The use of serum-free medium and its related genera of animal-free and protein-free media has grown significantly

in the last 15 years. This is particularly true in industrial applications where the use of serum presents a safety hazard as well as a source of unwanted contamination for the production of biopharmaceuticals. Serum-free medium is prepared without the use of animal serum, but may contain serum constituents or substitutes thereof. Animal-free medium is similar to serum-free except that the components are derived from non-animal sources. Recombinant proteins replace native proteins and the nutrients are obtained from synthetic, plant or microbial sources. In contrast, protein-free medium is defined as devoid of protein, although few formulations of these media can be 100% protein-free without loss of function. A better definition would be low protein where minimal quantities of small mass proteins are employed. The three types of media can be thought of as gradations of complexity. Serum-free media is the more complex composition designed for universal use in culturing mammalian cell lines. Animal-free and protein-free media formulations are less complex and more defined, but are limited to the cultivation of specific cell types. Thus, the selection of one medium over another will depend on the intended use. For most research applications, serum-free medium is the best choice as it provides broad-spectrum utility while giving good control of the composition. In contrast, in the manufacture of protein-based drugs, potential contaminants and production costs play a more significant role in the selection of a more defined medium composition. Serum-free medium has four basic advantages over serum containing medium. These include: 1] a simplified and better defined composition, 2] a reduced degree of contaminants, 3] elimination of a potential source of infectious agents, and 4] lower cost.

Simplified and Defined Composition

Serum is an ill defined component of medium. Typically used at 5% to 10% v/v, serum provides a range of factors which have proved necessary for the cultivation of mammalian and insect cells. Serum however can be derived from different sources and the composition of the serum can vary greatly. Advances in the manufacture and processing of serum has reduced this variability, but many researchers continue to find it necessary to qualify new lots of serum by comparing the growth of their cell lines in the new serum relative to a current lot. With a typical acceptance criteria of ?20%, the variance of whatever physiological function is being measured can be quite high. This leads to poor reproducibility of results between and within laboratories and stories abound of irreproducible results which can be directly attributed to the use of serum as a media component. A quantitative understanding of cellular physiology cannot be obtained with such ill-defined and variable conditions. Serum-free medium on the other hand is a more defined medium. While composed of many constituents, the composition is known and the level of each component precisely defined. Therefore, the variance seen with serum containing medium is eliminated giving a more controlled environment in which cells can be grown.

Reduced Range and Level of Contaminants

Medium prepared with serum at 10% has a protein concentration of 6,200 to 10,000 mg/L. A typical recombinant protein produced in mammalian cells is anywhere from a few mg/L to 1,000 mg/L. For a native protein, the level of accumulation can be even lower. The serum proteins thus become a major contaminant of any crude supernatant

in which the target protein accumulates. Further, if the target protein is functionally, biochemically or physically related to a serum protein, then it may prove difficult to separate the target protein from the serum protein. This could be disastrous for the production of a protein-based drug, but equally troubling to the researcher trying to study the functional and physical properties of the target protein. The protein concentration of serum-free media on the other hand is between 50 mg/L and 1,000 mg/L. Unlike serum, the composition is known and typically three proteins, albumin, transferin and insulin, comprise 80% to 90% of the proteins present. Consequently, the relative level of the target protein is significantly higher and with fewer contaminants. Thus, the target protein is easier to purify in fewer steps which gives higher recovery values. Drug discovery, physiological, or gene expression studies are simpler to perform with serum-free medium. Serum components are known to bind, degrade or otherwise interact with chemicals added to the medium. Complex associations are possible between the serum, the added effector and the cells. Thus, the effect elicited by the added chemical can be altered or eliminated by the serum factors. Serum-free medium by contrast has fewer possible interferents and where an interaction is observed, more readily controlled.

Elimination of Potential Source of Infectious Agents

Viral, bacterial and fungal contamination of serum has for some years been a concern by manufacturers of biopharmaceuticals. This has been a driving force behind the adoption of serum-free, animal-free and protein-free media formulations in the manufacturing process. More recently, transmissible spongiform encephalophathies described in animal-derived materials has lead to the elimination of any animal product for the manufacture of a pharmaceutical product. In the research setting, where more fastidious cell lines are employed, the complete elimination of animal-derived components is both impractical and unusable. Recombinant forms of some key components remain expensive and in some cases not available. Many of the commercially available animal-free and protein-free media were designed for specific cell types and will not support the growth of a wide range of cell types. Because the materials used for the production of serum-free media are for the most part highly purified, the risk of contaminating a culture with adventitious agents is greatly reduced or eliminated altogether. For nonpharmaceutical applications, serum-free medium remains a good balance between safety and efficacy.

Lower Cost and Availability

Serum can cost between $7 and $50 per liter of medium depending on the type and percentage of serum used. Fetal calf serum, the most commonly used source of serum, is also the most costly, with current prices (August 2002) averaging $456/L of serum. Therefore, serum can contribute significantly to the cost of the medium. Serumfree medium by contrast averages about $120/L with a range from $26/L for a DMEM derivative to $692/L for highly specialized bone marrow medium. Most of the serum-free media are supplied ready-to-use, so they are available without the need of preparing a multi-component reagent.

Disadvantages
The use of serum-free medium is routine for many cell types and many formulations are available in the literature or through a number of vendors. Nevertheless, there are a few drawbacks that should be considered when using serum-free medium. First, an investment in time is required to adopt a particular cell line to serum-free medium.

The cells will have to be weaned from serum slowly. Moreover, some cell lines may require the addition of growth factors specific to that cell type to overcome a deficiency in the particular medium employed. It is advisable to begin with a serum-free medium which has a source of growth factors, such as pituitary extract, which can be incrementally removed if necessary. The second limitation is that the low protein concentration of serum-free medium while an advantage for reducing potential sources of contamination removes proteins which play a role in shear protection and attachment to growth substratum. The BSA present in serum protects cells grown in suspension from shear damage. The addition of Pluronic F68 or polyethylene glycol may be needed in place of BSA. BSA also provides other transport functions and the replacement of native sources of albumin with recombinant sources is not straightforward. Alternative supplements may be needed. Many of these issues are resolved by the commercially available formulation and only become a concern when developing a custom medium. Attachment-dependent cell lines require an extracellular matrix on the growth substratum. Serum provides some the components for this matrix. Therefore, when using serum-free medium the substratum (plastic dishes) should be pre-coated with a fibronectin, laminin or another suitable alternative such as FNC Coating Mix (a fibronectin/collagen mixture manufactured by AthenaES, Baltimore, MD), Pronectin (a synthetic fibronectin polymer manufactured by Sanyo Chemical Industries, Kyoto, Japan) or Matrigel.

Summary
Serum-free medium is an excellent alternative to standard serum-containing media for the cultivation of cells. It has several marked advantages which include better definition of the composition, reduced contamination by adventitious and infectious agents, and lower cost. Further, the commercial availability of many variations of serum-free media has made it easy to obtain and employ. Consequently, the use of serum-free medium has now become a routinely used reagent in many laboratories for the culture of a wide variety of cell types.

About the Author

Sheldon E. Broedel, Jr., Ph.D. serves as the Chief Science Officer at AthenaES? . He has over 23 years of research experience, 15 of which were spent in the biotechnology industry. Dr. Broedel has broad expertise in a range of biology disciplines with over 20 products and patents to his credit. Currently, his research team is developing reagents designed to increase the production of recombinant proteins.

Tissue engineering refers to the application of the principles of engineering to cell culture for the construction of functional anatomical units- tissues/organs. The aim of tissue engineering is nothing but to supply the various body parts for the repair or replacement of damaged tissues or organs. It is now possible to grow skin cells, blood cells cardiac cells etc. by using the ability of stem cells to proliferate and differentiate. During the last decade, the tissue culture work in animals demonstrated that virtually any human tissue or organ can be grown in culture. This became possible only after it became known that the ability of cultured cells to undergo differentiation can be restored. Skin was the first organ to be cultured in artificial media and could be successfully used for transplantation following serious skin burns. For past few years some of the biotech companies like ATS (Advanced Tissue Science, USA), Biosurface Technology (BTI, Cambridge) and Organogenesis, are developing artificial skins to the stage of clinical trials. In the field of tissue replacement, focus of attention is the Artificial cartilage. As it is not vascularized, it is not rejected due to immunogenic response. This will have lots of implications in the treatment of sport related injuries and diseases like arthritis. Design and engineering of tissues The design and tissue engineering should essentially cause minimal discomfort to the patient. The damaged tissues should be easily fixed with the desired functions quickly restored. Another important factor controlling the designing of tissue culture is the source of donor cells. The cells from the patient himself, is always preferred as it considerably reduces the immunological complications. However under certain situations allogeneic cells (cells taken from a person other than the patient) are also used. The other important factors are the support material, its degradation products, cell adhesion characteristics etc. It was demonstrated in 1975 that human keratinocytes could be grown in the laboratory in a form suitable for grafting. A continuous sheet of epithelial cells can be grown now however there is still difficult to grow TE skin with the dermal layer with all the blood capillaries, nerves, sweat glands, and other accessory organs. Some of the implantable skin substitutes which are tissue engineering skin constructs with a limited shelf life of about 5 days are: a) Integra TM A bioartificial material composed of collagen-glycosaminoglycan and is mainly used to carry the seeded cells. b) DermagraftTM- This is composed of poly glycolic acid polymer mesh seeded with human dermal fibroblasts from neonatal foreskins. c) ApligrafTM- It is constructed by seeding human dermal fibroblasts into collagen gel with the placement of a layer of human keratinocytes on the upper surface.

These tissue constructs integrate into the surrounding normal tissue and form a good skin cover with minimum immunological complications. The urothelial cells and smooth muscle cells from bladder are now being cultured and attempts are on to construct TE urothelium. Some progress has also been made in the repair of injured peripheral nerves using tissue engineered peripheral nerve implants. The regeneration of the injured nerve occurs from the proximal stump to rejoin at distal stump. The regeneration process requires substances like(a) Conduct material- The conduct material is composed of collagen- glycosaminoglycans, PLGA (poly lactic- co- glycolicacid), hyaluronan and fibronectin and forms the outer layer. (b) Filling material- The filling material contains collagen, fibrin, fibronectin and agarose. This supports the neural cells for regeneration. and (c) Additives- A large number of other factors are also added e.g. growth factors, neurotrophic factors such as fibroblast growth factor (FGF), nerve growth factor (NGF). The other important applications of tissue engineering are in gene therapy, pseudo-organs and as model cell systems for developing new therapeutic approaches to human diseases.The attempts are on to create tissue models in the form of artificial organs using tissue engineering. The artificial liver is being created using hepatocytes cultured as spheroids and held suspended in artificial support system such as porous gelatin sponges, agarose or collagen. Some progress has been made in the area of creating the artificial pancreas using spheroids of insulin secreting cells which have been developed from mouse insulinoma beta cells. Three dimensional brain cell cultures have been used for the study of neural myelination, neuronal regeneration, and neurotoxicity of lead. The aggregated brain cells are also being used to study Alzheimers disease and Parkinsons disease. Thyroid cell spheroids are being used to study cell adhesion, motility, and thyroid follicle biogenesis. (Table 8.2 page 155, gupta) TABLE DEPICTING THE TECHNOLOGICAL GOALS AND AREAS OF RESEARCH IN TISSUE ENGINEERING

Growth of cells in three- dimensional systems Delivery systems for protein therapeutics Cell cultivation methods for culturing recalcitrant cells Expression of transgenic proteins in transplantable cells To develop vehicles for delivering transplantable cells Development of markers for tracking transplanted cells

Avoiding immunogenicity in transplantable cells Development of in vivo and ex vivo biosensors for monitoring cell behaviour during tissue production
DOWNSTREAM PROCESSING Downstream processing or downstreaming is the extraction and purification of the desired end products of fermentation processes. Such products might include cells, solvents or solutes. Various processes are available for the separation of cells from the fermentation broth in which they are grown, including flocculation, filtration, centrifugation, sedimentation or flotation. The procedure adopted depends on whether it is the cells, or the solution surrounding them, that contains the desired end products.

Classification
Tissue culture may be of two types based on medium. 1. Plant tissue culture 2. Animal tissue culture

Animal Tissue Culture

The term tissue culture refers to the culture of whole organs, tissue fragments as well as dispersed cells on a suitable nutrient medium. It can be divided into (1) organ culture and (2) cell culture mainly on the basis of whether the tissue organisation is retained or not. The beginning of animal tissue culture can be traced to 1880 when Arnold showed that leucocytes can divide outside the body. Later, in 1903, Jolly studied the behaviour of animal tissue explants immersed in serum, lymph or ascites fluid. In 1907, Harrison cultured frog tadpole spinal chord in a lymph drop hanging from a cover slip into the cavity on a microscopic slide; this is regarded as the turning point. A few years later, in 1913, Carrel developed a complicated methodology for maintaining cultures free from contamination, especially by bacteria. Subsequently, suitable culture media were developed and the techniques of cell culture were refined.

Culturing Animal Tissues- The steps

1. Animal tissue is obtained either from a particular specimen, or from a tissue bank of cryo-preserved (cryo = frozen at very low temperatures in a special medium)

2. Establishment of the tissue is accomplished in the required medium under aseptic conditions 3. Growing the cells / tissue requires an optimum temperature, and subculturing when required 4. Human cells, for example are grown at 37degrees and 5% CO2

Animal tissue/cell culture - differences from plant tissue culture

1. Animal cell lines have limited numbers of cell cycles before they begin to degrade 2. Animal cells need frequent subculturing to remain viable 3. Tissue culture media is not as fully defined as that of plants - in addition to inorganic salts, energy sources, amino acids, vitamins, etc., they require the addition of serum (bovine serum is very common, but others are used) 4. Animal tissue cultures can pose biohazard concerns, and cultures require special inactivation with hypochlorite (e.g. Janola,Chlorox, etc.) and then incineration

Uses of Animal Tissue Culture

1. Growing viruses - these require living host cells 2. Making monoclonal antibodies, used for diagnosis and research 3. Studying basic cell processes 4. Genetic modification & analysis 5. Knockout technology inactivating certain genes and tracing their effects 6. Providing DNA for the Human Genome Project (and other species genome projects)

Bibliography
1. Dodds, J.H., Roberts, L.W., 1995, Experiments in Plant Tissue Culture, 3rd ed., Cambridge University Press 2. Hartmann, H., Kester, D., et.al., 1997, Plant Propagation, 6th ed., Prentice Hall International 3. http://www.une.edu.au/agronomy/AgSrHortTCinfo.html 4. http://aggie-horticulture.tamu.edu /tisscult/pltissue/pltissue.html 5. http://www.liv.ac.uk/~sd21/tisscult/what.htm 6. http://user.school.net.th/~anoparp/bptc1.htm

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