Marrow Mesenchymal Stem Cells Cultivation, Cryopreservation and Induction of Differentiation of Neural Stem Cells in Vitro

Abstract: There exists a large number of adult stem cells in the marrow of mammals. It is convenient to get the experimental materials from mammals. Therefore, the marrow of mammals became the central issue of researches on adult stem cells. The marrow mesenchymal stem cells(MSCs) is one kind of bone marrow-derived multipotent stem cells, and it was studied more in recent years following after researches on hematopoietic stem cells. The MSCs has strong ability of multiplication and differentiation and it is easy to multiply and induced differentiation in vitro. Moreover, it has the characteristic of secreting varied cell facters to the extracellular space, and it is involved in the restructure of cell micro-environment. They have low immunogenicity when transplanted in vivo. Therefore, there are broad application prospects for MSCs in organ transplantation, autoimmune diseases and replacement therapy.In this article, SD(sprague-dawley) rats were used as MSCs donors. We investigated the method of isolation and multiplication at first. After getting the bilateral femurs and shinbones of SD rats, we cultured the bone marrow cells by adherent method, and then inoculated marrow cells by the method of density gradient centrifugation. The results showed that it could shorten the time of primary culture effectively with the density of 0.5~1×107cells/mL. The growth of passage cells was stable and the growth curves of P2, P4 and P6 were similar to each other. With immunofluorescence method, it was identified that the surface antigen CD29 and CD44 positive expressed while surface antigen CD34 of hematopoietic cell lines did not express. Double immunofluorescence techniques were used to identify the purity of the P3 cells, The respective positive rates of CD29 and CD44 were 97.96±0.35%and 98.14±0.19%. The above results completely demonstrated that this method was appropriate to gain a large quantity of high purified MSCs in vitro which have high biological activity.The cryopreservation of MSCs were studied based on the results above. Using LG-DMEM medium, fetal calf serum and DMSO as the main materials, we designed freezing protection solution that had different mixture ratios. P3 MSCs were stored under the condition of 1×106cells/mL density and -80℃for 7 days and then revived. The thawed rate was detected with trypan blue staining method. The results showed that the thawed rate of group F10(80%DMEM+10%FBS+10%DMSO)was 61.00±3.61%, which was notedly better than the other groups. The thawed MSCs could multiply in vitro and pass on from generation to generation normally. P4 MSCs were stored by this method, and revived after one, three, six months. The thawed rates were 61.67±4.41%, 61.67±3.33%, 65.00±11.54%, here was no significant difference among them (P>0.05). The ultiplication and subcultured of MSCs was normal after thawed. The immunofluorescence identification of MSCs subcultured after thawed showed that the surface antigen CD29 and CD44 positive expressed. It illustrated that the MSCs after cryopreservation still had biological activities.In addition, we did research on the influence of the paracrine interactions of MSCs on the differentiation of NSCs. The neonatal SD rats within 24h were used as NSCs donors, We isolated and cultured the hippocampus-derived NSCs, and then induced them to differentiate. NSCs surface antigen Nestin, neuron surface antigen NSE and fibrous astrocyte surface antigen GFAP were detected with the immunofluorescence method. The results indicated that the serum-free culture combined with basic fibroblast growth factor and epidermal growth factor could make NSCs showing good ability of continuing proliferation, passing from generation to generation stably, and the NSCs after passage were Nestin positive expressed. After removing the growth factor and adding 10%FBS, NSCs could differentiate into neuron that NSE positive expressed and astrocyte that GFPA positive expressed. Subsequently, NSCs were induced to differentiate in the MSCs culture solution, and using the above medium which added FBS 10% as a contrast. 7 days later, by means of immunofluorescence techniques, it was identified that the MSCs culture solution had stimulative effect on the differentiation of NSCs into neuron and astrocyte compared with the controls…
Key words: mrrow mesenchymal stem cells ; neural stem cells ; culture in vitro ; differentiation induction; SD rats

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