Isolation of Primary Liver Tumor Cells

  1. prepare 6-well plate at least 1hr before use (2ml PBS/2%FBS +2µl Laminin per well)
  2. Remove livers from mouse and wash liver tissue in 10ml PBS in 10cm plate.
  3. transfer liver tissue to a new10cm plate, add 5ml PBS/2%FBS, and Chop these pieces with a 5 razor blade cutting object, and then add 5ml 2mg/ml collagenase/ dispase solution(UCSF CCF)(stock 2mg/ml ) , incubate for 30~40min at 37°C.
  4. Sieve liquid through medical gauze into 50ml Falcon tube(usually use the 10ml from the plate plus 10ml PBS/2%FBS to wash the plate and tissue chunks with)
  5. spin ( 4°C, 1000rpm, 5 min ) and resuspend in 10ml PBS/2%FBS and filter through 70µm filter followed by 45µm filter. Bring up to 5~10ml and spin again( RT, 1000rpm, 2 min ) .
  6. resuspend in 5-10ml of 1×RBC Lysis butter, incubate on ice for 5-10min.
  7. bring up to 5~10ml PBS/2%FBS and spin ( RT, 1000rpm, 2 min ) .
  8. repeat step 7 for 2~3 times.
  9. resuspend the cells with Tm1 medium, and aliquot into 6-well plate.

Reagents

  1. Laminin: L2020-1MG, Store at -20°C, sigma
  2. sterile cotton gauze pad : 12-Ply; 3 x 3 in, #22-415-468 , Fisherbrand
  3. sterile cell strainer: 70µm nylon mesh, #22-363-548, Fisherbrand
  4. sterile cell strainer: 45 µm nylon mesh,#352340, BD falcon
  5. (5) 1 × RBC lysis buffer: 200ml , #00-4333-57,store at 4°C, eBioscience
  6. D-PBS/2%FBS : 500ml D-PBS+10ml FBS, store at 4°C
  7. Collagenase(2mg/ml): 50ml D-PBS+100mg Collagenase dispase, aliquot into 5ml of each tube. Store at -20°C, (CCF#ROCZR113), (Roche#10269638001)
  8. Tm2 medium: 500ml DMEM (10%FBS)+5ml 100×ITS+20µl EGF
  9. Tm1 medium: 50ml DMEM (10%FBS)+0.5ml 100×ITS+2µl EGF+2µl IGF2
  10. ITS:Insulin-Transferrin-Sel-G Supplement(100×), #41400-045, Invitrogen
  11. EGF: Recombinant Human EGF (1µg/µl) , #AF100-15, PeproTech
  12. IGFII: Recombinant Human IGF-II (0.2µg/µl) , #100-12, PeproTech