Microenvironmental characterization of pancreatic islets within their endogenous niche towards a bioengineered microtissue for β-cell replacement therapy

Abstract

Type 1 diabetes is characterised by insulin deficiency leading to chronic hyperglycaemia causing microvascular and macrovascular complications. Current treatment with glucose monitoring and insulin administration can cause recurrent serious hypoglycaemia significantly impacting on quality of life. Despite considerable advances towards an artificial pancreas, only pancreatic β-cell replacement therapy can restore truly normal glucose homeostasis. Existing clinical options for this are whole pancreas and isolated islet transplantation. Pancreas transplantation requires major abdominal surgery and can lead to dangerous early complications including pancreatitis, as the organ is particularly susceptible to post mortem stress prior to reimplantation. When successful, pancreas transplantation usually leads to insulin independence. Islet transplantation is a minimally invasive procedure, but many islets are also lost through post mortem stress associated with isolation and transplantation, negatively impacting on islet microenvironment, viability, mass and function. Long term insulin independence is rarely sustained. The overall aim of the work comprising this thesis was to explore tissue engineering approaches to provide isolated islets with sufficient replacement of the lost niche to maintain integrity, mass, viability and function and therefore transplant outcomes. The research question hereby was, how the culture of islets in proximity or contact with a hydrogel impacts on islet health with the hypothesis that the replacement of lost ECM via a hydrogel will improve islet integrity, viability and function after isolation and prior to transplant. Specific objectives were: 1. To characterise the islet niche in situ within human donor pancreas and analyse acute changes related to ischaemia, tissue processing and islet isolation. 2. To evaluate biocompatibility and impact on islet integrity, viability and function of extracellular matrix (ECM) replacement through a novel collagen-containing hydrogel. 3. To explore bioengineering approaches to reduce the impact of hypoxia on islets combined with a hydrogel ECM niche to form a tissue-engineered β-cell replacement product. Islet microenvironment, integrity and morphology in situ was analysed in a pancreas with minimal ischaemic damage by immunohistochemistry (IHC), immunofluorescence staining (IF) and electron microscopy (EM) analysis revealing peri-islet basement membrane (BM), peri-vascular BM, rich vascularisation and endocrine cell-to-cell connections. Impact of ischaemia on donor pancreata was analysed by EM analysis leading to development of the Newcastle EM Ischaemia Score (NEMIS). Characterisation of isolated islets with IHC, IF and EM showed loss of peri-islet BM and reduced integrity through the isolation process. Biocompatibility of a novel ECM replacement hydrogel comprising collagen I, alginate and fibrinogen (CAF) with a pseudoislet model derived from the MIN6 β-cell line and primary human islets was confirmed by propidium iodide (PI) viability staining and integrity scoring. Maintained metabolic function was confirmed by Seahorse flux analysis. Towards islet hypoxia reduction an electrospun nanofiber membrane to provide microchannels for oxygen delivery and dynamic culture to prevent islet clumping were assessed. Expression of hypoxia-induced gene signature was reduced in dynamic culture with maintained viability (PI staining), and improved integrity scores, but decreased ATP production (Seahorse flux analysis). As a final step a perifusion system was developed to facilitate ECM replacement simultaneously with decreased hypoxia. In conclusion, the quality of transplanted pancreatic tissue is negatively impacted through organ preservation and islet isolation. A new EM ischaemia score has been developed to inform better selection of donor organs. A novel collagen-containing hydrogel has a potential in replacement of islet ECM, but hypoxia remains an unresolved challenge to successful β-cells tissue engineering. Reducing hypoxia with dynamic culture enables maintained islet quality in vitro and an engineered perifusion system may enable preparation of novel β-cell microtissue for successful transplantation without the detriment of prolonged ex vivo hypoxia.