Wittrup:Making a cell line from tumor sections

*Protocol courtesy of Leah Schmidt, Jacks Lab @MIT*

Reagents:

Collagenase Type IV Worthington Biochemical Corp ls004186

Hyaluronidase Worthington Biochemical Corp ls002594

Collagenase Type I Worthington Biochemical Corp ls004194

Collagenase/Dispase Roche 10269638001

Protocol:

1. Finely dice tumors in plastic dishes without media, then transfer to a 15 ml Falcon tube (no filter yet)

2. Add 2 mL digestion media/tumor: For FACS samples, 125 U/ml Collagenase Type IV, 60 U/ml Hyaluronidas in PBS or RPMI + 1% serum; for cell lines, 125 U/ml Collagenase Type I, 60 U/ml Hyaluronidase, 2 mg/ml Collagenase/Dispase in PBS or RPI + 1% serum (can also add gentamicin for cell lines)

NOTE: Collagenase type I and Dispase tend to remove surface proteins, so don't use those for FACS samples

3. Incubate in digestion media ~ 1 hr on 37°C shaker, then pass the cells over a cell strainer

4. Wash with PBS and plate cells in DMEM+gentamicin, pen-strep, and 10% FBS

5. Next day, change media (gently)

6. In a few days to weeks, cells should grow out amidst many dead/senescent cells. Passage them until they look somewhat uniform.