Cryopreservation, Culture And Alteration Of Potassium Current During Differentiation Of Newborn Sd Rats Neural Stem Cells In Vitro

The discovery of neural stem cells (NSCs) were based on the study of hematopoiesis andneurodevelopment. It is shown that neural stem cells are widely distributed in embryonic andadult brain. The NSCs has strong ability of multiplication and it is easy to multiply andinduced differentiation in vitro. Moreover, it has low immunogenicity when transplanted invivo. Therefore, the NSCs has vast applied foreground in clinical care.In this article, newborn SD rats were used as experimental materials. We investigated themethod of isolation, culture and verification. After isolating hippocampus from SD rats, wecultureed the cells without FBS. The results showes that the growth of passage cells wasstable with the density of5×10~5n/mL. With immunofluorescence method, it was identifiedthat the surface antigen possitive expressed. The above results completely demonstrated thatthis method was appropriate to gain a method of cltured in vitro and appraisal system.The cryopreservation of NSCs were studied based on the results above. UsingDMEM/F12medium, DMSO and FBS as bases, we designed freezing protection solution thathad different mixture ratios, then stored in refrigerator80centigrade below zero one week,two weeks and one month. The recovery rate of the cells was detected with trypan bluestaining method. The results indicated that the recovery rate of group D10(70%DMEM/F12+20%FBS+10%DMSO) was (54.00±1.73)%,(59.00±1.16)%,(58.00±2.08)%,which was notedly better than the others. It illustrated that the NSCs after cryopreservationstill had biological activities.In addiition, we did research on the changes of potassium channel in vitro duringdifferent differentiation days. During the third generation in NSCs, we detected thedifferentiated cells by immunocytochemical stain. The changes of potassium channel in5th,10th,15th,20thinduced days were tested by whole-cell patch clamp technigues, respectively.The resultes indicated that removing the growth factors and adding10%FBS, NSCs coulddifferentiate into neuron and astrocyte, which surface antigen NSE and GFAP positiveexpressed. Neurons which were from NSCs could be detected potassium current, which was enhanced with the elongation of differentiation time. It was indicated that the neurons canbecome mature in electrophysiology functions after the differentiation of NSCs.