Dose-dependent effects of necrostatin-1 supplementation to tissue culture media of young porcine islets

Abstract

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.

RIPK1 and RIPK3 regulate TNFα-induced β-cell death in concert with caspase activity

“…Previous studies have investigated TNFα-induced NFκB activation and FADD signaling in β cells [ 6 , 7 ], but few have evaluated RIPK1, RIPK3 or MLKL [ 50 , 51 ], components of TNF receptor signaling known to promote cell death in other cell types [ 19 , 25 , 52 ]. Notably, a recent study found that inhibition of RIPK1 promotes porcine islet viability and β-cell abundance [ 50 ].…”

“…Previous studies have investigated TNFα-induced NFκB activation and FADD signaling in β cells [ 6 , 7 ], but few have evaluated RIPK1, RIPK3 or MLKL [ 50 , 51 ], components of TNF receptor signaling known to promote cell death in other cell types [ 19 , 25 , 52 ]. Notably, a recent study found that inhibition of RIPK1 promotes porcine islet viability and β-cell abundance [ 50 ]. In the present study, we found that NIT-1 RIPK1Δ cells are strongly protected from TNFα-induced cell death ( Figure 2 A,B,C), that this is associated with reduced caspase 3/7 activation ( Figure 2 D), and that cIAPs repress TNFα-induced cell death in a RIPK1-dependent manner ( Figure 2 E,F), a mechanism that has been described in other cell types [ 53 , 54 ].…”

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

“…After islet isolation, PPIs were cultured in islet maturation media (IMM) for 7 days [45]. Untreated islets cultured in IMM alone served as a control (n = 5), whereas Nec-1 treated islets were cultured in IMM supplemented with Nec-1 (100 µM; cat#ab141053, Abcam, Cambridge, UK) either immediately after islet isolation (D0 Nec-1, n = 5) or on day 3 (D3 Nec-1, n = 5) [16]. A full media change was performed on day 1, and a half media change was done every 48 h thereafter.…”

“…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.…”

Updateon xenotransplantation for May/June 2021

“…More recently, Lau et al. further investigated the dose efficiency to improve maturation of porcine islets, which was seen to significantly increase with a dose of 100 μm of nec‐1 introduced at day 3 of culture in order to provide a further boost in maturation and recovery of the cells 29 . 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.…”