An islet maturation media to improve the development of young porcine islets during in vitro culture

Abstract

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.

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

“…The current data showing a reduction in islet recovery after 7 days of culture in media without Nec-1 supplementation is in accordance with past studies, in which a substantial loss of young porcine islets could be observed throughout the prolonged in vitro tissue culture period [16,19]. In support of our results that lower doses of Nec-1 at 25 and 50 μM could not improve the recovery of PPIs on day 7 of tissue culture compared to control islets, previous findings have demonstrated that Nec-1 treatment on rat cardiomyocytes at a low dose of 30 μM failed to delay the opening of mitochondrial permeability transition pores while 100 μM Nec-1 treatment could significantly prolong the time to pore opening [43].…”

“…In contrast, the use of young porcine islets for clinical islet xenotransplantation could not only offer a cost-effective and scalable alternative, but has also been shown to lessen the need for exogenous insulin administration, heighten hypoglycemic awareness, and reduce HbA1C levels in multiple clinical trials [12,14,39,40]. Nevertheless, the prolonged culture period required for the functional maturation of young porcine islets is associated with a substantial islet loss [16,19]. To facilitate their clinical use, the tissue culture media must be optimized to preserve islet quantity and improve islet quality before transplantation.…”

“…As young porcine islets largely consist of immature endocrine cells and pancreatic progenitor cells that are capable of differentiation of proliferation during culture and after transplantation, various studies have explored the utilization of cytoprotective agents and growth factors to prevent islet loss and enhance islet development [17][18][19]41]. Previous studies have identified that Nec-1, a potent inhibitor of necroptosis, could reduce islet cell death and DAMP https://doi.org/10.1371/journal.pone.0243506.g008…”

“…Isolated islets were cultured in an islet maturation culture media supplemented with 50% Ham's F-12 medium (cat# 10-080, Corning Inc.), 50% medium 199 (cat# 50-051-PB, Corning Inc.), 10 mM HEPES (cat# H3375, Sigma-Aldrich), 5 mM L-glutathione (cat# G4251, Sigma-Aldrich), 0.6 mL/L ITS+3 (cat# I2771, Sigma-Aldrich), 10 mM nicotinamide (cat# N5535, Sigma-Aldrich), 100 ug/mL gentamicin sulfate (cat# 30-005-CR, Corning Inc.), 10 uM trolox (cat# 238813, Sigma-Aldrich), 200 U/L heparin (cat# 400-10, Sagent Pharmaceuticals), 0.1 mM pefabloc (cat# sc202041B, Santa Cruz Biotechnology), 2 mM L-glutamine (cat# 56-85-9, Alfa Aesar), 2.5 mM calcium chloride dihydrate (cat#C79-3, Fisher Scientific), 1000 U/L DNase (cat#D4263, Sigma-Aldrich), antibiotic/antimycotic solution (Corning Inc, cat#30004CI), and 10% porcine serum using T-150 untreated suspension flasks (cat #CLS430825, Corning Inc) for 7 days in a 37˚C, 5% CO 2 humidified incubator (cat#3110, Thermo Forma Series II 3120 Water Jacketed Incubators) [19]. A full media change was performed 24 hours post islet isolation, and a half media change was done every 48 hours thereafter.…”

“…Despite these advantages, young porcine islets require prolonged culture to reach optimal maturation before transplantation [16]. As a substantial loss of islets has been reported during this in vitro tissue culture period, the use of growth factors could assist to shorten culture time and accelerate islet development [16][17][18][19].…”