Dissociation of Human and Mouse Tumor Tissue Samples for Single-cell RNA Sequencing

Summary

This protocol outlines the procedure for rapidly dissociating human and mouse tumor samples for single-cell RNA sequencing.

Abstract

Human tumor samples hold a plethora of information about their microenvironment and immune repertoire. Effective dissociation of human tissue samples into viable cell suspensions is a required input for the single-cell RNA sequencing (scRNAseq) pipeline. Unlike bulk RNA sequencing approaches, scRNAseq enables us to infer the transcriptional heterogeneity in tumor specimens at the single-cell level. Incorporating this approach in recent years has led to many discoveries, such as identifying immune and tumor cellular states and programs associated with clinical responses to immunotherapies and other types of treatments. Moreover, single-cell technologies applied to dissociated tissues can be used to identify accessible chromatin regions T and B cell receptor repertoire, and the expression of proteins, using DNA barcoded antibodies (CITEseq).

The viability and quality of the dissociated sample are critical variables when using these technologies, as these can dramatically affect the cross-contamination of single cells with ambient RNA, the quality of the data, and interpretation. Moreover, long dissociation protocols can lead to the elimination of sensitive cell populations and the upregulation of a stress response gene signature. To overcome these limitations, we devised a rapid universal dissociation protocol, which has been validated on multiple types of human and murine tumors. The process begins with mechanical and enzymatic dissociation, followed by filtration, red blood lysis, and live dead enrichment, suitable for samples with a low input of cells (e.g., needle core biopsies). This protocol ensures a clean and viable single-cell suspension paramount to the successful generation of Gel Bead-In Emulsions (GEMs), barcoding, and sequencing.

Introduction

Although many advancements in cancer research have led to the development and FDA approval of agents targeting inhibitory receptors expressed on immune and tumor cells, known as checkpoint blockade inhibitors, therapy resistance and identifying mechanisms that drive patient response remain challenging¹. The complex challenge of characterizing tumor heterogeneity on its molecular diversity and the intricate interplay between tumor cells and the immune microenvironment necessitates new approaches to dissect this complex ecosystem at the single-cell resolution. Understanding the molecular intricacies within tumors and their microenvironments is pivotal for advancing therapeutic strategies and deciphering the complex biology underlying cancer progression². Single-cell RNA sequencing (scRNA-seq) has become increasingly popular because it provides high-resolution analysis of individual cells within complex tumor samples^3,4.

Tumor heterogeneity, encompassing genetic, epigenetic, and phenotypic diversities, poses a challenge in uncovering the intricacies of cancer biology⁵. The conventional methods of bulk RNA sequencing tend to obscure the unique expression profiles and phenotypes of heterogeneous cell populations present within tumors, as these methods average signals across entire cell populations⁴. In contrast, scRNA-seq allows the dissection of individual cell types, revealing diverse gene expression, repression patterns, and cellular states otherwise overlooked^4,6. The premise of scRNA-seq lies in its capacity to decode individual cells' genetic and molecular signatures, holding immense promise in discovering new cancer therapeutics⁷.

By deciphering the gene expression profiles of tumor cells and the surrounding stromal and immune cells, researchers can identify novel therapeutic targets and develop precision genetic medicine strategies tailored to individual patients⁶. Furthermore, dissecting the immune repertoire within the tumor microenvironment provides crucial insights into the interplay between tumor cells and immune effectors, paving the way for immunotherapeutic interventions³. Despite advances in using scRNAseq on clinical samples, several challenges during the processing steps can harm the cell's RNA integrity, viability, and quality of the data generated. Moreover, processing tissue with low cell viability can increase ambient RNA levels in the droplets generated during the encapsulation of individual cells, resulting in cross-contamination and incorrect annotation of cell types, while long dissociation protocols performed at 37 °C can lead to the upregulation of a stress response gene signature in multiple cell types^8,9,10.

The stepwise procedure (Figure 1A) outlined in this protocol commences with rapid mechanical and enzymatic dissociation of tumors, followed by filtration and the removal of dead cells, depending on the viability of each tumor sample. At present, this procedure has been verified in melanoma¹¹, colorectal¹², head and neck squamous cell carcinoma¹³, pancreatic and lung (unpublished data) tumor samples. These techniques ensure the generation of high-quality viable cell suspensions crucial for downstream analyses¹⁴. Notably, removing red blood cells, devoid of synthesized RNA, becomes imperative to purify the sample¹⁵. Subsequent evaluation of cell counts and the elimination of dead cells serve as a prerequisite for successful 10x Genomics Gel bead-in Emulsion (GEM) generation, barcoding, and increase the recovery of transcripts and sequenced cells without jeopardizing the exclusion of sensitive populations (e.g., neutrophils and epithelial cells)¹⁶. These steps are fundamental in unlocking the potential of single-cell resolution analysis within tumor samples^9,10, uncovering novel gene expression profiles and immune signatures necessary for driving innovative therapeutic interventions, and identifying new tumor vulnerabilities.

Protocol

This study complied with all institutional guidelines regarding human tissue sampling. Informed consent was received from patients, and identifiable sample data was anonymized. Samples are collected in the operation room, placed in a solution of RPMI, saline, or PBS, and confirmed cancerous via the pathology department before use in research. All steps, except when indicated, should be carried out at 4 °C or on ice. Work inside a biosafety hood when processing human tissue. See the Table of Materials for details on all materials, reagents, and instruments used in this protocol.

1. Obtaining the sample

1. Obtain a solid tumor sample in a tube of media and place it on ice.
   1. Instruct the operating room staff to transport the sample in a tube containing enough RPMI, saline, or PBS to completely submerge the tissue to avoid sample dehydration.
      NOTE: Ensuring the sample is not desiccated is important, as this will decrease viability.
2. Cool down all centrifuges to 4 ˚C and bring the thermal mixer to 37 ˚C.
3. Take out prepared human or mouse tumor dissociation enzymes (Table of Materials), thaw on the 37 ˚C thermal mixer, and move to ice once thawed.
4. Retrieve a 20 mL aliquot of RPMI.
5. Record relevant sample information (e.g., Patient ID, de-identifier, sample type).
6. Transfer the sample to a 60 mm tissue culture dish (placed on ice) by inverting the tube several times and pouring the contents into the dish to ensure all sample material is transferred. If the sample was not delivered in media upon arrival, add fresh RPMI media to the dish.
   1. If the sample is fragmented, spin the tube containing the sample at 450 × g, 4 ˚C for 5 min, discard the supernatant, resuspend the sample in 420 µL of RPMI, and move to section 2.

2. Mechanical and enzymatic digestion

1. Pipette 420 µL of media from the 60 mm sample dish to a 1.5 mL microcentrifuge tube. Attempt to include any sample fragments suspended in the media.
2. Transfer the sample tissue from the 60 mm dish to the 1.5 mL tube containing media using tweezers. Cut up the sample with clean scissors inside the tube so the pieces are maximum 2-3 mm in diameter.
3. Add digestion enzymes, prepared according to manufacturer specifications (Table of Materials), to the sample in the following volumes: 42 µL of Enzyme H (nonspecific proteases) for human samples, Enzyme D (nonspecific proteases) for murine samples, 21 µL of enzyme R (nonspecific proteases), and 5 µL of enzyme A (nuclease).
4. Add another 420 µL of media from the dish or up to the 1 mL mark on the tube. Do not exceed 420 µL of media.
5. Vortex the tube for 5 s and place it in a thermal mixer positioned vertically on its side (Figure 1B). Incubate at 37 ˚C, 450 rpm, for 15 min.
   NOTE: During incubation, take out all 10x Genomics materials needed for GEM generation, as these require 30 min to equilibrate to room temperature

3. Sample filtering

1. Place a 50 µm cell filter on top of a new 15 mL conical tube and prime the filter by passing 1 mL of fresh RPMI media through it into the tube.
2. After digestion, transfer the sample to the 15 mL tube using a 1,000 µL low-retention wide pipette tip. Wash the 1.5 mL tube with 1 mL of fresh media and transfer the media to the filter to ensure all sample material is passed through the filter.
3. Obtain a 1 mL plastic syringe and take out just the plunger (discard the remainder of the syringe). Using the back of the syringe plunger, grind and push the sample on the filter in a twisting motion.
4. Wash the filter with 5 mL of media and any remaining media from the dish that contained the sample (section 1) to collect any cells that might have detached from the tissue.
5. Place a 30 µm cell filter on top of a new 15 mL conical tube, prime the filter with 1 mL of media, and transfer the contents of the tube containing the dissociated sample through the filter.
   NOTE: This step will remove large debris that might clog or cause a wetting failure when running the sample using the 10x Genomics microfluidic chips.

4. Red blood cell lysis

1. Centrifuge the sample at 450 × g, 4 ˚C for 5 min. Aspirate the media, being careful not to disturb the pellet.
2. Resuspend in ACK Lysis buffer to remove red blood cells (RBCs) and record the volume used. The volume will depend on pellet size and the extent of RBCs in the pellet.
   NOTE: Resuspended ACK-sample solution should be colorless when enough ACK Lysis buffer has been added, as this will clear the red blood cells from the sample.
3. Centrifuge the sample tube at 450 × g, 4 ˚C for 5 min, and aspirate the supernatant.
4. Repeat steps 4.2-4.3 as needed until the sample pellet has no visible red color after centrifugation (Figure 2). Record any extra volumes of ACK Lysis buffer used in this step.
5. Resuspend the sample pellet in fresh media in preparation for counting.

5. Cell counting

1. Place 10 µL of trypan blue in a 1.5 mL tube. Gently mix the dissociated cells, take 10 µL of sample, and mix (1:1) with the trypan blue. Load 10 µL on each side of a hemocytometer and count the cells.
2. Record cell concentration, total live cell count, viability percentage, and final media volume after count.
3. Ensure cell count is between 700 and 1,200 cells/µL (7 × 10⁵- 1.2 × 10⁶/mL) for an optimal 10x Genomics run.
   1. If the sample is too concentrated, dilute it with media and recount it.
   2. If the sample is too diluted, centrifuge the sample at 450 × g, 4 ˚C for 5 min, and resuspend in a lesser volume of media.
   3. If cell viability is below 80%, perform a live-dead procedure to remove dead cells.
   4. If there are under one million cells, see section 6.
   5. If there are over one million cells and viability > 10%, see section 6.
   6. If there are over one million cells and viability < 10%, see section 7.
   7. If cell viability is above 80%, keep the processed cells on ice and proceed immediately to GEM generation according to the manufacturer's instructions.

6. Dead cell removal - Kit 1

NOTE: Work inside a biosafety hood when using the dead cell removal reagents, as these can easily get contaminated.

1. Centrifuge the sample at 450 × g, 4 ˚C for 5 min.
2. Prepare isolation media in a 15 mL conical tube and keep it at room temperature: 9.8 mL of PBS, 10 µL of CaCl₂, and 200 µL of heat-inactivated FCS (added in a Biosafety hood).
3. After centrifugation, aspirate the media from the pellet and resuspend in 1 mL of the isolation media.
4. Centrifuge the tube again at 450 × g, 4 ˚C for 5 min. Aspirate the supernatant and resuspend the pellet in 100 µL of isolation media.
5. Transfer 100 µL of sample in isolation media into one well of a 0.2 mL PCR strip-tube.
6. In a biosafety hood, add 10 µL of Annexin V Cocktail and 10 µL of Biotin Selection Cocktail to the well containing 100 µL of the sample in isolation media. Pipette mix the solution and allow it to sit at room temperature for 4 min.
7. After 4 min, vortex the dextran-coated magnetic particles for 30 s. Add 20 µL of the beads and 60 µL of isolation media to the sample (a total volume of 200 µL). If viability is lower than 40%, scale the amount of beads up to 40 µL while adjusting the volume of isolation media (up to 40 µL), keeping a total volume of 200 µL. Pipette mix and allow the solution to sit at room temperature for 3 min.
8. Place the strip tube onto a 10x Genomics Magnetic Separator (on the high setting) for 5 min at room temperature.
9. Transfer the liquid from the strip tube without disturbing the beads into a new 1.5 mL lo-bind microcentrifuge tube. Do not remove the strip-tube from the magnet.
10. Centrifuge the 1.5 mL tube containing the sample at 450 × g, 4 ˚C for 5 min. Aspirate the supernatant and resuspend the pellet in an appropriate volume of RPMI media based on the pellet size and count cells, as described in section 5. Repeat the dead cell removal procedure if viability remains below 80% (Figure 3 and Figure 4).
    NOTE: Do not resuspend cells in isolation media for downstream GEM generation, as the calcium chloride will interfere with the encapsulation process and the reverse transcription (RT) step.
11. Keep processed cells on ice if cell viability is above 80% and proceed immediately to GEM generation according to the manufacturer's instructions.

7. Dead cell removal - Kit 2

NOTE: Work inside a biosafety hood when using the kit reagents.

1. Place the microbeads and the 20x Binding Buffer stock solution inside a biosafety hood.
2. Centrifuge the sample suspension at 450 × g, 4 ˚C for 5 min.
3. Prepare 1x buffer solution under sterile conditions using 1 mL of the 20x Binding Buffer stock solution pipetted into 19 mL of DNAse-, RNAse-free distilled water.
4. Once the centrifugation is completed, aspirate the supernatant, add the microbeads, gently mix the sample, and incubate at room temperature for 15 min.
   1. When the cell count is 10⁷ or less, add 100 µL of the microbeads.
   2. For counts higher than 10⁷ total cells, adjust the microbeads for a final concentration of 100 µL per 10⁷ live cells.
5. During incubation, obtain one MACS positive selection column, MACS multistand, and the corresponding magnetic separator. Ensure the separator is leveled on the multistand and no other metal or magnetic object is near the stand, as the magnetic force is powerful and could affect dead cell removal efficiency. Place the column snugly into a separator holder with wings facing out.
6. After sample incubation, if the total resuspension volume is less than 500 µL, add 1x Binding Buffer until the total volume is 500 µL.
7. Place a 15 mL conical tube below the column and prime the column with 3 mL of 1x Binding Buffer.
8. Replace the conical tube with a new 15 mL conical for cell sample collection and add the sample to the column, allowing it to flow completely through the column.
9. Wash the column 4 x 3 mL of 1x Binding Buffer, only adding one wash at a time after the previous wash entirely flowed through the column.
10. After the fourth wash, centrifuge the conical tube containing the isolated cells for 5 min at 450 × g, 4 ˚C. Discard the column and the remaining 1x Binding Buffer.
11. Resuspend the sample solution in the appropriate amount of RPMI media based on pellet size and count the isolated cells using the method described in section 5 (Figure 3 and Figure 5).
    1. If viability remains below 80%, and the total number of cells is 10⁶ or less, repeat section 6.
    2. If cell viability is above 80%, keep processed cells on ice and proceed immediately to GEM generation according to the manufacturer's instructions.

Results

A melanoma biopsy suspended in RPMI was obtained and immediately placed on ice. The sample was transferred into a 1.5 mL microcentrifuge tube containing 420 µL of RPMI and digestion enzymes, minced into small pieces using scissors, and subjected to subsequent enzymatic digestion for 15 min in a 37 ˚C vertically positioned thermal mixer (Figure 1). Following digestion, the sample was filtered through a 50 µm sterile disposable filter, and the filter was washed with fresh RPMI t...

Discussion

This protocol describes human or murine tumor samples dissociation into a single-cell suspension for scRNA sequencing using the 10x Genomics microfluidic system. In processing tissues that often come from rare cancers or are part of ongoing clinical trials or long experiments for murine tumors, the sample must be optimally and carefully preserved between all steps. All steps should be carried out rapidly on ice or at 4 ˚C to keep the native cellular RNA profiles and prevent degradation, as working at room temperatur...

Materials

Name	Company	Catalog Number	Comments
1x PBS	Corning	21-040-CV
10 mL serological pipette	Corning	357551
1000 μL low-retention pipette tips	Rainin	30389213
1000 μL low-retention wide pipette tips	Rainin	30389218
1000 μL pipette tips	Rainin	30389212
10x Magnetic Separator	10x Genomics	120250
15 mL conical tubes	Corning	430052
2 mL aspirating pipette	Corning	357558
20 μL low-retention pipette tips	Rainin	30389226
20 μL pipette tips	Rainin	30389225
200 μL low-retention pipette tips	Rainin	30389240
200 μL pipette tips	Rainin	30389239
50 mL Polypropylene Conical Tube	Falcon	352098
60 x 15 mm Tissue Culture Dish	Falcon	353004
ACK Lysing Buffer	Gibco	A10492-1
BD 1 mL syringe	Becton, Dickinson and Company	3090659
Bright-Line Hemocytometer	Hausser Scientific	551660
Calcium Chloride Solution	Sigma Aldrich	2115-100ML
Cell Ranger	10x Genomics	N/A	https://www.10xgenomics.com/support/software/cell-ranger/latest
Cell counting slides for TC20 cell counter	Bio-Rad	1450015
CellTrics 30μm sterile disposable filters	Sysmex	04-004-2326
CellTrics 50μm sterile disposable filters	Sysmex	04-004-2327
Dead Cell removal Kit	Miltenyi Biotec	130-090-101	Store at -20 °C; kit 2
Dissecting Forceps, Fine Tip	VWR	82027-386
DNA LoBind Microcentrifuge tubes, 1.5 mL	Eppendorf	22431021
EasySepTM Dead Cell Removal (Annexin V) Kit	STEMCELL Technologies	17899	Store at 4 °C; Kit 1
Eppendorf centrifuge 5425R	Eppendorf	5406000640
Eppendorf centrifuge 5910R	Eppendorf	2231000657
Eppendorf Easypet 3 Electronic Pipette controller	Eppendorf	EP4430000018
Eppendorf Thermomixer F1.5 Model 5384	Eppendorf	EP5384000012
FBS	Gibco	26140-079	Store at 4 °C, use in a Biological hood
German Stainless Scissors	Fine Science Tools	14568-12
Leica Dmi1 Microscope	Leica Microsystems	454793
LS Column	Miltenyi Biotec	130-042-401
MACS Multistand	Miltenyi Biotec	130-042-303
Pipet-Lite LTS Pipette L-1000XLS+	Rainin	17014382
Pipet-Lite LTS Pipette L-200XLS+	Rainin	17014391
Pipet-Lite LTS Pipette L-20XLS+	Rainin	17014392
Quadro MACS Magnet	Miltenyi Biotec	130-091-051
RPMI Medium 1640 (1x)	Gibco	21870-076	Store at 4 °C
TempAssure 0.2 mL PCR 8-Tube strips	USA Scientific	1402-4700
Trypan blue stain 0.4%	Thermo Fisher Scientific	T10282
Tumor Dissociation Kit, Human	Miltenyi Biotec	130-095-929	Store at -20 °C, prepare enzymes according to kit instructions: Reconstitute lyophilized Enzyme H vial in 3 mL of RPMI 1640 Reconstitute lyophilized Enzyme R vial in 2.7 mL of RPMI 1640 Reconstitute lyophilized Enzyme A vial in 1 mL of Buffer A supplied with the kit.
Tumor Dissociation Kit, Mouse	Miltenyi Biotec	130-096-730	Store at -20 °C, prepare enzymes according to kit instructions: Reconstitute lyophilized Enzyme D vial in 3 mL of RPMI 1640 Reconstitute lyophilized Enzyme R vial in 2.7 mL of RPMI 1640 Reconstitute lyophilized Enzyme A vial in 1 mL of Buffer A supplied with the kit.
UltraPure Distilled Water	Invitrogen	10977-015	Store at 4 °C