TITLE: Single cell digestion of tumor tissue
AUTHORS: Shubhangi     Agarwal, donna.peehl  , Renuka   Sriram

[DESCRIPTION]
The purpose of this protocol is to provide details on how to obtain a single-cell suspension from a patient-derived xenograft tumor. 

[STEPS]
SECTION: Preparation of Medium
1. Prepare digestion medium 
DMEM supplemented with
a. 10 % volume  FBS,
b.  200 units/ml of type I collagenase, 
c. 1 units/ml DNase I, 
d. 2 Micromolar (µM) Y-27632 and 
e. 100 ug/ml Gentamicin

SECTION: Preparation of Medium
2. Prepare storage medium
DMEM supplemented with
a. 10 % volume  FBS, and 
b. 100 ug/ml Gentamicin
SECTION: Harvest Tumor Tissue
3. Prepare an ice bucket with ice and 50 ml falcon tubes with 35-45 mL HBS. 

SECTION: Harvest Tumor Tissue
4. Euthanize the tumor-bearing mouse and extract the tumor. 
SECTION: Harvest Tumor Tissue
5. Blot the tumor on a Kim-wipe to get rid of excess blood.

SECTION: Harvest Tumor Tissue
6. Weigh the tumor and note the weight. 
SECTION: Harvest Tumor Tissue
7.  Place the fresh tumor tissue in a falcon tube containing 35-45 mL HBS. 

SECTION: Tissue digestion process (performed in BSL2 hood)
10. Transfer the tissue from the falcon tube into the petri dish containing HBS. 
SECTION: Tissue digestion process (performed in BSL2 hood)
9. 
Prepare a petri dish containing fresh HBS and place it on ice. 
SECTION: Tissue digestion process (performed in BSL2 hood)
11. Wash tumor multiple times with 10 mL HBS and remove excess HBS. Repeat this process two more times. 

SECTION: Tissue digestion process (performed in BSL2 hood)
12. Mince the tumor with scissors or scalpels in the petri dish. 
 

SECTION: Tissue digestion process (performed in BSL2 hood)
13. Add digestion medium to the petri dish containing tumor tissue and add enough volume to thoroughly cover the entire tumor. 
SECTION: Tissue digestion process (performed in BSL2 hood)
14. Place the petri dish in an incubator at 37 °C over 120 min- 240 min hrs.  
 


SECTION: Tissue digestion process (performed in BSL2 hood)
15. 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. 
SECTION: Tissue digestion process (performed in BSL2 hood)
17. Discard the supernatant and incubate the digested tissue with 5 mL  Red Cell Lysis Buffer for 3 min. 

SECTION: Tissue digestion process (performed in BSL2 hood)
20. 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. 
SECTION: Tissue digestion process (performed in BSL2 hood)
21. 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.  
SECTION: Tissue digestion process (performed in BSL2 hood)
22. Centrifuge the solution containing tumor cells at 300 x g, 4 °C for 5 mins. 


SECTION: Tissue digestion process (performed in BSL2 hood)
24. Perform Trypan Blue dye exclusion test and note the live and total cell count. 
SECTION: Harvest Tumor Tissue
8. Transfer the falcon tube into a BSL2 hood. 
SECTION: Tissue digestion process (performed in BSL2 hood)
23. Discard the supernatant and resuspend the cell pellet in fresh DMEM prepared in step 2.  
SECTION: Tissue digestion process (performed in BSL2 hood)
25. Prepare cryogenic vials with 900 µL  of 1 M  live cells and 100 µL  of DMSO per cryovial. 

SECTION: Tissue digestion process (performed in BSL2 hood)
26. Place the prepared cryovials in a freezing container and place the container in -80 °C  freezer for 24 hours.  

SECTION: Tissue digestion process (performed in BSL2 hood)
27. After 24 hours, transfer the cryovials into liquid nitrogen storage for long-term storage. 
SECTION: Tissue digestion process (performed in BSL2 hood)
19. Discard the supernatant and resuspend the cell pellet in 50 mL fresh DMEM prepared in step 2.  

SECTION: Tissue digestion process (performed in BSL2 hood)
18. Centrifuge the above solution at 300 x g, 4 °C for 5 mins. 
SECTION: Tissue digestion process (performed in BSL2 hood)
16. Transfer the above solution into a 50 ml falcon tube and centrifuge the above solution at 300 x g, 4 °C for 5 mins.