Acinar-to-Ductal Metaplasia Induction: A Method to Study Acinar Cell to Ductal Cell Differentiation in 3D Ex Vivo Culture

Abstract

Source: Fleming Martinez, A. K. et. al. Mimicking and Manipulating Pancreatic Acinar-to-Ductal Metaplasia in 3-dimensional Cell Culture. J. Vis. Exp. (2019).

This video describes the technique of differentiating pancreatic acinar cells to ductal cells in ex vivo 3D culture. This helps to understand the mechanisms regulating the acinar-to-ductal metaplasia (ADM), a key process in the early development of pancreatic cancer.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 1. Preparation of Materials, Solutions, and 3-dimensional Matrix Bases Cut 500 µm and 105 µm polypropylene meshes into 76 mm by 76 mm squares. Fold each square in half twice to create a smaller folded square. Place one 500 µm and one 105 µm mesh square into an autoclavable pouch. Autoclave polypropylene mesh s…

Representative Results

Figure 1: Representative results of primary acinar cells plated within collagen after stimulation with TGF-α. Primary acinar cells from a non-transgenic mouse were infected with GFP-adenovirus, embedded in collagen I or basement membrane matrix, and stimulated with or without TGF-α (50 ng/mL). Twenty-four hours post-infection with GFP adenovirus, images were captur…

Materials

5% CO2, 37 °C Incubator	NUAIRE	NU-5500
50 ml tubes	Falcon	352070
Beaker, 600 mL	Fisherbrand	FB-101-600
Adenovirus, Ad-GFP	Vector Biolabs	1060
Dexamethasone	Sigma	D1756	Create a 4 mg/ml solution by dissolving powder in methanol, aliquoting and storing at -20 °C
Fetal Bovine Serum	Sigma	F0926-100mL
Forceps	Fine Science Tools	11002-12
Glass slide, 8-well	Lab-Tek	177402
Ice bucket, rectangular	Fisher Scientific	07-210-103
Matrigel	Corning	356234	Referred to as 'basement membrane matrix' in the manuscript.
PBS	Fisher Scientific	SH30028.02
Penicillin-Streptomycin	ThermoFisher Scientific	15140122
Plate, 12-well	Corning Costar	3513
Plate, 24-well plate	Corning Costar	3524
Plate, 6-well	Falcon	353046
Polypropylene Mesh, 105 µm	Spectrum Labs	146436
Polypropylene Mesh, 500 µm	Spectrum Labs	146418
Scissors	Fine Science Tools	14568-12
Sodium Bicarbonate (Fine White Powder)	Fisher Scientific	BP328-500
Sodium Hydroxide	Fisher Scientific	S318-500
Soybean Trypsin Inhibitor	Gibco	17075029
Type I Rat Tail Collagen	Corning	354236
PIPETMAN Classic P10, 1-10 μl	Gilson	F144802
Pipettes, 5 ml	Falcon	357543
Pipet tips, 10 µl	USA Scientific	1110-3700
Pipet tips, 1000 µl	Olympus Plastics	24-165RL
Pipet tips, 200 µl	USA Scientific	1111-1700
Pipettes, 25 ml	Falcon	357525
Pipet-Aid	Drummond
Pipettes, 10 ml	Falcon	357551
PIPETMAN Classic P200, 20-200 μl	Gilson	F123601
PIPETMAN Classic P1000, 200-1000 μl	Gilson	F123602
PIPETMAN Classic P20, 2-20 μl	Gilson	F123600
Waymouth MB 752/1 Medium (powder)	Sigma	W1625-10X1L
Weigh boat, hexagonal, medium	Fisherbrand	02-202-101