Recent advances in the design of implantable insulin secreting heterocellular islet organoids.

Islet transplantation has proved one of the most remarkable transmissions from an experimental curiosity into a routine clinical application for the treatment of type I diabetes (T1D). Current efforts for taking this technology one-step further are now focusing on overcoming islet donor shortage, engraftment, prolonged islet availability, post-transplant vascularization, and coming up with new strategies to eliminate lifelong immunosuppression. To this end, insulin secreting 3D cell clusters composed of different types of cells, also referred as heterocellular islet organoids, spheroids, or pseudoislets, have been engineered to overcome the challenges encountered by the current islet transplantation protocols. ß-cells or native islets are accompanied by helper cells, also referred to as accessory cells, to generate a cell cluster that is not only able to accurately secrete insulin in response to glucose, but also superior in terms of other key features (e.g. maintaining a vasculature, longer durability in vivo and not necessitating immunosuppression after transplantation). Over the past decade, numerous 3D cell culture techniques have been integrated to create an engineered heterocellular islet organoid that addresses current obstacles. Here, we first discuss the different cell types used to prepare heterocellular organoids for islet transplantation and their contribution to the organoids design. We then introduce various cell culture techniques that are incorporated to prepare a fully functional and insulin secreting organoids with select features. Finally, we discuss the challenges and present a future outlook for improving clinical outcomes of islet transplantation.

An all-aqueous approach for physical immobilization of PEG-lipid microgels on organoid surfaces.

Emulsion-based generation of hydrogel particles has been widely explored for numerous applications in fields such as biomedical, food, and drug delivery. Water-in-water emulsion (w/w) is an organic solvent-free approach and exploits solely aqueous media to generate nano- or microparticles. This strategy is environment-friendly and favorable for biomedical applications where biocompatibility is the ultimate criterion. Hence, PEG-based microgels can be synthesized with desired size and functionality using w/w emulsion technique. To estimate the influence of emulsification parameters on size and stability of PEG-lipid microgels, optimizations using three independent input variables were carried out: (i) ultrasonication power, (ii) ultrasonication duration, and (iii) duration of light exposure. Physical immobilization of microgels on islet-organoids was achieved through hydrophobic interactions. Cell function and viability were assessed thoroughly after microgel immobilization. Microgel size is dependent on ultrasonication parameters and microgel stability is vastly determined by the duration of light exposure. Immobilization of microgels with 5 mM lipid moiety promoted coating of islet-organoids. Coated organoids retained their function and viability without significant adverse effects. This is important for understanding fundamental aspects of PEG-lipid microgels using w/w emulsion, useful for possible drug/gene delivery applications to increase treatment efficiency and ultimately lead to clinical translation of PEG microgels for biomedical applications.

Sensitivity Study for the Key Parameters in Heterospheroid Preparation with Insulin-Secreting ß-Cells and Mesenchymal Stem Cells.

The outcome of islet transplantation in clinics has been determined by the success of tissue engraftment. The strong immune attack that occurs upon transplantation of ß-cells plays a central role as this attack results in the failure of transplanted tissue. To improve tissue engraftment, deleterious effects of immune reactions should be minimized for ß-cell function and survival. Here, we report a systematic analysis of the effect of insulin-secreting ß-cell (MIN6) and mesenchymal stem cell (MSC) number and size on the function of ß-cells and present immune protection potential of heterospheroid structures through MSCs and synthetic scaffolds. We prepared 3D heterospheroids with MSCs and MIN6 cells through a hanging-drop approach. To precisely estimate the influence of critical parameters on heterospheroid size and insulin secretion function of ß-cells, we prepared heterospheroids using two independent input variables: (i) initial cell number in each droplet and (ii) MIN6:MSC ratio. We studied the influence of initial cell numbers of 200 and 500, and six different MIN6:MSC ratios (1:0, 0:1, 1:1, 2:1, 5:1, and 10:1) for the preparation of heterospheroids through the hanging drop. Next, we used PEG hydrogels as a semipermeable physical barrier to improve immune protection from cytokines. Through encapsulation of our heterospheroids within PEG hydrogel, we were able to observe sustained ß-cell survival and insulin secretion despite exposure of heterospheroids with proinflammatory cytokines. Insulin secretion was further promoted with glucagon like peptide-1 (GLP-1) incorporation within PEG hydrogel structure. This study is significant to demonstrate the synergistic effects of MIN6-MSC and scaffold-MIN6 interactions and to improve therapeutic efficacy of islet transplantation. Overall, this study comprehensively presents the optimum conditions for the preparation of MIN6-MSC spheroids, utilizes MSCs and GLP-1 functional PEG hydrogels as a scaffold to retain insulin secretion function and further demonstrates protection of heterospheroids exposed to proinflammatory cytokines.