Protocol to study ductal progenitor-like cells from the adult human pancreas using 3D suspension and methylcellulose-based culture systems.

The pancreas is comprised of two compartments, the exocrine and endocrine. The exocrine acinar cells produces and secreted digestive enzymes that are carried by ductal cells to the duodenum, whereas the endocrine islets of Langerhans regulate blood sugar levels. In both type 1 and type 2 diabetes, specific gluocose-sensing, insulin secreting cells in the islets are either destroyed or become dysfunctional, making it necessary to monitor blood sugar levels and inject insulin when necessary. The FDA has approved allogeneic (or from another individual) islet cell transplantation to treat patients with severe forms of diabetes. Over the last twenty years, allogeneic islet cell transplantation has proven an effective method to restore a patient’s ability to regulate their own blood sugar levels without needing to inject insulin. Thus, many laboratories, including ours, are studying alternative sources of beta-like cells to supplement the currently limited supply of allogeneic islets. We and others have shown that a small population of pancreatic ductal cells has the potential to form functional, beta-like cells. These rare ductal cells are termed progenitor-like cells, because they can self-renew (make more of themselves) and differentiate (make other, mature pancreatic cell types, including the beta cells). Here, with support from CIRM, we present our detailed protocols using good manufacturing practice (GMP)-compatible methods to grow and study these progenitor-like ductal cells from donated human pancreatic tissue. These protocols were used in our previously published article in iScience (https://doi.org/10.1016/j.isci.2024.109237), which demonstrated that extracellular matrix (ECM) proteins were necessary to stimulate the activation and proliferation of ductal cells cultured using our suspension platform. Overall, our studies and reported protocols contribute to the field of regenerative medicine aimed at using primary pancreatic tissue to restore functional islet cells to diabetic patients who have lost the ability to regulate their own blood sugar levels. We add mechanistic insight into how the environment signals activate and support a rare population of ductal progenitor-like cells, which can improve clinical translation of these cells for therapeutic purposes.