Tina:Inactivated MEF cells preparation

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Cell information[edit]

  • Purpose: To generate inactivated MEFs
  • Cells: P3 untreated CF-1 MEFs from GlobalStem (cat# GSC-6001)
  • Batch #1:
    • Date: 6/15/2012 ~ 6/28/2012
    • Cells: GlobalStem GSC-6001, lot#
    • Thaw one vial of CF-1 MEF in 1*75T flasks on 6/15. Passage cells in four 75T flasks on 6/20. Passage cells in four P150 dishes on 6/23. Change media on 6/25.
    • Mitomycin C treatment: Add 100ul 1mg/mL mitomycin C to 10ml media per P150 dishes on 6/28. Incubate 3 hours at 37゚C.
    • Cell count: total cell number = 26.25*10e6
    • Final freezing concentration: 2.4*10e6 per vial (for one 6-well plate). Total cryovial number: 11 vials.
    • Check MEF quality and quantity: Thaw one vial on 7/2 in two 6-well plate. Observe on 7/3. Cell quality is OK. Survival rate looks ok. However, according to cell density, cells should be thaw in four 35mm dishes per vial next time.
  • Batch #2:
    • Date: 6/27/2012 ~ ongoing work
    • Cells: GlobalStem GSC-6001, lot#94301077
    • Thaw two vial of CF-1 MEF in four 75T flasks and two P100 dishes on 6/27. Passage cells into seven P150 dishes on 7/1.
    • Mitomycin C treatment: Add 100ul 1mg/mL mitomycin C to 10ml media per P150 dishes on 7/5. Incubate 3 hours at 37゚C.
    • Cell count: total cell number = 8.75*10e6
    • Final freezing concentration: 2.2*10e6 per vial (for one 6-well plate). Total cryovial number: 4 vials.
    • Fold of expansion is not ideal. The seeding density of MEFs on 7/1 was too high. Cells may not have enough space to proliferate. Better also perform cell count at first passaging.
    • Check MEF quality and quantity: Thaw one vial on 7/9 in two 6-well plate. Observe on 7/10. Cell quality is OK. Survival rate looks good. However, according to cell density, cells should be thaw in nine 35mm dishes per vial next time.