ES Cell Culture for Blastocyst Injection.doc

ES Cell Culture for Blastocyst Injection:

Media: Filter sterilize all media.

LIF Media (250mL; 10%FBS)

  250 mL GMEM
  2.5 mL Amino Acids
  2.5 mL L-glutamine
  2.5 mL Sodium Pyruvate
  0.28 mL 2-mercaptoethanol (1000x stock)
  25 mL FBS
  0.5 mL LIF

10% FBS Media w/o LIF (25mL)

  25 mL GMEM
  0.25 mL Amino Acids
  0.25 mL L-glutamine
  0.25 mL Sodium Pyruvate
  0.028 mL 2-mercaptoethanol (1000x stock)
  2.5 mL FBS

Blast Injection Media

  10 mL GMEM
  1.0 mL FBS
  0.275 mL 1M HEPES

Cell Culture:

  D0: Thaw a vial of cells.  
  Resuspend in 10mL LIF Media 
  Pellet
  Wash with 5mL LIF Media
  Pellet
  Resuspend in 10mL LIF Media and plate onto a gelatinized T25

  D1: Feed cells with 10mL LIF Media or split (1:10) if needed
 
  D2: Feed cells with 10mL LIF Media or split (1:5) if needed

  D3: Feed cells with fresh LIF Media as early as possible
  10:00-10:30 Trypsinize cells off of T25
  Resuspend in 9 mL 10% FBS Media w/o LIF
  Split 1:5 into a new T25
  Pellet 4mL of cells. Resuspend in 2mL Blast Injection Media and Count
  Dilute cells to 1x106 cells/mL with Blast Injection Media

*I provide Paul with 1-2 mL of cells at 1x106 cells/mL. *He also needs the excess Blast Injection Media.