| Background: Bone-derived msenchymal stem cells (MSCs) owns powerful capability of osteogenesis. Furthermore, it could be easily isolated and auto-transplanted. As a result, MSC is the optimum seed cell for bone tissue engineering. However, the widely adopted methods of MSCs culture have a obvious shortage: the growth of cultured MSCs is so slow that limits it to clinical therapy. For this reason, it is necessary to improve the method in order to promote cultured MSCs proliferation.Objection: Utilizing the conditioned medium of hematopoietic stem cells (HSCs) to culture rabbit MSCs(rMSCs). Meanwhile, selected total marrow adherent and density gradient centrifugation to culture rMSCs from same individual. Compared the growth, the purify and the osteogenesis capability of rMSCs under three culture condition to investigate the effect of HSC environment on MSCs in vitro.Methods: Total marrow was isolated from adult rabbit and divided into total marrow adherent group (T group), density gradient centrifugation group (D group) and conditioned medium group (C group). rMSCs in T group and in D group were cultured in high-glucose DMEMwith 15% FCS, 37℃, 5% CO2 by total marrow adherent or by density gradient centrifugation respectively. Obtained the conditioned medium of HSC to culture rMSCs in C group. The growth of rMSCs in three group were compared by drawing growth curves and cell cycling analysis. Cell surface markers were identified by flow cytometer to compare the purify of rMSCs in three group. The osteogenesis capability was tested by DEX inducing experiment and immunocytochernistry.Result: In T group, the density of spindle-shape adhered cells was low and there were a lot of un-adhered HSCs or blood cells mixed. The float cells were removed after several changing medium. The adhered cells grew rapidly and formed clones .The rMSCs could be passaged 9-10 days after seeding. The growth speed of passaged rMSCs decreased alongwith the increasing times of passaging and the cells with aging phenotype increased gradually. The cells could be passaged 5-6 times at last. In D group, the adhered cells were homogeneous spindle-shape with cloning proliferation. 12-14 days after seeding, cells could be passaged and the changing of passaged rMSCs was similar with in T group. In C group, the primary cells proliferated rapidly and could be passaged 5-7 days after seeding. The aging phenotype cells appeared later than those in T group or in D group. The cells could be passaged 10-12 times eventually.By cell cycling analysis, for primary cells, the percentages of Go/Gi period in T group and in C group were significantly lower than in D group. Correspondingly, the percentages of S and G2/M period in former were significantly higher than in later. For P4 cells, the percentages of G0/G1 period in C group were significantly lower than in other two groups and the percentages of S and G2/M period were significantly higher than in later.By flow cytometer analysis, most of rMSCs in three groups were CD34", Strol-1 + and SH2+. There were no significant differences between three groups.After DEX inducing, the osteocalcin positive cells in three groups significantly increased. The positive rate in three groups were not significantly different.Conclusion: Comparing with density gradient centrifugation, both total marrow adherent and HSC conditioned medium methods could accelerate the proliferation of primary rMSCs. The HSC conditioned medium can promote the growth of rMSCs, contribute to maintain the proliferation capability and enable to increase the maximum passaging times. For purity and osteogenesis capability aspect, the rMSCs cultured by total marrow adherent, by density gradient centrifugation and by HSCs conditioned medium methods are alike.
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