Controlled aggregation of primary human pancreatic islet cells leads to glucose‐responsive pseudoislets comparable to native islets

Abstract

Clinical islet transplantation is a promising treatment for patients with type 1 diabetes. However, pancreatic islets vary in size and shape affecting their survival and function after transplantation because of mass transport limitations. To reduce diffusion restrictions and improve islet cell survival, the generation of islets with optimal dimensions by dispersion followed by reassembly of islet cells, can help limit the length of diffusion pathways. This study describes a microwell platform that supports the controlled and reproducible production of three-dimensional pancreatic cell clusters of human donor islets. We observed that primary human islet cell aggregates with a diameter of 100–150 μm consisting of about 1000 cells best resembled intact pancreatic islets as they showed low apoptotic cell death (<2%), comparable glucose-responsiveness and increasing PDX1, MAFA and INSULIN gene expression with increasing aggregate size. The re-associated human islet cells showed an a-typical core shell configuration with beta cells predominantly on the outside unlike human islets, which became more randomized after implantation similar to native human islets. After transplantation of these islet cell aggregates under the kidney capsule of immunodeficient mice, human C-peptide was detected in the serum indicating that beta cells retained their endocrine function similar to human islets. The agarose microwell platform was shown to be an easy and very reproducible method to aggregate pancreatic islet cells with high accuracy providing a reliable tool to study cell–cell interactions between insuloma and/or primary islet cells.

Reestablishment of Glucose Inhibition of Glucagon Secretion in Small Pseudoislets

“…Similar to what is seen in the murine cells, dispersed human islets reassociate to form pseudoislets (19,25). Glucagon secretion, which is normally inhibited in the islet in response to 11 mmol/L glucose, is significantly increased in the dispersed state (Fig.…”

“…Taken together, these data highlight the importance of pseudoislet culture time with regard to function. Pseudoislets, prior to full recovery, may remain in a dysfunctional dispersed state rather than an isletlike state (19,25). …”

“…This is of particular relevance because it has long been thought that size is a critical component to islet function, and pseudoislet dimensions must be comparable to intact islets for proper hormone secretion (14,16–18,22,25,38). Although large pseudoislets have been used in transplantation and β-cell mass replacement studies, this model can also be used to interrogate the function of the other islet cell types.…”

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

“…)), and agarose microwell (Hilderink et al. ), may affect function of pseudoislets. 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.…”

Nanoemulsion-induced enzymatic crosslinking of tyramine-functionalized polymer droplets

“…[41][42][43] For example, controlled aggregation of cells is key to bottom-up engineering of islets of Langerhans for the treatment of diabetes. 44,45 To investigate the potential of nanoemulsion-induced enzymatic crosslinking for 3D cell cultures, we set out to encapsulate the pancreatic beta cell line MIN6 to form insulin producing microtissues. First, we aimed to identify the smallest amount of H2O2 containing nanoemulsion that still resulted in robust cell encapsulating microcapsules, as even a small excess of H2O2 (order 1 to 10 mg/l) has been proven detrimental to cell functioning.…”