Original Article

Adipose-Derived Stem Cells: An Optimized

Protocol for Isolation and Proliferation
Mahdieh Ghiasi1, Reza Tabatabaei Qomi1, Naser Kalhor1, Mohsen Sheykhhasan1*

Stem cell laboratory, Stem cell laboratory, The Academic Center for Education, Culture and Research (ACECR)-Qom Branch, shabnam
avenue, isar square, Qom, Iran
1

Article Information

ABSTRACT

Background and Aims: Current advances in the researches on the stem cells has
opened new approaches for their apply in tissue engineering and clinical trials. The
most common sources of stem cells are adult and embryonic stem cells. Due to
ethical issues, embryonic stem cells use in research has been hotly debated. Unlike
embryonic stem cells, adult stem cell have not ethical problem for clinical and research.
The purpose of present study was to stem cells isolation and proliferation from the
human dipose tissue.
Materials and Methods: In this study, stem cells were successfully isolated from
human adipose tissue by digestion with type I collagenase enzymes. In summary,
adipose tissue were digested by type I collagenase enzyme. Subsequently, the cell
solution was centrifuged for mature adipocytes and debris elimination and obtained
sedimentation was cultured in culture medium contains Dulbecco's Modified Eagle's
Medium (DMEM) via 1% penicillin/streptomycin antibiotics and 10% fetal bovine
serum (FBS) at 37C, 5% CO2 and 95% humidity.
Results: in this study, ADSCs were successfully isolated and proliferated. Human
ADSCs were able to divide in our culture mediums.
Conclusion: The results of the current study were suggesting that this adipose-derived
stem cell isolation protocol provides an effective and improved method for isolation
and proliferation of these cells in order to tissue engineering application.

Received: 19 Jul 2015

Accepted: 26 Sep 2015
Plagiarism software: Turnitin

Keywords:

Adipose-derived stem cells,

Optimized protocol,
Cell isolation,
Cell proliferation

Mahdieh Ghiasi

INTRODUCTION
Recent studies have shown that the one of the most
regenerative system in the body is indicated by stem cells.
These cells are available in different part of the body, such
as blood, adipose and etc.1,2 These cells have attractive
properties such as multilineage differentiation and selfrenewal capability.3 Adult stem cells, such as adiposederived stem cells (ADSCs) are one of the most common
stem cells because these cells have not ethically challenge.
ADSCs are usually isolated by enzymatic digestion
of different tissue such as abdominal subcutaneous
adipose.2,3 Mesenchymal stem cells are well-known by
self-renewal capability, to differentiate into different
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DOI:
10.5530ami.2016.1.25

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cell types, to adhere to flask bottom and to expression

of variety surface markers. 4,5 MSCs observed and
isolated from all post-natal tissue and organ.6 Recently,
the development of stem cell technology has provided
significant hope to treat or prevent a disease and
tissue damages and finally supplied huge progress in
regenerative medicine and tissue engineering.79
Stem cell technology is an advanced approach to the
therapy of diseases and damages that uses suitable cell
sources.10 Studies using this method have been suitable
alternative to conventional clinical method.11 Stem cells
are cells with the capability to divide and make a lot of
identical stem cells or to specialize and type specific cells
of bodily tissues.12 Recently, the development of stem
cell technology has provided significant hope for tissue
engineering and regenerative medicine.13 Try to optimize
of isolation and expansion adult stem cells protocols is a
critical step in the expansion of regenerative medicine
methods for tissue repair or regeneration for the therapy
of diseases and damages.14-28 Adipose-derived stem cells
supply a plentiful and suitable source of adult stem
cells for regenerative medicine and tissue engineering

Corresponding Author:
Mohsen Sheykhhasan, Stem cell laboratory, The Academic Center for Education, Culture and Research (ACECR)- Qom Branch, shabnam avenue,
isar square, Qom, Iran. Fax: +982532700154,Telephone: +982532700152, Email: [email protected]

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Ghiasi, et al. : Adipose-derived stem cells

application.29-32 Several methods have been used during

the years to treat a variety of body damages and lesions.33-35
Among them, recently, the emerging progress in the field of
stem cell technology is to treat the diseases and lesions by using
different cell sources, which provide an appropriate support
for repair of lesions and stimuli for tissue regeneration.36 This
will ensure the availability of autologous ADSCs for research,
trial and clinical applications in the future.37 ADSCs have an
appropriate cell source for regenerative medicine.10,13

and were incubated in trypsin/EDTA at 37C for 3min. After

the cells reached confluence at the third passage, ADSCs were
frozen according to current study protocol. Cold freezing
medium contains about 50% fetal bovine serum, 10% dimethyl
sulfoxide (DMSO) and 40% DMEM. The viable cells were
preserved in a final concentration of up to 1 106 cells/ml
at-196C in liquid nitrogen tank for a long time.

In this study, adipose-derived stem cells were successfully

isolated by enzymes digestion.

In the current study, we succeeded in isolating and

proliferating stem cells from the subcutaneous adipose
tissue using an easy and effective protocol. In this work, cell
adhesion observed 2-3day after initiation of the primary
culture. Also, isolated cells having a spindle-shape and
gradually they become elongated. At last, these cells showed
a typical fibroblast morphology (fibroblast-like shape)
under inverted phase-contrast microscope. The cells were
started to adhesion and proliferation. Two weeks after cell
isolation, ADSCs reached to up to 80% confluent rate. The
cells had a higher proliferation rate in the next passages to
passage three and obtained at about 90% confluence after
the seven days (Figure3). These cells had a rapid expansion
in vitro condition. Also, ADSCs displayed positive staining
for the mesenchymal surface markers CD44, CD90, CD105
(Figure4). ADSCs at passage four that showed high-level of
CD44, CD90, CD105 expression. More than 70% of ADSCs
expressed the mesenchymal-specific markers (CD44, CD90,
CD105). In contrast, only a small proportion (less than
5%) of ADSCs expressed the hematopoietic marker CD34
(Figure5 & Table1).

The purpose of this in vitro study was to establish an

improved protocol for ADSC isolation and proliferation.
We also compared the properties of different protocols in
isolation and proliferation of adipose-derived stem cells.

MATERIALS AND METHODS

Human abdominal subcutaneous adipose sample was
obtained from 3 patients undergoing of elective surgery
after obtaining written consent from patients (Figure1). Cell
isolation was performed from the adipose tissue samples.
Initially, the adipose tissue sample was mechanically
segmented and connective tissue and blood vessel were
removed carefully from this sample. The obtained pieces
were rinsed with phosphate buffered saline (PBS) several
times and were digested with typeI collagenase (1.5mg/1g
adipose tissue) at 37C for 45-60min. Then, the cell suspension
was centrifuged at 1800rpm for 10min and the cell pellet was
cultured in Dulbeccos Modified Eagles Medium (DMEM)
supplemented with penicillin streptomycin and 10% FBS,
and incubated at 37C with 5% CO2 and 95% humidity. The
medium was changed at least twice per week until the cells
reach up to 80% confluence. In this method, A large number
(approximately 200,000) of adipose-derived stem cells (ADSCs)
were obtained per gram of adipose tissue (Figure2a-j). Cells
were passaged at 80% confluence by washing twice with PBS

RESULTS

Table1: Flow cytometry analysis of adiposederived

stem cells(ADSCs) at passage 4
Surface markers
CD105
CD90
CD44
CD34

ADSCs at passage 4
74.90.2
72.92.5
70.40.7
4.40.5

Data are presented as the meanstandard error of mean(SEM), N=5

Figure 1. Schematic image demonstrating of the human abdominal subcutaneous adipose tissue and its components

117

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Ghiasi, et al. : Adipose-derived stem cells

d
j

Figure 2: different steps of adipose-derived stem cells isolation (a-j). This schematic describes the isolation process of adipose-derived stem cells from harvested
whole human adipose tissue

Figure 3: Human adipose-derived stem cells at different passages. (a) Passage 0, (b) Passage 1, (c) Passage 2 and (d) Passage 3

Figure 4: Schematic image of ADSCs specific surface markers (CD44, CD90,

CD105)

ADSCs were successfully stored more than six months in

liquid nitrogen tank (Figure6).

DISSCUSION
In this study, we showed the potential of this protocol
for adipose-derived stem cells isolation. In the present
study, in primary isolated cells, more than 70% of the
cell population positively expressed mesenchymal
surface markers (CD 44, CD90, and CD105). Also, these
cell populations negatively expressed hematopoetic
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marker (CD34). The characterization of isolated cells

was confirmed as ADSC by flow cytometry method.
These results were supported by many studies result
that reported previously.24,10,38 Our current method was
developed by using of a simple protocol of washing
adipose tissue. Also, in this method the processing time
was modified in comparison with the enzymatic methods.
In this work, we succeeded in isolating and proliferating
these cells in the try to use them in tissue engineering
application in the future.
The stem cells of the adipose tissue can represent a critical
source of cells with multipotential properties.36,39,40 The
optimization of the isolation and culture conditions may
increase the comparability of the results from different
research laboratories.14 Although, the essential elements
in optimal isolation of stem cells are the type of used
enzymes and or materials and duration of different
steps and the centrifugation process4,5 but no unique and
optimized isolation protocol for ADSCs has been accepted
overall.41

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Figure 5: Expression of ADS cells specific markers (CD44, CD90, CD105) on primary culture of ADSCs by flow cytometry method. CD34 antibody was used as
negative marker

Katz et al were developed a novel protocol foradipose-derived

stem cells isolation with modifying in collagenase digestion
and tissue washing steps that were largelycorroborated our
present study.36
Such as our protocol, The recent protocols for human adiposederived stem cell isolation have been used a collagenase
digestion followed by centrifugation process to separate the
stromal vascular fraction (SVF) from pre-adipocytes.22
In some of the protocols, collagenase enzyme such as
0.075% collagenase typeI, 0.3% collagenase typeI and 0.6%
collagenase type I was replaced with trypsin.11,12,14 Some
protocols such developed protocol by shah et al was used
a non-enzymatic method.21,23,27,44
Figure 6: Schematic image demonstration of successful storage of ADSCs in
liquid nitrogen tank

The primary techniques to isolate ADSCs were pioneered

by rodbell et al utilizing rat adipose tissue. 29 This
classic method to isolate human adipocyte progenitors
was modified by Van, Roncari, Deslex, Hauner and
others.20,32,42,43
Different methods were assessed for ADSCs isolation.8-32
Most Common methods for ADSCs isolation is consisted
of washing the adipose tissue, digestion with collagenase
and subsequently centrifugation process steps.31
The first important step in isolation adipose-derived stem
cells is washing process that this process can be affected on
ADSCs heterogeneity.21
Different ADSCs isolation protocols were used in variance
components such as Ca2+-and Mg2+-free Hanks balanced
salt solution containing sodium HEPES, Phosphate Saline
Serum (PBS) and Normal Saline Serum in washing step.27, 28
In the current study, we used to Phosphate Saline Serum
(PBS) for washing the adipose tissue sample.
119

For example, in a study, a mechanical strategy was used for

isolating adipose-derived stem cells from a conventional
liposuction method by raposio et al.14 In this study, no
enzymes such as collagenase and animal-derived materials
were used. Also, Developed protocol by baptista et al was
reply on mechanical shaking incubation plus to red blood
cell lysis solution, simultaneously.3
ADSCs proliferation can be highly influenced by basal
medium, glucose concentration, Quality of FBS, cell plating,
and cell density.45 Cheng et al were used the KNAC medium
culture for growth of human ADSCs with a lower calcium
rate.10 This experiment that use of K-NAC medium for cell
growth have been significantly increased in cell proliferation
ability.

CONCLUSION
In conclusion, our findings propose that this protocol can
be utilized for the isolation of ADSCs, particularly when
purpose of study related to tissue engineering strategies.
Also, this protocol can be easier, cheaper, and faster method
than other current procedures.

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Ghiasi, et al. : Adipose-derived stem cells

ACKNOWLEDGEMENTS
We thank The Academic Center for Education, Culture
and Research (ACECR)-Qom Branch as the financial and
executive sponsor.

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How to cite this article: Ghiasi M, Qomi RT, Kalhor N, SheykhhasanM.
Adipose-derived stem cells: An optimized protocol for isolation and
proliferation. Acta Medica International. 2016;3(1):116-121.
Source of Support: Academic Center for Education, Culture and
Research (ACECR) - Qom Branch as the financial and executive sponsor,
Conflict of Interest: None declared.

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