferable.

Spin-off. It is anticipated that research on cultured meat will have a significant spin-off,
primarily for the biomedical industry. Four areas of spin-off can be discerned: 1)
Generation of stem cells from pigs (and other farm animals); 2) Production of tissue
culture media that do not contain animal products; 3) Increased knowledge on aspects of
tissue engineering; 4) Specific know-how on (industrial scale) bioreactors.

Ad 1) Generation of porcine stem cells. The generation of stem cell lines from farm
animal species also has the potential of generating genetically modified animals. Murine
embryonic stem cells can be used to introduce specific gene modifications in mice and, as
a result, numerous genes have been altered in mice. This has been a powerful method to
help understand the functions of many genes and in 2007 the Nobel Prize in Physiology
or Medicine was awarded to the discoverers of these techniques. Gene targeting is
currently not common in mammals other than in mice, but the development of stem cell
lines from farm animal species could facilitate to create pigs or cows with targeted gene
modifications. Stem cells from farm animals can therefore be used for the generation of
transgenic animals with improved production traits or disease resistance. Similarly
transgenic farm animals, particularly pigs, can be extremely useful large animal model
systems for human medicine. The pig is a more useful model for human medicine than
the currently predominantly used mouse models, because pigs and humans have a
comparative anatomy and physiology. For instance, the organ dimensions and life-span
of pigs are more similar to those of man than those of rodents. It is therefore anticipated
that porcine stem cells can be used to generate genetic models for human diseases. In

39
addition, stem cells can be used for the generation of animals that can be used in organ
transplantation research or therapy (animal models or xenotransplantation).

Ad 2) Production of tissue culture media that do not contain animal products. Currently
most media used for the culture of mammalian cells contain fetal bovine serum. This
serum, containing basic components to nurture cells, including hormones and growth
factors, is derived from bovine fetuses of at least 3 months old by means of cardiac
puncture. The availability of (affordable) culture media that do not contain animal
products, i.e. for which no animals have been used will significantly reduce the use of
animals. Indeed it has been proposed that fetal bovine serum should be replaced in cell
culture both on scientific (batch differences) and moral grounds44 . For safety reasons, it
is crucial that cells and tissues used for human regenerative medicine have not been in
contact with animal products. The development of animal-product free culture media will
therefore also be beneficial for regenerative human medicine.

Ad 3) Increased knowledge on aspects of tissue engineering. Since the culture of muscle

tissue from stem cells is in essence similar to tissue engineering as is being developed for
the biomedical industry, the results and knowledge obtained are interchangeable.
Knowledge on coatings, scaffolds, culture conditions and so forth is also advantageous
for the in vitro culture of 3-dimensional human tissue to be used for regeneration
studies. In addition, knowledge on in vitro culture of (engineered/transgenic) porcine
tissues can be helpful for those interested in the use of porcine tissue/organs for
xenotransplantation.

Ad 4) Specific know-how on (industrial scale) bioreactors. Many stem cell and tissue
engineering studies are conducted with the ultimate goal of using in vitro cultured stem
cells and tissues for regenerative medicine. It would be extremely beneficial for human
medicine if tissue regeneration would become a commonplace therapy and, if this
happens, bioreactors will be needed for tissue generation. The knowledge obtained from
cultured meat studies can be used for the optimization of these bioreactors. Other cells,
including plant and bacterial cells, can also be cultured using bioreactor technology.
These can be used for the generation of food products other than cultured meat, but also
for the generation of biofuel.

It has to be noted that the worldwide resources for human (regenerative) medicine are
much higher than for cultured meat. Therefore, it is anticipated that, also in the future,
knowledge transfer will be largely from the human medicine field to the cultured meat
field.

40
41
Experts’ opinions (9): Potential spin-offs of cultured meat research

• If one understands how to propagate muscle stem cells and how to differentiate these into
muscle cells, combined with the knowledge on muscle tissue engineering and muscle tissue
formation, this knowledge can be helpful in basic research to minimize the use of animals.
• Cheap growth media for other tissue engineering applications, including those in biomedicine.
Perhaps also insights into muscle tissue engineering that support medical therapies.
• Mostly in basic biology. Maybe also spin-off to medicine but more likely the other way around,
because most probably much more funding will go to the medical endeavor to grow human
organs for transplantation.
• As a spin-off, there is the strong potential that the role that technology can play in production
of food, morality and sustainability is re-evaluated.
• Cultured meat is a spin-off of medical research into the differentiation of stem cells in body
tissues.
• The technology will have an impact on tissue engineering and regenerative medicine.
• Fundamental knowledge on tissue engineering. Intellectual Property.
• As the development of the technology will demand contributions from several disciplines the
spinoffs are likely to be considerable:
o Chemical/biotechnological process plant construction and optimization
o Materials technology
o Biosensor technology
o Instrument manufacturing
o Advanced multivariate analysis methodology
o Mathematical modeling of biological tissues and organs from gene to phenotype
o Stem cell creation from domesticated animals
o Large scale stem cell culturing
o Cell co-culture methodologies and issues
o Methodologies for directed stem cell differentiation into skeletal muscle
o Bioprinting technology (myoblast sheets)
o The developmental biology of muscle tissue
o Experimental and theoretical biophysics of muscle tissue including the extracellular matrix
o The developing and mature muscle tissue phenotype (in the wide sense)
o Large scale genotyping technologies
o Muscle tissue phenotyping technologies (in the wide sense)
o (Large scale) 3-d cell (co-)culturing technology
o Neutral lipid / flavor /muscle cell fatty acid biology
o Enzymology of relevance to food processing
o Cell culture diseases and associated diagnostic methodology
o Production economist and process engineer to estimate costs under various regimes
o Environmental economist with experience in life-cycle analysis to estimate environmental
impacts relative to conventional meat
o Food market analyses to assess consumer acceptance of in vitro meat

Policy making

Commercial production of cultured meat is as yet not possible, since knowledge is still
lacking on the fundamental science level, as well as on the technological and societal
levels. For one, suitable, well-characterized, stem cells from farm animal species are not
yet available. In addition, there is a critical lack of information concerning culture
conditions that would keep these stem cells undifferentiated and culture conditions that
would steer these cells efficiently to skeletal muscle cells, bioreactors, etc. There is still
too little known about consumer's judgment influences, consumer's choices etc. For
reasons of credibility and for marketing strategies, it seems appropriate that a good
communication exists between scientists/developers/industry and consumers (societal
embedding).

42
For cultured meat to become realistic it is recommended that on a short term the focus
must be on these lacunas in knowledge and lack of existing technology. With respect to
long-term goals, if the technology has been established, attention should be directed
towards scaling up of the bioreactors, not only for the culture of stem cells and
production of cultured meat, but also for the production of culture media. The
involvement of companies would be essential at this stage. During the whole process,
transparency towards the consumers and societal embedding need to be warranted.

Experts’ opinions (10): Investments in cultured meat research and technology

• Ball park - €25-50 million to establish labs and hire a team of ~5 scientists for 2-3 years.
• Realistic timelines on this research are unfortunately still very long: I estimate 15-20 years of
intensive research at the very least. I would guess that for properly setting up research on in
vitro meat, annually €4-$6 million over a 15-20 year period is needed.
• My guess is over €10 million will be needed to establish proof of principle. Then commercial
firms would invest many millions to scale-up the technology.
• As a start at least €10 million is required to initiate research and development.
• If a broad but very concerted and tightly controlled R&D programme is made, I would think
around €100 million is needed over a 3-4 year period before a pilot plant can be set up where
the development to the next stage can be taken.
• About €30-50 million.
• Somewhere between €20-50 million.

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44
Summary

The global population is estimated to increase with 50% during the next 40 years. This
population increase will be accompanied with an almost doubling of the greenhouse gas
emissions if no actions are taken. It is anticipated that also the global meat consumption
will double during the next 40 years if societies worldwide become more affluent. Meat
production requires a relatively high proportion of land, energy, and fresh water use.
Moreover, livestock contributes significantly to the emission of greenhouse gases and, in
many countries, to the pollution of water and soil. An obvious solution to the problem
would be to consume less products of animal origin. This may, certainly in Western
societies where the consumption of (animal) proteins is very high, be part of the solution.
Replacement of dietary animal (vertebrate) proteins by plant, fungal, or even insect
proteins can be another part of the solution.
Yet another possibility is to culture large amounts of muscle cells derived from stem cells
of farm animal species and to produce cultured meat (in vitro meat). The advantage of
this technology is that for the making of most products of animal origin no animals are
needed. Products prepared with the latter technology may combine a favorable ecological
footprint with similar nutritional values as conventional products. Ultimately, cultured
meat products could be made with similar sensory qualities of some of the conventional
products. The present report is a survey of the current state of the required technologies,
the life cycle assessment, ethical and societal issues, and economical aspects.
The first requirement for developing cultured meat technologies is a suitable, bona fide,
stem cell line that can proliferate indefinitely. Both embryonic and adult stem cells may
be suitable although adult stem cells must have a minimal self-renewal capacity since
most adult stem cell types cannot be cultured indefinitely. One of the aims of the Dutch
‘In vitro meat’ project (2005-2009), funded by SenterNovem (an agency of the Dutch
Ministry of Economic Affairs), was to generate a suitable stem cell line (embryonic and
adult) from pigs. Adult stem cells have indeed been generated from porcine muscle but
these cells do not very effectively differentiate into skeletal muscle cells. The generation
of a suitable ‘starter stem cell’ from farm animals and its growth and efficient
differentiation into muscle fibers still requires much research. Fortunately, the fast
developments in stem cell biology will enable more directed research in this area.
A second requirement for the production of cultured meat is the availability of culture
media that do not contain products of animal origin. Most media for culturing mammalian
or avian cells contain animal serum. It is clear that for cultured meat production serum-
free media must be used, a requirement that is also important if stem cells are used in
regenerative medicine. Different media must be developed for growing the stem cells and
for the differentiation media to produce muscle cells. A lot of progress has been made in
recent years in the development of serum-free media and indeed several media are
commercially available that do not contain animal products. The challenge is to produce
the right culture media at prices that are compatible with food production.
A third requirement for cultured meat technologies to become successful is the formation
of a three-dimensional structure of fused muscle cells (myofibers). In the absence of a
blood flow, that provides oxygen and nutrients to the cells, the diffusion rates of these
compounds limit the culturing of many layers of cells. Scaffolds or hydrogels may be
used to circumvent this problem provided that these materials are edible and not from
animal origin. Studies have been carried out as part of the ‘In vitro meat’ project to
determine the optimal three-dimensional environment for muscle cells for culturing and
differentiation. In addition, biochemical and biophysical stimuli (electrical and
mechanical) were studied that may be important in the differentiation and maturation
towards muscle tissue. While knowledge is accruing about the factors that are important
for culturing muscle tissue, a lot of basic knowledge is still lacking. Nevertheless, the
technology to grow small pieces of meat (as ingredients in complex food products) may
be developed within a relatively short term, provided that the above-mentioned

45
requirements (suitable cells and medium) are met. The development of the technologies
to produce larger pieces of meat may take considerably longer.
A fourth requirement is that cultured meat must be produced at prices that are
comparable with factory gate prices for cheap meat such as chicken meat. A study
commissioned by the ‘International In Vitro Meat Consortium’ indicated that cultured
meat (from a three-dimensional culture) can be produced for €3500/tonne (compared to
€1800/tonne for chicken meat and €3550/tonne for beef). It should be noted that several
assumptions had to be made, since the technology to produce cultured meat has not
been developed yet. Nevertheless, it is encouraging that the applied economic model
predicts production costs that are comparable with that of current meat production.
A fifth requirement is that the production of cultured meat should have a favorable
ecological footprint compared to the conventional production of meat. A preliminary life
cycle assessment of cultured meat by dr. Tuomisto (University of Oxford) and dr.
Teixeira de Mattos (University of Amsterdam) indicated that cultured meat production
has a much lower environmental impact than conventional meat production. It was
estimated that the average energy use is 45-60% lower; greenhouse gas emissions are
80-95% lower; land use is 98% lower and water use is 90-98% lower. Only poultry meat
has a lower energy use per kilogram than cultured meat. Although also for this study
several assumptions had to be made it is clear that the ecological footprint of the
production of cultured meat is substantially lower than that of conventional meat.
A sixth requirement for the production of cultured to become successful is consumer
acceptance. Until now no scientific study has been conducted about the ethical and
societal aspects of cultured meat production and consumption. From the start of the ‘In
vitro meat’ project in 2005 the research of the Dutch consortium has received a lot of
media attention from all over the world. The coverage by the media provoked numerous
discussions on the internet and, as discussed by Holmes and Dacey in their publication in
the Journal of Agricultural and Environmental Ethics in 2008, the majority of the opinions
is favorable towards cultured meat technology. One should be careful to draw
conclusions from these anecdotes. Therefore, it is deemed of utmost importance to carry
out research with respect to the ethics and societal aspects of cultured meat.
It is anticipated that research on cultured meat will have a significant spin-off, primarily
for the biomedical field. Four areas of spin-off can be discerned: 1) Generation of stem
cells from pigs (and other farm animals); 2) Production of tissue culture media that do
not contain animal products; 3) Increased knowledge on aspects of tissue engineering;
4) Specific know-how on (industrial scale) bioreactors. It has to be noted that the
worldwide resources for human (regenerative) medicine are much higher than for
cultured meat. Therefore, it is anticipated that, also in the future, knowledge transfer will
be largely from the human medicine field to the cultured meat field.
In the framework of our study we interviewed national and international experts from
companies, research institutes and universities about many aspects of the development
of cultured meat. Most experts indicated that there is an urgent need for dietary protein
sources other than meat and that cultured meat may be an interesting option to produce
meat in a sustainable way. However, a few experts never heard of cultured meat and
were skeptical about developing this technology. The experts indicated several
technological challenges; these challenges were also recognized by the authors and
comprise an important part of this report. The experts were unanimous that at this point
in time the traditional food companies will not invest in research on such a completely
different commodity as cultured meat. All experts mentioned that, at this stage,
investments in the technology should be done with public money. Although most experts
see an important role for the Dutch scientists in cultured meat research, it is preferable
that both funding and research efforts are coordinated internationally. The experts
estimate that €10-€100 million is required to initiate an R&D program.
It can be concluded that commercial production of cultured meat is as yet not possible,
since crucial knowledge is still lacking on the biology and technology. Furthermore,

46
knowledge is lacking with respect to ethical and societal issues. Companies will only
invest if the technology has demonstrated to be viable on a small scale. It is
recommended that on a short term the focus must be on filling these gaps in knowledge.
During the whole process, communication with consumers and societal embedding is
essential.

Nederlandse samenvatting

Geschat wordt dat de wereldbevolking gedurende de volgende 40 jaar met 50% zal
toenemen. Deze toename van de wereldbevolking zal vergezeld gaan met een
verdubbeling van de uitstoot van broeikasgassen als er niet ingegrepen wordt.
Wereldwijd zal ook de consumptie van vlees in de komende 40 jaar verdubbelen
vanwege het feit dat, naast de bevolkingsgroei, mensen ook meer te besteden hebben en
de consumptie van vlees bij veel volkeren geassocieerd is met welvaart. De productie
van vlees vereist relatief veel land, energie en water. Bovendien draagt de veehouderij
verhoudingsgewijs sterk bij aan de emissie van broeikasgassen en in veel landen ook aan
de verontreiniging van bodem en oppervlaktewater. Een voor de hand liggende oplossing
zou de vermindering van de consumptie van dierlijke producten zijn. Dit zou zeker een
deel van de oplossing in de Westerse wereld zijn waar de consumptie van (dierlijke)
eiwitten hoog is. De vervanging van dierlijke eiwitten (uit gewervelden) in het dieet door
eiwitten uit planten, schimmels of zelfs insecten zou een ander deel van de oplossing
zijn.
Een heel andere oplossing is om niet de dierlijke eiwitten zelf maar de bron van dierlijke
eiwitten te vervangen. Spiercellen, verkregen uit stamcellen van landbouwhuisdieren,
zouden op grote schaal gekweekt kunnen worden. Aangezien vlees voornamelijk uit
spiercellen bestaat kan op deze wijze ‘kweekvlees’ (in vitro meat) geproduceerd worden.
Het grote voordeel is dat met deze technologie geen dieren meer nodig zijn voor de
productie van vlees en vleesproducten. Kweekvleesproducten zouden op een
milieuvriendelijke wijze vervaardigd kunnen worden terwijl de voedingswaarde
vergelijkbaar is met die van conventionele producten. In eerste instantie zou de nieuwe
technologie gericht kunnen zijn op het maken van ingrediënten voor samengestelde
producten terwijl op de lange termijn smakelijke producten ontwikkeld kunnen worden
die volledig vervaardigd zijn met de kweekvleestechnologie. Dit rapport is een studie
naar de huidige kennis op het gebied van 1) de technologieën die nodig zijn voor de
ontwikkeling van kweekvlees; 2) de milieugerichte levenscyclusanalyse; 3) de
economische haalbaarheid en 4) ethische en maatschappelijke aspecten.
Een eerste vereiste voor de ontwikkeling van kweekvleestechnologieën is een geschikte
stamcellijn die bij voorkeur oneindig door kan groeien. Zowel embryonale als niet-
embryonale (‘volwassen’) stamcellen kunnen gebruikt worden hoewel de meeste
volwassen stamcellen niet oneindig in kweek kunnen worden gehouden. Deze cellen
moeten daarom voldoen aan bepaalde eisen wat betreft hun delingscapaciteit. Eén van
de doelstellingen van het Nederlandse ‘In vitro meat’ SenterNovem project (2005-2009)
was het verkrijgen van een geschikte stamcellijn (zowel een embryonale als volwassen
lijn) van het varken. Volwassen stamcellen zijn inderdaad verkregen uit varkenspieren
maar deze cellen bleken, in tegenstelling tot muizenstamcellen, niet op efficiënte wijze
naar skeletspiercellen te differentiëren. Het verkrijgen van een geschikte ‘startstamcel’
uit landbouwhuisdieren en de groei en efficiënte differentiatie in spiervezels vereist nog
veel onderzoek. Aangezien de ontwikkelingen in de stamcelbiologie snel gaan is het
mogelijk steeds gerichter onderzoek te doen naar stamcellen van landbouwhuisdieren.
De recente opheldering van de sequentie van het varkensgenoom is ook belangrijk in dit
verband. Een tweede vereiste voor de ontwikkeling van kweekvleestechnologieën is de
beschikbaarheid van kweekmedia die geen producten van dierlijke oorsprong bevatten.
De meeste media die gebruikt worden om cellen van zoogdieren en vogels te kweken
bevatten dierlijk serum. Vanwege de aard en veiligheid van het product is het duidelijk
dat voor de bereiding van kweekvlees serumvrije media gebruikt moeten worden,

47
hetgeen overigens ook een eis is als stamcellen gebruikt worden in de regeneratieve
geneeskunde. Verschillende media moeten ontwikkeld worden voor het opgroeien van
stamcellen en voor de differentiatie en groei van spiercellen. De afgelopen jaren is veel
vooruitgang geboekt met het ontwikkelen van serumvrije media en media die geen
dierlijke producten bevatten zijn nu commercieel verkrijgbaar. De uitdaging is nu de
juiste media te maken voor de productie van kweekvlees tegen een zo gunstig mogelijke
prijs.
Een derde vereiste om de productie van kweekvlees succesvol te maken is de vorming
van een driedimensionale structuur van een aantal lagen gefuseerde spiercellen
(spiervezels). In de afwezigheid van een bloedstroom, die voedingstoffen en zuurstof
voor de cellen beschikbaar maakt, is het niet mogelijk veel cellagen te stapelen. Een
steigerwerk (‘scaffold’) of een hydrogel zou een oplossing zijn voor dit probleem waarbij
de gebruikte materialen eetbaar moeten zijn en niet van dierlijke oorsprong. Ook zijn
andere technologieën denkbaar om een driedimensionale structuur van spiervezels te
maken. In het ‘In vitro meat’ project is onderzoek uitgevoerd om de optimale
driedimensionale omgeving te creëren om spiercellen te kweken en te laten
differentiëren. Bovendien werd onderzocht op welke wijze de differentiatie en rijping van
spiercellen gestimuleerd kan worden. Hiervoor werden de cellen zowel biochemisch als
fysisch (elektrisch en mechanisch) gestimuleerd. Ondanks het feit dat al veel bekend is
over de factoren die van belang zijn om spierweefsel te kweken ontbreekt er nog veel
kennis. Desalniettemin zal de technologie om kleine stukjes vlees te maken (als
ingrediënt voor samengestelde producten) binnen relatief korte termijn ontwikkeld
kunnen worden als de juiste cellen en media beschikbaar zijn. De ontwikkeling van
technologieën om grotere stukken vlees te maken zal echter aanzienlijk meer tijd in
beslag nemen.
Een vierde vereiste is dat kweekvlees geproduceerd moet kunnen worden tegen prijzen
die vergelijkbaar zijn met de prijzen van gangbaar vlees, zoals goedkoop kippenvlees.
Een studie, die in opdracht van het ‘International In Vitro Meat Consortium’ is uitgevoerd,
wees uit dat kweekvlees geproduceerd kan worden voor €3500/ton (vergeleken met
€1800 voor kippenvlees en €3550 voor rundvlees). Een kanttekening hierbij is dat
verschillende aannames gemaakt moesten worden aangezien de kweekvleestechnologie
nog niet ontwikkeld is. Het is echter bemoedigend dat het toegepaste economische
model productiekosten voorspelt die vergelijkbaar zijn met de huidige productiekosten
van vlees.
Een vijfde vereiste is dat de productie van kweekvlees duurzaam moet zijn en minder
schadelijk voor het milieu dan de huidige vleesproductie. De voorlopige resultaten van
een kwantitatieve, milieugerichte levenscyclusanalyse door dr. Tuomisto (University of
Oxford) en dr. Teixeira de Mattos (Universiteit van Amsterdam) geven aan dat de
productie van kweekvlees veel minder schadelijk is voor het milieu dan conventionele
vleesproductie. De berekeningen wezen uit dat het gemiddeld energiegebruik voor de
productie van kweekvlees 45-50% lager is, de emissie van broeikasgassen 80-95%
lager, landgebruik 98% lager en watergebruik 90-98% lager. Hoewel ook voor deze
studie verschillende aannames gemaakt moesten worden is het duidelijk dat de
ecologische voetafdruk van kweekvlees aanzienlijk kleiner is dan die van conventioneel
vlees.
Een zesde vereiste om de productie van kweekvlees succesvol te maken is
consumentenacceptatie. Tot op heden is er geen wetenschappelijk onderzoek uitgevoerd
naar de ethische en maatschappelijke aspecten van de productie en consumptie van
kweekvlees. Vanaf de start van het ‘In vitro meat’ project in 2005 heeft het onderzoek
van het Nederlandse consortium veel media-aandacht gehad vanuit de hele wereld. Deze
aandacht heeft veel discussies uitgelokt (o.a. op internetfora). In een artikel van Holmes
en Dacey in het tijdschrift ‘Journal of Agricultural and Environmental Ethics’ in 2008
werden deze discussies besproken. Het bleek dat de meerderheid van de meningen
positief is ten opzichte van kweekvleestechnologie. Het zou echter onjuist zijn op grond
van anekdotisch onderzoek verregaande conclusies te trekken. Het is daarom van groot

48
belang om gedegen wetenschappelijk onderzoek te verrichten naar de ethische en
maatschappelijke aspecten van kweekvlees.
Het is de verwachting dat kweekvleesonderzoek een aanzienlijke spin-off kan hebben
voor het biomedische veld. Er kunnen minimaal 4 gebieden geïdentificeerd worden die
van dit onderzoek zouden kunnen profiteren: 1) Het verkrijgen van stamcellen van het
varken (en van andere landbouwhuisdieren); 2) De productie van serumvrije
kweekmedia voor cellen en weefsels; 3) Kennis over bepaalde aspecten van ‘tissue
engineering’; 4) Specifieke ‘know-how’ op het gebied van bioreactoren. Er moet gezegd
worden dat er veel meer middelen voor onderzoek op het gebied van de (regeneratieve)
geneeskunde zijn, zodat meer kennis van het domein van de geneeskunde naar de
ontwikkeling van kweekvleestechnologie zal gaan dan vice versa.
Ten behoeve van dit rapport werden nationale en internationale experts geïnterviewd
over verschillende aspecten van de ontwikkeling van kweekvlees. Deze experts zijn
werkzaam bij bedrijven, onderzoeksinstituten en universiteiten. De meeste experts
gaven aan dat er een sterkte behoefte bestaat aan alternatieve bronnen voor
dieeteiwitten en dat gekweekt vlees een mogelijke manier is om op duurzame wijze in
deze behoefte te voorzien. Enkele experts hadden echter nog nooit gehoord van
kweekvlees en waren sceptisch over de technologie. De experts noemden verschillende
technologische uitdagingen. Deze uitdagingen werden ook gezien door de auteurs en
vormen een belangrijk deel van dit rapport. De experts waren eensgezind over het feit
dat, met de huidige kennis, de traditionele voedingsindustrie niet zal investeren in een
product als kweekvlees. Alle experts waren de mening toegedaan dat op dit moment
investeringen in de technologie met publieke middelen zouden moeten worden bekostigd.
De meeste experts zien een belangrijke rol voor Nederlandse wetenschappers in
onderzoek en ontwikkeling van de technologie. Toch zal het de voorkeur verdienen om
op termijn het onderzoek internationaal te coördineren, uit te voeren en te bekostigen.
Volgens schattingen van de experts zou met het starten van een R&D programma €10-
100 miljoen gemoeid zijn.
Er kan geconcludeerd worden dat de commerciële productie van kweekvlees nu nog niet
mogelijk is omdat er nog lacunes zijn in de kennis wat betreft biologie en technologie.
Bovendien is er nog geen onderzoek uitgevoerd naar de ethische en maatschappelijke
aspecten van de productie en consumptie van kweekvlees. Bedrijven zullen pas grote
investeringen doen als het op kleine schaal mogelijk is een product te maken.
Aanbevolen wordt daarom dat op korte termijn de aandacht gericht moet zijn om de
ontbrekende kennis te verwerven. De technologie kan vervolgens verder ontwikkeld
worden door een gezamenlijke inspanning van overheid en het bedrijfsleven. Gedurende
het gehele traject is het essentieel de consumenten goed voor te lichten en voor een
maatschappelijk draagvlak te zorgen.

49
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51
Appendices

Interviewed experts

Name Function
J. Chon Director, Upstream Process Development, PERCIVIA (joint venture
between Crucell and DSM Biologics), Cambridge, UK
R.G. Dennis Associate Professor, Dept. of Biomedical Engineering, University of
North Carolina, Chapel Hill, NC, USA
R.J. Hamer Programme Director, TI Food and Nutrition; Professor,
Agrotechnology and Food Sciences Group, WUR, Wageningen
T.H. van Head Section Matrix Biochemistry, Nijmegen Centre for Molecular
Kuppevelt Life Sciences, Radboud University, Nijmegen
J. Maat Director External Research at Unilever, Vlaardingen and Managing
Director TI Food & Nutrition, Wageningen
J. Matheny Director of New Harvest and Johns Hopkins School of Public Health,
Baltimore, MD, USA
C. Medlow Technical Manager SAFC Biosciences, Sigma-Aldrich Corporation
C.L. Mummery Professor of Developmental Biology, Chair of Department of
Anatomy and Embryology, Leiden University Medical Center,
Leiden
S. Omholt Professor at the Norwegian University of Life Sciences (UMB) and
Director of the Centre for Integrative Genetics (CIGENE), Aas,
Norway
M.F.W. te Pas Senior Researcher Genomics and Bioinformatics, Animal Production
Systems, Animal Sciences Group, WUR, Lelystad
J. Teixeira Professor, Department of Biological Engineering, Universidade de
Minho, Braga, Portugal
H. Tobi Associate Professor Research Methodology, Wageningen UR
H.A.P. Urlings Director Quality Assurance, VION NV and Professor Chain directed
animal production, Animal Nutrition Group, WUR
J. Vereijken Expert Industrial Proteins, Agrotechnology & Food Sciences Group,
WUR, Wageningen
P. Verstrate Director Operations, Stegeman, Deventer
C. van der Weele Assistant Professor, Applied Philosophy Group, Social Sciences,
WUR, Wageningen
S. Welin Professor Biotechnology, Culture, Society, Department of Medical
and Health Sciences, Linköpings University, Linköping, Sweden
G. Zijlstra Senior Scientist, DSM Pharmaceutical Products, R&D Department,
DSM Biologics Company BV, Groningen

Statements of the experts are personal views and do not reflect the opinion of the
organizations that they represent.

52
Alternative (animal) cell systems for protein production

Since the early sixties, when research in 'single cell protein' production was at its peak, we know
that eukaryotic cells in general - in contrast to the cells of prokaryotes - are suitable as a edible
source of food for humans. The latter, apart from the toxic products they may contain, like
endotoxin, generally have a too high nitrogen content, because of the relatively high content of
ribosomes - and therefore RNA - so that uric acid, and by consequence kidney stones, are formed
in the human consumer [1]. A well-known exception to this rule are some cyanobacteria, like
Spirulina platensis, which - in part because of its slow growth rate - has been in use for centuries
as a traditional source of food in tropical regions of Africa [2]. It should be added, however, that
bacterial biomass from various species - when present in large amounts - readily elicits allergic
reactions [3].
The human diet should ideally contain a balanced amount of molecules from multiple
classes, most notably, sugars, amino acids, fatty acids, vitamins and minerals. The first three of
these are usually derived from their polymeric precursors: polysaccharides, proteins and lipids. All
eukaryotic cells contain these components, in varying proportion. Furthermore, when cells are
consumed as a 'tissue', specific components may be over-represented, like (phospho)lipids in brain
tissue, fatty acids in seeds and fibrous cell wall components in plants. The fibrous material
constitutes a class of its own, because it may function 'catalytically' in the food-intake process, i.e.
by having a positive, stimulatory effect with being consumed itself.
Besides a balanced overall elemental composition, for a healthy human diet specific
attention should be paid to the relative enrichment of the (l-)amino acids. This is because humans
are unable to synthesize a limited number (8) of these themselves, so that these have to be
provided directly through food intake [4]. The best known example is l-lysine, but several more are
essential or important*. Therefore the advise generally is given that, if a diet is vegetarian, it
should be composed of a combination of products from two differen plant species, i.e. a monocotyl,
like corn or wheat (for: methionine, valine, threonine, phenylalanine, leucine en isoleucine) and a
dicotyl, like beans (for: valine, threonine, phenylalanine, leucine, isoleucine, tryptophan en lysine).
The suitability of a food ingredient can be expressed as its essential amino acid index (EAAI; i.e.
amino acid content relative to that in eggs, or: LAAC, the 'limiting amino acid concept'; see e.g.
[5]).
In many eukaryotic cells, particularly in those of muscle, the amino acid composition is
much more similar to the optimal composition for human nutrition than the one of other cell types,
i.e. they are relatively enriched in the essential amino acids, in particular in l-lysine. Hence the
advise to make meat an intrinsic part of the human diet. However, this optimality in amino acid
composition may be offset by a less optimal composition of one or more of the other key
ingredients, like e.g. (unsaturated) fatty acids or vitamins. Because of the above, the (non-
)suitability of a specific cell type as a major source of amino acids for the human diet can only be
decided within the context of the overall composition of that particular diet and the WHO has
guidelines as to the minimum content of specific foodstuffs with respect to the essential amino
acids [6].
The above considerations lead to the conclusion that almost all toxin-free eukaryotic cell
types are suitable to form part of the ingredients of the human diet. This then includes microbial-,
plant-, and animal cells and tissues. This being said, it is clear that consumer preference will have
a dominant impact on the choice from this range of possibilities. The recent extensive publicity on
Dutch TV on the use of insects as a suitable protein source is an illustration that in a significant
fraction of the Dutch population, consumer preference begins to move away from traditional meat
sources.
Very recently additional insight has been obtained into the mechanism by which the amino
acid composition of our diet may have effects on our health, in particular related to aging [7]. This
mechanism is based on variation in the rate of gene transcription which modulates the level of
expression of DNA repair enzymes (see also [8]). Accordingly, our diet directly affects the amount
of DNA damage inflicted upon our genomes. These insights may in the future raise the interest of
the consumer in specifically engineered food (to be obtained either through genetic- or through
physiological engineering) in which the (amino acid) composition has been optimized with respect
to its effect on health (see e.g. also [9]).
From a technical point of view there is quite a difference between large-scale culturing of a
free-living unicellular eukaryotic microorganism and that of a mammalian myocyte that can only
grow on a solid support, with many eukaryotic cell types intermediate between these two
extremes. A good example are the insect cells that are extensively used for the heterologous
overproduction of human proteins. This is routinely performed with the Sf 21 cell line that was
originally prepared from the ovaries of Spodoptera frugiperda [10]. Scale-up of growth of these
cells in chemically defined media is straightforward up to the m3 scale and the technical facilities

53
required do not much extend beyond those for single-celled (eukaryotic) microorganisms. This is
largely due to the considerable shear-stress resistance of these cells. Besides shear-stress
resistance, two other major factors in the scale-up of the growth of animal cells is their
dependence on (i) specific growth factors and (ii) a solid support.

Literature
1. Giesecke, D., S. Gaebler, and W. Tiemeyer, Purine Availability and Metabolism in Dogs Fed
Single-Cell Protein or Rna. Journal of Nutrition, 1982. 112(10): p. 1822-1826.
2. Colla, L.M., et al., Production of biomass and nutraceutical compounds by Spirulina platensis
under different temperature and nitrogen regimes. Bioresour Technol, 2007. 98(7): p. 1489-
93.
3. Sikkeland, L.I., et al., Complement activation and cytokine response by BioProtein, a bacterial
single cell protein. Clin Exp Immunol, 2007. 148(1): p. 146-52.
4. Berg, J.J., Tymoczko, J.L., Stryer, L., Biochemistry. Sixth Edition ed. 2006, New York: W.H.
Freeman and Company.
5. Woodham, A.A. and E.M. Clarke, Nutritive value of mixed proteins. 2. As determined by net
protein utilization and protein efficiency ratio tests. Br J Nutr, 1977. 37(3): p. 309-19.
6. Moon N.J., H.E.G., Glatz B.A., Conversion of cheese whey and whey permeate to oil and
single-cell protein. Dairy Science, 1978. 61: p. 1537-1547.
7. Evenblij, M., Veroudering en DNA schade. Mediator, 2009. 20(3): p. 8-9.
8. Garinis, G.A., et al., Persistent transcription-blocking DNA lesions trigger somatic growth
attenuation associated with longevity. Nature Cell Biology, 2009. 11(5): p. 604-U370.
9. Gudbrandsen, O.A., et al., Dietary single cell protein reduces fatty liver in obese Zucker rats.
Br J Nutr, 2008. 100(4): p. 776-85.
10. Chan, L.C., P.F. Greenfield, and S. Reid, Optimising fed-batch production of recombinant
proteins using the baculovirus expression vector system. Biotechnol Bioeng, 1998. 59(2): p.
178-88.

*essential amino acids: Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Threonine,

Tryptophan, Valine

*semi-essential (in young children): Tyrosine, Cysteïne, Histidine, Arginine

54
List of speakers at the first in vitro meat symposium

This symposium was held at the Norwegian Food Research Institute (Matforsk), Aas,
Norway, hosted by the Norwegian University of Life Sciences (UMB) and the Norwegian
Food Research Institute (Matforsk), April 9-11 2008. The authors of this report are not
included.

Expertise: Founder of New Harvest (www.new-harvest.org)

Jason Matheny, New Harvest & Johns Hopkins University, Baltimore, MD, USA

Expertise: Myogenic cells/Biotechnology

Prof. Dr. Tor Erling Lea
Prof. Dr. Stig W. Omholt
Norwegian University of Life Sciences, Aas, Norway

Expertise: Market motivation

Prof. Dr. Elizabeth L. DeCoux, Florida Coastal School of Law, Jacksonville, FL, USA

Expertise: Large scale fractionation of cell extracts

Dr. Arild Johannessen, International Research Institute of Stavanger, Stavanger, Norway

Expertise: Synthetic serum-free media for cell cultures

Dr. Kjell Bertheussen, University of Tromsø, Tromsø, Norway

Expertise: Bioreactors for tissue engineering

Prof. Dr. Jose Teixeira
Dr. Manuela Gomes, University of Minho, Braga, Portugal

Expertise: Muscle tissue engineering

Robert G. Dennis, UNC NCSU, University of North Carolina, Chapel Hill, NC, USA

Expertise: Experimental and theoretical biophysics of muscle tissue

Dr. Poul Nielsen, Department of Engineering Science, The University of Auckland,
Auckland, New Zealand

Expertise: Large and complex bio-engineering plants

Dr. Gunnar Kleppe, Norwegian Bioindustry Association, Oslo, Norway

55
Participants in a European FP7 application (2009)
IMPROVESS (In vitro Meat PROduction is Vital for Environmental and Societal
Sustainability); Call FP7-KBBE-2009-3; Activity 2:2 Fork to farm ; Subactivity KBEE-2-2-
3 Sustainable food and feed processing. The authors of this report are not included.

Expertise: Biotechnology and society; Bioethics

Prof. Dr. Stellan Wellin
Prof. Dr. Anders Norgen
Dr. Maria Hilling
Linköping University, Linköping, Sweden

Expertise: Ethics in science and technology

Prof. Dr. Mathias Kaiser
National Committee for Research Ethics in Science and Technology
Bergen University, Bergen, Norway

Expertise: Food technology

Dr Johanna Berlin
Katarina Lorentzon
Dan Melin
SIK, The Swedish Institute for Food and Biotechnology, Göteborg, Sweden.

Expertise: Biophysics
Dr. Julie Gold
Prof. Dr. Peter Apell
Department of Applied Physics, Chalmers University of Technology, Göteborg, Sweden

Expertise: Innovation and development of polysaccharide products for

biomedical applications.
Dr. Peter Fyhr
Dr. Bo Ekman
Magle. Magle is a privately held company with administrative headquarter in Lund,
Sweden and its pharmaceutical manufacturing plant in Kristianstad, Sweden

Expertise: Function of markets and institutions in Europe

Dr. Georg Licht
Dr. Mark O. Sellenthin
Industrial Economics and International Management at the Centre for European Economic
Research (ZEW), Mannheim, Germany

Expertise: muscle differentiation

Dr. Marinus F.W. te Pas
Animal Science Group of WageningenUR, Lelystad, The Netherlands

56
Publications about in vitro meat

links can be found on the following webpages:

http://invitromeat.org/content/view/27/45/

and

http://www.new-harvest.org/resources.htm

57
About the authors

Henk P Haagsman (1954) graduated from Utrecht University in 1978 with a degree in
chemistry. He defended his thesis in January 1983. From 1978 onwards he teaches at
the Faculty of Veterinary Medicine. In 1982 he started to work on the pulmonary
surfactant system and studied mechanisms that cause alterations in the surfactant
system in toxic and diseased states. As an experimental system he used isolated type II
pneumocytes. This work was supported by a grant from the Netherlands Asthma
Foundation. In 1985 he was the recipient of the Constantijn en Christiaan Huygens
Award bestowed by the Netherlands Organisation for Scientific Research (NWO). This
award, intended to save excellent scientists for the academia and given to only a few
biochemists, enabled the recipients to do research and to teach for five years at
universities of their choice. From 1986 to 1988 he worked as a visiting scientist at the
Cardiovascular Research Institute, Faculty of Medicine, University of California at San
Francisco, USA. Here he started his work on the structure and function of pulmonary
surfactant proteins in the laboratory of Professor J.A. Clements. After his return he
continued working on these proteins. His publications on this subject include more than
90 scientific papers. In 1998 he was appointed Professor of Meat Science at the Faculty
of Veterinary Medicine of Utrecht University. From 1998 until 2003 he also had an
appointment at the TNO Nutrition and Food Research Institute. From 2001-2005 he was
a member of the University Council. Gradually the emphasis on applied meat research
and regulation of muscle homeostasis shifted towards innate host defence and stem cell
biology. His credo is: “Healthy animals, Healthy food, Healthy people”.

Klaas J Hellingwerf (1950) graduated in 1975 from the University of Groningen with a
degree in chemistry. He obtained his PhD degree in 1978 at the University of Amsterdam
and subsequently became an assistant professor at the Department of Microbiology of
the University of Groningen. In 1982 he was a visiting scientist at the Department of
Physics and Biology of the University of California at San Diego, and in 1987 he was a
visiting scientist at the Institute for Zoology of the Chinese Academy of Sciences, Beijing,
China. From 1988 he is professor in general Microbiology at the University of
Amsterdam. His research interests are signal transduction mechanisms and bioenergetics
of pro- and eukaryotic microorganisms.

Bernard AJ Roelen (1968) studied Biology at Utrecht University where he graduated in

1992. He then worked as a PhD student at the Hubrecht Laboratory in Utrecht under the
supervision of dr. Christine Mummery, and obtained his PhD degree in 1997.
Subsequently he worked as a junior scientist at the same Hubrecht Laboratory until
2001. From 2002-2002 he worked as a post-doctoral researcher at Harvard University in
Boston and after that at the Netherlands Cancer Institute in Amsterdam. In 2003 he was
appointed assistant professor at the Faculty of Veterinary Medicine of Utrecht University.
His research subjects are development of germ cells and stem cells.

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