Isolation, culture and cell viability testing of rabbit bone marrow mesenchymal stem cells

doi:10.3969/j.issn.2095-4344.2013.01.010

Abstract

Abstract:

BACKGROUND: Most of recent studies on isolation and purification of bone marrow mesenchymal stem cells adopt single methods, and there is no systematic and comprehensive evaluation on cell viabilities during amplification.
OBJECTIVE: To establish an effective method of purifying rabbit bone marrow mesenchymal stem cells and to test the cell viabilities.
METHODS: Rabbit bone marrow mesenchymal stem cells were isolated by density gradient centrifugation and adherent cultivation methods. Rabbit bone marrow mesenchymal stem cells were identified from the morphology, cell phenotype and osteogenic and adipogenic capacity. Cell viabilities were evaluated using growth curve, seeding efficiency and colony-forming efficiency.
RESULTS AND CONCLUSION: The primary cells were spindle-shaped and circular. Most of the cells attached to the dish after seeding for 48 hours. After 8 or 10 days, cells reached 80%-90% confluency and the subculture cycle was 3 to 5 days. Flow cytometry demonstrated that cells were negative for CD14, CD34 and CD45, but they were positive for CD29, CD44 and CD90. Calcium nodules were observed by alizarin red S staining. A large amount of adipose-derived stem cells were observed by oil red O staining. Cell viability of passages 1 and 3 was stronger than that of passage 5. These findings suggest that density gradient centrifugation combined with adherent cultivation can be used to effectively isolate and purify bone marrow mesenchymal stem cells and well maintain the proliferation and differentiation potential of bone marrow mesenchymal stem cells. Cell viability will be decreased to different extents during cell proliferation.
Key Words: stem cells; bone marrow-derived stem cells; bone marrow mesenchymal stem cells; isolation and purification; identification; growth curve; adherence rate; clone formation rate; National Natural Science Foundation of China; stem cell photographs-containing paper

Key words: stem cells, bone marrow-derived stem cells, bone marrow mesenchymal stem cells, isolation and purification, identification, growth curve, adherence rate, clone formation rate, National Natural Science Foundation of China, stem cell photographs-containing paper

CLC Number:

R394.2

Kong Gen-xian, Jiang Zhi-xin, Sha Hang, Zhang Qing-hua. Isolation, culture and cell viability testing of rabbit bone marrow mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2013, 17(1): 62-67.

Figures/Tables 7

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