Sample HIM tumor Protocol ( Click to Download )

Day 1

  1. Thaw 1-2 vials of 639L RM organoids CP (Age 28) and resuspend in 10 ml of RMFC, pellet and remove supernatant, resuspend each in 20 ml RMFC and plate at 37C for 1-2 hours in a 15 cm dish.
  2. Remove non-adherent cells and PBS wash to a tube, use an 18G needle to break up clusters (8X pass).  Spin down cells 1200 rpm 4’. Wash once with cold PBS + 0.1% BSA, spin down.
  3. Resuspend cells in 5 ml T-EDTA (0.05% Trypsin), pipet up and down w/ p1000 for 1 min, incubate at 37C for 5 min, repeat.
  4. Add 20 ml RMFC + 0.05 mg/ml Dnase I (add 120 ml of 5 mg/ml stock, Roche), pipet up and down to break up clumps.
  5. Filter through 40 mm mesh, pellet cells and resuspend in 5 ml MC.
  6. Resuspend at 1×106 per ml ml in MEGM.

Day 2

  1. Trypsinize and count RMF-EG/HGF; resuspend at 1 x 106/ml, keep on ice
  2. Break up clusters of virally infected cells with p1000
  3. Collect infected cells to 15 ml conical tube + PBS wash of wells
  4. Pellet cells, wash 1X with PBS
  5. Count, resuspend at 1 x 106/ml in MEGM, keep on ice
  6. Prepare 1:1 collagen: matrigel solution, keep on ice
  7. Mix RMFs + epithelials together, pellet
  8. Resuspend in col:mat, transfer to epi tubes and keep on ice until injection
    | Cell line | Count | Need |
    RMF-EG/HGF 4.5 x106
    SV40/Ras 500K
    LgT/Ras 700K
    LgT/PVRL4/Ras 700K

1:1 collagen: matrigel

Mix 700 µl 4.3 mg/ml Rat tail collagen + 500 µl 0.2 N GAA

Add 180 µl of 0.01 N NaOH  = pH 5.5 approx.

Mix 500 µl of (~2 mg/ml) Collagen + 500 ul Matrigel

Add ~ 25 µl 0.01 N NaOH to get pH 7.0 approx.

Infection Glands RMFs Epis Col:Mat
SV40/Ras Min 5
LgT/Ras Min 7
LgT/PVRL4/Ras Min 7

Part 4: Injections, 8 mice

  1. SV40/Ras (2 glands)
  2. SV40/Ras (2 glands
  3. LgT/Ras (2 glands)
  4. LgT/Ras (2 glands)
  5. LgT/Ras (2 glands)
  6. LgT/PVRL4/Ras (2 glands)
  7. LgT/PVRL4/Ras (2 glands)
  8. LgT/PVRL4/Ras (2 glands)