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....

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. ....

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

List of materials used in this article

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