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Suspension Cultured Human Adipose-Derived Stem Cells maintain the Stemness Properties
+1,2 Wang, Y H; 3Chou, P J; 3Yeh, M L; 2Wang, G J; 2Ho, M L; 4,5,6Chen, C H
+ School of Dentistry, College of Dental Medicine, Kaohsiung Medical University, 2 Orthopaedic Research Center, College of Medicine, Kaohsiung
1
Medical University, 3 Institute of Biomedical Engineering, National Cheng Kung University, 4 Department of Physical Medicine and Rehabilitation,
Kaohsiung Medical University Hospital, 5 Department of Physical Medicine and Rehabilitation, Faculty of Medicine,6 Department of Physical Medicine
and Rehabilitation, Kaohsiung Municipal Ta-Tung Hospital, Kaohsiung, Taiwan, R.O.C
[email protected]
differentiation of hADSC, and offer an better approach of cell culture for
ABSTRACT INTRODUCTION:
tissue engineering application.
Human adipose-derived stem cells (hADSCs) have become popular
choice for stem cell source for tissue regeneration. It is because adpose
tissue is easy accessibility and abundance.HADSCs are less ethical
concern and have multipotency to different into osteoblast, adipocyte,
chondrocyte and myocyte [1]. For regular monolayer culture (2-D
culture), the cell number is restricted by the limited surface area [2]. A
more efficient approach to maintain large number of stem cell would be
a great benefit for stem cell application in tissue engineering. To develop
a new culture system to cultivate stem cells in vitro is very important.
Here, hADSCs were cultured in the ultra-low attachment surface plates
(ULASP) to mimic a suspension culture system which may suit to
Fig.1 The cell morphology analysis. monolayer culture (a, c, e) and
culture large number of stem cells. The purpose of this study is to
suspension culture (b, d, f). (scale bar = 100 μm).
investigate the proliferation and differentiation ability of suspension
cultured hADSCs.
METHODS:
HADSCs (5×105 cells ml-1) were cultured in the suspension culture,
which was the Ultra-Low Attachment Surface Plates (ULASP) condition.
Cell morphology (day1, 3, & 5) was observed by the light microscopy.
The mean diameter of cell aggregates was measured by using Image-Pro
Plus software and size distribution of aggregates (day1, 3, & 5) was
determined by over twenty photograph images analysis at least 500
aggregates. The viability of cell aggregates (day 2, 4, & 7) was evaluated
with double staining with fluorescein calcein AM and ethidium
homodimer-1 (EthD-1) stain. For histological analyses were used to
evaluate the differentiation ability (day14) of cell aggregates of
induction medium including osteogensis, chondrogensis and adipogensis
in suspension culture. Cell aggregates were observed by staining with
hematoxylin and eosin, alizarin red s, sarfranin o and oil red o. Further,
QRT-PCR assays (day3, 5, & 7) were discussed for the expression of
osteogenic genes (BMP2, ALP, Runx2 and Osteocalcin) of aggregates in
suspension culture. Statistical differences were put into practice using
independent Student’s t test and one-way ANOVA with Tukey Post hoc
test. A value of p<0.05 was defined as significant difference.
RESULTS SECTION:
After 1day suspension culture, hADSCs formed many cell aggregates
(Fig 1). The size distribution of these aggregates was majorly within 50
to 200 μm (diameter) for 1 to 5 days culture (Fig 2). And, most of cell
aggregates were alive after 7 days culture (Fig 3). In addition, hADSCs
maintained differentiation ability in suspension culture. By
histomorphometric analysis, hADSCs aggregates could be effectively
induced into adipocytes, osteoblasts and chondrocytes (Fig 4). For
osteogenic differentiation, the expression of osteogenic marker genes,
including BMP2, ALP, runx2 and osteocalcin, exhibited significantly
higher expression level in suspension culture than in monolayer culture
(Fig 5, p<0.05, n=3). Our results indicated that suspension culture not
only maintains cell alive but also maintains hADSCs differentiation
ability.
DISCUSSION:
The results showed that hADSCs could maintain cell viability in
suspension culture system and cells formed aggregates. The ability of
cell aggregation allow hADSCs to form 3-D spheroid may be a critical
step for cell survive in suspension culture. Cell aggregation may provide
an environment for hADSCs to overcome the anchorage-independent
grow. Additionally, hADSCs formed aggregates in suspension culture
could maintain the characteristic of multilineage differentiation.To our
surprise, for osteogenic differentiation, the suspension cultured hADSCs
have stronger expression level of osteogenic marker genes. The
signaling transduction mechanism of suspension cultured hADSCs will
require further investigation. Finally, our study conclude that suspension
culture can maintain cell viability and contribute to the effective
Fig.2 The size distribution of cell aggregates in suspension culture.
Fig.3 Cell viability of hADSCs aggregates by staining with calcein AM
and EthD-1. (scale bar = 100 μm).
Fig.4 Cell differentiation activity of cell aggregates by
histomorphometric analysis. Oil red O (a); Alizarin red S (b) and
Sarfranin O (c).
Fig.5 Comparison of the osteogenic marker genes expression between
monolayer culture and suspension culture. BMP2 (a); Runx2 (b); ALP
(c); Osteocalcin (d).
REFERENCES:
[1] Locke, M., J. Windsor, and P.R. Dunbar ANZ J Surg, 235-44, 2009.
[2] Ryu, J.H., et al. Biotechnol Lett, 1363-7, 2003.
Poster No. 1765 • ORS 2011 Annual Meeting