ES cell culture

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  1. cell culture supplies and reagentss
    1. Environment: cell culture requires a sterile environment, so it needs a separate room with the air purification system and a UV lamp to sterilize the entire room.
    2. Equipments: carbon dioxide incubator, clean benches, refrigerator, water bath, inverted microscope, centrifuge, laboratory table, pipettes, etc..
    3. Experiment supplies: disposable cell culture dishes or cell culture flask, 15ml or 50ml centrifuge tubes, cotton flower, sealing film, etc..
    4. Reagents: good quality reagents.
    5. Reagent preparation:
      1. DMEM medium: deionized water containing 1.34% DMEM powder, 0.22% NaHCO3
      2. MEF medium: DMEM medium containing 10% FBS, 1000u/ml penicillin, 1000g/ml streptomycin
      3. ES medium: DMEM medium containing 15% FBS, 1×106 U penicillin, 1×106 g of Streptomycin, 1mM pyruvate, 0.1mM none essential amino acids, 2mM L-glutamine, 0.1mM 2-mercapto-ethanol, 1000u/ml leukemia inhibitory factor
      4. D-Hank's: 0.8% NaCl, 0.04% KCl, 0.035% NaHCO3, 0.006% KH2PO4, 0.005325% Na2HPO4, 0.001% Phenol-red
      5. 0.1% gelatin: D-Hank's solution containing 0.1% gelatin
      6. ES cell freezing medium: 90% ES medium, 10% DMSO
      7. MEF cell freezing medium: 90% MEF medium, 10% DMSO
      8. Feeder cell freezing medium: 90% FBS, 10% DMSO
      9. Mitomycin C: according to the supplier's manual
  2. cell culture step

    Embryonic stem cells (ES cell) is the most commonly culture stem cells. Here is a protocol for mouse embryonic stem cell culture.

    1. Thawing mouse embryonic fibroblasts (mouse embryonic fibroblasts, MEF)

      Embryonic stem cells are grown at 37℃/ 5% CO2 / 95% humidity in dishes coated with a feeder layer of mitotically inactivated mouse embryonic fibroblasts. Here is how to culture mouse embryonic fibroblasts to prepare the feeder layer.

      1. Thaw a cryovial of MEF cells by quickly warming it in a 37℃ waterbath.
      2. Aseptically transfer the cell suspension to a sterile tube containing several ml of warm MEF medium, gently mix and pellet the cells by centrifugation at 1000×g for 5min.
      3. Aspirate off the supernatant (removing DMSO in freezing medium) and resuspend cells into 10ml of warm MEF medium and plate out in a 10cm dish,incubating at 37℃ with 5% CO2 in the humidified incubator.
      4. Medium should be changed everyday or must be changed when it turns orange.
      5. In about 4 days, upon subconfluence, cells can be passaged or frozen or used for preparing feeder cell layer.
    2. preparing the feeder cell layer
      1. Aspirate the MEF medium, add 10ml fresh MEF medium with 110μl mitomycin C solution in dark, incubating the cells in the carbon dioxide incubator with 37℃, 5% CO2 after mixing gently for 2 hours.
      2. The medium containing mitomycin C can be re-used (add the medium with mitomycin C directly into dishes and incubate at 37℃ with 5% CO2 for 5h in the humidified incubator) within a week, so the mitomycin C medium can be collected in a sterile 15 ml tube and kept in 4℃ without light. Wash the MEF cells extensively with PBS (without bivalent cations) for 2~3 times and dislodge the cells with 1ml trypsin/EDTA for about 3 min in the humidified incubator.
      3. Add 1ml MEF medium to stop trypsin/EDTA digestion, and collect the cells by low-speed centrifugation (1000 rpm for 5 min).
      4. Remove the supernatant, and resuspend the cells with 1ml MEF medium. Plate the cells onto tissue culture dishes pretreated with 0.1% gelatin, in 10ml MEF medium. Incubate the cells in the humidified incubator at 37℃ with 5% CO2 . (Pretreating with gelatin: add about 5ml 0.1% gelatin to a 10cm dish, and incubate the dishes at 37℃ with 5% CO2 in the humidified incubator. If the color of gelatin changes into yellow from red, remove the gelatin).
      5. The cells can be used after they adhere to the dish. Feeder Cell Layer may be used for up to 1 week, and the medium should be replaced with ES cell culture medium immediately before being used. Check for the integrity of feeder cell monolayer).
    3. Thawing mouse embryonic stem cells (ES cells)
      1. Thaw a cryovial of ES cells by quickly warming it in a 37℃ waterbath.
      2. Aseptically transfer the cell suspension to a sterile tube containing several ml of warm ES medium, gently mix and pellet the cells by centrifugation at 1000×g for 5min.
      3. Aspirate off the supernatant (removal of DMSO in freezing medium) and resuspend cells into 10ml of warm ES medium and plate out in a 10cm feeder plate, incubating at 37℃ with 5% CO2 in the humidified incubator.
      4. Medium should be changed everyday or must be changed when it turns orange .
      5. In about 3~4 days, upon subconfluence,cells can be passaged or frozen or used for doing experiments.
    4. Passagee of mouse embryonic stem cells
      1. Aspirate off medium, wash the cells 2 or 3 times with PBS (without bivalent cations), add 1ml of trypsin/EDTA and incubate at 37℃ with 5 %CO2 in the humidified incubator about 5min until cells float off.
      2. Add 1ml of MEF medium to stop digesting, collect cells in a tube and centrifuge at 1000×g for 5min.
      3. Aspirate off the supernatant, resuspend cells in appropriate volume of ES medium and distribute cells into several feeder plates depending on plate format and splitting ratio (Splitting ratios for ES cells can vary from 1:1 to 1:10).
    5. Freezing mouse embryonic stem cells
      1. Aspirate off medium, wash the cells 2 or 3 times with PBS (without bivalent cations), add 1ml of trypsin/EDTA and incubate at 37℃ with 5 %CO2 in the humidified incubator about 5min until cells float off.
      2. Add 1ml of MEF medium to stop digesting, collect cells in a tube and centrifuge at 1000×g for 5min.
      3. Aspirate off the supernatant, resuspend cells in appropriate volume of pre-cooled freezing medium and transfer into freezing vials (1ml per vial). Gradient cooling: 4℃ for 20min, -20℃ for 20min, -80℃ for over night and transfer cells to a liquid nitrogen for long term storage.
  3. Notes
    1. Mitomycin C must be used in dark to avoid decomposition.
    2. ES cells tend to grow together, and then chose appropriate size of dishes to thaw them.
    3. Avoid overgrowing, otherwise ES cells will differentiate.
    4. To avoid the contamination caused by water or serum, the serum should be checked for mycoplasma, and tested for contamination.
    5. It is better to refeed cells with fresh medium about 3 hours before passaging or freezing to ensure cells' status and remove the dead cells and impurities.