ES细胞培养-实验方法.doc

ES细胞培养-实验方法ES cell culture media and solutions ES细胞培养需要较高的实验条件，培养血清要用纯度较高的（ES级别）的胎牛血清，为防止细胞分化，需要在培养皿底部铺种滋养层细胞（Feeder cells）并在培养基中加入白细胞抑制因子（LIF）培养细胞的平皿和吸管均为一次性聚乙烯材料A) DMEM with high glucose (B) 0.1 mM non-essential amino acids (100×stock,aliquoted ,stored at 4℃)(C) 1 mM sodium pyruvate (100×stock,aliquoted ,stored at 4℃)(D) 10-4M β­mercaptoethanol (100×stock,aliquoted ,stored at-20℃)(E) 2mM L­glutamine (100×stock,aliquoted ,stored at-20℃)(F) 15%FBS 要用纯度较高的（ES级别）的胎牛血清(G) Penicillin and streptomycin (final concentration 50 μg/ml each)(H) 1000 U/ml LIF 白细胞抑制因子，抑制ES 细胞分化 Preparation of EMFI feeder layers Regents Frozen vials of primary embryo fibroblasts Tissue culture dishes PBS without Ca2+ and Mg 2+0.05% tripsin in saline /EDTADMEM +10%FBS Mitomycin C (stock 1 mg/ml in PBS stored in dark at 4℃ and used within two weeks ,mitomycin C is toxic ;wear gloves and use caution when handling )Methods1. Thaw a frozen vial EMFI cells quickly at 37℃.2. Add cells to 10 ml DMEM +10%FBS and centrifuge (270g,5 min)3. Decant supernatant , resuspend the cell pellet gently in 10 ml DMEM +10%FBS, and split onto five 150 mm plates each containing a total of 25 ml DMEM +10%FBS .Mix well.注意摇动混匀，不要单纯只按一个方向摇动，以使细胞较均匀地分布于平皿中，。

4. Incubate cells at 37℃ ,5% CO2 .5. When the cells form a confluent monolayer (approx. three days )each plate should either be:(a)Thrypsinized ,split onto five additional 150 mm dishes ,and grown until they form a confluent monolayer ,or (b)Directly treated with mitomycin C （丝裂霉素）to inhibit cell growth and division .因为ES细胞与滋养细胞共培养时，未经丝裂霉素处理的滋养细胞增殖很快，会与ES细胞竞争养分，此步丝裂霉素处理可使滋养细胞失去增殖，但仍保持存活6. Remove the medium from the confluent plates and 10 ml DMEM + 10%FBS containing 100μl mitomycin C (1mg/ml stock).Swirl plates to ensure an even distribution of medium.7. Incubate cells at 37℃,5% CO2 for 2-2.5h.8. Wash the monolayer of cells twice with 10 ml PBS per dish .9. Add 5 ml trypsin /EDTA to each plate .10. incubate 37℃,5% CO2 until the cells come off the plate .11. Add 10 ml DMEM +10%FBS to each plate and break any cell aggregates by gently pipetting.12. Centrifuge cells (270 g,5min)and resuspend the pellet in DMEM+10%FBS.13. Count the cells and dilute to a concentration of 2 ×105 cells/ml.14. Plate the cells immediately onto tissue culture dishes containingDMEM+10%FBS for the appropriate cell densities and volumes of medium for different plate sizes.15. Allow feeders to attach at least 2h ,but preferably overnight ,before adding ES cells. 16. Change the medium to ES cell medium immediately before adding ES cells .Mitomycin C treated EMFI feeders can be used for up to seven days with medium changes every three to four days.Preparatiooon of a stock of mitomycin C treated EMFI cells Reagents正常的滋养细胞贴壁生长于培养皿底面，呈梭形，应当均匀分布，并将皿底完全覆盖。

1. thaw a frozen via of EMFI cells quickly at 37℃.2. Add cells to 10 ml DMEM +10% FBS and centrifuge (270 g,5min).3. Decant supernatant , resuspend the cell pellet gently in 10 ml DMEM +10%FBS, and split onto five 150 mm plates each containing a total of 25 ml DMEM +10%FBS .Mix well.4. incubate cells at 37℃ ,5% CO2 . 5. When the cells form a confluent monolayer (approx. three days )each plate should trypsinized ,split onto five additional 150 mm dishes ,and grown until they form a confluent monolayer 6. Remove the medium from the confluent plates and 10 ml DMEM + 10%FBS containing 100μl mitomycin C (1mg/ml stock).Swirl plates to ensure an even distribution of medium.7. Incubate cells at 37℃,5% CO2 for 2-2.5h.8. Wash the monolayer of cells twice with 10 ml PBS per dish .9. Add 5 ml trypsin /EDTA to each plate .10. incubate 37℃,5% CO2 until the cells come off the plate .(5-10min).11. Add 10 ml DMEM +10%FBS to each plate and break any cell aggregates by gently pipetting.12. Centrifuge cells (270 g,5min)and resuspend the pellet in freezing medium .Freezing all the cells from each plate in one freezing vial in 1 ml of 1×freezing medium and store at -70℃ for one day .Transfer the vials to liquid nitrogen.13. to make feeder plates thaw a frozen vial of mitomycin C treated EMFI cells quickly at 37℃14. Add cells to 10 ml DMEM +10%FBS and cenrifuge (270 g,5min).15. Decant supernatant , and resuspend the cell pellet gently in 30 ml DMEM +10%FBS.16. Seed cells directly into tissue culture plates. Depending of the size of the plates required put 10 ml /100mm plate, 5ml /60 mm plate,5ml/60 mm plate, or 1.5ml/35mm plate.17. Allow feeders to attach preferably overnight ,before adding ES cells or at least 2h if using gelatinized plates .18. Change the medium to ES cell medium immediately before adding ES cells . Growth of ES cells on feeders plates Equipment and reagents 100mm dishes containing feeder layers ES cell medium briefly pre-warmed to 37℃0. 05% trypsin in saline /EDTAPBS without Ca2+ and Mg2+Gelatin ,0.1% solution in water ,autoclaved Method。