A Preliminary Study Of BMSCs Characteristics And Its Transplantation Mediated By Gelatin Sponge Into Nervous System

PartⅠThe preliminary study of BMSCs isolation, culture, identification and characteristicsObjective:To study the isolation, culture, identification and other major characteristics of BMSCs, and offer cells resource for stem cells transplantation mediated by gelatin sponge in the next stage experiment.Methods: BMSCs were isolated and purified from rat bone marrow using differential adhesion method. The surface markers of BMSCs were identified by flow cytometry. The oncogenicity potential were detected through comparing the growth curves of BMSCs with C6, a glioma cell line. The immunofluorescence method was used to study the spontaneous differentiation of BMSCs into nerve cells, and transwell test to observe the migration characteristics of BMSCs toward damaged nervous tissue. At last, we studied the convenient conservation method including cryopreservation and resuscitation.Results: Mix cells run-out from rat bone marrow cavity showed a great quantity of small round cells with single or agglomerate homogeneous distribution observed under the microscope. At 4 to 8 hours, part of the cells sagginged and spread to the bottom of culture dish. At 24 hours, a great part of cells transformed gradually into flattening and fusiform. At the 3rd day, all the no-adherent cells were removed through culture medium change. With amplification unceasingly of the adherent cells, at the 10th to 14th day we observed that a large number of cells assemble to plate and agglomerative growth with different shapes, such as large flat, polygon, starlike and fusiform shape. With passage increasing to the 5th generation, the cellular shapes and types trend to uniform. The results of flow cytometry revealed that the surface markers CD31, CD34 and CD45 were negative, and CD71 positive. Compare with C6 cell line, BMSCs grow was depressive visibly. The results of immunofluorescence discovered that BMSCs could differentiate into nerve cells and expressed nerve cell markers GFAP and NSE spontaneously. Transwell test revealed that BMSCs showed strong migration capability toward injured nerve tissue (P<0.05). Through convenient cryopreservation method the BMSCs can survival at least 3 month under -80℃condition, at least half a year in liquid nitrogen. Through resuscitation, the cell quantity will have a big lost than before cryopreservation. However these survival BMSCs can achieve a large number and enough density in dish after a period of culture.Conclusions: Through differential adhesion culture, which was mostly common used in many cell lines culture, we can achieve a stable and purified BMSCs line, and that had been identified by flow cytometry in this sdudy. There was no oncogenicity that was detected in the 10th passage BMSCs. In vitro, BMSCs could differentiate into neurons and glia cells spontaneously. Moreover, it possessed strong migration capability toward injured nerve tissue. BMSCs could be keeped for a long time through conveniently cryopreservation and resuscitation methods, and could provid the cells resource for later application.PartⅡThe preliminary study of BMSCs transplantation into nervous system through gelatin spongeObjective:To study the BMSCs survival rate and characteristics changes after carried by gelatin sponge, and conform whether BMSCs carried by gelatin sponge can coexistence with nerve cells.Methods:Firstly, we viewed the shape change and the adherent cells quantity of BMSCs when they were transplanted into gelatin sponge (GS-BMSCs). Then, the cell growth curves were drawn and oncogenicity was detected in both GS-BMSCs and BMSCs groups, and theirs cell shape and the rate of neurons was also be studied. Lastly, we observed whether GS-BMSCs can coexistence with nerve cells.Results:Compare with control group, there was no conspicuous change in cell shape and the quantity of adherent cells in GS-BMSCs group, most of them showed thin and fusiform, and few were flat, which were presumed belong to a group of ageing cells. BMSCs could adherented in the middle or margin of the gelatin sponge, and most parts gathered in the middle of gelatin sponge. There was no significant deviation of the number of no-adherent cells between two groups (P>0.05). And there was significant change in growth curve also. After induced 6 to 8 hours, the cell body started to shrink and stretch, parts of axons connected with each other, till form network structure. The expression of neuroglia cell marker GFAP and neuron cell marker NSE could be detected by immunofluoresc- ence assay. Through flow cytometry method there is no significant deviation in NSE positive rate (P>0.05). After 12 to 24 hours most of the cells could adherence and stretch, and whereafter connected into network. The originally generated nerve cells could be cultured more than 14 to 30 days in vitro. When co-culture some days, we could see that the two type cells can syntrophism and propagation.Conclusions: BMSCs packaged through gelatin sponge can adherent, survival, and no obvious characteristics changes. Its oncogenicity and differentiation potential also have no markly changes. And the BMSCs packaged by gelatin sponge can coexistence with nerve cells.