1、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-DMEM Media (F-DMEM)DMEM (GIBCO 11960-044) 450ml FCS GIBCO 16000-044 50ml 10%Pen/Strep 2.5ml 50IU/mlL-glut GIBCO 25030-081 5ml 2uM/ml人类胚胎干细胞培养基HESC-MediaKO-DMED GIBCO 10829-018 480ml KOSR GIBCO 10828-028 120ml 20%10mM NEAA GIBCO 11140-05
2、0 6ml 0.1mML-glut(0.2M) GIBCO 25030-081 6ml 2mMPen/Strep 3ml 55mM BME GIBCO 21985-023 1.1ml 0.1mMITS GIBCO 41400-045 6ml 4-80C 避光保存。用前每50毫升加200-400ng bFGF细胞冻存培养基FBS-DMSOFCS GIBCO 16000-044 9ml 90%DMSO SIGMA D2650 1ml 10%细胞消化液 Trypsin-EDTA0.25% Trysin-1mM EDTA GIBCO 25200-056 2ml 0.05%,0.2mMPBS(-) 14
3、190-144 8ml MEF 细胞有丝分裂终止液 Mito CMitomycin C 0.2mg 0.05mg/mldd-H2O 15230-162 4ml 避光保存。Mito C 的终浓度为10ug/ml 即40 吸 ul 工作液加到 2ml F-DMEM 中。.0.1% Gelatin(用于培养皿的包被)1% Gelatin 40ml 0.1%ddH2O GIBCO 15230-162 360ml 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)
4、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0.5d。3)取怀孕12.514.5d 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
5、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 30sec。4) 加入等体积的培养液终止消化。室温下静止5-10min 。5) 弃上清液,留下鼠胚组织块沉淀物。
6、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%CO2、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，
7、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510min。11） 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 8001000rpm 5min 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35 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56次, 即重复消化鼠胚组织块56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。)将收集到的单细胞悬液离心，8001000rpm 5min。)弃上清 ,加入
8、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%CO2、饱和湿度培养箱内培养。每天观察。11)培养23d 后,细胞连成片,即可消化。消化过程同组织块培养法)步骤。12)以1:21:3 比例传代培养,2 3d 传一代。采用35代细胞作为饲养细胞。（3）培养结果在培养 MEF 初始的 12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.
9、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25 cm2 的细胞 加1ml FBS-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HESC Medium、PBS(+) 、PBS(-) 、Mito C、Trypsin-EDTA离心管、移液管Mito C 处理（以25 cm2培养瓶为例）1. 需要铺皿前一天，全换液。.2. 留2ml 培养基。3. 加40ul Mito C（ 10ug/ml） 。4. 培养
10、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将细胞转移到一15ml Falcon 管中；4加入5ml ES 培养基（用培养基冲洗冻存
11、管） ；5离心200g 5min；6弃上清，用2ml ES 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：11 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15ml Falcon 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1、Stem Cell Analysis 干细胞分析（功能、表型、核酸含量、边群）London Research
12、Institutebrief introduction：Stem Cell Analysis ,FACS Laboratory, London Research Institute干细胞分析，包括以下四方面内容：Functional AnalysisPhenotypingNucleic Acid contentSide Population analysis2、Production of ES Cell Chimeras by Aggregation wtih Eight-Cell Stage Embryos Nagy Lab，Mount Sinai Hospitalbrief introdu
13、ction：Production of ES Cell Chimeras by Aggregation wtih Eight-Cell Stage EmbryosNagy Lab，Mount Sinai Hospital3、Production of Completely ES Cell-Derived Fetuses by Aggragation with Tetraploid EmbryosNagy Lab，Mount Sinai Hospitalbrief introduction：Production of Completely ES Cell-Derived Fetuses by A
14、ggragation with Tetraploid EmbryosNagy Lab，Mount Sinai Hospital4、Picking ES Cell Colonies and Transferring Them to 24-Well Culture Dishesbrief introduction：Picking ES Cell Colonies and Transferring Them to 24-Well Culture DishesFrom the Laboratory of Dr. Allan Bradley Baylor College of Medicine, Hou
15、ston, Texas5、Preparation of Feeder Layers And SNL Stocks Baylor College of Medicinebrief introduction：Preparation of Feeder Layers And SNL Stocks ,Allan Bradleys Lab, Baylor College of Medicine, Houston, Texas6、Preparing 48-Well Plate FeedersBaylor College of Medicinebrief introduction：Preparing 48-
16、Well Plate FeedersFrom the Laboratory of Dr. Allan Bradley ,Baylor College of Medicine, Houston, Texas7、Freezing Embryonic Stem (ES) Cell Clones in 96-Well Plates 冻存 96 空板中的胚胎干细胞克隆Baylor College of Medicinebrief introduction：Freezing Embryonic Stem (ES) Cell Clones in 96-Well Platesthe Laboratory of
17、 Dr. Allan Bradley ,Baylor College of Medicine, Houston, Texas8、In Vitro Differentiate ES cells into glial cells and neurons 胚胎干细胞体外分化为神经元和神经胶质细胞(Nagy Lab，Mount Sinai Hospital)brief introduction： Differentiate ES cells into glial cells and neurons ,Nagy Lab，Mount Sinai Hospital胚胎干细胞体外分化为神经元和神经胶质细胞9、
18、In Vitro Differentiation of ES Cells into Cystic Embryoid Bodies 胚胎干细胞体外分化为囊胚体(Nagy Lab，Mount Sinai Hospital)brief introduction：In Vitro Differentiation of ES Cells into Cystic Embryoid Bodies ,Nagy Lab，Mount Sinai Hospital胚胎干细胞体外分化为囊胚体10、In Vitro Differentiation of ES Cells into Cardiac Muscle 胚胎干细
19、胞体外分化成心肌细胞 (Nagy Lab，Mount Sinai Hospital )brief introduction：In Vitro Differentiation of ES Cells into Cardiac MuscleNagy Lab，Mount Sinai Hospital11、Isolation of Primary Fibroblasts from Mouse Embryos 从小鼠胚胎分离纤维原细胞 Baylor College of Medicinebrief introduction：Isolation of Primary Fibroblasts from Mo
20、use Embryos ,Allan Bradleys Lab, Baylor College of Medicine, Houston, Texas12、ES Cell Subcloning Protocol 胚胎干细胞亚克隆(NIH Stem Cell Unit)brief introduction：ES Cell Subcloning Protocol, NIH ，Stem Cell Unit13、ES and TS cell freezing/thawing 胚胎干细胞冻存复苏brief introduction：ES and TS cell freezing/thawing, Nag
21、y Lab,Samuel Lunenfeld Research Institute, Room 881 ,Mount Sinai Hospital该实验室专著于小鼠遗传学以及其跟人类疾病关系14、ES Cell Culture and Manipulation (The Cancer Center at Washington University)brief introduction：ES Cell Culture and Manipulation (The Cancer Center at Washington University)15、ES / MEF cell culture and
22、electroporation of targeting construct 胚胎干细胞培养及电穿孔brief introduction：This website is an online resource for those interested in murine transgenesis. Within this site you will find information pertaining toresearch and techniques employed in the field of transgenics, including both DNA microinjection
23、 and embryonic stem cell injection.16、Electroporation of ES Cell 胚胎干细胞电穿孔Mount Sinai Hospitalbrief introduction：Electroporation of ES Cell ,Nagy Lab,Samuel Lunenfeld Research Institute, Room 881 ,Mount Sinai Hospital胚胎干细胞电穿孔，该实验室专著于小鼠遗传学以及其跟人类疾病关系17、Culture of Human Embryonic Stem Cells (hESC)人胚胎干细胞
24、培养NIH Stem Cell Unitbrief introduction：Culture of Human Embryonic Stem Cells (hESC),NIH Stem Cell Unit人胚胎干细胞培养，来自 NIH Stem Cell Unit 的材料，该网址下还有许多 NIH 关于干细胞的资料。18、Analysis of ES Cell Clones by Mini-Southern 分析胚胎干细胞克隆（Baylor College of Medicine）brief introduction：Analysis of ES Cell Clones by Mini-Sou
25、thern ,Allan Bradleys Lab, Baylor College of Medicine, Houston, Texas,Mini-southern 法分析胚胎干细胞克隆19、General ES Cell Protocols 胚胎干细胞基本操作（Baylor College of Medicine）brief introduction：EMBRYONIC STEM CELLS PROTOCOL,Allan Bradleys Lab, Baylor College of Medicine, Houston, Texas20、Stem Cell Protocols 很全的干细胞
26、操作University of Wisconsinbrief introduction：Stem Cell Protocols Thomson Lab, University of Wisconsin内容很全的干细胞基本操作;主要包括以下内容：Media and reagentsThawing ES CellsSplitting ES Cells on MEFs (stem cell photos)Freezing ES CellsMatrigel Aliquoting and PlatingSplitting ES Cells on MatrigelEmbryonic Bodiesedite
27、d by sdimmuno Feeder-Free Culture of hESCsMEF Conditioned mediumPlate MEFs (p4 or p5) onto gelatin-coated plates at a density of 1x106 cells/10cm culture dish.The next day, wash cells with PBS and add 10ml normal hESC medium containing 4ng/ml bFGF. This is collected the next day and each subsequent
28、day for 7 days.CM may be stored frozen for several months.Before use, add 4ng/ml bFGF to the medium and filter.Culture of hESCsCoat 6-well tissue culture plates (Falcon) with 5% Matrigel in DMEM/F12 overnight at 4C using 1.5ml Matrigel suspension per well.Allow the Matrigel plate to warm to room tem
29、perature in the hood for approx 30min prior to use. DO NOT WARM IN INCUBATOR.Remove hESC cells from MEFs using collagenase treatment and sediment as usual. Aspirate the Matrigel and plate triturated colonies in conditioned medium supplemented with an additional 4ng/ml bFGF or in normal hESC medium c
30、ontaining 100ng/ml bFGF.Feed cells every day up to 7 days. Note: Colonies can grow bigger and more densely on Matrigel without losing morphology than on MEFs.PassagingWash cells once with PBS and incubate at 37C with 1ml/well of 2mg/ml dispase in DMEM/F12.Colonies should detach intact within 1015 mi
31、nutes upon tapping smartly on the side of the plate. DO NOT SCRAPE.Remove the colonies in dispase to a 15ml tube and rinse wells with an additional 1ml/well growth medium.Sediment and wash twice more as usual.Triturate VERY gently as colonies grow flat and dissociate readily in dispase. Note:Small colonies and single cells do not survive well on Matrigel especially in 100ng/ml bFGF only.Plate as usual on fresh Matrigel
