Acinar-to-Ductal Metaplasia Induction: A Method to Study Acinar Cell to Ductal Cell Differentiation in 3D Ex Vivo Culture
Published: April 30, 2023
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…
Declarações
The authors have nothing to disclose.
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 |
Citar este artigo
Acinar-to-Ductal Metaplasia Induction: A Method to Study Acinar Cell to Ductal Cell Differentiation in 3D Ex Vivo Culture. J. Vis. Exp. (Pending Publication), e20310, doi: (2023).