Mimicking and Manipulating Pancreatic Acinar-to-Ductal Metaplasia in 3-dimensional Cell Culture

Abstract

The differentiation of acinar cells to ductal cells during pancreatitis and in the early development of pancreatic cancer is a key process that requires further study. To understand the mechanisms regulating acinar-to-ductal metaplasia (ADM), ex vivo 3D culture and differentiation of primary acinar cells to ductal cells offers many advantages over other systems. With the technique herein, modulation of protein expression is simple and quick, requiring only one day to isolate, stimulate or virally infect, and begin culturing primary acinar cells to investigate the ADM process. In contrast to using basement membrane matrix, the seeding of acinar cell clusters in collagen I extracellular matrix, allows acinar cells to retain their acinar identity before manipulation. This is vital when testing the contribution of various components to the induction of ADM. Not only are the effects of cytokines or other ectopically administered factors testable through this technique, but the contribution of common mutations, increased protein expression, or knockdown of protein expression is testable via viral infection of primary acinar cells, using adenoviral or lentiviral vectors. Moreover, cells can be re-isolated from collagen or basement membrane matrix at the endpoint and analyzed for protein expression.

Hyperinsulinemia acts via acinar insulin receptors to initiate pancreatic cancer by increasing digestive enzyme production and inflammation

“…This would result in decreased ADM and PanIN formation, as in the PK- Insr f/f pancreata. To test this hypothesis, we utilized an 3D ex vivo model of ADM formation from wild-type acinar cells (Figure 8D) (Fleming Martinez and Storz, 2019; Means et al, 2005a). Wild-type acinar clusters grown in collagen for 5 days maintained their acinus morphology, as previously shown (Means et al, 2005a).…”

“…Primary pancreatic acinar cells were isolated from pancreata of wild-type female mice at the age of 6-8 weeks. The acinar cell isolation procedure was adapted from the protocol described by Martinez and Storz (Fleming Martinez and Storz, 2019). Briefly, the mouse pancreas was harvested and washed three times in 1x HBSS, which were then minced into 1-5mm pieces.…”

Organotypic Culture of Acinar Cells for the Study of Pancreatic Cancer Initiation

“…The protocol for the isolation and the culture of acinar cells has been optimized by several laboratories over the past decades [ 38 , 39 , 40 , 41 ], but still hinges on a light enzymatic digestion of minced pancreata to preserve the tri-dimensional organization of the acini. Digestion time is variable [ 39 ] and results in a heterogeneous mixture of microscopic tissue explants. Cocktails of Ser/Thr proteases inhibitors are added in order to prevent autodigestion of the explants (due to stress-induced release of proteases from acinar cells).…”

“…The protocol for the isolation and the culture of acinar cells has been optimized by several laboratories over the past decades [38][39][40][41], but still hinges on a light enzymatic digestion of minced pancreata to preserve the tri-dimensional organization of the acini. Digestion time is variable [39] and results in a heterogeneous mixture of microscopic tissue explants.…”

Organotypic Culture of Acinar Cells for the Study of Pancreatic Cancer Initiation

“…The protocol for the isolation and the culture of acinar cells has been optimized by several laboratories over the past decades [37][38][39][40], but still hinges on a light enzymatic digestion of minced pancreata to preserve the tri-dimensional organization of the acini. Digestion time is variable [38] and results in a heterogeneous mixture of microscopic tissue explants. A gradient sedimentation step enriches the preparation for acini, which sink after slow centrifugation, although that does not impede the accidental co-isolation of other cell populations.…”