Single cell digestion of tumor tissue

Prepare digestion medium

DMEM supplemented with

a. 10% volume FBS,

b. 200units/ml of type I collagenase,

c. 1units/ml DNase I,

d. 2micromolar (µM) Y-27632 and

e. 100ug/ml Gentamicin

Prepare storage medium

DMEM supplemented with

a. 10% volume FBS, and

b. 100ug/ml Gentamicin

Prepare an ice bucket with ice and 50 ml falcon tubes with 35-45mL HBS.

Euthanize the tumor-bearing mouse and extract the tumor.

Blot the tumor on a Kim-wipe to get rid of excess blood.

Weigh the tumor and note the weight.

Place the fresh tumor tissue in a falcon tube containing 35-45mL HBS.

Transfer the falcon tube into a BSL2 hood.

Prepare a petri dish containing fresh HBS and place it on ice.

Transfer the tissue from the falcon tube into the petri dish containing HBS.

Wash tumor multiple times with 10mL HBS and remove excess HBS. Repeat this process two more times.

Mince the tumor with scissors or scalpels in the petri dish.

Add digestion medium to the petri dish containing tumor tissue and add enough volume to thoroughly cover the entire tumor.

Place the petri dish in an incubator at 37°C over 2h 0m 0s- 4h 0m 0s hrs.

Periodically assess the digestion under the microscope and stop once small clusters or clumps of cells are released from the tissue and before complete digestion to single cells occurs.

Transfer the above solution into a 50 ml falcon tube and centrifuge the above solution at 300x g,4°C,0h 0m 0s for 5 mins.

Discard the supernatant and incubate the digested tissue with 5mL Red Cell Lysis Buffer for 0h 3m 0s.

Centrifuge the above solution at 300x g,4°C,0h 0m 0s for 5 mins.

Discard the supernatant and resuspend the cell pellet in 50mL fresh DMEM prepared in step 2.

Pass the above solution containing tumor cells through a 70 um filter and collect the solution containing tumor cells that passed through the filter in a fresh 50 ml falcon tube.

Pass the above solution containing tumor cells through a 40 um filter and collect the cells that passed through the filter in a fresh 50 ml falcon tube.

Centrifuge the solution containing tumor cells at 300x g,4°C,0h 0m 0s for 5 mins.

Discard the supernatant and resuspend the cell pellet in fresh DMEM prepared in step 2.

Perform Trypan Blue dye exclusion test and note the live and total cell count.

Prepare cryogenic vials with 900µL of 1M live cells and 100µL of DMSO per cryovial.

Place the prepared cryovials in a freezing container and place the container in -80°C freezer for 24 hours.

After 24 hours, transfer the cryovials into liquid nitrogen storage for long-term storage.