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Cell and Tissue BankingInternational Journal for Banking,Engineering and Transplantationof Cells and Tissues IncorporatingAdvances in Tissue Banking ISSN 1389-9333 Cell Tissue BankDOI 10.1007/s10561-012-9304-6

Adipose tissue can be generated in vitroby using adipocytes from human fattissue mesenchymal stem cells seeded andcultured on fibrin gel sheet

Cong Toai Tran, Duy Thao Huynh,Ciro Gargiulo, Le Bao Ha Tran, MinhHang Huynh, Khanh Hoa Nguyen, LuisFilgueira & D. Micheal Strong

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ORIGINAL PAPER

Adipose tissue can be generated in vitro by usingadipocytes from human fat tissue mesenchymal stem cellsseeded and cultured on fibrin gel sheet

Cong Toai Tran • Duy Thao Huynh • Ciro Gargiulo •

Le Bao Ha Tran • Minh Hang Huynh • Khanh Hoa Nguyen •

Luis Filgueira • D. Micheal Strong

Received: 14 November 2011 / Accepted: 20 February 2012

� Springer Science+Business Media B.V. 2012

Abstract The current study has developed an inno-

vative procedure to generate ex novo fat tissue by

culturing adipocytes from human fat tissue mesenchy-

mal stem cells (hFTMSCs) on fibrin gel sheet towards

applications in medicine and cosmetology. Fibrin gel

has been obtained by combining two components

fibrinogen and thrombin collected by human peripheral

blood. By this procedure it was possible to generate

blocks of fibrin gel containing adipocytes within the

gel that show similar features and consistency to

human fat tissue mass. Results were assessed by

histological staining methods, fluorescent immune-

histochemistry staining as well photos by scanning

electron microscopy (SEM) to demonstrate the adhe-

sion and growth of cells in the fibrin gel. This result

opens a real possibility for future clinical applications

in the treatment of reconstructive and regenerative

medicine where the use of stem cell may eventually be

a unique solution or in the field of aesthetic medicine

where autograft fat stem cells may grant for a safer and

better outcome with long lasting results.

Keywords Mesenchymal stem cells � Adipocytes �Fibrin gel � Fat tissue mass

Introduction

Nowadays, scientists have identified different variety

of sources from which it may be obtained multipotent

mesenchymal stem cells (MSCs), such as bone

marrow (BM), umbilical cord blood (UCB), peripheral

blood, placenta and adipose tissue. Among those, due

to its qualities and great availability adipose tissue is

probably the higher and more attractive source of

MSCs (Unguryte et al. 2010). Fat tissue is very

common and abundant in the body, particularly rich in

MSCs and very easy to collect with a drastically low

invasive procedure. hFTMSCs have the same charac-

teristics and features of those from BM and UCB,

(Locke et al. 2009; Zuk et al. 2002; Gimble and Guilak

2003), however results either from our study or

published researches have confirmed that fat tissue

compared to BM and UCB contains more MSCs, for

C. T. Tran (&) � D. T. Huynh � M. H. Huynh �K. H. Nguyen

Department of Histo-pathology, Embryology, Genetics

and Biotechnology for Tissue Transplants, Pham Ngoc,

Thach Medical University, Ho Chi Minh City, Vietnam

e-mail: toaiphd@yahoo.com

C. Gargiulo � L. Filgueira

University of Western Australia School of Anatomy

and Human Biology, Crawley, WA, Australia

L. B. H. Tran

Laboratory Research and Application of Stem Cells,

University of Natural Sciences, Ho Chi Minh City,

Vietnam

D. M. Strong

Department of Orthopaedics and Sport Medicine,

University of Washington School of Medicine, Seattle,

WA, USA

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DOI 10.1007/s10561-012-9304-6

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instance, from 1 g of tissue it is possible to collect

5 9 104–5 of MSCs c.ca which is 500-folds larger than

1 g of MSCs obtained from BM (Mizuno 2009; Strem

et al. 2005). hFTMSCs express all the specific markers

of the MSCs genre with the same authentic capacity to

differentiate into multiple cell types (Zuk et al. 2002),

such as osteocells, chondrocytes, hepatocytes, neu-

rons, insulin secreting cells, keratinocytes and adipo-

cytes (Wilson et al. 2011; Gimble. 2003; Entenmann

and Hauer 1996; Toai et al. 2010). This project

particularly focused on obtaining adipocytes from

hFTMSCs to be used as fat tissue producers that could

be extensively used in clinical for different medical

purposes.

The main important aspect was to have an appro-

priate scaffold capable to contain adipose cells, that

allows cell growth and support differentiation (Cheryl

et al. 2006). Modern reconstructive strategies to repair

defected tissues such as breast, skin, cartilages and

bones are based on the use of implants and filler

(Gomillion and Burg 2006a, b; O’Brien et al. 2004;

Einhorn 1995; Ogawa 2006). However, there is no a

single filler material or implant which may be suitable

for all different needs (Gomillion and Burg 2006a, b).

This has literally pushed medical scientists towards

new and alternative solutions to be used in reconstruc-

tive and regenerative medicine, tools that would

definitely combine the high feasibility of bio-materials

and the incredible ductility of stem cells (O’Brien et al.

2004; Einhorn 1995; Chun et al. 2009; Gomillion and

Burg 2006a, b; Toai et al. 2010; Kucerova et al. 2007).

As a result, we are witnessing an enormous quantity of

data coming from either in vitro or in vivo investigation

of hFTMSCs alone or in combination with bio-

material, these types of materials that have been

studied for the application as substrate or carrier are

cell membrane collagen, fibronectin, fiberglass, cera-

mic, coral, or scaffolds from plastic chemical synthesis

(Zhang et al. 2007; Kucerova et al. 2007; Strem et al.

2005; Gomillion and Burg 2006a, b; Zuk et al. 2002;

Wang et al. 2009; Ogawa 2006; Tuan and Chen 2006;

Rebellatto et al. 2008; Mizuno 2009; Banas et al.

2007). However, there is just a little pure biological

material capable of carrying and supporting cell

growth and cell differentiation. Thus, we focused in a

particular product that could combine the softness of

gel and a bio-compatibility of human tissue easy to

manage, insert and safe. Therefore, we were looking at

two main components in the peripheral blood, fibrin-

ogen and thrombin, and we eventually ended up to a

procedure that allows us obtain by combining together

these two components to generate a fibrin gel to be used

as cell supporter and carrier. This material has many

applications in medicine (Samir et al. 2003; Buckley

et al. 1999; Stechison 1992; Saltz et al. 1991) and could

be used in cell culture (Lee 2008; Krasna et al. 2005).

Materials and methods

Collection of fat tissue

Adipose tissue has been obtained from healthy donors

in sterile conditions at hospital operating room and

transferred by an apposite tube containing a specific

medium composed DMEM/F12, FBS (10%), Gent-

amycine (50 lg/ml), to the laboratory. Blood donors

were tested for HIV, HBV, HCV and VDRL and

samples were proceeded to isolate mesenchymal stem

cells (Figs. 1, 2).

Fig. 1 The process of receiving and processing fat tissue. a Adipose tissue collected in sterile conditions. b Cut the fat into small

pieces. c Adipose tissue is incubated with the enzyme mixture

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Isolation of cells

Adipose tissue has been placed into petri dishes and

rinsed three times with PBS solution containing

penicillin/streptomycin and washed one time with

PBS solution with no antibiotics. Afterwards tissue

was transferred into a different petri dish and

immerged into a basic culture medium that is

composed as follow: DMEM/F12, FBS (10%), Gent-

amycine (50 lg/ml), HEPES (15 mm), NaHCO3

(14 nM), Biotin (33 lm), D-Panto (17 lm), penicillin

(100 U/ml) and streptomycin (0.1 mg/ml), it was

removed the excess connective tissue and washed

from blood.

Sample were manually fragmented into small tissue

blocks and immerged into an enzymatic solution of

dispase-collagenase (ratio 3:1, v/v) and incubated at

temperatures 37�C/5%CO2 for 90 min. Samples were

centrifuged at 3,000 rpm for 5 min, it was removed all

floating material above the solution and the sediment

on the bottom was collected. The collected material

was immerged into a basic culture medium, cells were

counted by trypan blue and cultured into T-25 cm2

flask and incubated at 37�C/5%CO2. Medium has been

replaced every 3 days.

Adipocytes from hFTMSCs

hFTMSCs were subcultured 2 times the culture

medium has been replaced by a pre-adipocytes

differentiation culture medium that includes, basic

culture medium with insulin (66 nM), Triiodo-L-

thyronine (1 nM), Human transferrin (10 lg/ml).

After 3 days, the medium was replaced by an

adipocytes differentiation culture medium composed

of Isobutyl-methylxanthin (0.5 mM), Hydrocortisone

(100 nM) and Dexamethasone (0.1 nM).

After 3 weeks of cell culture, cells were collected

and identified by observing morphological changes

through inverted microscope, by cyto-chemical stain

Oil Red and Nile Red.

How to obtain fibrin gel

Fibrin gel was obtained by combining two components

fibrinogen and thrombin, extracted from peripheral

blood of healthy and consent donors. Blood has tested

negative for HIV, HBV, HCV and VDRL.

Collection of fibrinogen (Hartman et al. 1992)

The blood was centrifuged at 3,000 rpm for 5 min. It

was collection 10 ml of serum that was centrifuged at

3,000 rpm for 5 min. The solution was filtered by

using a filter with a diameter of 0.20 lm (Minisart

Sartorius�) and placed into a new sterile tube and

incubated into a refrigerator at 4�C for 1 h, and moved

overnight into a different refrigerator at -20�C.

Collection of thrombin (Quick 1966)

Blood sample was centrifuged at 3,000 rpm for 5 min

and 10 ml of serum was collected. Serum was

centrifuged at 3,000 rpm for 5 min. The solution

was collected and filtered by a diameter 0.20 lm

Minisart Sartorius� filter into a new sterile tube and

incubated at 4�C for 1 h and overnight at -20�C. Then

the tube was thawed at a temperature of 4�C, PBS

Fig. 2 Isolated mesenchymal stem cells from adipose tissue. a Cell growth after 3 days of culture. b Cell growth after 7 days of

culture. c Cells were stained with giemsa after 10 days culture

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solution was added (ratio 1:9). Acetic acid was added

1% to adjust the pH at 5.3. The solution was allowed to

stand for 30 min to precipitate. Afterwards it was

centrifuged at 3,000 rpm for 5 min. The sediment on

the bottom was removed and collected. PBS was

added (ratio 1:9). Na2CO3 0.1 M was used to adjust to

pH 7.0. The whole was placed in a thermostatic bath at

37�C for 15 min and CaCl2 (0.01 M) was added to

create clots. Clots were removed and discharged,

while the remaining liquid that is thrombin was

collected and preserved at a temperature of -20�C.

Seeding hFTMSCs onto fibrin gel

Fibrin gel is formed by the combination of fibrinogen

and thrombin (ratio of 1:1). hFTMSCs at second

passage were collected by enzymatic digestion, a

solution of trypsin–EDTA (0.25–0.02%). Cells were

centrifuged at 3,000 rpm for 5 min, to cells deposited

at the bottom were added a fibrinogen solution. The

cell-fibrogen solution is moved to a 30 cm diameter

petri dishes and additional thrombin was poured (ratio

1:1), the whole was manually mixed and let rested for

few minutes up to it became an homogeneous compact

formation. After 1 day, the cell culture medium was

replaced with pre-adipocytes differentiation culture

medium. After 3 days, culture medium was replaced

by adipocytes differentiation culture medium. After

3 weeks of culture, the cells are tested by inverted

microscope, Oil Red staning, H&E staining for fibrin

gel.

Results

Flowcytometry for hFTMSCs

Results showed that cell lines are negative for the

marker: CD14, CD45, HLA-DR and positive for the

marker: CD13, CD44, CD73, CD90, CD105 and

CD166. Thus, we have isolated and successfully

cultured mesenchymal stem cells isolated from adi-

pose tissue (Fig. 3) .

Differentiated mesenchymal stem cells

into adipocytes

After 21 days, hFTMSCs cultured in adipogenic

medium were observed under inverted microscope to

evaluate and change in cell shape, cells had changed

into a typical oval-round shape with the characteristic

cytoplasmic lipid droplet accumulation.

Oil Red staining results confirmed that the cyto-

plasm of cells containing lipid particles are captured

by red color, lipid particles accounted for nearly all of

the cell cytoplasm (Fig. 4).

Nile Red staining confirmed that the cells cyto-

plasm contains lipid particles and are very specific.

This is the best effective method to identify cells as fat

cells (Fig. 5).

Overall, the obtained results are conclusive and

confirmed that hFTMSCs are able to completely

differentiate into adipocytes at least in in vitro culture.

Create blocks of tissue from MSCs and fibrin gel

Results generated fibrin gel

When mixed fibrinogen and thrombin (ratio of 1:1)

they are able to form a block of gel within 5 min, the

final result is a fibrin gel that is bright yellow in color

and very tough and resilient in consistency (Fig. 6).

Results of cell culture on fibrin gel

After 21 days culture, hFTMSCs cultured on fibrin gel

by using an adipogenic culture medium are completely

differentiated into adult adipocytes (Fig. 7).

Oil Red staining directly performed onto cells

cultured on fibrin gel showed that hFTMSCs are

completely differentiated into adipocytes with the

typical round shape and the very characteristic cyto-

plasm containing lipid particles captured by bright red

with oil red dye (Fig. 8).

Then, fibrin gels containing adipocytes were fixed

in 10% neutral buffer saline solution and stained H&E.

The results show that cells survive and grow well on

fibrin gel, within the cell cytoplasm are visible lipid

droplets (Fig. 9).

Discussion and Conclusion

Presently, the adipose tissue is considered the same as

an active functional endocrine system, actively

involved in hormone regulation and homeostatic

balance (Wisse 2004; Tuan and Chen 2006; Lidong

et al. 2006). It has revealed great potentials in terms of

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Fig. 3 Cells was assessed

by flow cytometry. a The

cell is negative for three

markers, including CD 45,

HLA-DR, and CD 14. b The

cell is positive for six

markers, including CD 13,

CD 44, CD 90, CD 166, CD

73, and CD 105

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limitless availability of MSCs and thus therapeutic

opportunities (Gomillion and Burg 2006a, b; Zuk et al.

2002; Wang et al. 2009; Ogawa 2006; Kucerova et al.

2007; Zhang et al. 2007; Rebellatto et al. 2008). In

fact, adipose MSCs show to belong to the big family of

MSCs, sharing qualities and features that have made

Fig. 4 Mesenchymal cells are stimulated to differentiate into

fat cells. a Mesenchymal stem cells used as negative controls.

b After the cells differentiate into fat cells. c Differentiated cells

after being stained with Oil Red dyes, cells positive for catching

red–orange dye, bright cytoplasm. d After staining with Oil Reddye in the cell cytoplasm that appeared many lipid droplets,

dominating the cell cytoplasm

Fig. 5 Results of Nile Red staining for adipocytes after

differentiation. a Negative control samples, mesenchymal stem

cells. b Adipocytes are differentiated after staining with Nile

Red, the results showed that the cytoplasm of many cells appear

red–orange lipid particles, lipid particles accounted for nearly

all the cell cytoplasm, the nucleus was push offset to one side of

the cell

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Fig. 7 Create a fibrin gel containing fat cells. a fibrin gel was

observed under inverted microscope. b fibrin gel was fixed and

stained H&E, the results show that gel is formed that contains

many small holes and cavities, suitable for cell adhesion and

development within the block of gel. c Cell growth inside the gel

were taken under inverted microscope, cells grow and distrib-

uted into several layers within the gel. d Cell adhesion and

growth on the surface of gel blocks

Fig. 6 The resulting fibrin gel. A Two components fibrinogen

and thrombin were isolated separately, (a) fibrinogen and

(b) thrombin. B The combination of fibrinogen and thrombin

with a 1:1 by volume, within 1–5 min, a block of gel quickly

formed, a yellow light, elastic and supple

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these cells so specials and one of the greatest

expectation in all medical world (Hartman et al.

1992; Locke et al. 2009; Unguryte et al. 2010; Zuk

et al. 2002; Wang et al. 2009; Zhu et al. 2008;

Kucerova et al. 2007; Zhang et al. 2007; Rebellatto

et al. 2008; Sathishkumar et al. 2011). Moreover, fat

tissue is easy to collect through a very low invasive

procedure with almost zero complications for the

donor (Quick 1966; Zhang et al. 2007; Kucerova et al.

2007; Strem et al. 2005; Gomillion and Burg 2006a, b;

Zuk et al. 2002; Wang et al. 2009; Ogawa 2006;

Rebellatto et al. 2008). The need of reconstructive

surgical procedures is increasing and extremely high is

the demand of repairing damaged tissues due to

diseases, injuries or congenital defections (Gomillion

and Burg 2006a, b; Mizuno 2009; Tuan and Chen

2006; Ogawa 2006). Modern reconstructive strategies

to repair damaged tissues such as breast, skin,

cartilages and bones are based on the use of implants

and filler (Kalmoz et al. 2006; Gomillion and Burg

2006a, b; O’Brien et al. 2004; Einhorn 1995; Ogawa

2006; Toai et al. 2010). However, enormous efforts

have been made to find newer, more effective and

safer solutions to be used in reconstructive and

regenerative medicine, solution that more and more

tend combine the high feasibility of bio-materials and

the incredible plasticity and infinite potential of stem

cells (O’Brien et al. 2004; Einhorn 1995; Chun et al.

2009; Gomillion and Burg 2006a, b; Toai et al. 2010;

Kucerova et al. 2007). As a result, there is a huge

amount of data from either in vitro or in vivo

investigation of hFTMSCs alone or in combination

Fig. 8 Results of differentiated mesenchymal stem cells into

adipocytes on fibrin gel. a Cell development and distribution in

the gel, the cell’s cytoplasm divided into several branches, well

developed in the gel. Cytoplasm of some cells contain many

lipid particles. b Cells differentiate into adipocytes, adhesion

and growth on the surface of gel. Nearly all cells had

differentiated into adipocytes with cytoplasm filled with lipid

particles. c Cells were stained with oil red dye directly on fibrin

gel

Fig. 9 Results of H&E staining for fibrin gel containing

adipocytes. a Gel containing adipocytes were photographed at

9100 magnification with inverted microscope. b Gel containing

adipocytes were photographed at 9200 magnification with

inverted microscope. The results show that cells grow in the gel

was stained H&E with the cells arrested purple, around the

nucleus of the cell have gaps and do not color it was droplets of

lipid within the cytoplasm of the cell

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with bio-material (Zhang et al. 2007; Kucerova et al.

2007; Strem et al. 2005; Gomillion and Burg 2006a, b;

Zuk et al. 2002; Wang et al. 2009; Ogawa 2006; Tuan

and Chen 2006; Rebellatto et al. 2008; Mizuno 2009;

Banas et al. 2007). hFTMSCs are extensively used in

liver, heart, bone, cartilages defections or used as

vector in anticancer therapy gaining a great consensus

in the field of cosmetic surgery (Zhang et al. 2007;

Kucerova et al. 2007; Strem et al. 2005; Gomillion and

Burg 2006a, b; Zuk et al. 2002; Wang et al. 2009;

Ogawa 2006; Tuan and Chen 2006; Rebellatto et al.

2008; Mizuno 2009; Banas et al. 2007). However, little

has been seen regarding the use of hFTMSCs together

with a human derived bio-material. This current study

has certainly confirmed all those instances, hFTMSCs

have clearly revealed to be useful as much as their

counterpart from hBM or hUCB, great plasticity,

enormous proliferative rate and a great natural capac-

ity to home, growth and differentiate in a bio-material

such as fibrin gel completely generated by human

donor. On the other hand, the human fibrin gel shows

to be an ideal environment where these cells may

eventually switch into adipocytes generating a product

with the consistency and characteristic certainly

comparable to human fat tissue. Moreover, this final

bio-material is absolutely safe and compatible since it

has obtained by the own patient blood and fat tissue.

To conclude, we have made success of adipocytes

sheet from human fat tissue mesenchymal stem cell

seeded and cultured on fibrin gel. We are sure that it

may eventually be of a great help in clinical field,

whether we think of reconstructive and regenerative

procedures or cosmetic application.

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