ES细胞培养实验方法

1、ES 细胞培养 -实验方法ES细胞培养-实验方法ES cell culture media and solutionsES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 mMio n-esse ntial ami no acids (100 x stock,aliquoted,stored at 4 C)(C) 1 mM sodium p

2、 yruvate (100 x stock,aliquoted ,stored at 4 C)(D) 104M P - mercaptoethanol (100 x stock,aliquoted ,stored at-20 C)(E) 2mM L-glutamine (100 x stock,aliquoted ,stored at-20卩 g/ml each)(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(H) 1000 U/ml LIF 白细胞抑制因子，抑制 ES 细胞

3、分化Preparation of EMFI feeder layersRegentsFrozen vials of primary embryo fibroblastsTissue culture dishesPBS without Ca2+ and Mg 2+0.05% tripsin in saline /EDTAand used within use caution whenDMEM +10%FBSMitomycin C (stock 1 mg/ml in PBS stored in dark at 4 two weeks ,mitomycin C is toxic ;wear glov

4、es and handling )Methods1. Thaw a frozen vial EMFI cells quickly at 37C .g,5 min)2. Add cells to 10 ml DMEM +10%FBS and centrifuge (2703. Decant supernatant , resuspend the cell pellet gently in 10 ml DMEM +10%FBS, andsplit onto five 150 mm plates each containing a total of25 ml DMEM+10%FBS.Mix well

5、. 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 C ,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 mmdishes ,and grown until they form a confluent monolayer ,or(b) Directly treated

6、with mitomycin C(丝裂霉素) to inhibit cellgrowth 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。6.7.8.9.10.11.Remove the medium from the confluent plates and 10 ml DMEM + 10%FBS containing 100 卩 l mitomycin C (1mg/ml stock).Swirl plates to ensure an even distri

7、bution of medium.Incubate cells at 37 C,5% CO2 for 2-2.5h.Wash the monolayer of cells twice with 10 ml PBS per dish .Add 5 ml trypsin /EDTA to each plate . incubate 37 C,5% CO2 until the cells come off the plate .Add 10 ml DMEM +10%FBS to each plate and break any cell aggregates by gently pipetting.

8、12. Centrifuge cells (270 g,5min)and resuspend the pellet in DMEM+10%FBS.13. Count the cells and dilute to a concentration of 2x 105 cells/ml.14. Plate the cells immediately onto tissue culture dishes containing DMEM+10%FBS for the appropriate cell densities and volumes of medium for different plate

9、 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.Reagents 正常的滋养细胞

10、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。1.2.3.Preparatiooon of a stock of mitomycin C treated EMFI cellsthaw a frozen via of EMFI cells quickly at 37C .Add cells to 10 ml DMEM +10% FBS and centrifuge (270 g,5min).Decant supernatant , resuspend the cell pellet gently in 10 ml DMEM +10%FBS, and split onto fiv

11、e 150 mm plates each containing a total of 25 ml DMEM +10%FBS .Mix well.4.5.6.7.8.9.incubate cells at 37 C ,5% CO2 .Whenthe 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 monolayerRe

12、move the medium from the confluent plates and 10 ml DMEM +10%FBS containing 100 卩 l mitomycin C (1mg/ml stock).Swirlplates to ensurean even distribution of medium.Incubate cells at 37 C ,5% CO2 for 2-2.5h.Wash the monolayer of cells twice with 10 ml PBS per dish .Add 5 ml trypsin /EDTA to each plate

13、 .10.incubate 37 C,5% CO2until the cells come off the plate .(5-10min).ES 细胞培养 -实验方法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 fr

14、eezing vial in 1 ml of 1X freezing mediumand store at -70 C for one day .Transfer the vials to liquid nitrogen.13. to makefeeder plates thaw a frozen vial of mitomycin C treated EMFI cells quickly at 37 c14. Add cells to 10 ml DMEM +10%FBS and cenrifuge (270 g,5min).15. Decant supernatant , and resu

15、spend the cell pellet gently in 30 mlDMEM +10%FBS.16.Seed cells directly into tissue culture plates. Depending of the size of the plates required put 10 ml /100mmplate, 5ml /60 mmplate,5ml/60 mm plate, or 1.5ml/35mm plate.17. Allow feeders to attach preferably overnight ,before adding EScells or at

16、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 platesEquipment and reagents 100mm dishes containing feeder layersES cell medium briefly pre-warmed to 37 0. 05% trypsin in saline /EDTAPBS without Ca2+ and

17、 Mg2+Method1.Gelatin ,0.1% solution in water ,autoclavedQuickly thaw one vial of frozen ESby warming in your hand or at 37c ,and transfer cells to a 12 ml tube containing 10 ml of EScell mediumbefore all the ice has disappeared .2.3.Centrifuge at 270 g for 5min .Aspirate supernatant and resuspend pe

18、llet in 10 ml ES cell medium and plate on a 100 mm dish with a feeder layer.Changethe mediumthe next day by swirling the mediumin dish to collect debris ,then aspirate .Add fresh mediumgently to the side of the plate so that the feeder layer is not disturbed.On the second day (cell should be just su

19、bconfluent) wash cells twice with PBS and add 2 ml trypsin/EDTA.Incubate for approx .5 min at 37 c until cells begin to come off the plate .4.5.6.ES 细胞培养 -实验方法7.8.9.Gently agitate the plate and observe under a microscope .When trypsinization is complete ,cells should detach as small clumps ,not as a

20、 single sheet or single cells.Add 5 ml ES cell medium and gently pipette the cells up and down to break cells clumps,If the cells are sticky ,gently pipette before adding medium.Transfer to a sterile 12 ml tube and pellet cells in centrifuge (5 min,270 g).10. Aspiratesupernatant and gently resuspend

21、 cell pellet in 5-7 mlmedium .11. Add 1 ml of the cell sus pen tion (about 2-5x 10 6 cells ) to a fresh100mm feeder layer dish containing 9 ml ES cell medium .Disperse EScells evenly by pipetting gently and rocking the plate prior to incubating at 37C, 5%CO12. Change the medium the next day and pass

22、 cells every second day as described above.正常的未分化的ES细胞应当呈球状细胞团附着于滋养细胞层表面，细胞团的边 缘与滋养细胞层因有空间距离而形成折光的亮边, 如果细胞有分化, 则细胞团的 球形边缘部分或全部平坦化, 与底层滋养细胞之间的亮边消失, 分化了的细胞不 应再使用。Long term freezing of ES cell stocksMethod短期保存可以放在-70 C,但长期保存应转入液氮。 1.2.3.4.5.Trypsinize cells from a 100 mm dish .Pellet cells by centrifug

23、ation and respension in 3 ml precooled freezing medium .Immediately aliquot 1 ml of the cell suspention into freezing vialson ice.Immediately transfer vial to a Styrofoam box pre-cooled to - 70C ,orslow-cool container, and then into a - 70C frezer. It is importantto work quickly .After 24 h transfer

24、 the tubes on dry ice to liquid nitrogen.Sandard electroporation ES cellsEuipment and reagentsElectroporation apparatus :Bio-Rad GenePulser and Capacitance Extender ES cell medium2+ 2+PBS without Ca 2+ and Mg2+100 mm gelatinized platesGeneticin (G 418)1.2.3.4.5.6.7.8.9.On the second day after passin

25、g recently thawed ES cells ,trypsinize cells ,but trypsinize for longer to obtain single cells.After pipetting the cells gently in 3 ml trypsin to break up the clumps ,add 7 ml medium and pipette gently up and down again . Pre-plate the cells by incubating them in the dishes for 15 -20 min to allow

26、feeder cells to re- attach to the pl ate .Harvest the ES cells by carefully mixing and withdrawing medium ,and transfer cells to a 50 ml Falcon tube ,combining the cells from two to five plate .Pellet the cells for 5 min at 270 gAspirate the supernatant and resuspend the cells in a minimal volume of

27、 PBS (1 ml /100mm plate starting culture ).Keep cells on ice . Count the ESCells using a haemocytometer and adjust cell concentration to 7-10 X 10 6 cells/ml with sterile PBS .Mix 0.8 ml of the cell suspention with 25 -40 卩 g of linearized vector DNA ,and transfer into an electropration cavette whic

28、h has been pre-cooled on ice .Set up the electr opo rati on con diti onsin adva nee :240V,500 卩 F for theBio-Rad Gene Pulser using the Capacitance Extender.10. Transfereach cuvette holder with electrodes facing the outputleads.Deliver the electric pulse.11. Remove each cuvette from the cuvette holde

29、r and place on ice for 20 min .12. Remove the cells from each cuvette and dilute in appropriate volume of ES cell medium (15-20ml/cuvette).Cells from several cuvettes can be combined.13. Transfer 10 ml resuspended ES cells .Disperse cells evenly by rocking the plate.14. Change medium the next mornin

30、g.15. Two days afer the electroporation,begin drug selection16. Change the medium every day if working with gancyclvir,since it canbreak down and becometoxic to the cells,otherwise every two days when using G418 selection only . Widespread cell death should be apparent afer two to three days of drug

31、 selection.初次采用的G418的浓度为200ug/ml,虽然也能看到大批的细胞死亡，但阴性 对照(不加 DNA 而用 TE 的电转)亦见大批细胞死亡。17. Afterabout six to eight days of selection ,individual drug -resistant colonies should have appeared and be large enough to pickand subclone for screening by Southern blot analysis or by PCR and to freeze for storage.

32、Growing drug resistant clonesMethod 1.Prepare two sets of 96-well plates,one containing 35卩 l of trypsin/EDTA and one gelatin coated . 我们没有明胶处理的培养皿，而是预先在 皿中铺种滋养细胞层，但滋养细胞过于稀疏，造成ES细胞发生分化，以后实 验应当避免，应使滋养细胞稠密完全Circle all visible ES cell colonies that will be picked on the bottom each plate with a marker

33、by holding plate up to light or using an inverted microscope.WashEScell containing plates twice with PBS.Leave cells in PBSduring picking .After picking ,replace PBS with ES medium and return plate to incubator if additional ,smaller colonies will be picked on subsequent days.P ick the 1.5-2 mmdrug

34、- resista ntEScell clones that form after abouteight days of selection with a drawn-out Pasteur pipette or a yellow Gilson tip under Ca dissecting microscope .Pick colonies of similar size ,or take an equivalent portion of large colonies .挑取细胞克隆时将细胞间的显微镜置于超净台中， 一半镜身(物镜)在隔离玻璃 里面，一半镜身(目镜)在外面，紫外照射消毒。操作

35、时需要几个人，以加快速 度。注意 ES 细胞的形态应当保持球形细胞团，而发生分化的细胞克隆应当舍弃。5.2.3.4.6.7.Transfer each colony in a minimal volume of PBS into one well of a Vsha ped 96-well p late con tai ning 35 卩 l of 0.05% tryp sin in sali ne /EDTA at room temperature .Usually every other row is used and 48 colonies picked at one time .Aft

36、er picking 48 colones ,incubateCO2 ,until cell clumps breaks up.Stop the reaction by adding 100 卩 lwell using multichannel pipettor. G418plate for approx.10 min at 37C ,5%of ES mediumcontaining G418to each 的浓度宜提高至 400-600 ug/ml.Mix cells gently by pipetting up and down and transfer to gelatinized 96

37、-well tissue culture plate.Washeach V-well from the trpsin plate with another 100 卩 l of medium per well and add to same well of the 96-well gelatinized plate .10.Change the medium the next day, and then daily thereafter.11.If the colonies have not grow n to80% con flue nee in two to threedays ,tryp

38、late the cells using the following procedure:(a)(b)8.9.(c)Aspirate the medium and wash the cells with PBS.Add 35 卩 l of trypsin /EDTA and incubate for 5 min at 37 until the cells lift off the dish.Add 200 卩 l ES cell medium and break colonies up by gently pipetting up and down .Change the medium 12-

39、24 h later.C ,or(d)12. Repeat steps 1-11 on second and third picking days ,using original plates of electroporated cells .13. Whenthe colonies in 96-well plates have reach at least 80%confluenceyou should passage them and freeze your clones and prepare replica for DNA analysis.Freezing drug resistant clones and preparation of replicaplates for Southern blot analysisMethod1.2.3.4