Protocol for isolating single cells from human pancreatic cancer tissues and analyzing major immune cell populations using flow cytometry

Summary

Here, we present a protocol for collecting, dissociating, isolating, staining, and analyzing immune cells from pancreatic cancer tissues for flow cytometry. The isolated cells can also be used for single-cell RNA sequencing and other related procedures.

For complete details on the use and execution of this protocol, please refer to Zhang et al. (2023).¹

Graphical abstract

Highlights

• •Isolation of high-viability single cells from human pancreatic cancer tissue
• •Enrich immune cells using Percoll
• •Purify immune cells to make them suitable for FACS and scRNA-seq
• •Identification of major immune cell populations in pancreatic cancer using FACS

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Here, we present a protocol for collecting, dissociating, isolating, staining, and analyzing immune cells from pancreatic cancer tissues for flow cytometry. The isolated cells can also be used for single-cell RNA sequencing and other related procedures.

Before you begin

Institutional permissions

The following plan outlines the steps for pancreatic cancer sample collection, tissue mechanical dissociation, single-cell dissociation, and flow cytometry staining. Before the experiment, it is necessary to prepare key equipment and reagents to achieve a higher cell yield and viability. All experiments were conducted in accordance with the approval of the Ethics Committee of the First Affiliated Hospital, School of Medicine, Zhejiang University, and the collection of tumor samples was performed while ensuring that it did not interfere with the pathological diagnosis. Additionally, we have also applied this protocol in liver cancer and other solid tumors, as well as in murine solid tumor models.

Pancreatic cancer tumor tissue collection

The pancreatic cancer samples were obtained from pancreaticoduodenectomy specimens. The tissue should be collected as soon as possible while avoiding the area of thermal injury caused by the ultrasonic scalpel after the tumor tissue is removed. The tissues were immersed in tissue preservation solution and transported on ice to the laboratory for further processing, ensuring an adequate quantity and high vitality of cells. The tissue collection procedure was conducted under the guidance of a pathology expert.

Reagent and equipment preparation

• 1.Prepare dissecting instruments, including scissors, surgical knives, and forceps, and sterilize them using appropriate methods.
• 2.Set the incubator to 37°C and the centrifuge to 4°C. Prepare 50 mL of PBS containing 2% FBS and pre-chill it on ice.
• 3.Prepare tissue digestion solution by dissolving 6 mg of Type IV collagenase, 6 mg Dispase II, and 0.1 mg DNase in 10 mL of RPMI medium containing 2% FBS and then sterilize the solution by filtering it through a 0.22 μm pore size filter. Pre-warm the solution to room temperature (15°C–25°C) before use.

CRITICAL: Due to the fact that tissue dissociation at 37°C takes approximately 1 h, it is necessary to perform sterile processing of the digestive solution in order to minimize microbial contamination and enhance cell vitality.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Anti-Human CD45 Antibody (clone: HI30) 1:100	BD	Cat#: 563716
Anti-human CD16 Antibody (clone: B7311) 1:100	BD	Cat#: 561313
Anti-human CD11b Antibody (clone: 1CRF44) 1:100	BD	Cat#: 562721
Anti-human CD11c Antibody (clone: B-ly6) 1:100	BD	Cat#: 563026
Anti-human CD4 Antibody (clone: L200) 1:100	BD	Cat#: 563094
Anti-human CD8 Antibody (clone: RPA-T8) 1:100	BD	Cat#: 557750
Anti-human TCR-γδ Antibody (clone: B1) 1:100	BD	Cat#: 740415
Anti-human CD3 Antibody (clone: UCHT1) 1:100	BD	Cat#: 555916
Human BD Fc Block 2.5 μg/mL	BD	Cat#: 564220
Anti-human CD68 Antibody (clone: Y1/82A)	BioLegend	Cat#: 333808
Anti-human CD56 Antibody (clone: 5.1H11)	BioLegend	Cat#: 362510
Anti-human HLA-DR Antibody (clone: L243)	BioLegend	Cat#: 307650
Anti-human CD19 Antibody (clone: HIB19)	BioLegend	Cat#: 302226
Anti-human CD14 Antibody (clone: HCD14)	BioLegend	Cat#: 325604
Chemicals, peptides, and recombinant proteins
Dispase II	Gibco	Cat#: 17105041
Collagenase, Type IV	Gibco	Cat#: 17104019
DNase I	Merck	Cat#: DN25
Percoll	Merck	Cat#: P4937-500ML
RPMI Medium 1640	GE HealthCare	Cat# SH30027.0
1XPBS	HyClone	Cat#SH30256.01
10XPBS	HyClone	Cat#SH30256.01
RBC lysis buffer	BD	Cat# 555899
MACS Tissue Storage Solution	Miltenyi	Cat#130-100-008
Percoll 1 L	GE	Cat#17-0891-09
Cytofix Cytoperm kit	BD	Cat#554714
FBS	Gibco	Cat#16000-044
Other
100 mm × 20 mm tissue-culture-treated culture dish	Corning	Cat#430167
15-mL centrifuge tube	NEST	Cat#601052
50-mL centrifuge tube	NEST	Cat#602052
40-mm nylon cell strainer/70-mm nylon cell strainer	Falcon	Cat#352340
1000-mL pipette tips	Rainin	Cat#30389218
200-mL pipette tips	Rainin	Cat#30389241
Scalpel	APPLYGEN	Cat#TB6298-1
0.22-μm syringe filter	Millipore	Cat#SLGP033RB
Sterile Pasteur pipette	ZDAN	Cat#ZD-H03
Constant-temperature shake	FAITHFUL	Cat#FS-50B
Centrifuge with a rotor	Eppendorf	Cat#5910ri

Materials and equipment

Digestion dissociation solution

Reagent	Final concentration	Amount
Dispase II	0.6 mg/mL	6 mg
Collagenase, Type IV	0.6 mg/mL	6 mg
DNase I	0.01 mg/mL	0.1 mg
FBS	2%	1 mL
RPMI Medium 1640		9 mL
Total		10 mL

Note: Store the buffer at 4°C for up to 1 day or −20°C for 1 month

Wash buffer I

Reagent	Final concentration	Amount
FBS	2%	1 mL
RPMI Medium 1640		49 mL
Total		50 mL

Note: Store the buffer at 4°C for up to 1 day or −20°C for 1 month

Wash buffer II

Reagent	Final concentration	Amount
FBS	2%	1 mL
1XPBS		49 mL
Total		50 mL

Note: Store the buffer at 4°C for up to 1 day or −20°C for 1 month

100% Percoll solution

Reagent	Final concentration	Amount
10XPBS	1×	5 mL
Percoll		45 mL
Total		50 mL

Note: Store the buffer at 4°C for up to 1 week

36% Percoll solution

Reagent	Final concentration	Amount
1XPBS		32 mL
100% Percoll	36%	18 mL
Total		50 mL

Note: Store the buffer at 4°C for up to 1 week

1× Perm/wash solution

Reagent	Final concentration	Amount
10× Perm/wash solution	1×	1 mL
ddH2O		9 mL
Total		10 mL

Note: Store the buffer at 4°C for up to 1 week

Step-by-step method details

Collection of human pancreatic cancer tumor tissue

Timing: 15 min

This section describes the steps to obtain the required pancreatic cancer tissue samples.

• 1.After the pancreatic cancer is resected, under the guidance of a pathologist, use a sterile scalpel to excise a portion of the tumor tissue (Figure 1A). The appropriate tissue block volume is approximately 1 cm × 0.5 cm × 1 cm.
• 2.Rinse the surface stains with pre-chilled 1XPBS and remove burnt and fatty tissues.
• 3.Immerse the tissue in a 15 mL centrifuge tube containing approximately 5 mL of tissue preservation solution, place it on ice, and bring it back to the laboratory for further processing.

Figure 1.

Collection and cleaning of pancreatic cancer tissue

(A) Resected pancreatic cancer specimens and sampling sites.

(B) The harvested pancreatic cancer tissue is removed and cleaned of any attached fat, blood stains, and burned tissue, The volume of tissue blocks required for tissue digestion is approximately 1 cm × 0.5 cm × 1 cm.

Mechanical dissociation of pancreatic cancer tissue

Timing: 10 min

This section describes the steps of mechanical dissociation of pancreatic cancer tissue for further digestion.

• 4.Thoroughly wash blood stains and remove fatty connective tissue by using pre-cooled 2% FBS-PBS (wash buffer II). Transfer the tissue to a 100 mm × 20 mm tissue-culture-treated culture dish containing approximately 2 mL of digestion solution (Figure 1B).
• 5.Use forceps to secure the tumor tissue and cut the tumor tissue with a scalpel, ensuring that the size is smaller than 1 mm × 1 mm × 1 mm (Figure 2A).
• 6.Transfer the tissue-digestion mixture to a 50 mL centrifuge tube, and continue adding digestion solution until the volume reaches approximately 15 mL.

Figure 2.

The mechanical and enzymatic dissociation of pancreatic cancer tissue

(A) The pancreatic cancer tissue is minced into particles smaller than 1 mm × 1 mm × 1 mm.

(B) After digestion and dissociation, pancreatic cancer tissue presents as a translucent mixture with dispersed tissue particles.

Single-cell digestion and dissociation of pancreatic cancer tissues

Timing: 60 min

This section introduces the steps of enzymatic digestion of pancreatic cancer tissue samples after mechanical dissociation and obtaining preliminary cell suspensions.

• 7.
  Transfer the centrifuge tube containing the mixture of the tissue-digesting solution to the shaker.

  • a.
    Tilt or place it flat, and fix it in position.
  • b.
    Set the speed to 150 rpm, the temperature to 37°C, and the time to 1 h (Figure 2B).
• 8.
  Filter the supernatant using a 40 μm or 70 μm filter.

  • a.
    During this process, grind the tissue debris using a 1 mL syringe plunger.
  • b.
    Rinse the filter with cold 2% FBS-1640 (wash buffer II).

    Note: The volume of the filtered cell suspension is approximately 20 mL.

  • c.
    Store the filtrate on ice (Figure 3).

Figure 3.

Collection of cell suspension after digestion and dissociation

(A) Filter the digestion system using a 70 μm filter, meanwhile grind with a 1 mL syringe plunger.

(B) The final volume of the filtered cell suspension is approximately 20 mL.

(C) Tissue residue on the filter after grinding and filtration.

Cell purification and flow cytometry

Timing: 60 min–90 min

This section describes the purification of immune cells from cell suspensions and further flow cytometry staining steps.

• 9.Centrifuge the filtrate obtained in step 8 at 300 g for 5 min at 4°C.
• 10.
  Discard the supernatant after centrifugation.

  • a.
    Gently aspirate the 36% Percoll solution with a Pasteur pipette and carefully resuspend the pellet to a volume of approximately 10 mL.
  • b.
    Centrifuge at 300 g for 5 min at 4°C, with the acceleration set at 8 and deceleration at 1.

Note: After centrifugation, the sample will present a layered structure, with most of the fat and stromal cells in the upper layer, the middle layer consisting of Percoll, and the lower layer mainly composed of immune cells (Figure 4A).

CRITICAL: It is necessary to set a lower deceleration speed on the centrifuge to achieve a stable and distinct separation of different components.

• 11.
  Discard the supernatant after centrifugation.

  • a.
    Use a pipette to gently aspirate 1 mL of the RBC lysis buffer to resuspend the cell suspension.
  • b.
    Transfer the cell suspension to a new 15 mL centrifuge tube and continue to add RBC lysis buffer to bring the volume to 10 mL.
  • c.
    Let it sit at room temperature (15°C–25°C) for 10 min (Figure 4B).

CRITICAL: The purpose of replacing the centrifuge tube is to prevent impurities adhering to the tube walls from reintroducing into the cell suspension, which may lead to a decrease in cell yield.

• 12.Centrifuge cell suspension at 300 g for 5 min at 4°C and resuspend the cells in an appropriate volume (40–200 μL) of wash buffer II. The volume of the resuspension is determined by the desired cell quantity for subsequent experiments (Figure 4C).
• 13.
  Resuspend the cells in 100 μL of wash buffer II.

  • a.
    Take 10 μL of the cells and dilute them 10 times.
  • b.
    Take 10 μL of the diluted cells and mix them with an equal volume of 0.4% trypan blue.
  • c.
    Use a cell counter to determine the viability and concentration of cells (Figure 5).
• 14.Select approximately 3 × 10⁶ cells for flow cytometry, transfer them into a 1.5 mL EP tube, and centrifuge at 500 g for 5 min at 4°C.
• 15.Discard the supernatant and resuspend the cells with 50 μL 2.5 μg/mL Fc blocker, Incubate the cells on ice for 10 min.
• 16.
  Prepare the staining cocktail by diluting the surface protein antibody with wash buffer II at a ratio of 1:100.

  • a.
    Centrifuge the cell suspension at 300 g for 5 min at 4°C.
  • b.
    Discard the supernatant, and gently resuspend the cell pellet in 100 μL of the staining cocktail.
  • c.
    Incubate the cells on ice for 20 min, avoiding exposure to light.
• 17.Add 200 μL of wash buffer II to the cell suspension, centrifuge the cell suspension at 300 g for 5 min at 4°C, and discard the supernatant. Repeat this step once.
• 18.Resuspend the cell pellet in 100 μL of fixation/permeabilization solution and incubate on ice for 20 min.
• 19.Add 200 μL of 1× Perm/wash solution to the cell suspension, centrifuge the cell suspension at 300 g for 5 min at 4°C, and discard the supernatant.
• 20.
  Prepare the staining cocktail by diluting the Intracellular protein antibody with 1× Perm/wash solution at a ratio of 1:100.

  • a.
    Gently resuspend the cell pellet in 100 μL of the staining cocktail.
  • b.
    Incubate the cells on ice for 20 min, avoiding exposure to light.
• 21.Wash cells twice with 200 μL 1× Perm/wash solution.
• 22.Resuspend cells in 200 mL PBS for analysis on a five-laser flow cytometer. The primary immune group is shown in Figure 6.

Figure 4.

Cell suspension purification

(A) After resuspending cells using 36% Percoll and centrifuging, the immune cells primarily accumulate in the bottom layer.

(B) Transfer the cell pellet into a new 15 mL centrifuge tube and proceed with the red blood cell lysis process.

(C) The pale yellow or white cells aggregate at the bottom of the centrifuge tube after complete red blood cell lysis and centrifugation.

Figure 5.

Cell viability and concentration

(A) Microscopic examination shows 93.54% cell viability and 9.26 × 10⁵/mL concentration, The concentration of the original cell suspension is 9.26 × 10⁶/mL.

(B) The range of cell diameter distribution.

Figure 6.

Gating strategy for major immune cell populations in pancreatic cancer

(A) cell populations include tumor-associated macrophages (TAM) (CD45⁺CD11b⁺CD68⁺), myeloid-derived suppressor cells (MDSC) (CD45⁺CD11b⁺CD68^-CD66b⁺), B cells (CD45⁺CD19⁺), CD4⁺T cells (CD45⁺CD3⁺CD4⁺), CD8⁺cytotoxic T cells (CTL) (CD45⁺CD3⁺CD8⁺ ), γδT cells (CD45⁺CD3⁺TCRγδ T⁺), natural killer cells (NK) (CD45⁺CD3^-CD16⁺CD56⁺), dendritic cell (DC) (CD45⁺HLA-DR⁺CD11c⁺) and natural killer T cell (NKT) (CD45⁺CD3⁺CD16⁺CD56⁺).

Expected outcomes

After the steps of tissue purification, pancreatic cancer samples appear as solid tissue blocks of pale yellow or white color (Figure 1B). Tissue mechanical dissociation results in the formation of white granular tissue clusters (Figure 2A). Following digestion dissociation, a translucent liquid mixed with tissue particles is obtained (Figure 2B). The product of the purification step using Percoll results in an Innermost layer of a cellular pellet containing red blood cells (Figure 4A). After complete lysis of the red blood cells, a white or pale-yellow cellular pellet can be observed at the bottom of the centrifuge tube (Figure 5C). Using the current single-cell isolation protocol, we are able to separate and enrich immune cells from pancreatic cancer. Over 10⁶ cells can be isolated from approximately 1 cm³ of pancreatic cancer tissue. The yield of cells may vary depending on factors such as the patient’s treatment history, sample collection time, and storage conditions. The viability of cells, assessed through Trypan blue staining, is typically at least 80%. Flow cytometry analysis of CD45⁺ cells reveals that they constitute over 70% of the total cell population. Moreover, the isolated single cells are suitable for cell sorting and single-cell RNA sequencing, offering further insights into the tumor immune landscape.

Limitations

While this method proposes a strategy for isolating single cells from pancreatic cancer tissue and purifying immune cells, there are still limitations that need to be addressed. The extended digestion and dissociation time, as well as the mechanical grinding procedure, may release more cells but could also decrease cell viability. To apply this method to other tissue types, the digestion and dissociation times should be optimized to strike a balance between cell quantity, purity, and viability. Furthermore, when using the dissociated cells for another downstream process like cell sorting and single-cell RNA-seq, additional filtration steps are necessary to meet the requirements for downstream analysis, as cell fragments and clumping can adversely affect the results.

Troubleshooting

Problem 1

The pancreatic cancer tissue has a harder texture, making it difficult for the surgical knife to completely crush it, resulting in a lower cell yield (related to step 5).

Potential solution

• •Pancreatic cancer tissue is highly fibrotic, resulting in increased toughness, which is more pronounced in tissues with a history of previous chemotherapy. To achieve better dissociation, it is recommended to perform initial cutting in a culture dish before transferring the tissue to a 1.5 mL EP tube containing approximately 1 mL of a digestion system. The tissue can then be further minced with scissors to improve the dissociation effect.

Problem 2

After 1 h of digestion in the digestion system, there is a significant amount of flocculent suspension and sometimes it aggregates into clusters (related to step 7).

Potential solution

• •The amount of DNase I in the digestive system may be insufficient, and the concentration of DNase I in the digestive system can be increased 2–3 times.

Problem 3

There are large tissue particles in the cell suspension after grinding and filtering steps (related to step 8).

Potential solution

• •Grinding of the syringe plunger may cause damage to the filter, thus causing large tissue particles to penetrate the filter. Continue to filter the suspension with a new 40/70 μm filter for 1–2 times.

Problem 4

After the step of lysing RBCs, the top of the cell pellet appears red (related to step 11).

Potential solution

• •After the red blood cell lysis and centrifugation step, red blood cell debris will be deposited on top of the cell pellet, which can be carefully aspirated and discarded using a pipette.

Problem 5

After cell purification, the cell pellet is difficult to resuspend, or visible aggregates form after resuspension (related to step 9- step 12).

Potential solution

• •Cell fragments or dead cells may aggregate into clumps. Wash the cells with washing buffer II and filter the cell suspension through a 40/70 μm filter.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the technical contact, Jianpeng Sheng (shengjp@zju.edu.cn) or the lead contact, Xueli Bai (shirleybai@zju.edu.cn).

Materials availability

This study did not generate new unique reagents.

Data and code availability

This study did not generate datasets or code.