Nov 30, 2021

Public workspaceSingle cell digestion of tumor tissue

DOI
dx.doi.org/10.17504/protocols.io.bvrun56w
  • 1University of California, San Francisco
Shubhangi Agarwal
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.bvrun56w
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 11, 2021
Last Modified: November 30, 2021
Protocol Integer ID: 50708
Keywords: Single cell digestion
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

MaterialsSourceCatalog Number
DMEMGibco11965092
Fetal bovine serum (FBS)Global Life Sciences SolutionsSH3039603
Type I collagenase Gibco17100017
DNase I Thermo Scientific89836
ROCK Inhibitor (Y-27632) Sigma AldrichSCM075
GentamicinGibco15750060
HEPES-buffered saline (HBS)Gibco15630080
Red Cell Lysis BufferRoche11814389001
Cell Freezing Medium-DMSO 1×Sigma AldrichC6164
ScissorsKent ScientificINS600393
Scalpels with blade no.10 Fisher Scientific12-460-451
Petri dishesMillipore SigmaP5481
15 mL conical tubesCorning430055
50 mL conical tubesCorning 430829
70 μm cell strainersCorning431751
40 μm cell strainers Corning431750
Pipettes tips (1000 ul)Millipore SigmaAXYT1000B
Trypan Blue dyeSigma Aldrich93595
Corning CoolCellCorning432000
Cryogenic vialNalgeneV5007

Preparation of Medium
Preparation of Medium
Prepare digestion medium
DMEM supplemented with
a. Concentration10 % volume FBS,
b. Concentration200 units/ml of type I collagenase,
c. Concentration1 units/ml DNase I,
d. Concentration2 micromolar (µM) Y-27632 and
e. Concentration100 ug/ml Gentamicin

Prepare storage medium
DMEM supplemented with
a. Concentration10 % volume FBS, and
b. Concentration100 ug/ml Gentamicin
Harvest Tumor Tissue
Harvest Tumor Tissue
Prepare an ice bucket with ice and 50 ml falcon tubes with Amount35-45 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 Amount35-45 mL HBS.

Transfer the falcon tube into a BSL2 hood.
Tissue digestion process (performed in BSL2 hood)
Tissue digestion process (performed in BSL2 hood)
6h 3m
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 Amount10 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 Temperature37 °C 95% air/5% CO2 over Duration02:00:00 - Duration04:00:00 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 Centrifigation300 x g, 4°C for 5 mins.
Discard the supernatant and incubate the digested tissue with Amount5 mL Red Cell Lysis Buffer for Duration00:03:00 mins .

3m
Centrifuge the above solution at Centrifigation300 x g, 4°C for 5 mins.
Discard the supernatant and resuspend the cell pellet in Amount50 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 Centrifigation300 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 Amount900 µL of Amount1 M live cells and Amount100 µL of DMSO per cryovial.

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

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