Background Necroptosis has been demonstrated to be a primary mechanism of islet cell death. This study evaluated whether the supplementation of necrostatin‐1 (Nec‐1), a potent inhibitor of necroptosis, to islet culture media could improve the recovery, maturation, and function of pre‐weaned porcine islets (PPIs). Methods PPIs were isolated from pre‐weaned Yorkshire piglets (8‐15 days old) and either cultured in control islet culture media (n = 6) or supplemented with Nec‐1 (100 µM, n = 5). On days 3 and 7 of culture, islets were assessed for recovery, insulin content, viability, cellular composition, GLUT2 expression in beta cells, differentiation of pancreatic endocrine progenitor cells, function, and oxygen consumption rate. Results Nec‐1 supplementation induced a 2‐fold increase in the insulin content of PPIs on day 7 of culture. When compared to untreated islets, Nec‐1 treatment doubled the beta‐ and alpha‐cell composition and accelerated the development of delta cells. Additionally, beta cells of Nec‐1‐treated islets had a significant upregulation in GLUT2 expression. The enhanced development of major endocrine cells and GLUT2 expression after Nec‐1 treatment subsequently led to a significant increase in the amount of insulin secreted in response to in vitro glucose challenge. Islet recovery, viability, and oxygen consumption rate were unaffected by Nec‐1. Conclusion This study underlines the importance of necroptosis in islet cell death after isolation and demonstrates the novel effects of Nec‐1 to increase islet insulin content, enhance pancreatic endocrine cell development, facilitate GLUT2 upregulation in beta cells, and augment insulin secretion. Nec‐1 supplementation to culture media significantly improves islet quality prior to xenotransplantation.

Background: The use of pancreata from pre-weaned piglets has the potential to serve as an unlimited alternative source of islets for clinical xenotransplantation. As pre-weaned porcine islets (PPIs) are immature and require prolonged culture, we developed an islet maturation media (IMM) and evaluated its effect on improving the quantity and quality of PPIs over 14 days of culture. Methods: PPIs were isolated from the pancreata of pre-weaned Yorkshire piglets (8-15 days old). Each independent islet isolation was divided for culture in either control Ham's F-10 media (n = 5) or IMM (n = 5) for 14 days. On day 3, 7 and 14 of culture, islets were assessed for islet yield, isolation index, viability, insulin content, endocrine cellular composition, differentiation of beta cells, and insulin secretion during glucose stimulation. Results: In comparison to control islets, culturing PPIs in IMM significantly increased islet yield. PPIs cultured in IMM also maintained a stable isolation index and viability throughout 14 days of culture. The insulin content, endocrine cellular composition, and differentiation of beta cells were significantly improved in PPIs cultured in IMM, which subsequently augmented their insulin secretory capacity in response to glucose challenge compared to control islets. Conclusions: Culturing PPIs in IMM increases islet yield, isolation index, viability, insulin content, endocrine cellular composition, differentiation of endocrine progenitor cells toward beta cells, and insulin secretion. Due to the improved islet quantity and quality after in vitro culture, the use of IMM in the culture of PPIs will assist to advance the outcomes of clinical islet xenotransplantation.

Previous studies have shown that necrostatin-1 (Nec-1) supplementation improved the viability of murine islets following exposure to nitric oxide, increased the survival of human islets during hypoxic culture, and augmented the maturation of pre-weaned porcine islets (PPIs) after 7 days of tissue culture. A limitation of these studies is that only one concentration of Nec-1 was used, and no studies have determined the optimal dose of Nec-1 for PPIs. Thus, the present study examined the effects of Nec-1 on PPIs at four different doses—0, 25, 50, 100, and 200 μM—after 7 days of tissue culture when supplemented on day 3. PPIs were isolated from pancreata of pre-weaned Yorkshire piglets (8–15 days old) and cultured in a specific islet maturation media added with Nec-1 on day 3 of tissue culture at 4 different doses—0, 25, 50, 100, and 200 μM (n = 6 for each dose). After 7 days of tissue culture, islets were assessed for recovery, viability, endocrine cellular content, GLUT2 expression in beta cells, and insulin secretion after glucose challenge. Nec-1 did not affect the viability of both intact islets and dissociated islets cells during tissue culture regardless of doses. Islets cultured in media supplemented with Nec-1 at 100 μM, but not 25, 50, or 200 μM, had a significantly higher recovery, composition of endocrine cells, GLUT2 expression in beta cells, and insulin secretion capacity than control islets cultured in media without Nec-1 supplementation. Moreover, culturing islets in 200 μM Nec-1 supplemented media not only failed to improve the insulin release but resulted in a lower glucose-induced insulin stimulation index compared to islets cultured in media added with 100 μM Nec-1. Xenotransplantation using porcine islets continues to demonstrate scientific advances to justify this area of research. Our findings indicate that Nec-1 supplementation at 100 μM was most effective to enhance the in vitro maturation of PPIs during tissue culture.

Introduction Islet recovery from within alginate‐based microcapsules is necessary for certain analytical assays like flow cytometry; however, this technology has not been widely characterized. In this study, we explore the ability of EDTA, EGTA, and sodium citrate to induce reverse alginate polymerization via chelation and assess the toxicity of each chelator on pancreatic islets. Methods EDTA, EGTA, and sodium citrate were used to dissolve single‐layered Ba2+ alginate encapsulated islets and the rate of capsule breakdown calculated from analysis of imaging data. The effect of chelator exposure on islet viability and recovery was assessed using flow cytometry, while glucose‐stimulated insulin release (GSIR) assay was used to measure effects on islet function. Results EGTA demonstrated the most rapid microcapsule dissolving rate followed by EDTA and sodium citrate. Islet recovery was significantly better when encapsulated islets were treated with EDTA than EGTA and Na+ citrate. A decrease in viability and increase in apoptotic cells were observed when encapsulated islets were treated with Na+ citrate compared to islets treated with EDTA and EGTA. Islets treated with EDTA and EGTA demonstrated comparable stimulation index values to non‐treated control. Conversely, islets treated with Na+ citrate exhibited significantly decreased SI values compared to control. All chelator groups showed significantly lower insulin secretion than non‐treated islets. Conclusion Islet recovery from alginate microcapsule is possible using common chelators like Na+ citrate, EDTA, and EGTA. Chelation of encapsulated islets using EDTA demonstrated the most efficient dissolving capabilities with the least toxicity toward islet recovery and health.

Background Necrostatin‐1 (Nec‐1) supplementation to tissue culture media on day 3 has recently been shown to augment the insulin content, endocrine cellular composition, and insulin release of pre‐weaned porcine islets (PPIs); however, its effects were only examined for the first 7 days of tissue culture. The present study examined whether the addition of Nec‐1 on day 3 could further enhance the in vitro development and function of PPIs after 14 days of tissue culture. Methods PPIs were isolated from 8‐ to 15‐day‐old, pre‐weaned Yorkshire piglets and cultured in an islet maturation media supplemented with Nec‐1 on day 3. The recovery, viability, insulin content, endocrine cellular composition, GLUT2 expression in beta cells, differentiation and proliferation potential, and glucose‐stimulated insulin secretion of PPIs were assessed on days 3, 7, and 14 of tissue culture (n = 5 on each day). Results Compared with day 7 of tissue culture, islets on day 14 had a lower recovery, GLUT2 expression in beta cells, proliferation capacity of endocrine cells, and glucose‐induced insulin stimulation index. Prolonging the culture time to 14 days did not affect islet viability, insulin content, proportion of endocrine cells, and differentiation potential. Conclusion The growth‐inducing effects of Nec‐1 on PPIs were most effective on day 7 of tissue culture when added on day 3. Our findings support existing evidence that the in vitro activities of Nec‐1 are short‐lived and encourage future studies to explore the use of other novel growth factors during prolonged islet tissue culture.

Pre-weaned porcine islets (PPIs) represent an unlimited source for islet transplantation but are functionally immature. We previously showed that necrostatin-1 (Nec-1) immediately after islet isolation enhanced the in vitro development of PPIs. Here, we examined the impact of Nec-1 on the in vivo function of PPIs after transplantation in diabetic mice. PPIs were isolated from pancreata of 8–15-day-old, pre-weaned pigs and cultured in media alone, or supplemented with Nec-1 (100 µM) on day 0 or on day 3 of culture (n = 5 for each group). On day 7, islet recovery, viability, oxygen consumption rate, insulin content, cellular composition, insulin secretion capacity, and transplant outcomes were evaluated. While islet viability and oxygen consumption rate remained high throughout 7-day tissue culture, Nec-1 supplementation on day 3 significantly improved islet recovery, insulin content, endocrine composition, GLUT2 expression, differentiation potential, proliferation capacity of endocrine cells, and insulin secretion. Adding Nec-1 on day 3 of tissue culture enhanced the islet recovery, proportion of delta cells, beta-cell differentiation and proliferation, and stimulation index. In vivo, this leads to shorter times to normoglycemia, better glycemic control, and higher circulating insulin. Our findings identify the novel time-dependent effects of Nec-1 supplementation on porcine islet quantity and quality prior to transplantation.

For the advancement of porcine xenotransplantation for clinical use in type 1 diabetes mellitus, the concerns of a sustainable and safe digestion enzyme blend must be overcome. Incorporating good manufacturing practices (GMP) can facilitate this through utilizing GMP-grade enzymes. In conjunction, still taking into account the cost-effectiveness, a wide concern. We evaluated how GMP-grade enzyme blends impact our piglet islets and their long-term effects.  Preweaned porcine islets (PPIs) were isolated from 8- to 10-day-old pigs. Digestion enzyme blends, collagenase type V (Type V), collagenase AF-1 GMP-grade with collagenase NB 6 GMP-grade (AF-1 and NB 6), and collagenase AF-1 GMP-grade with collagenase neutral protease AF GMP-grade (AF-1 and NP AF) were compared. Islet quality control assessments, islet yield, viability, and function, were performed on days 3 and 7, and cell content was performed on day 7.  GMP-grade AF-1 and NB 6 (17,209 ± 2,730 islet equivalent per gram of pancreatic tissue [IE/g] on day 3, 9,001 ± 1,034 IE/g on day 7) and AF-1 and NP AF (17,214 ± 3,901 IE/g on day 3, 8,833 ± 2,398 IE/g on day 7) showed a significant increase in islet yield compared to Type V (4,618 ± 1,240 IE/g on day 3, 1,923 ± 704 IE/g on day 7). Islet size, viability, and function showed comparable results in all enzyme blends. There was no significant difference in islet cellular content between enzyme blends.  This study demonstrated a comparison of GMP-grade collagenase enzyme blends and a standard crude collagenase enzyme in preweaned-aged porcine, a novel topic in this age. GMP-grade enzyme blends of AF-1 and NB 6 and AF-1 and NP AF resulted in substantially higher yields and as effective PPIs compared to Type V. In the long run, considering costs, integrity, and sustainability, GMP-grade enzyme blends are more favorable for clinical application due to high reproducibility in comparison to undefined manufacturing processes of standard enzymes.

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