The effects of necrostatin‐1 on the in vitro development and function of young porcine islets over 14‐day prolonged tissue culture

Abstract

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.

Updateon xenotransplantation for May/June 2021

“…To further test the efficacy of nec‐1 in prolonged exposure to see the effect on neonatal and adult porcine islet cell composition, they tested this over an extended 14‐day culture period and undertook flow cytometric analysis of the preps at two time points. They focused their analysis of the cells at 7 and 14 days culture points and demonstrated that enhanced attributes previously seen in the cell culture does not continue to significantly increase past day 7, and proliferation, islet recovery, and GLUT2 expression significantly decreases between day 7 and 14; however, the use of nec‐1 in culture did continue to have an enhanced effect on endocrine composition but was not significantly different between day 7 and 14 where the alpha and delta cells continued to increase 15 …”

“…The media in which the cells are cultured is of significant importance indicating that additives during cell culture not only benefit cell proliferation during the culture phase by reducing the fragility from mechanical stress during the isolation process, 14 as demonstrated by Lau et al. with hypoxia‐related cell death, 15 but also inhibiting apoptosis and increasing revascularization of the cells following transplantation 16,17 …”

Necrostatin-1: a promising compound for neurological disorders

“… Jo et al (2015) also found that Necrostatin-1 improves the survival of mouse oocytes. Numerous studies show that Necrostatin-1 promotes the maturation, development, and graft function of neonatal porcine islets ( Lau et al, 2020a , b , 2021 ), providing an effective strategy for the future application of islet grafts ( Qin et al, 2022 ). Emerging evidence suggests that Necrostatin-1 has potential radical scavenging activities ( Ushijima and Monzaki, 2023 ).…”

Necrostatin-1 Supplementation to Islet Tissue Culture Enhances the In-Vitro Development and Graft Function of Young Porcine Islets

“…Previously, we determined that culturing pre-weaned porcine islets (PPIs) in media supplemented with Nec-1 immediately after islet isolation for 7 days significantly augmented islet insulin content, proportion of endocrine cells, and in vitro insulin secretion [15]. Our recent studies have further shown that 100 µM of Nec-1 was the most effective dose to enhance the in vitro maturation of PPIs for up to 7 days during tissue culture [16,17]. However, these investigations lack a detailed comparison of the optimal timing to supplement Nec-1 to islet tissue culture and its impact on the in vivo function of PPIs.…”