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KMID : 1161520140180040228
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Animal Cells and Systems
2014 Volume.18 No. 4 p.228 ~ p.236
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Expansion, characterization, and differentiation of rabbit bone marrow-derived mesenchymal stem cells in serum-free medium
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Jin Lifang
Ji Shaohui
Shen Mei
Zhang Jianlong
Han Jiwei
Ni Jian
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Abstract
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Mesenchymal stem cells (MSCs) derived from rabbit bone marrow are routinely investigated in cardiovascular and orthopedic models for regenerative medicine application. However, classical medium used for generating rabbit MSCs (rbMSCs) was supplemented with fetal bovine serum (FBS), which would raise several safety concerns and inconsistencies in the generation of MSCs. Here, we tested a serum-free medium for culture of rbMSCs through the investigation of multiple parameters including cell morphology, expansion, phenotype, and trilineage differentiation. RbMSCs were isolated using the gradient centrifugation method, and expanded in StemPro® MSC SFM in the presence of substrates and basic fibroblast growth factor (bFGF). Cells were then characterized by phase contrast microscopy observation, cell proliferation analysis, immunocytochemistry staining, RT-PCR, and qPCR analysis. Similar to that achieved with FBS-containing culture medium, the viability and proliferation of rbMSCs were improved in serum-free medium supplemented with bFGF and pre-coated with CELLstartTM. Serum-free expanded rbMSCs showed the fibroblastic spindle-shape morphology, and were positive for CD29, CD44, and CD73, and negative for CD34, CD45, and CD166. After the corresponding differentiation, rbMSCs from serum-free condition were demonstrated to differentiate into adipogenic, osteogenic, and chondrogenic lineages. In addition, differences were also observed between rbMSCs cultured with or without serum in terms of their morphology, proliferation, and gene expression levels of early differentiation transcription factors. In conclusion, this study demonstrates that rbMSCs can be expanded in a serum-free condition while maintaining the original characteristics of MSCs, which provides a useful tool to understand the basic biological properties of rbMSCs in culture.
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KEYWORD
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mesenchymal stem cells, serum-free medium, differentiation, rabbit
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