A method for the purification of single A, B and D cells and for the isolation of coupled cells from isolated rat islets

Abstract

A method has been developed for the purification of single A, B and D cells and for the isolation of coupled islet cells. Isolated rat islets were dissociated by repeated pipetting in the presence of trypsin and ethylene glycol bis (beta-aminoethyl ether) N,N' tetra-acetic acid (EGTA), and then filtered through nylon and Percoll; the cell preparation consisted of 70% single and 30% coupled cells. Sizing of the cells led to the recognition of a small-islet cell population (35%; cell volume 200-600 micrometer 3) composed of A and D cells, and a large-islet cell population (65%; cell volume 600-1500 micrometer 3) identified as B cells. Differences in sedimentation velocity formed the basis for the islet cell separation by counterflow elutriation. Single islet cells eluted prior to coupled cells and were distributed over A and D cell-enriched fractions I and II (65% A, 25% B, 10% D) and the B cell-enriched fraction III (93% B). The slightly different densities of A (d = 1.068), B (d = 1.065) and D (d = 1.070) cells allowed a subsequent purification by density gradient centrifugation resulting in a final 10- to 30-fold enrichment in either A, B or D cells. Most coupled islet cells were recovered in fraction IV, occurred mainly as doublets and were composed of 90% B cells and 7% D cells; the multiple pseudopods, which characterize isolated D cells, might contribute to the coupling tendency of the D cells. It is concluded that the purified A, B and D cell fractions and the coupled islet cell preparations offer a direct approach to the study of individual islet cell types and their intercellular communication.

The biosociology of pancreatic B cells

“…Techniques for the purification of islet cells have been developed in the rat, where standardised methods exist for the reproducible isolation of pancreatic islets [7,15] and for their dissociation into single and viable cells [15,16]. They take advantage of the differences which (Table 1).…”

“…Both in volumetric and surface analysis, insulin-containing B cells were found to be two-to three-fold larger than the other islet cell types (Fig.2, [ ty than islet non-B cells. Centrifugal elutriation, which separates cells according to their sedimentation velocity [18][19][20], distributed the islet cells over a first single cell fraction enriched in islet non-B cells, a second single cell fraction composed of more than 90 percent B cells and a third fraction of mostly structurally coupled cells [16]. The knowledge that islet B cells were separable on the basis of their larger size [16,17] led to attempts to purify islet cells by flow cytometry, with the cellular light scatter activity as separation parameter.…”

“…Centrifugal elutriation, which separates cells according to their sedimentation velocity [18][19][20], distributed the islet cells over a first single cell fraction enriched in islet non-B cells, a second single cell fraction composed of more than 90 percent B cells and a third fraction of mostly structurally coupled cells [16]. The knowledge that islet B cells were separable on the basis of their larger size [16,17] led to attempts to purify islet cells by flow cytometry, with the cellular light scatter activity as separation parameter. In our experience, light scatter alone discriminates poorly between islet B and non-B cells and can therefore not be adequately used for their isolation [21,22].…”

“…Other laboratories also were unsuccessful in purifying islet non-B cells by light scatter flow cytometry, but reported on the ability of this technique to isolate islet B cells [23][24][25][26], albeit at a lower cell yield and a lower degree of purification than with other methods [15,16,211. Differences in cell density can be employed to purify glucagon-containing A cells from islet cell preparations which are already enriched in islet non-B cells [16].…”

“…Differences in cell density can be employed to purify glucagon-containing A cells from islet cell preparations which are already enriched in islet non-B cells [16]. Isopycnic gradient centrifugation of elutriated islet non-B cells yields preparations with more than 90% A cells between densities 1.067 and 1.07 g/ml, but the final cell yield is less than 20% [16].…”