Optimization of the method to culture rat embryonic neural stem cells in vitro / 中国组织工程研究

ABSTRACT

AIM:Neural stem cells can be induced to differentiate into various types of neural cells such as neurons and neuroglia cells,but the technique of depuration and cultivation does not consummate.This article determines the optimal culture technique of neural stem cells by different culture concentrations and passage methods. METHODS:Experiments were conducted from May to December 2006 at Laboratory of Transplantation Immunity of Sichuan University.①Clean pregnant female rats(embryonic age range from 12-16 days)and the disposition of animal met ethical standard.②The cerebral cortex of rat embryos were collected,and digested with trypsin and ethylenediamine tetraacetic acid mixture to obtain signal cell suspension.They were cultured in serum-free medium (DMEM/F12 medium containing B27,basic fibroblast growth factor and epidermal growth factor).The 3~(rd)passage cells were collected,and incubated at 1?10~7 L~(-1),1?10~8 L~(-1),1?10~9 L~(-1),1?10~(10)L~(-1),respectively.In addition,neural stem cells were collected 7-10 days after primary culture to harvest formative cell masses.Mechanical blow refers to soft blowing with haustorial tube from thick to thin after centrifugation,or sterile syringe with No.5 pinhead blowing cells when the blow was 5 times.Bubble production was avoided during the operation.Trypsin aspiration combined with mechanical blow refers to trypsin was added after centrifugation,at 37℃for 10 minutes,the neural stem cells were lightly blown with haustorial tube polished with flame or blown with sterile syringe with No.5 pinhead,and then fetal bovine serum was added to stop digestion.③The growth characteristic of the 3~(rd)passage cells at different culture concentration was observed and proliferation was measured at days 1,3,5 and 7 by MTT assay.The neural clone spheres of subcultured was counted to determine the optimal passage way.Immunofluorescence was carried out to detect nestin(special marker to neural stem cells),BrdU,neurone specific enolase,glial fibrillary acidic protein. RESULTS:①Growth characteristics and identification of rat embryonic neural stem cells in vitroThe dissociated neural stem cells from the cerebral cortex of rat embryos were continuously harvested and purified by suspension cultures to get the daughter cell clone.Nestin positive cells could be found in the neurospheres and after attachment they could differentiate into neurone specific enolase and glial fibrillary acidic protein positive cells,and immunofluorescence showed a great of BrdU-positive cells.②The effect of different incubated number of neural stem cells on proliferationWhen the neural stem cells planted at the concentration of 1?10~9 L~(-1),the growth rate of the cells was the highest of all concentrations.The number of clone spheres exceeded others at concentration 1?10~7 L~(-1),1?10~8 L~(-1)and 1?10~10 L~(-1)(P