es细胞培养-实验方法

es细胞培养-实验方法 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?) -4(D) 10M β?mercaptoethanol (100×stock,aliquoted ,stored at-20?) (E) 2mM L?glutamine (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 /EDTA DMEM +10%FBS and used within Mitomycin C (stock 1 mg/ml in PBS stored in dark at 4? two weeks ,mitomycin C is toxic ;wear gloves and use caution when handling ) Methods 1. 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% CO 2 . 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% COfor 2-2.5h. 2 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% COuntil the cells come off the plate . 2 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. 513. Count the cells and dilute to a concentration of 2 ×10 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 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. incubate cells at 37? ,5% CO4.2 . 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% COfor 2-2.5h. 2 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% COuntil the cells come off the plate .(5-10min). 2 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 /EDTA PBS without Ca2+ and Mg2+ Gelatin ,0.1% solution in water ,autoclaved Method 1. Quickly thaw one vial of frozen ES by warming in your hand or at 37?,and transfer cells to a 12 ml tube containing 10 ml of ES cell medium before all the ice has disappeared . 2. Centrifuge at 270 g for 5min . 3. Aspirate supernatant and resuspend pellet in 10 ml ES cell medium and plate on a 100 mm dish with a feeder layer. 4. Change the medium the next day by swirling the medium in dish to collect debris ,then aspirate .Add fresh medium gently to the side of the plate so that the feeder layer is not disturbed. 5. On the second day (cell should be just subconfluent) wash cells twice with PBS and add 2 ml trypsin/EDTA. 6. Incubate for approx .5 min at 37? until cells begin to come off the plate . 7. Gently agitate the plate and observe under a microscope .When trypsinization is complete ,cells should detach as small clumps ,not as a single sheet or single cells. 8. 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. 9. Transfer to a sterile 12 ml tube and pellet cells in centrifuge (5 min,270 g). 10.Aspirate supernatant and gently resuspend cell pellet in 5-7 ml medium . 611.Add 1 ml of the cell suspention (about 2-5 ×10 cells ) to a fresh 100mm feeder layer dish containing 9 ml ES cell medium .Disperse ES cells evenly by pipetting gently and rocking the plate prior to incubating at 37?,5%CO。 2 12.Change the medium the next day and pass 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 stocks Method 短期保存可以放在- 70?,但长期保存应转入液氮。 1. Trypsinize cells from a 100 mm dish . 2. Pellet cells by centrifugation and respension in 3 ml precooled freezing medium . 3. Immediately aliquot 1 ml of the cell suspention into freezing vials on ice. 4. Immediately transfer vial to a Styrofoam box pre-cooled to - 70?,or slow-cool container, and then into a - 70? frezer. It is important to work quickly . 5. After 24 h transfer the tubes on dry ice to liquid nitrogen. Sandard electroporation ES cells Euipment and reagents Electroporation apparatus :Bio-Rad Gene Pulser and Capacitance Extender ES cell medium 2+2+ PBS without Ca and Mg 100 mm gelatinized plates Geneticin (G 418) 1. On the second day after passing recently thawed ES cells ,trypsinize cells ,but trypsinize for longer to obtain single cells. 2. 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 . 3. Pre-plate the cells by incubating them in the dishes for 15 -20 min to allow feeder cells to re –attach to the plate . 4. 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 . 5. Pellet the cells for 5 min at 270 g 6. Aspirate the supernatant and resuspend the cells in a minimal volume of PBS (,1 ml /100mm plate starting culture ).Keep cells on ice . 7. Count the ES Cells using a haemocytometer and adjust cell concentration 6 to 7-10×10cells/ml with sterile PBS . 8. Mix 0.8 ml of the cell suspention with 25 -40 μg of linearized vector DNA ,and transfer into an electropration cavette which has been pre-cooled on ice . 9. Set up the electroporation conditions in advance :240V,500μF for the Bio-Rad Gene Pulser using the Capacitance Extender. 10.Transfer each cuvette holder with electrodes facing the output leads.Deliver the electric pulse. 11.Remove each cuvette from the cuvette holder 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 morning. 15.Two days afer the electroporation,begin drug selection 16. Change the medium every day if working with gancyclvir,since it can break down and become toxic 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 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.After about six to eight days of selection ,individual drug – resistant colonies should have appeared and be large enough to pick and subclone for screening by Southern blot analysis or by PCR and to freeze for storage. Growing drug resistant clones Method 1. Prepare two sets of 96-well plates,one containing 35 μl 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 2. Circle all visible ES cell colonies that will be picked on the bottom each plate with a marker by holding plate up to light or using an inverted microscope. 3. Wash ES cell containing plates twice with PBS .Leave cells in PBS during picking .After picking ,replace PBS with ES medium and return plate to incubator if additional ,smaller colonies will be picked on subsequent days. 4. Pick the 1.5-2 mm drug –resistant ES cell clones that form after about eight days of selection with a drawn-out Pasteur pipette or a yellow Gilson tip under ?a 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. Transfer each colony in a minimal volume of PBS into one well of a V shaped 96-well plate containing 35μl of 0.05% trypsin in saline /EDTA at room temperature .Usually every other row is used and 48 colonies picked at one time . 6. After picking 48 colones ,incubate plate for approx.10 min at 37?,5% CO2 ,until cell clumps breaks up. 7. Stop the reaction by adding 100μl of ES medium containing G418 to each well using multichannel pipettor. G418的浓度宜提高至400-600 ug/ml. 8. Mix cells gently by pipetting up and down and transfer to gelatinized 96-well tissue culture plate. 9. Wash each V-well from the trpsin plate with another 100 μl 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 grown to ,80% confluence in two to three days ,tryplate the cells using the following procedure: (a) Aspirate the medium and wash the cells with PBS. (b) Add 35 μl of trypsin /EDTA and incubate for 5 min at 37 ?,or until the cells lift off the dish. (c) Add 200 μl ES cell medium and break colonies up by gently pipetting up and down . (d) Change the medium 12-24 h later. 12.Repeat steps 1-11 on second and third picking days ,using original plates of electroporated cells . 13.When the colonies in 96-well plates have reach at least 80% confluence you should passage them and freeze your clones and prepare replica for DNA analysis. Freezing drug resistant clones and preparation of replica plates for Southern blot analysis Method 1. Prepare the required number of sets of two 96-well gelatinzed flatbottom plates with 200 μl ES cell medium ,and one 96-well V-bottom plate containing 50μl cold 2freezing medium ,keep on ice. 2. Remove medium from each well of the 96-well plate containing growing ES cell clones . 3. Wash cells twice with 200 μl PBS. 4. Add 50 μl trypsin and incubate for 10 min at 37 ?,5% CO2. 5. Observe cells under an inverted microscope .If all cells have detached,stop trypsinization by adding 50μl of ES cell medium ,then mix well for pipetting. 6. Working quickly ,transfer 50μl of cell suspention to the 96 V-well master plate,which contains 50μl of pre-cooled 2× freezing medium in each well,mix by pipetting up and down. 7. Layer 100μl of cold mineral oil on the top of each well containing ES cells in freezing medium . 8. Seel plate by wrapping Parafilm around the outside edge where the top and bottom meet wrap plate in aluminium foil ,and transfer in a pre-cooled Styrofoam box .to -70 ? freezer. 9. Mix the remaining cell suspension and transfer 25 μl into each of the two gelatinized 96-well replica plates containing 200μl of medium per well .Return replica plates to incubator. 10.Change the medium daily on replica plates .Perform DNA extraction when cells are overly confluent The goal is to obtain the maximum number of cells.