Culturing Mouse Embryonic Stem Cells

Mouse embyonic stem (ES) cells grow very well in culture. Log-phase growth has a doubling time on the order of 18 hours. It is necessary to culture ES cells in the presence of LIF (leukemia inhibitory factor) in order to prevent their spontaneous differentiation and loss of pluripotency. ES cells like to grow in clumps piled on top of each other. A healthy, non-differentiated culture of ES cells will show discrete large "patches" of cells with individual cells not being distinguishable within the patch. Additionally, the patches should show sharp, bright borders under a phase contrast microscope on low power indicative of their 3-dimensional piled-up nature. Non-healthy and/or differentiated ES cells will show flat monolayers of individually distinguishable cells that appear dull under phase contrast. ES cell cultures having trouble can sometimes be helped back to a fully differentiated state by allowing the cells to grow without splitting but with frequent medium changes to very high density on the plate before splitting. The idea is that the undifferentiated cells which can grow in 3-dimensional piles will be enriched relative to the differentiated monolayer cells that will tend to get "crowded out".

All manipulations must be carried out in a laminar flow tissue culture hood.

Preparing a tissue culture plate for receiving ES cells

1. Completely coat the bottom of a tissue culture plate with a 0.1% gelatin solution
   
   • eg, use 3 ml for a 10 cm diameter plate
     
   • make sure the bottom of the plate is completely covered by tilting the plate back and forth a few times
     
   • let the gelatin sit on the plate for a minute or two
     
   • store sterile 0.1% gelatin solution at room temperature
     
2. Completely aspirate off the gelatin solution but do not allow the plate to dry out
   
   • leaving too much gelatin on the plate will "drown" the ES cells
     
3. Add an appropriate volume of ES cell media to the plate
   
   • 8 - 10 ml for a 10 cm plate works well
     

Thawing frozen ES cells

1. remove the vial of cells from frozen storage and wipe down the vial with a 70% ethanol solution
   
2. open and then reclose the vial briefly to allow air pressure to equilibrate
   
   • skip this step if using a sealed glass vial
     
3. wearing gloves, hold the vial of cells in your hand until partially thawed
   
4. mix the partially thawed cells by inverting the vial a few times
   
5. dump the partially thawed cells into the ES cell medium on a prepared tissue culture plate (see 'Preparing a tissue culture plate for receiving ES cells' above)
   
6. thaw the cells completely by swirling in the medium on the plate
   
7. place immediately in a 37 C humidified tissue culture incubator with 5% CO₂
   

Splitting ES cells

1. remove the plate of ES cells from the incubator to a laminar flow tissue culture hood
   
2. aspirate the medium from the plate
   
3. add an appropriate volume of trypsin/EDTA solution to the cells and tilt the plate back and forth several times to ensure even treatment
   
   • 2 ml for a 10 cm plate works well
     
4. when the majority of the cells detatch from the plate with gentle rocking (usually a minute or two), add at least two volumes of tissue culture medium to the trypsinized cells and pipette up and down several times to disperse the cell clumps
   
   • do not allow cells to sit in the trypsin/EDTA solution longer than necessary as they will lyse.
     
   • cells will be stable after dilution into medium as the serum in the medium stops the action of the trypsin
     
5. add an appropriate volume of unclumped trysinized ES cells to the medium on a prepared tissue culture plate and replace in the incubator
   
   • split ratios of 10:1 work well. 20:1 is doable if necessary. splits of greater than 20:1 are not recommended.
     

Freezing ES cells

1. trypsinize the cells from a 50% confluent 10 cm plate as described above in 'Splitting ES cells'
   
2. centrifuge the unclumped ES cells dispersed in medium for 5 minutes at 500 g in a clinicial centrifuge
   
3. aspirate the trypsin/EDTA/medium from the cell pellet
   
4. resuspend the cell pellet completely in 1 ml of 90% ES medium / 10 % DMSO
   
5. add to a labeled freezer vial
   
6. freeze slowly by either using a freezing container at -80 C or by placing directly in the vapor phase of a liquid nitrogen freezer
   
   • do NOT place cells directly in the liquid phase of a liquid nitrogen freezer
     
7. short-term (several days) at -80 C is ok. for long-term storage (more than one week) store in a liquid nitrogen freezer
   
   • either liquid or vapor phase storage works fine
     
   • cells stored in a liquid nitrogen freezer should be stable for several years
     

---

Solutions

ES Medium

• 500 ml DME - high glucose
  
• 75 ml ES-cell qualified fetal bovine serum
  
• 6 ml 100x penn/strep (10000 U / ml each stock)
  
• 6 ml 100x non-essential amino acids (10 mM each stock)
  
• 6 ml 100x L-glutamine (200 mM stock)
  
• 6 ml diluted 2-mercaptoethanol
  
  • diluted is 21.6 μl of stock 2-mercaptoethanol in 30 ml of PBS without Ca or Mg
    
• 60 μl LIF (stock is 10⁷ U / ml)
  
  • available as "ESGRO" from Chemicon
    

mix thoroughly, store at 4 C for up to several weeks

Trypsin/EDTA solution

• 0.2% trypsin
  
• 1 mM EDTA
  

in PBS without Ca or Mg
store at 4 C for up to several weeks, or freeze at -20 C for long-term storage

---

Comments