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实验室内部的人胚胎干细胞protocol人类胚胎干细胞H1和H9维持方案
一、试剂配制饲养层细胞培养基Fibroblast-DMEMMediaF-DMEMDMEMGIBCO11960-044450mlFCSGIBCO16000-04450ml10%Pen/Strep
2.5ml50IU/mlL-glutGIBCO25030-0815ml2uM/ml人类胚胎干细胞培养基HESC-MediaKO-DMEDGIBCO10829-018480mlKOSRGIBCO10828-028120ml20%10mMNEAAGIBCO11140-0506ml
0.1mML-glut
0.2MGIBCO25030-0816ml2mMPen/Strep3ml55mMBMEGIBCO21985-
0231.1ml
0.1mMITSGIBCO41400-0456ml4-80C避光保存用前每50毫升加200-400ngbFGF细胞冻存培养基FBS-DMSOFCSGIBCO16000-0449ml90%DMSOSIGMAD26501ml10%细胞消化液Trypsin-EDTA
0.25%Trysin-1mMEDTAGIBCO25200-0562ml
0.05%
0.2mMPBS-14190-1448mlMEF细胞有丝分裂终止液MitoCMitomycinC
0.2mg
0.05mg/mldd-H2O15230-1624ml避光保存MitoC的终浓度为10ug/ml即40吸ul工作液加到2mlF-DMEM中.
0.1%Gelatin用于培养皿的包被1%Gelatin40ml
0.1%ddH2OGIBCO15230-162360ml高压消毒
二、培养方案一饲养层细胞的培养
1、主要实验材料
(1)培养液为F-DMEM
(2)小鼠周龄为7~8w的性成熟母小鼠和周龄为8w的种公鼠
2.小鼠胚胎成纤维细胞MEF的培养
(1)小鼠胚胎的准备1性成熟母小鼠与种公鼠按1公(♂):2母(♀)比例合笼2每天早上观察母小鼠阴道口有乳白色或蛋黄色冻胶状物阴道栓即确定为怀孕见栓当天上午定为怀孕的
0.5d3取怀孕
12.5~
14.5d母鼠断颈处死,无菌条件下暴露子宫4用镊子提起近子宫颈端分离子宫系膜剪断子宫角5取出整个子宫在无菌滤纸上吸干血迹置于有PBS或无血清DMEM平皿内用PBS洗涤三次弃除表面残余血迹6沿子宫系膜侧剪开子宫取出带有胎膜的胚胎置于另一盛有PBS或无血清DMEM平皿内充分洗涤弃除表面红细胞7用小镊子撕破胎膜取出胎鼠弃除胎膜用PBS洗涤三次8去除胚胎头部、内脏和四肢,将躯干部置于另一盛有PBS或无血清DMEM平皿内用PBS至少洗涤三次充分弃除红细胞
(2)小鼠胚胎成纤维细胞MEF的培养二培养方法有两种:组织块培养法和单细胞悬液培养法组织块培养法1用无菌眼科小剪刀或刮胡刀片将鼠胚躯干剪切成1mm3以下的碎块吸置于离心管内2室温下静置5~10min弃上层液,留下胚胎组织碎块3向装有鼠胚组织碎块的离心管内加入1ml消化液轻轻吹吸30sec4加入等体积的培养液终止消化室温下静止5-10min5弃上清液留下鼠胚组织块沉淀物6)加入5ml培养液重新悬浮鼠胚组织块移入培养瓶内每5个鼠胚的组织块可使用1个100ml的培养瓶7补加适量的培养液使组织块悬浮液能均匀分布于培养瓶表面37℃、5%CO
2、饱和湿度培养箱内培养8培养开始的前两天不要晃动培养瓶第三天见组织块附着培养瓶表面周围生长出细胞补充培养液或更换培养液继续培养每天观察9待细胞连成一片时即可消化消化时弃培养液用PBS洗涤一次;加适量消化液(以能覆盖细胞层为度),在室温下作用大约30sec;弃消化液,让残余消化液继续作用轻敲培养瓶底当细胞层出现裂隙时,加入培养液终止消化或在倒置显微镜下观察,当细胞与细胞之间分开即可终止消化10)补加培养液,轻轻吹打均匀,吸置离心管内,静置5~10min11)上层为单细胞悬液下层为组织块沉淀将上层单细胞悬液轻轻吸置另一离心管内弃组织块12)以800~1000rpm5min离心,弃上清加入培养液重新悬浮细胞沉淀以1:3比例传代采用3~5代细胞作为饲养细胞单细胞悬液培养法1~5)步骤同组织块培养法1~5步骤6重复组织块培养法3)~4)步骤5~6次即重复消化鼠胚组织块5~6次每次消化后都收集上层液即单细胞悬液最后一次消化后弃组织块7将收集到的单细胞悬液离心,800~1000rpm5min8弃上清加入培养液轻轻吹吸制备成均匀的单细胞悬液9移置培养瓶内5个鼠胚制备出来的单细胞悬液可以使用3个100ml的培养瓶10补加适量的培养液,吹吸均匀37℃、5%CO
2、饱和湿度培养箱内培养每天观察11培养2~3d后细胞连成片即可消化消化过程同组织块培养法9步骤12以1:2~1:3比例传代培养2~3d传一代采用3~5代细胞作为饲养细胞
(3)培养结果在培养MEF初始的1~2代时杂细胞(非成纤维细胞的其他组织细胞)较多细胞形态多样;在第三代开始时杂细胞逐渐减少小鼠胚胎成纤维细胞为梭状;但连成片时由于受杂细胞的影响形态不够典型三)细胞冷冻
1.当细胞90%-100%融合时尤其细胞少量堆聚可冷冻
2.处理方法同传代培养1-8,每25cm2的细胞加1mlFBS-DMSO重悬细胞
3.每个冷冻管编号加1ml细胞悬液
4.置入40C1小时,放入1cm厚的泡沫盒中置入-800C过夜,置入液氮长期保存四)灭活饲养层细胞并备饲养层皿试剂和材料HESCMedium、PBS+、PBS-、MitoC、Trypsin-EDTA离心管、移液管MitoC处理(以25cm2培养瓶为例)
1.需要铺皿前一天,全换液.
2.留2ml培养基
3.加40ulMitoC(10ug/ml)
4.培养皿底以
0.1%Gelatin溶液覆盖预处理
5.3小时后,去培养基,处理方法同传代方法
6.重悬细胞,细胞计数,以、
7.0×104/cm2(MEF)铺皿
7.置于培养箱常规培养
8.15分钟后,取出皿,吹打皿中央,重新置于培养箱中培养
五、复苏及冻存hES细胞细胞被冻存在10%二甲基亚砜(DMSO)中防止结晶的形成,结晶的形成会损害细胞然而,二甲基亚砜对细胞有毒性,快速的进行细胞复苏是很重要的步骤1.从液氮中取出一管细胞;2.将冻存管置于37℃水浴中2分钟(或放到管内溶液恰好完全溶解);3.将细胞转移到一15mlFalcon管中;4.加入5mlES培养基(用培养基冲洗冻存管);5.离心200g×5min;6.弃上清,用2mlES培养基重悬细胞,吹打10次;7.接种在明胶包被(见下文)的6孔或6cm组织培养皿;8.孵育冻存细胞冻存液90%HS和10%二甲基亚砜步骤1.1×PBS洗细胞并留少许PBS在培养皿内;2.用细胞刮刀收集细胞;3.将细胞转入15mlFalcon管内并离心200g×5min;4.弃去上清并将细胞重悬于冷的冻存液中5.分装于冻存管内,每管1ml;将冻存管装入Nalgen冻存盒内6.置-80℃过夜,第二天移入液氮转载自丁香园
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