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Application of a novel B cell line MIN6 to a mesh-reinforced polyvinyl alcohol hydrogel tube and three-layer agarose microcapsules: an in vitro study.

This study examines the function of a novel B cell line (MIN6) enclosed in hybrid bioartificial pancreas with mesh-reinforced polyvinyl alcohol hydrogel tube (MRPT) or with improved, three-layer agarose microcapsules. MIN6 was established from insulinomas obtained by targeted expression of the simian virus 40 T-antigen gene in transgenic mice. MIN6 retains the ability to secrete insulin in response to physiological glucose concentrations. The MRPT and the three-layer agarose microcapsules, which were developed to act as an artificial pancreas, were readily permeated by insulin, glucose, and other nutrients. Both can immunoisolate enclosed MIN6 cells from the recipient's humoral and cellular immunosystems, which causes a xenogeneic rejection response. MIN6 cells (5.0 x 10(6) or 1.5 x 10(6)) were enclosed in MRPT or in a hundred three-layer microcapsules and subjected to an in vitro perifusion study or a static incubation study to observe the insulin release from each bioartificial pancreas in response to glucose stimulation. In vitro study revealed that the insulin secretion in response to 16.7 mM glucose stimulation was twice that with 3.3 mM glucose stimulation with both MRPT and the three-layer agarose microcapsules. The present study demonstrates that MIN6 effectively functions as a bioreactor for the hybrid bioartificial pancreas. The application of MIN6 cells to the hybrid bioartificial pancreas may offer a solution to the current serious dearth of organs.

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PubMedDate not supplied

Gelatin density gradient for isolation of islets of Langerhans.

Isolation of islets of Langerhans (islets) has been performed by means of collagenase digestion of the pancreatic tissue combined with density gradient separation of islets from unwanted exocrine tissues. An enormous number of islets are necessary for clinical islet transplantation. The density gradient used for isolation of a large number of islets should satisfy several requirements in addition to those for the conventional density gradients, such as high viscosity for creating fine interfaces with a large area, easy sterilization, and low cost. This study is concerned with the development of a new density gradient made of low-molecular-weight gelatin. We isolated islets from the hamster pancreatic tissue using the gelatin density gradients. The yield and purity of islet and its insulin release function were compared with those of islets isolated using Ficoll and Ficoll-Conray density gradients that have been conventionally used. The new gelatin density gradient can separate islets from the unwanted exocrine tissue as effectively as the Ficoll density gradient and more effectively than the Ficoll-Conray density gradients. The islets collected using the gelatin gradient retain ability of insulin release increase in response to glucose stimulation, similar to those isolated by the Ficoll-Conray gradient and more than those collected by the Ficoll gradient. In addition, the gelatin effectively inhibited enzyme activities, that is, collagenase and proteolytic enzymes released from the exocrine tissue, and thus it can inhibit overdigestion of islets during their density gradient isolation. The gelatin gradient satisfies most of the additional requirements for islet isolation from the pancreatic tissue of large animals mentioned above. Although several factors, such as molecular weight of gelatin, osmolality of the gradient, and centrifugal conditions, still remain to be optimized, our results suggest that the gelatin gradient has potentiality to isolate islets from the pancreatic tissue of a large animal.

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