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Products for this protocol

• Dimethyl sulfoxide (DMSO) (e.g., Sigma)
   
  
• Feeder-conditioned medium (feeder-CM)
   
  Prepare mitomicin-treated mouse embryonic fibroblasts (MEF) and culture in TS medium for 72 hours. Collect the medium. Spin to remove the floating cells and debris, filter (0.45 µm), and store at -20°C in aliquots. Thaw each aliquot as needed and store it at 4°C; do not refreeze. Use the MEF to prepare two more batches of feeder-CM, then discard the cells. MEF are used up to 10 days after mitomycin treatment.
   
  
• Feeder-conditioned medium (feeder-CM) FGF4 stock solution (1000X, 25 µg/ml)
   
  
  • Human recombinant fibroblast growth factor-4 (FGF4; Sigma)
     
    
  • 0.1% (w/v) BSA fraction V in PBS
     
    To make a 1000x, 25 µg/ml stock solution of FGF4, resuspend lyophilized FGF4 in its vial with 1.0 ml of 0.1% (w/v) bovine serum albumin (BSA) in phosphate-buffered saline (PBS). Mix well, divide into 100-µl aliquots, and freeze at -80°C. Thaw each aliquot as needed and store at 4°C. Do not refreeze.
     
    
• Heparin stock solution (1000X, 1.0 mg/ml) (Sigma, 10,000 units)
   
  Resuspend in PBS and store at -80ºC. The stock can be also prepared as 10,000X (10 mg/ml) solution.
   
  
• Mouse Embryo Fibroblast (MEF) feeder cells
   
  Use half the density usually used for ES cells (i.e., 1 x 105 cells/ml)
   
  
• PBS/0.1%(w/v) BSA fraction V (Sigma) for FGF4 stock solution
   
  
  • 10X Stock
     
    
  • NaCl 80 g
     
    
  • KCl 2 g
     
    
  • Na₂HPO₄ 14.4 g
     
    
  • KHa₂PO₄ 2.4 g
     
    
  If necessary, PBS may be supplemented with the following:
   
  
  • CaCL₂·2H₂O 1.33 g
     
    
  • MgCL₂·6H₂O 1.0 g
     
    To prepare 1 L of 10X PBS, dissolve the reagents listed above in 800 mL of H₂O. Adjust the pH to 7.4 (or 7.2, if required) with HCl, and then add H₂O to 1 L. Dispense the solution into aliquots and sterilize them by autoclaving for 20 min at 15 psi (1.05 kg/cm²) on liquid cycle or by filter sterilization. Store PBS at room temperature.
     
    
• 0.1% Trypsin/EDTA
   
  
• TS cells
   
  
• TS medium
   
  
  • RPMI 1640, 500 ml
     
    
  • FBS, 130 ml (20% final)
     
    
  • Penicillin and Streptomycin (100x stock, 50 µg/ml each final)
     
    
  • Sodium pyruvate, 6.5 ml (100 mM stock, 1 mM final)
     
    
  • ß-mercaptoethanol, 6.5 ml (10 mM stock, 100 µM final)
     
    
  • L-glutamine, 6.5 ml (200 mM stock, 2 mM final)
     
    

• Incubator preset to 37°C, 5% CO₂, 95% air
   
  
• Microscope, inverted
   
  
• Tissue culture dishes
   
  

1. To culture TS cells on MEF:
    
   
   1. Prepare TS+F4H medium by adding 10 µl of 1000X FGF4 (final concentration 25 ng/ml) and 10 µl of 1000X heparin (final concentration 1 µg/ml) to 10 ml of TS medium.
       
      
   2. Culture the TS cells in TS + F4H medium on half the density of MEF feeder cells as usually used for ES cells, in a standard tissue culture incubator (37ºC, 5% CO₂).
       
      
   3. Change the medium every other day and passage the cells (1:10 - 1:20) every fourth day or when the culture has reached ~80% confluency. Passaging TS cells at higher densities may lead to precocious differentiation.
       
      
   4. Trypsinize the cells to very small clumps with some single cells. A complete single-cell suspension is not required, and it may even be detrimental to the culture. It is usually sufficient to trypsinize for 3-4 minutes at 37ºC with some pipetting up and down.
       
      
2. To culture TS cells on tissue culture plastic:
    
   
   1. Prepare 70cond medium by adding 3 ml of TS medium to 7 ml of feeder-CM.
       
      
   2. Prepare 70cond + F4H medium by adding 10 µl of 1000X FGF4 (final concentration 25 ng/ml) and 10 µl of 1000X heparin (final concentration 1 µg/ml) to 10 ml of 70cond medium.
       
      
   3. Culture the TS cells in 70cond + F4H medium. TS cells grow well on standard tissue culture dishes without MEF if the medium is supplemented with feeder-CM. The dishes do not need to be gelatin coated.
       
      
   4. Feed and passage the cells as described in Step 1.iii and 1.iv.
       
      When switching from MEF to feeder-CM, it may be desirable to remove the MEF immediately. The different adherence rates of MEF (fast) and TS cells (slow) can be used to obtain a pure TS cell population. Passage the cells to a new plate and incubate the culture for 1 hour at 37°C/5% CO₂. Remove the supernatant and plate onto another dish. This population of cells should consist almost entirely of TS cells. Such differential plating enriches for stem cells at the expense of differentiated trophoblast cells.
       
      
3. TS cells are frozen and thawed using procedures similar to those for ES cells (Freezing and Thawing of Embryonic Stem (ES) Cells Using Cryovials), except that a higher serum content is used. A 2X freezing medium is freshly prepared and kept on ice before being added to the cell suspension in equal volume. It consists of 50% FBS, 20% DMSO, and 30% TS medium (already containing 20% FBS).