Neural Stem Cells Vitrification And Its Effect On The Biological Properties

The interests in the possibilities of treating intractable neurological diseases and the damaged mammalian central nervous system (CNS) by transplantation of ex vivo expanded neural stem cells (NSCs) are dramatically increasing now. As a result, development of effective cryopreservation protocols and consummate biochemical examining after vitrification seems to be much more important.According to the changes of cell volume after CPA loading, penetration balance time was examined. The NSCs single cell suspension (0.4mL) were injected into 0.8mL of mixed solution containing 1.5×CPA through six-step method, which had been pre-equilibrated at temperatures of 0℃and 23℃, respectively. Then the cells were quickly dropped into liquid nitrogen for one day storage. The CCK-8 kit and Trypan blue stain were both used to detect cellular viability and survival rate. The flow cytometry and the Hoechst/Ca/PI triple staining were adopted to detect the cellular survival rate. Apoptosis assays kit and immunocytochemistry assay were performed to detect the apoptosis for single cell and multi-differentiation potency after vitrification. Moreover, a kind of cryoprotective agent with yolk in the absence of permeable component DMSO has been designed. The water permeability of single cell suspension was also determined experimentally.The results indicated that the CPA loading at 0℃could offer a little bit higher cell viability and more perfect reproduction than that at room temperature. The cell survival rates for the loadings at 0℃and room temperature were 76.3% and 72.5%, respectively. Comparing with the control group, reproduction of NSCs after vitrification will delay about 3-4 days. The flow cytometry and the Hoechst/Ca/PI triple staining were performed to visually detect the cellular survival rate, somewhat different from the Trypan blue stain case. Apoptosis assays kit shows that early apoptosis rate is 12.4% and late apoptosis rate is 34.6% after vitrification. The results of immunocytochemistry assay demonstrated that the sternness and multipotency of NSCs was decreased with the passage times, there were only 25% expressing nestin positive, but there were no significant difference between control and vitrification.According to the improved empirical formula and experimental data, the calculated water permeability was 0.2133. However, the effect of neurospheres long term preservation of 14 months was unsatisfactory and the survival rate was only 19%, but the situation of cell proliferation reproduction seems well after several days culture. In addition, yolk was not an active ingredient to substitute DMSO for vitrification.