Beruflich Dokumente
Kultur Dokumente
DOI 10.1007/s10561-012-9304-6
ORIGINAL PAPER
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
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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.
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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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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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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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