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Cell Tissue Bank (2013) 14:97–106 DOI 10.1007/s10561-012-9304-6

ORIGINAL PAPER

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 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

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

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

Fibrin gel Fat tissue mass

Mesenchymal stem cells Adipocytes

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

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

5 9 10 45 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).

to isolate mesenchymal stem cells (Figs. 1 , 2 ). Fig. 1 pieces. c Adipose tissue

Fig. 1

pieces. c Adipose tissue is incubated with the enzyme mixture

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

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Cell Tissue Bank (2013) 14:97–106 99 Fig. 2 Isolated mesenchymal stem cells from adipose tissue. a

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%), 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%CO 2 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 cm 2 flask and incubated at 37 C/5%CO 2 . 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

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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). Na 2 CO 3 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 CaCl 2 (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

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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 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

CD 14. b The cell is positive for six markers, including CD 13, CD 44, CD

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102 Cell Tissue Bank (2013) 14:97–106 Fig. 4 Mesenchymal cells are stimulated to differentiate into fat

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

appeared many lipid droplets, dominating the cell cytoplasm Fig. 5 Results of Nile Red staining for

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

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.

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redorange lipid particles, lipid particles accounted for nearly all the cell cytoplasm, the nucleus was push offset to one side of the cell

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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Cell Tissue Bank (2013) 14:97–106 103 Fig. 6 The resulting fibrin gel. A Two components fibrinogen

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

of gel quickly formed, a yellow light, elastic and supple Fig. 7 Create a fibrin gel

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

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104 Cell Tissue Bank (2013) 14:97–106 Fig. 8 Results of differentiated mesenchymal stem cells into adipocytes

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

were stained with oil red dye directly on fibrin gel Fig. 9 Results of H&E staining

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

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

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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

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

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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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