Chapter 24 Sequential Dissociation of the Exocrine Pancreas into Lobules, Acini, and Individual Cells

Abstract

Publisher Summary The chapter outlines the procedure for the sequential dissociation of the exocrine pancreas into lobules, acini, and individual cells. Dispersion of the pancreas into isolated single cells involves sequential enzymic digestion of stromal collagen and basement membrane, Ca2+ chelation to disrupt cell-cell junctions (that is, desmosomes and tight junctions), and mechanical shearing to complete the separation of gap and tight junctions. Filtration and washing of the tissue digest yield a purified preparation free of cell debris, collagenase, and subcellular organelles liberated in the final steps of the procedure. The protocol for tissue dissociation described allows the investigator to control the extent of dissociation of pancreatic exocrine tissue into minilobules, single acini, and separated cells, and to analyze how the progression of dissociation affects cell structure and function. Treatment of the tissue with purified collagenase and with mild mechanical shearing forces was found to be sufficient to dissociate the tissue into minilobules and single acini, whereas an additional step of Ca2+ chelation was found to be essential to break down the junctional elements between cells in order to obtain separated cell.