Objective: This research is designed to explore the methods of isolation and culture for endothelial progenitor cells from bovine bone marrow, characteristic, induced differentiative capacity in vitro.
Material and methods: Main experimental reagents contain DMEM/F12, fetal bovine serum, percoll lymphocyte separating, Trypsin 1: 250, VEGF, bFGF, GF-1, EDTA and so on. Cultivation system is DMEM/F12 with 10% FBS and VEGF 10 ng/mL, cultured under 37°C, 5% CO2, saturated humidity. Cell viability is measured by trypan blue solution exclusion test. Immunofluorescent detection is used to detected cell surface markers and double swallows, while bovine chromosome is analyzed by karyotyping.
Results: We find that the majority of bovine endothelial progenitor cells (EPCs) are fibrous shaped. Frozen survival of bovine EPCs before and after cryopreservation is 95.2±0.14% and 80.9±0.30% respectively; cryopreservation affects little on the viability of bovine EPCs. Immunofluorescent detection of the cell surface markers CD34, CD133 and flk present positive, which can confirm that the cell cultured in vitro are EPCs. Then Dil-ac-LDL and FITC-UAE-1 uptake assays are carried out. Eventually, bovine EPCs are induced to differentiate into endothelial cells and smooth muscle cells respectively, demonstrating the multi-lineage differentiation potential of bovine EPCs in vitro.
Conclusion: EPCs can be got with proper culture system. The little cell cryopreservation effect and stronger induced differentiation potential in vitro imply that EPCs can be applied in genetic resources conservation and reuse.
Key words: Bovine; Bone marrow; EPCs; Culture
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