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A mixture of alginate and polyethylene glycol acrylate was investigated as a system for the encapsulation of islets of Langerhans. This system showed dual crosslinkability: the alginate was ionically crosslinked by multivalent cations, and the PEG was covalently crosslinked by photoactivated free radical polymerization. The major advantage of the dually crosslinkable system was the chemical stability of the resultant gels due to the presence of covalent bonds that maintain the integrity of the gel as opposed to reversible ionic linkages that were the only mode of crosslinkage in previous generations of alginate-based encapsulation systems. The physical aspects of gelation of such alginate/PEG compositions were investigated. Diffusion of dextrans of known molecular weights through these gels was studied in order to shed light on the hydrogel porosity and permeability. In vitro viability and function tests demonstrated that these gels were biocompatible. Islets encapsulated in these systems were healthy and retained both viability and insulin secretory function.

The development of techniques for separating islets from acinar cells on a mass scale is a prerequisite to successful clinical attempts at islet transplantation. We have identified and partially characterized two blood group-reactive monoclonal antibodies (McAb), CAC1 and CAC2, with specific binding activity to acinar cells but not islets. These McAb are of the IgM subclass, and were found on immunocytochemical analysis to possess broad interspecies cross-reactivity, binding to antigens expressed in the acinar tissue of rats, dogs, and man. The antigens that these McAb recognize are glycolipid in nature and thus were not denatured by collagenase digestion of the pancreas. These properties, together with their ability to effect complement-mediated lysis, may make the McAb generally useful reagents in islet isolation and purification.

A simple, rapid method of islet purification is important in large-scale human islet isolation. We have previously identified monoclonal antibodies specific for acinar cells, but not islets, and described an immunologic method of purification by selective lysis of the acinar cells. An attractive alternative to lysis of the acinar cell is depletion by a magnetic immunomicrosphere technique. We report in this study a rapid, reproducible method of rat islet purification utilizing magnetic microspheres coated with acinar-cell-specific monoclonal antibodies. Pancreatic digestion with collagenase followed by depletion of acinar cells with the magnetic immunomicrospheres (MIMS) yields large numbers of intact islets. We compared the islets thus obtained with hand-picked (HP) islets (control) for yield, purity, in vitro insulin secretory capacities, and in vivo functional viability. The islet yield with the MIMS method (n = 35) was 72.7% that obtained with the HP method (n = 6) (378 +/- 8 vs. 519 +/- 31 islets per pancreas). The purity of the MIMS-isolated islets was 84 +/- 1.9%, ranging from 75-95%. Static glucose stimulation showed excellent function (2-3-fold increase of insulin release over basal levels) with no statistical difference in insulin secretion between MIMS and HP islets. Under microscopic examination, both groups revealed a well-preserved structure with healthy endocrine cells. When 1321 +/- 59 MIMS islets were transplanted into streptozotocin-induced diabetic rats (n = 10), normoglycemia (less than 200 mg/dl) was restored in all recipients following transplantation, and 100% of them remained normoglycemic on day 120 postgrafting. In summary, a rapid, consistent, and simple method of isolating viable, purified rat islets is described. The broad interspecies crossreactivity of the McAb suggests that this technique may be generally useful for islet purification in large mammalia, including man.

We have developed two immunological methods of islet purification using anti-acinar cell monoclonal antibodies (McAb). Pancreatic digestion with collagenase followed by depletion of acinar cells with McAb and complement or magnetic immunomicrospheres (MIMS) yields large numbers of intact islets. We compared the islets thus obtained with either handpicked (HP) islets, or islets separated on density gradients alone, for yield, purity, in vitro in sulin secretory capacities, and in vivo functional viability. Exposure of canine acinar tissue to the cytotoxic McAb resulted in a seven-fold enrichment in islet concentration, with preparations of 65% to 87% purity. Islet yield, however, was impaired by the procedure of McAb treatment, reducing the average yield from the control digestate of 40,000 to 60,000 islets/pancreas by 40% to 45%. Using the MIMS method in the rat model, the islet yield was 73% that obtained by the HP method (378 +/- 8 vs 519 +/- 31 per pancreas). The purity of the MIMS isolated islets was 84 +/- 1.9%, ranging from 75% to 95%. Analysis of the complement and MIMS treatments by in vitro static glucose tests and the capacity to restore normoglycemia after isografting in streptozotocin-induced diabetic rats indicates that the functional integrity of the islets was not affected by the purification process.

Identification, separation and purification of human islets from acinar cells following collagenase digestion of the pancreas remains a difficult problem. We have identified and characterized two monoclonal antibodies, CBL3 and TT62, with specific binding activity to human islets but not acinar cells. The antigens these monoclonal antibodies recognize are lipid in nature and thus unaffected by collagenase digestion of the human pancreas. These properties allow isolated human islets to be identified by fluorescein isothiocyanate-labeled CBL3 and TT62, making these monoclonal antibodies potentially useful reagents in islet identification and purification.