Pancreatic Tissue Dissection to Isolate Viable Single Cells

Summary

Pancreatic metaplastic cells are precursors of malignant cells that give rise to pancreatic tumors. However, isolating intact viable pancreatic cells is challenging. Here, we present an efficient method for pancreatic tissue dissociation. The cells can then be used for single-cell RNA sequencing (scRNA-seq) or for two- or three-dimensional co-culturing.

Abstract

The pancreas includes two major systems: the endocrine system, which produces and secretes hormones, and the exocrine system, which accounts for approximately 90% of the pancreas and includes cells that produce and secrete digestive enzymes. The digestive enzymes are produced in the pancreatic acinar cells, stored in vesicles called zymogens, and are then released into the duodenum via the pancreatic duct to initiate metabolic processes. The enzymes produced by the acinar cells can kill cells or degrade cell-free RNA. In addition, acinar cells are fragile, and common dissociation protocols result in a large number of dead cells and cell-free proteases and RNases. Therefore, one of the biggest challenges in pancreatic tissue digestion is recovering intact and viable cells, especially acinar cells. The protocol presented in this article shows a two-step method that we developed to meet this need. The protocol can be used to digest normal pancreata, pancreata that include pre-malignant lesions, or pancreatic tumors that include a large number of stromal and immune cells.

Introduction

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancer types¹. Clinical evidence supports the notion that PDAC develops from exocrine-system cells, including acinar cells, over many years, driven by mutations in the KRAS proto-oncogene².

Pancreatic tumors include many different cell types, and it has been demonstrated that malignant cells count for only 20%-50% of the tumor mass³. Different cell types interact with the epithelial cells, support their transformation, and enhance tumor formation and growth. Early events cause acinar metaplasia, which gives rise to microscopic lesions called pancreatic intraepithelial neoplasia (PanINs), which can in some cases develop into PDAC⁴.

There is a critical need to investigate these interactions and target pivotal signals. Single-cell RNA-sequencing (scRNA-seq) is a powerful method that reveals gene expression at a single-cell resolution, thereby tracking the changes that epithelial cells undergo, thus enabling the exploration of pancreatic cancer development.

Tissue dissection and digestion to single cells is the first stage in a scRNA-seq experiment. Several factors make pancreatic tissue digestion especially challenging: i) acinar cells account for more than 90% of the pancreas and acinar cells contain large amounts of digestive enzymes, including proteases and RNases that reduce the quality of RNA-based libraries; (ii) acinar cells are very sensitive and may lyse if standard protocols are used; (iii) acinar cells express a small number of genes at very high levels. Therefore, if these cells are lysed during the experiment, this can contaminate the observed gene expression profile of other cells; (iv) pancreatic tissue recovered from tumors is desmoplastic, making it hard to dissect without damaging the cells. Thus, even though maintaining high viability of all the cell types is required, the large number and sensitivity of acinar cells add additional complexity. These factors impose difficulties in achieving a single-cell suspension that is more than 80% viable and has no clumps, as is required for scRNA-seq experiments.

Here, we developed a protocol using trypsin C and collagenase P, along with frequent tissue monitoring. This supports dissociation to single cells while retaining high viability to support the success of scRNA-seq experiments^5,6.

Protocol

The joint ethics committee (Institutional Animal Care and Use Committee) of the Hebrew University (Jerusalem, Israel) and Hadassah Medical Center (Jerusalem, Israel) approved the study protocol for animal welfare (MD-18-15417-5 "Tissue dynamics in pancreatic cancer in mice"), and the protocol presented here complied with all relevant ethical regulations for animal testing and research. The Hebrew University is an Association for Assessment and Accreditation of Laboratory Animal Care International-accredited institute.

NOTE: The mouse strain stock #007908, stock #019378, and stock #008179 were obtained from Jackson's laboratory. PRT (Kras+/LSL-G12D; Ptf1a-Cre^ER; Rosa26^LSL-tdTomato) mice were created by crossing the above strains. Mice from both genders, between 6 weeks and 15 months of age, were used for the study. Tamoxifen was prepared by dissolving the powder in corn oil. Adult mice (6-8 weeks of age, females and males), were injected with tamoxifen subcutaneously on days 0 and 2 at a dose of 400 mg/kg and examined twice a week following the injection. It was not possible to measure tumors as they were internally located; therefore, euthanasia was performed if abnormal clinical signs were observed according to the ethical protocol. Mice were euthanized at different time points post-tamoxifen induction, using isoflurane and cervical dislocation.

1. Pancreatic dissection

NOTE: For optimal yield during extraction and to ensure good cell viability, rapid dissection is critical. To shorten the time required for pancreas isolation, all instruments and equipment must be ready on ice before euthanizing the mouse.

1. Euthanize the mouse by CO₂ asphyxiation and verify using cervical dislocation. From this step on, all procedures must be performed with sterile dissecting instruments.
2. Fix the mouse and spray the abdomen with 70% ethanol. Make a V-shaped incision of 2.5 cm in the genital area with scissors and forceps and proceed upward to fully open the abdominal cavity.
3. Locate the stomach on the left side of the mouse. Locate the pancreas, which is near the spleen. Separate the pancreas from the stomach and duodenum using two forceps (without tearing). Continue and separate the pancreas from the small intestine, jejunum, and ileum.
4. Move the pancreas to the right side of the mouse. Separate the remaining connections between the pancreas and the thoracic cavity with forceps to completely detach the pancreas and attached spleen.
5. Remove the pancreas and spread it out for examination in a Petri dish on ice.
   NOTE: Care must be taken during this step to remove only the pancreas, and not remove mesenteric fat tissue or other adjacent tissue along with the pancreas, to avoid cellular contamination.

2. Enzymatic and mechanical dissociation of the pancreas

1. Prepare the following buffers in advance.
   1. Dissociation buffer 1: 4 mL of trypsin C + 6 mL of phosphate buffered saline (PBS) (see Table 1) for each sample.
   2. Dissociation buffer 2: 9 mL of Hanks′ balanced salt solution (HBSS) 1x; 4% bovine serum albumin (BSA); 1 mL of collagenase P (10 mg/mL); 200 µL of trypsin inhibitor (10 mg/mL); and 200 µL of DNase I (10 mg/mL) (see Table 1).
   3. Wash buffer: 50 mL of HBSS 1x; 2 g of BSA; 1 mL of trypsin inhibitor (10 mg/mL); and 1 mL of DNase 1 (10 mg/mL).
   4. Enzyme activity stop solution: HBSS 1x containing 5% fetal bovine serum (FBS) and 150 g of DNase I (0.2 mg/mL).
2. Place the pancreas in a 50 mL tube on ice. Rinse the pancreas in 10% FBS in HBSS 1x. The fatty tissue will float and the pancreas will sink. This is an easy way to visualize and quickly remove the contaminating white adipose tissue still attached to the pancreas.
3. Transfer the mouse pancreatic tissue to a sterile Petri dish containing 5 mL of HBSS 1x on ice. Cut the pancreas into small pieces of 1 to 3 mm³ using Noyes scissors and a scalpel (Figure 2A). In case of more than one sample, the samples should be kept on ice in 10% FBS/HBSS 1x.
4. Transfer the tissues to a centrifuge tube. Centrifuge at 350 x g at 4 °C for 5 min. Aspirate and discard the supernatant to remove cell fragments and blood cells.
5. Resuspend the pieces in dissociation buffer 1 containing 0.02% trypsin C-0.05% EDTA for 10 min at 37 °C with agitation (180 rpm). Immediately wash with 10% FBS/Dulbecco's modified Eagle's medium (DMEM). Centrifuge for 5 min at 350 g at 4 °C.
6. Wash again by resuspending the pellet in 10 mL of wash buffer and centrifuging at 350 x g for 5 min at 4 °C before the next dissociation step.
7. Incubate the pancreas in dissociation buffer 2 for 15 min at 37 °C with agitation (180 rpm).
8. After 15 min, perform mechanical dissociation by vigorously pipetting the pancreatic fragments up and down in sterile pipettes of decreasing size (25, 10, and 5 mL serological pipettes) 10 times and bring back to 37 °C.
   1. After an additional 5 min, repeat the mechanical dissociation and use light microscopy to monitor the dissociation according to the amount of single-cell suspension. Usually if at this stage, less than 90% of the suspension consists of isolated single cells, a longer incubation time is needed. Use trypan blue to monitor cell viability.
   2. Continue with the incubation and take samples to detect the dissociation every 5 min.
      NOTE: The total incubation time depends on the tissue and can vary between samples. The incubation and tissue dissociation should end when 90% of the cells are separated into single cells or when a reduction in viability is detected.
9. After the pancreatic tissue is well dissociated (corresponding to the disappearance of pancreatic fragments and the increasing turbidity of the solution) (Figure 2), stop the enzymatic reaction by washing twice with enzyme activity stop solution for 5 min at 4 °C. From this step, keep the cell suspension on ice.
10. Pass the cell suspension through a 70µm nylon mesh and check the cell viability under the microscope. Smaller sizes of nylon mesh may reduce cell viability.
11. Resuspend and wash the pellet with 5-10 mL of ice-cold buffered wash solution. Count the cells.
12. If several red blood cells are observed, treat the cells with a red blood cell lysis buffer for 2 min at room temperature. In cases where clumps are observed, the sample should be treated again with trypsin, as described in step 2.5. Viability is detected with trypan blue under the microscope, and if viability is less than 80%, live cells should be isolated using the magnetic-activated cell sorting (MACS) dead cells removal kit with MACS MS columns (see Table 1).

Results

In a recently published work⁵, we applied the protocol described above to explore the early stages of PDAC development using a mouse model. The mouse was genetically engineered to include the cassettes Ptf1a-CreER, LSL-Kras-G12D, LSL-tdTomato⁷, which allow the expression of constitutively active KRAS in acinar cells after tamoxifen injection.

After cervical dislocation (according to the mouse ethi...

Discussion

In this article, we present a protocol for pancreatic tissue dissociation. The protocol is simple, easy to use, and provides a tool to isolate viable single cells from pancreatic tissue at different stages during the malignancy process, including solid tumors. In previous studies, different types of collagenases were used to digest the pancreas^8,9. Using a very potent collagenase, such as collagenase D, results in a large population of immune cells and a lower pe...

Materials

Name	Company	Catalog Number	Comments
Reagent or Resource
70 µm nylon mesh	Corning	cat##431751
BSA	Sigma Aldrich	cat# A7906
Collagenase P	Roche	cat# 11213857001
Critical Commercial Assay
DAPI	Sigma Aldrich	cat#MBD0015
Dnase I	Roche	cat# 10104159001
Experimental Models: Organisms/Strains
Fetal Bovine Serum South American	ThermoFisher	Cat#10270106
Hanks' Balanced Salt Solution	Biological industries	cat#02-018-1A
KRASLSL-G12D mice	Jackson Laboratory	JAX008179
MACS dead cells removal kit	Milteny Biotec	cat#130-090-101
PBS	Biological industries	cat#02-023-1A
Ptf1a-CreER mice	Jackson Laboratory	JAX019378
Ptf1a-CreER; Rosa26LSL-tdTomato mice	Jackson Laboratory	JAX007908
Trypsin C-EDTA 0.05%	Biological industries	cat# 03-053-1A
Trypsin inhibitor	Roche	cat#T6522