Alginate has been used to protect transplanted pancreatic islets from immune rejection and as a matrix to increase the insulin content of islet progenitor cells. The throughput of alginate bead generation by the standard extrusion and external gelation method is limited by the rate of droplet formation from nozzles. Alginate bead generation by emulsion and internal gelation is a scaleable alternative that has been used with biological molecules and microbial cells, but not mammalian cells. We describe the novel adaptation of this process to mammalian cell immobilization. After optimization, the emulsion process yielded 90 ± 2% mouse insulinoma 6 (MIN6) cell survival, similar to the extrusion process. The MIN6 cells expanded at the same rate in both bead types to form pseudo-islets with increased glucose stimulation index compared to cells in suspension. The emulsion process was suitable for primary pancreatic exocrine cell immobilization, leading to 67 ± 32 fold increased insulin expression after 10 days of immobilized culture. Due to the scaleability and broad availability of stirred mixers, the emulsion process represents an attractive option for laboratories that are not equipped with extrusion-based cell encapsulators, as well as for the production of immobilized or encapsulated cellular therapeutics on a clinical scale.

Encapsulation of insulin-producing cells in alginate beads could improve the treatment of type 1 diabetes by reducing or eliminating the need for immunosuppression. We have recently adapted an emulsion and internal gelation process to β-cell encapsulation. This process has the advantages of being well suited for m(3)/h production rates and allowing the use of increased alginate concentrations. Compared with 1.5% alginate beads generated by a standard extrusion process, 5% alginate emulsion-generated beads demonstrated greater in vitro stability and greater volumetric exclusion of antibody-sized pullulan. When βTC3 cells were transplanted into streptozotocin-induced allogeneic diabetic mice, a significant decrease in the blood glucose levels was seen within 2 days with the 5% emulsion-generated beads but not until >16 days with the 1.5% extrusion-generated beads. This was correlated with higher cell survival and lower graft-specific plasma immunoglobulin levels. These results suggest that higher-concentration alginate beads generated by emulsion and internal gelation have improved graft immunoprotection. The emulsion process is a promising and scalable technology for cellular therapies requiring immune isolation.

Cell encapsulation in alginate beads has been used for immobilized cell culture in vitro as well as for immunoisolation in vivo. Pancreatic islet encapsulation has been studied extensively as a means to increase islet survival in allogeneic or xenogeneic transplants. Alginate encapsulation is commonly achieved by nozzle extrusion and external gelation. Using this method, cell-containing alginate droplets formed at the tip of nozzles fall into a solution containing divalent cations that cause ionotropic alginate gelation as they diffuse into the droplets. The requirement for droplet formation at the nozzle tip limits the volumetric throughput and alginate concentration that can be achieved. This video describes a scalable emulsification method to encapsulate mammalian cells in 0.5% to 10% alginate with 70% to 90% cell survival. By this alternative method, alginate droplets containing cells and calcium carbonate are emulsified in mineral oil, followed by a decrease in pH leading to internal calcium release and ionotropic alginate gelation. The current method allows the production of alginate beads within 20 min of emulsification. The equipment required for the encapsulation step consists in simple stirred vessels available to most laboratories.

Aims/HypothesisWe previously identified the transcription factor Myt3 as specifically expressed in pancreatic islets. Here, we sought to determine the expression and regulation of Myt3 in islets and to determine its significance in regulating islet function and survival.Methods Myt3 expression was determined in embryonic pancreas and adult islets by qPCR and immunohistochemistry. ChIP-seq, ChIP-qPCR and luciferase assays were used to evaluate regulation of Myt3 expression. Suppression of Myt3 was used to evaluate gene expression, insulin secretion and apoptosis in islets.ResultsWe show that Myt3 is the most abundant MYT family member in adult islets and that it is expressed in all the major endocrine cell types in the pancreas after E18.5. We demonstrate that Myt3 expression is directly regulated by Foxa2, Pdx1, and Neurod1, which are critical to normal β-cell development and function, and that Ngn3 induces Myt3 expression through alterations in the Myt3 promoter chromatin state. Further, we show that Myt3 expression is sensitive to both glucose and cytokine exposure. Of specific interest, suppressing Myt3 expression reduces insulin content and increases β-cell apoptosis, at least in part, due to reduced Pdx1, Mafa, Il-6, Bcl-xl, c-Iap2 and Igfr1 levels, while over-expression of Myt3 protects islets from cytokine induced apoptosis.Conclusion/InterpretationWe have identified Myt3 as a novel transcriptional regulator with a critical role in β-cell survival. These data are an important step in clarifying the regulatory networks responsible for β-cell survival, and point to Myt3 as a potential therapeutic target for improving functional β-cell mass.