Establishment of cell line from murine liver tumor

 Collagenase:

Collagenase IV, Life Technologies (Gibco), cat #. 17104-019 1g.
1 mg/ml DMEM without FCS!! Sterile filter (0.22µm) and aliquot (10ml per aliquot)

Growth medium : DMEM + 10 % FCS + PS

Procedure 

1.       Collect tumor tissue in cold PBS. Wash with PBS to get rid of blood if any.

2.       Mechanically disrupt liver tumor tissues with scalpels.

         (if minced tissue has too much blood it’s good to give the PBS washes)

3.    Add collagenase and let it digest for 30 minutes, 37°C, shaking. If needed: vortex and digest      

       for more 10 to 15 min or add fresh collagenase and digest for more time. 

         (It can be done in 37°C incubator )   

4.     When solution appears cloudy and with smaller tissue pieces take out from 37°C incubator and    

      Let remaining pieces sink by gravity. Take out the supernatant. 

       (if there are any small pieces left after digestion for hour just  resuspend these pieces also in DMEM + 10 % FCS +   

       PS and plate in separate 10 cm dish ) 

5.       Filter the supernatant through 70-µm sterile nylon gauze.

6.      Centrifuge at 300g for 5 min 

7.      Discard the supernatant, resuspend the pellet in 10 ml Growth medium and plate in 10 cm dish (Plate         approximately 1x 10⁶ cells per 10 cm dish)

8.       Change the medium after 48 hours 

       (* If you don’t see cell attached after 48 hours then to get rid of debris take off sups, replenish

       medium. let sups stand for 5 min, take off & discard cleared supernatant. Re-plate pellet 

       (same dish). Potentially repeat the procedure the following day. )


#Practical Biology #LIFE technologies