Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

Summary

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

Abstract

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 10⁶ acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

Introduction

A frequently encountered problem for research laboratories working on exocrine pancreatic tissue is the difficulty of cultivating acinar cells in vitro for a period of time long enough to allow a long-term experiment.

One factor impeding development of such culture systems is the intrinsic sensitivity of pancreatic tissue to experimental manipulation due to the high content in glycolytic, proteolytic, and lipolytic enzymes, which literally digest the pancreatic tissue when they are released during the isolation of pancreatic cells.

A second factor is the remarkable in vitro plasticity of acinar ....

Protocol

All procedures were approved by an ethic committee under regulatory of governmental authority ("Comité d'Evaluation Commun au Centre Léon Bérard, à l'Animalerie de transit de l'ENS, au PBES et au laboratoire P4" (CECCAPP)). Mice were maintained in a specific pathogen-free animal facility at the "Plateforme AniCan, Centre Léon Bérard" (Lyon, France) and handled in compliance with the institutional guidelines.

A schematic representation of the procedure is shown in Figure 1.

1. Pancreas Dissection and Dilaceration (Day 0)

Results

Figure 1 schematizes the "dispersed" acini method for primary acinar cells isolation. The critical steps, which have to be strictly respected during the protocol, are described in the discussion part.

To facilitate its removal, the pancreas has to be collected from the abdomen along with the attached spleen (Figure 2). Both organs need to be cut apart, and the residual fat tissue that could be still attached to the pancreas must be removed (Step 1.6).

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Discussion

In this protocol, we describe a procedure for isolating pancreatic acinar cells. This method makes possible to isolate more than 20 x 10⁶ acinar cells per animal in less than 1 hr. Thanks to its rapid and simple implementation (as many as 10 pancreases can be independently processed per experiment in parallel), this protocol appears as a good compromise between existing isolation methods ^3-5,9-12,17 .

Critical steps/Trouble-shooting

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.2 μm filter	Dutscher	146560
10 ml serological pipettes	Beckton Dickinson	357551
100 μm filter	Beckton Dickinson	352360
100 mm Petri dish	Beckton Dickinson	353003
1000 μl filter tips	Starlab	S1122-1830
20 μl filter tips	Starlab	S1120-1810
200 μl filter tips	Starlab	S1120-8810
25 ml serological pipettes	Beckton Dickinson	357535
5 ml serological pipettes	Beckton Dickinson	357543
50 ml polypropylene tube	Beckton Dickinson	352070
6-well plate	Beckton Dickinson	353046
Acetic acid 100%	VWR BDH Prolabo	20104.298
Collagenase IA	Sigma-Aldrich	C2676
Curved forceps, Dumont #7	World Precision Instruments	14188	To sterilize before use
Dissecting scissors, straight	World Precision Instruments	14393	To sterilize before use
Epidermal Growth Factor, human	Promokine	C-60180
Ethanol absolute (AnalaR Normapur)	VWR BDH Prolabo	20821.310
Fetal Bovine Serum	Lonza	14-801F
Forceps, Dumont #5	World Precision Instruments	14098	To sterilize before use
Hank’s Balanced Salt Solution 1x	Gibco	14025050
HEPES 1 M (pH 6.98-7.30)	Lonza	17-737F
Incubator O2/CO2	Sanyo	MCO-19M
Inverted microscope	Nikon	Eclipse TS100
Matrigel	Beckton Dickinson	356234
Microbiological Safety Cabinet, level II	Faster	SafeFast Elite 212 S
Noyes scissors, sharp/sharp tips, German	World Precision Instruments	500228-G	To sterilize before use
Penicillin-Streptomycin mixture	Gibco	15140122
Phosphate Buffer Saline 10x	Gibco	14200067
Pipet-Aid	Drummond Scientific Company	Pipet-Aid XP
Pipetman P1000	Gilson	F123602
Pipetman P20	Gilson	F123600
Pipetman P200	Gilson	F123601
Refrigerated centrifuge	Eppendorf	5810R
Scalpel	Paramount Surgimed Ltd.	Disposable Scalpel Size 23
T25 flask, 25 cm2	Sigma-Aldrich	Z707481
Trypsin inhibitor, from Glycine Max	Sigma-Aldrich	T6522
Type I collagen	Beckton Dickinson	354236
Waymouth’s medium	Gibco	31220-023