Isolation, Culture And Chondrogenic Induction Of Human Bone Marrow Mesenchymal Stem Cells

In addition to hematopoietic stem/progenitor cells (HSPCs), the adult bone marrow (BM) also contains mesenchyaml stem cells (MSCs). BM-derived MSCs have attracted a great interesting for a lot of investigators because they retain the multipotentiality of differentiation into osteogenic, chondrogenic, adipogenic, tendongenic, myogenic, cardionyogenic and neural cells etc., and BM-derived MSCs also can be served as a cell feeder layer to support the growth of hematopoietic stem cells. MSCs are easy to be isolated, cultured and expanded in vitro due to their characteristic of adhering to the tissue culture plastic. Therefore, MSCs have been considered as prospective adult stem cell in tissue engineering, cell transplantation, gene therapy and clinic use.MSCs have active self-proliferative and multi-differentiate capacity. They display a stable phenotype and remain as a monolayer in vitro. The cells express several surface epitopes and most of them are in the G0/G1 phase.In present studies, MSCs from human BM were isolated by Ficoll density centrifugation and cultured expanded in DMEM-LG containing FBS. MSCs are adherent and fibroblastic, and maintained similar morphology with passaging. Flow cytometry analysis indicated that MSCs were universally positive for CD29, CD166, and negative for CD34, CD45, CD117, and HLA-DR. At passage 3, cell cycle status analysis by measuring DNA content revealed that a small population of the cells was engaged in proliferation (S+G2+M=20%), and more than 80% of cells were in G0/G1 phase. The number of cells in G0/G1 phase decreased with passage. In passage culture, it is proper for the cells plated at the density of 5000 cells/cm2 to propagate. The DMEM-LG culture medium is the most proper medium for MSCs to adhereto the tissue culture plastic and proliferate. The MSCs of passage 1 to 3 were froze in liquid nitrogen by two-step frozen procedure , after 60-90 days, we thawed the frozen MSCs and found the cells can proliferate about 4-6 passages in vitro, 10 passages especially for sample 17. It suggested that the frozen MSCs still have good expansion ability. At passage 1 to 3, four cases was induced by the combination of TGF-β1 and Vitaraine C in vitro for two weeks and expressed articular cartilage matrix-procollagen II mRNA. MSCs can be passaged in vitro about 7~10 passages.