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Cell Tissue Bank (2013) 14:97106

DOI 10.1007/s10561-012-9304-6

ORIGINAL PAPER

Adipose tissue can be generated in vitro by using


adipocytes from human fat tissue mesenchymal stem cells
seeded 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 / Published online: 6 March 2012
Springer Science+Business Media B.V. 2012

Abstract The current study has developed an innovative procedure to generate ex novo fat tissue by
culturing adipocytes from human fat tissue mesenchymal 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 immunehistochemistry staining as well photos by scanning

electron microscopy (SEM) to demonstrate the adhesion 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
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

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 characteristics 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

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


5 9 1045 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, neurons, insulin secreting cells, keratinocytes and adipocytes (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 appropriate 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; OBrien 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 reconstructive and regenerative medicine, tools that would
definitely combine the high feasibility of bio-materials
and the incredible ductility of stem cells (OBrien 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 biomaterial, these types of materials that have been

Cell Tissue Bank (2013) 14:97106

studied for the application as substrate or carrier are


cell membrane collagen, fibronectin, fiberglass, ceramic, 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, fibrinogen 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%), Gentamycine (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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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

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%), Gentamycine (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 37C/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 37C/5%CO2. Medium has been
replaced every 3 days.

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 4C for 1 h, and moved
overnight into a different refrigerator at -20C.
Collection of thrombin (Quick 1966)

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-Lthyronine (1 nM), Human transferrin (10 lg/ml).
After 3 days, the medium was replaced by an

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 4C for 1 h and overnight at -20C. Then
the tube was thawed at a temperature of 4C, PBS

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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
37C 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 -20C.
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 trypsinEDTA (0.250.02%). 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 adipose tissue (Fig. 3) .
Differentiated mesenchymal stem cells
into adipocytes
After 21 days, hFTMSCs cultured in adipogenic
medium were observed under inverted microscope to

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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 cytoplasm 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 cytoplasm 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 cytoplasm 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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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

redorange dye, bright cytoplasm. d After staining with Oil Red


dye 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

redorange lipid particles, lipid particles accounted for nearly


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

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

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103

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 15 min, a block of gel quickly


formed, a yellow light, elastic and supple

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 distributed into several layers within the gel. d Cell adhesion and
growth on the surface of gel blocks

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Fig. 8 Results of differentiated mesenchymal stem cells into


adipocytes on fibrin gel. a Cell development and distribution in
the gel, the cells 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

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; OBrien 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 (OBrien 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

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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 capacity 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.

References
Banas A, Teratani T, Yamamoto Y, Takeshita F, Quinn G,
Okochi H, Ochiya T (2007); Adipose tissue derived mesenchymal stem cells as source of hepatocytes; American
Association of for the study of liver diseases, pp 219228
Buckley RC, Breazeale EE, Edwand JA, Brzezienski MA
(1999) A simple preparation of autologous fibrin glue for
skin-graft fixation. Plast Reconstr Surg 103(1):202206
Chun Y, Lee TM, Chiu KH, Shaw SY, Yang SY (2009) A comparative study of the physical and mechanical properties of
three natural corals based on the criteria for bone-tissue
engineering scaffold. J Mater Sci Mater Med 20:12731280
Einhorn TA (1995) Enhancement of fracture healing. J Bone
Joint Surg 77:940956

105
Entenmann G, Hauer H (1996) Relationship between replication
and differentiation in cultured human adipocyte precusor
cells. Am J Physiol 270:C1011C1016
Gimble JM (2003) Adipose tissue-derived therapeutics. Expert
Opin Biol 3:705713
Gimble J, Guilak F (2003) Adipose-derived adult stem cells:
isolation, characterization, and differentiation. Cytotherapy 5:362369
Gomillion CT, Burg KJL (2006a) Stem cells and adipose tissue
engineering. Biomaterials 27:60526063
Gomillion CT, Burg KJL (2006b) Stem cells and adipose tissue
engineering. Biomaterials 27:60526063
Hartman AR, Galanakis DK, Honig MP et al (1992) Autologous
whole plasma fibrin gel. Arch Surg 127:357
Kalmoz LP, Kolbus A, Wick N, Mazal PR, Eisenbock B, Burjak
S, Meissl G (2006) Cultured human epithelium: human
umbilical cord blood stem cells differentiate into keratinocytes. Burns 32:1619
Krasna M, Planinsek F, Knezevic M, Arnez ZM, Jeras M (2005)
Evaluation of a fibrin-based skin substitute prepared in a
defined keratinocyte medium. Int J Pharm 291(12):3137
Kucerova L, Altanerova V, Matuskova M, Tyciakova S, Altaner
C (2007) Adipose tissue derived human mesenchymal stem
cells mediated prodrug cancer gene therapy. Cancer Res
67(13):63046313
Lee OK (2008) Fibrin glue as a vehicle for mesenchymal stem
cell delivery in bone regeneration. J Chin Med Assoc
71(2):5961
Lidong G, Shaoqing L, Yunfang W, Huimin Y, Daqing L, Lijuan H, Cixian B, Fang Y, Xue N, Shuangshuang S, Xuetao
P (2006) In vitro differentiation of human adipose derived
mesenchymal stem cells into endothelial-like cells. Chinese Science Bullettin 51(15):18631868
Locke M, Windsor J, Dunbar PR (2009) Human adipose-derived
stem cells: isolation, characterization and applications in
surgery. ANZ J Surg 79:235244
Mizuno H (2009) Adipose derived stem cells for tissue repair
and regeneration: ten years of research and literature
review. J Nippon Med Sch 76:5666
OBrien FJ, Farrell E, Waller MA, Connell I, OMahoney D,
McGarry JP, Murphy BP, McHugh P, Campbell VA, Prendergast PJ (2004); Scaffolds and cells: preliminary biomechanical analysis and results for the use of a collagen gag
scaffold for bone tissue engineering. In: Prendergast PJ,
McHugh PE (eds), Topics in bio-mechanical engineering,
pp 167183
Ogawa R (2006) The importance of adipose derived stem cells
and vascularized tissue regeneration in the field of tissue
transplantation. Curr Stem Cell Res Ther 1:1320
Quick AJ (1966) Haemorrhagic diseases and thrombosis, p 442
Rebellatto CK, Aguiar AM, Moretao MP, Senegaglia AC,
Hansen P, Barchiki F, Oliveira J, Martins J, Kuligowski C,
Mansur F, Christofis A, Amaral VF, Brofman PS, Goldbenberg S, Nakao LS, Correa A (2008) Dissimilar differentiation of mesenchymal stem cells from bone marrow,
umbilical cord blood and adipose tissue. Exp Biol Med
233:901913
Saltz R, Sierra D, Feldman D, Dimick Alan, Marcia, Vasconez
LO (1991) Experimental and clinical application of fibrin
glue. Plast Reconstr Surg 88(6):10161017

123

106
Samir S, Pradeep J, Jyoti S (2003) Preparation of two component Fibrin Glue and its clinical evaluation in skin grafts
and flaps. Indian J Plast Surg 36(1):1417
Sathishkumar S, Mohanashankar P, Boopalan PRJV (2011) Cell
surface protein expression of stem cells from human adipose tissue at early passage with reference to mesenchymal
stem cell phenotype. Int J Med Med Sci 3(5):129134
Stechison MT (1992) Rapid polymerizing fibrin glue from
autologous or single donor blood: preparation and indications. J Neurosurg 76(4):626628
Strem BM, Hicok KC, Isabella Wulur MZ, Alfonso Z, Schreiber
RE, Fraser JK, Hedrick MH (2005) Multipotential differentiation of adipose tissue-derived stem cells. Keio J Med
54(3):132141
Toai TC, Gargiulo C, Thao DU, Tuan HM, Thuy TTT, Van PH,
Filgueira L, Strong DM (2010) In vitro culture and differentiation of osteoblasts on coral scaffold from human
Bone Marrow Mesenchymal Stem cells; Cell and Tissue
Banking, pp 115
Tuan RS, Chen FH (2006) Cartilage; stem cells and gene based
therapy; A Botler, J Lear; Springer, vol 12, pp 179189
Unguryte A, Bernotiene E, Venalis A (2010) Human mesenchymal adipose stromal cells from mature adipocyte fraction. Cent Eur J Biol 5:4758

123

Cell Tissue Bank (2013) 14:97106


Wang Q, Steigelman MB, Walker JA, Chen S, Hornsby PJ,
Bohnenblust ME, Wang HT (2009) In vitro osteogenic
differentiation of adipose stem cells after lentiviral transduction with green fluorescent protein. J Craniofac Surg
20(6):21932199
Wilson A, Butler PE, Seifalian AM (2011) Adipose-derived
stem cells for clinical applications: a review. Cell Prolif
44:8698
Wisse B (2004) The inflammatory syndrome: the role of adipose
tissue cytokines in metabolic disorders linked to obesity.
J Am Soc Nephrol 15:27922800
Zhang DZ, Gai LY, Liu HW, Jin QH, Huang JH, Zhu XY (2007)
Transplantation of autologous adipose derived stem cells
ameliorates cardiac function in rabbits with myocardial
infarction. Chin Med J 120(4):300307
Zhu Y, Liu T, Song K, Fan X, Xuehu M, Zhanfeng C (2008)
Adipose-derived stem cell: a better stem cell than BMSC.
Cell Biochem Funct 26:664675
Zuk P, Zhu M, Ashjian P, De Ugarte DA, Huang JI, Mizuno H,
Alfonso ZC, Fraser JK, Benhaim P, Hedrick MH (2002)
Human adipose tissue is a source of multipotent stem cells.
Mol Biol Cell 13:42794295