Islet Isolation Research
The Islet Transplant Research Group (ITRG) is led by Professor Paul Johnson. Our team performs innovative research aimed at optimising all stages of the human islet isolation and transplantation process. This includes: the optimisation of islet extraction, enhancement of islet activity by preconditioning prior to transplantation and the isolation of islet cells from the immune system using encapsulation devices. Islet segregation from the immune system is a critical step in allowing islet transplantation to become available to children, as it relinquishes the need for lifelong immunosuppression.
Extensive Human Tissue Biobank
The ITRG benefits from the exclusive use of a comprehensive human tissue biobank, containing pancreatic tissue and islet samples for research use, collected from a wide range of donors. Biopsies are obtained from all organs that are received by the facility, which have the appropriate written research consent. This has allowed our group to build a power bioresource, which contains an extensive tissue sample portfolio that spans over a decade. Such samples are an invaluable asset for our research.
Some of our current and ongoing research projects are highlighted below.
Characterising the pancreatic extracellular matrix to enhance pancreas digestion
The main prerequisite for any successful islet transplantation procedure is the requirement to isolate a sufficient number of intact and viable islets from the donated human pancreas. The extraction of islets from their native environment occurs during the critical pancreas digestion phase of the isolation process. This involves the infusion of commercially available collagenase based enzymes through the main pancreatic duct. The infused enzymes then become distributed at the regional interface that separates the islet from the general exocrine tissue (the ‘islet-exocrine interface’). Aided by mechanical shaking, the enzymes digest the pancreatic extracellular matrix (ECM), to release ‘free’ (devoid of exocrine tissue) islets. As the majority of the ECM is collagen derived, we used commercially available collagenase for this purpose, as these enzymes are capable of breaking down collagen. In addition, we add a supplementary protease, which has shown to be beneficial for enhancing pancreas digestion.
Although it is possible to isolate sufficient numbers of islets from pancreases retrieved from older donors, isolating large numbers of islets from donors aged under 35 is an extreme technical challenge. Central to this issue, is the inability for the commercially available enzymes to digest the pancreatic ECM in this donor group. This is likely to result from changes in quantity and composition of the proteins that make up the ECM (collagens, laminins etc). However, such assumptions have not been studied in detail at the molecular level. By defining donor age specific changes in the protein structure of the pancreas, we have the potential to allow the digestion enzymes to be tailored to the specific protein composition within younger donors. This will allow us to improve isolation success rates from the full pool of donors.
Our group have characterised age related differences in the pancreatic ECM using a range of novel high-throughput technologies, including Raman microspectroscopy and liquid chromatography tandem mass spectrometry. Our findings suggest that pancreases from younger donors have an increased expression of collagens, compared to those from older donors. We have also shown that age affects the way in which collagenase digests the matrix. Our ongoing experiments are therefore investigating how increasing the concentration of collagenase used in the isolation of islets from younger donors may help to increase pancreas digestion from this donor type. Research within this subtheme is led by Dr Rebecca Spiers.
Optimising islet survival and function after transplantation (Re-establishing of the peri-islet pancreatic matrix)
Effective and highly active enzyme blends quickly disperses all exocrine, ductal and vascular components of the pancreas. The dissociation of the non-endocrine structures includes also the islet basement membrane which is the most relevant peri-islet barrier serving as a communication interface between islets, blood vessels, nerves and acinar cells, to maintain the functional and structural integrity of native islets. The loss of the islet basement membrane has significant implications on the survival of isolated islets outside their native environment. Our current work is directed to replace the natural islet basement membrane by recombinant extracellular matrix proteins, namely collagen, laminin and nidogen. We could demonstrate that these matrix proteins are highly efficient to protect islet integrity in a hypoxic harmful environment as found after transplantation. Due to the enormous complexity of the native islet basement membrane, more efforts are required in the future to transfer our findings into a clinical setting. Dr Daniel and Dr Heide Brandhorst lead this research subtheme.
Minimising the immunosuppressive treatment of the islet graft recipient
The successful implantation of isolated islets in an allogeneic patient with type 1 Diabetes mellitus essentially requires life-long immunosuppression to prevent rejection of the islet transplant by the recipient’ s immune system and to protect ongoing function of the graft. Several clinical trials revealed that a potent immunosuppression, which is effective to support islet graft survival in the recipients, is associated with severe adverse events, which affect the large majority of diabetic patients treated with islet transplants. Therefore, risks and benefits of allogeneic islet transplantation has to be carefully balanced for any potential recipient particularly concerning the progression of diabetic complications. The only present concept to maintain islet graft survival without continuous immunosuppression is the implantation of isolated islets in immuno-protective devices, such as microcapsules or macroencapsulation devices. Currently, we are involved in a multidisciplinary approach that is dedicated to optimize the isolation from the activated immune system but also to implement factors and conditions that are most relevant for survival and function of macroencapsulated islets, such as oxygen supply and the presence of an extracellular matrix. Initial findings suggest that this concept is very efficient to improve survival of islets when implanted in immuno-protective macrodevices. Dr Daniel Brandhorst and Dr Heide Brandhorst lead this research subtheme.
Strategies to Improve the Oxygen Supply to Microencapsulated Islets.
Brandhorst D. et al, (2020), Transplantation, 104
Donor age significantly influences the Raman spectroscopic biomolecular fingerprint of human pancreatic extracellular matrix proteins following collagenase-based digestion.
Spiers RM. et al, (2019), Acta Biomater, 99, 269 - 283
Bradshaw CJ. and Johnson PRV., (2019), Surgery (United Kingdom), 37, 216 - 220
Comparison of Clostripain and Neutral Protease as Supplementary Enzymes for Human Islet Isolation.
Brandhorst H. et al, (2019), Cell Transplant, 28, 176 - 184