Acinar-to-Ductal Metaplasia Induction: A Method to Study Acinar Cell to Ductal Cell Differentiation in 3D Ex Vivo Culture
Trascrizione
– The exocrine portion of the pancreas constitutes acinar cells and ductal cells. Acinar cells synthesize and secrete digestive enzymes into their lumen. The ductal cells line the tube that delivers these enzymes to the small intestine. To adapt to genetic and environmental stress, acinar cells undergo morphological and functional conversion into duct-like cells, termed acinar-to-ductal metaplasia, or ADM.
At times, ADM may lead to the onset of pancreatic cancer. To induce ADM ex vivo, prepare a chilled collagen cell suspension by combining primary acinar cells with collagen gel kept on ice to prevent matrix solidification. Treat the suspension with a test ADM stimulant or inhibitor of interest.
Distribute the suspension into a culture plate and incubate to facilitate collagen solidification and embed the cells within. Next, pipette an additional volume of media containing the test ADM stimulant or inhibitor of interest over the solidified collagen cell layer.
Incubate the plate for an appropriate duration. With acinar-to-ductal metaplasia induction, pyramidal-shaped primary acinar cells transdifferentiate to form cells with duct cell-like morphology. In the following protocol, we will demonstrate the ex vivo induction of acinar-to-ductal metaplasia in primary murine acinar cells using TGF alpha as a stimulant.
– To make a collagen gel, combine 7 milliliters of type-1 rat tail collagen, 700 microliters of 10 times Waymouth's medium, and 466.6 microliters of 0.34 molar sodium hydroxide in a 50 milliliter tube on ice to make collagen gel. In a 50 milliliter tube, combine equal volumes of cell suspension and collagen gel after three hours' incubation and gently mix. Plate 2,000 microliters one well at a time in a six-well plate while keeping the plate on ice, and swirl to evenly distribute the collagen cell layer.
To solidify the medium, incubate the plate in a cell culture incubator at 37 degrees Celsius and 5% carbon dioxide for 30 minutes. Then add 1.5 milliliters per well of 1 times Waymouth's complete medium with desired stimulus or inhibitors.
Change the medium the following day and then every other day. Depending on the stimulus used, acinar-to-ductal metaplasia, or ADM, can be observed between day three and day five.
