J Vet Sci.  2012 Dec;13(4):413-417.

Suitability of autologous serum for expanding rabbit adipose-derived stem cell populations

• Bahn JJ
• Chung JY
• Im W
• Kim M
• Kim SH

Abstract

Adipose-derived stem cells (ASCs) are believed to have potential use for treating many illnesses. Most cells, including ASCs, are generally cultured in medium containing fetal bovine serum (FBS). However, FBS, which could induce an immune response or infection, is not recommended for clinical applications. In the present study, we evaluated the morphology, proliferation rate, and characterization of rabbit ASCs grown in medium containing autologous serum (AS) and compared these cells to ones cultured with FBS. Morphological changes were monitored by microscopy and flow cytometry. Proliferation rates were assessed with cell counting and ASC phenotypes were characterized by flow cytometry using representative surface markers (CD44 and CD45). Expression of epidermal growth factor, brain-derived neurotrophic factor, and vascular endothelial growth factor was measured by reverse transcription-polymerase chain reaction. Results of our study showed that ASCs had a greater expansion rate in AS without developing morphological heterogeneity than cells grown in FBS. AS-cultured ASCs expressed representative growth factors, CD44 but not CD45, similar to cells cultured in FBS. Expression levels of some growth factors were different between AS and FBS. In conclusion, our findings indicated that AS could potentially be used as a culture medium supplement for the expansion of autologous ASCs.

Keyword

adipose-derived stem cells; autologous; bystander effect; rabbit

MeSH Terms

Brain-Derived Neurotrophic Factor
Bystander Effect
Cell Count
Epidermal Growth Factor
Flow Cytometry
Intercellular Signaling Peptides and Proteins
Microscopy
Phenotype
Population Characteristics
Stem Cells
Vascular Endothelial Growth Factor A

Figure

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  Fig. 1 Morphology and proliferation rate of adipose-derived stem cells (ASCs). (A) Rabbit ASCs were grown in medium containing 5% autologous serum (AS) (left panels) or 5% fetal bovine serum (FBS) (right panels) until passage 3, ×100. Scale bar = 50 µm. (B) Forward scatter vs. side scatter dot plot of AS- (green dot) or FBS-treated (black dot) ASCs. (C) ASCs grown in AS or FBS at passage 3 were counted at 48, 72, and 96 h (n = 4) after seeding at the same density. PC: primary culture, P3: passage 3. *p < 0.05 according to Mann-Whitney U-test.

• 

  Fig. 2 Surface marker expression of the ASCs. Expression of two surface markers on ASCs cultured in AS or FBS was analyzed by flow cytometry. Detached ASCs were treated with anti-CD44 and anti-CD45 antibodies. The positive population was evaluated relative to a baseline determined with a negative control.

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  Fig. 3 Expression of growth factors by the ASCs. Rabbit ASCs were grown for three passages in 5% AS or 5% FBS, and an RT-PCR analysis was performed. (A) mRNA expression of growth factors, including epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), and brain-derived neurotrophic factor (BDNF), was measured in cells cultured under both conditions. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. (B) The graph shows relative mRNA expression levels. Relative values were defined as each expression level normalized to that of GAPDH. *p < 0.05 according to Mann-Whitney U-test.

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