Nov 30, 2021
Single cell digestion of tumor tissue
- Shubhangi Agarwal1,
- donna Peehl1,
- Renuka Sriram1
- 1University of California, San Francisco
Protocol Citation: Shubhangi Agarwal, donna Peehl, Renuka Sriram 2021. Single cell digestion of tumor tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.bvrun56w
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: June 12, 2021
Last Modified: November 30, 2021
Protocol Integer ID: 50708
Keywords: Single cell digestion, single cell digestion of tumor tissue, single cell digestion, derived xenograft tumor, xenograft tumor, tumor tissue, cell suspension, derived xenograft, tissue
Abstract
The purpose of this protocol is to provide details on how to obtain a single-cell suspension from a patient-derived xenograft tumor.
Materials
| Materials | Source | Catalog Number | |
| DMEM | Gibco | 11965092 | |
| Fetal bovine serum (FBS) | Global Life Sciences Solutions | SH3039603 | |
| Type I collagenase | Gibco | 17100017 | |
| DNase I | Thermo Scientific | 89836 | |
| ROCK Inhibitor (Y-27632) | Sigma Aldrich | SCM075 | |
| Gentamicin | Gibco | 15750060 | |
| HEPES-buffered saline (HBS) | Gibco | 15630080 | |
| Red Cell Lysis Buffer | Roche | 11814389001 | |
| Cell Freezing Medium-DMSO 1× | Sigma Aldrich | C6164 | |
| Scissors | Kent Scientific | INS600393 | |
| Scalpels with blade no.10 | Fisher Scientific | 12-460-451 | |
| Petri dishes | Millipore Sigma | P5481 | |
| 15 mL conical tubes | Corning | 430055 | |
| 50 mL conical tubes | Corning | 430829 | |
| 70 μm cell strainers | Corning | 431751 | |
| 40 μm cell strainers | Corning | 431750 | |
| Pipettes tips (1000 ul) | Millipore Sigma | AXYT1000B | |
| Trypan Blue dye | Sigma Aldrich | 93595 | |
| Corning CoolCell | Corning | 432000 | |
| Cryogenic vial | Nalgene | V5007 |
Troubleshooting
Preparation of Medium
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
Prepare storage medium
DMEM supplemented with
a. 10 % volume FBS, and
b. 100 ug/ml Gentamicin
Harvest Tumor Tissue
Prepare an ice bucket with ice and 50 ml falcon tubes with 35-45 mL 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 35-45 mL HBS.
Transfer the falcon tube into a BSL2 hood.
Tissue digestion process (performed in BSL2 hood)
6h 3m
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 10 mL HBS and remove excess HBS. Repeat this process two more times.
Mince the tumor with scissors or scalpels in the petri dish.
Note
For larger tissue (larger than 1 cm3), use multiple rounds of mincing
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 95% air/5% CO2 over 02:00:00 - 04:00:00 hrs.
Note
Vigorously mix the tissue every 10-15 minutes using a pipette to aid dissociation.
6h
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 300 x g, 4°C for 5 mins.
Discard the supernatant and incubate the digested tissue with 5 mL Red Cell Lysis Buffer for 00:03:00 mins .
3m
Centrifuge the above solution at 300 x g, 4°C for 5 mins.
Discard the supernatant and resuspend the cell pellet in 50 mL 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 300 x g, 4°C 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 900 µL of 1 M live cells and 100 µL 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.