Bioengineered human pseudoislets form efficiently from donated tissue, compare favourably with native islets in vitro and restore normoglycaemia in mice

Abstract

Aims/hypothesisIslet transplantation is a treatment option that can help individuals with type 1 diabetes become insulin independent, but inefficient oxygen and nutrient delivery can hamper islet survival and engraftment due to the size of the islets and loss of the native microvasculature. We hypothesised that size-controlled pseudoislets engineered via centrifugal-forced-aggregation (CFA-PI) in a platform we previously developed would compare favourably with native islets, even after taking into account cell loss during the process.MethodsHuman islets were dissociated and reaggregated into uniform, size-controlled CFA-PI in our microwell system. Their performance was assessed in vitro and in vivo over a range of sizes, and compared with that of unmodified native islets, as well as islet cell clusters formed by a conventional spontaneous aggregation approach (in which dissociated islet cells are cultured on ultra-low-attachment plates). In vitro studies included assays for membrane integrity, apoptosis, glucose-stimulated insulin secretion assay and total DNA content. In vivo efficacy was determined by transplantation under the kidney capsule of streptozotocin-treated Rag1−/− mice, with non-fasting blood glucose monitoring three times per week and IPGTT at day 60 for glucose response. A recovery nephrectomy, removing the graft, was conducted to confirm efficacy after completing the IPGTT. Architecture and composition were analysed by histological assessment via insulin, glucagon, pancreatic polypeptide, somatostatin, CD31 and von Willebrand factor staining.ResultsCFA-PI exhibit markedly increased uniformity over native islets, as well as substantially improved glucose-stimulated insulin secretion (8.8-fold to 11.1-fold, even after taking cell loss into account) and hypoxia tolerance. In vivo, CFA-PI function similarly to (and potentially better than) native islets in reversing hyperglycaemia (55.6% for CFA-PI vs 20.0% for native islets at 500 islet equivalents [IEQ], and 77.8% for CFA-PI vs 55.6% for native islets at 1000 IEQ), and significantly better than spontaneously aggregated control cells (55.6% for CFA-PI vs 0% for spontaneous aggregation at 500 IEQ, and 77.8% CFA-PI vs 33.4% for spontaneous aggregation at 1000 IEQ; p < 0.05). Glucose clearance in the CFA-PI groups was improved over that in the native islet groups (CFA-PI 18.1 mmol/l vs native islets 29.7 mmol/l at 60 min; p < 0.05) to the point where they were comparable with the non-transplanted naive normoglycaemic control mice at a low IEQ of 500 IEQ (17.2 mmol/l at 60 min).Conclusions/interpretationThe ability to efficiently reformat dissociated islet cells into engineered pseudoislets with improved properties has high potential for both research and therapeutic applications.Electronic supplementary materialThe online version of this article (10.1007/s00125-018-4672-5) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

Delivery of shRNA via lentivirus in human pseudoislets provides a model to test dynamic regulation of insulin secretion and gene function in human islets

“…Although perifusion was not preformed, a bioengineered microwell that recently became commercially available was reported to allow the formation of human pseudoislets with robust GSIS using a similar simple step as ours (Yu et al. ) and may further improve first‐phase GSIS compared with a low attachment well we used in the current study.…”

“…; Yu et al. ), human pseudoislets mirror the original intact islets well for both nonendocrine and endocrine cells. When sorted mouse beta, alpha, and delta cells were used, alpha and delta cells were incapable to form aggregates implicating beta cells are critical for the formation of pseudoislets (Reissaus and Piston ).…”

“…; Yu et al. ) and indicated that the ratio of insulin/glucagon cells in the original intact islets and pseudoislets is similar (Zuellig et al. ).…”

“…demonstrated that human pseudoislets made by controlled aggregation of dispersed islet cells, an approach employed by us as well, improve insulin secretion corrected by cell number highlighting the advantage of pseudoislets over dispersed islet cells for the efficiency of insulin secretion (Yu et al. ). Secretory profile of human pseudoislets to KCl and glibenclamide was also similar to that of fresh islets further supporting the utility of human pseudoislets to dissect the regulation of insulin secretion in human islets.…”

In Vitro Platform for Studying Human Insulin Release Dynamics of Single Pancreatic Islet Microtissues at High Resolution

“…In order to obtain uniform islet spheroids, isolated islets can be dissociated in primary‐cell suspensions that feature physiological proportions of the various islet endocrine cell types, and the dissociated cells can then be reaggregated in controlled ratios 35–38. However, the formation of human pseudoislets featuring physiological GSIS response remains challenging.…”