Functional islets and where to find them

This thesis explores methods and strategies to most optimally isolate viable, functional pancreatic islets from donor pancreases. After analyzing the current challenges within the field, the manuscript examines the applicability of donation after circulatory death pancreases for islet isolation. The results of these isolations are discussed in the next chapter. A novel strategy to judge suitability of all donor pancreases is presented hereafter. The Islet Isolation Outcome Prediction Score is a multifactorial donor risk index in predicting obtained islet equivalents after isolation. After an organ is allocated and accepted, it must be preserved before it arrives at the isolation facility. To optimize this process, the use of hypothermic machine perfusion is explored in a feasibility study. Next, a novel islet isolation technique, the PancReatic Islet Separation Method is presented. This method can vastly improve isolation duration, efficiency, and standardization. Lastly, the results of the thesis are discussed.LUMC / Geneeskund

Clinical use of donation after circulatory death pancreas for islet transplantation

Due to a shortage of donation after brain death (DBD) organs, donation after circulatory death (DCD) is increasingly performed. In the field of islet transplantation, there is uncertainty regarding the suitability of DCD pancreas in terms of islet yield and function after islet isolation. The aim of this study was to investigate the potential use of DCD pancreas for islet transplantation. Islet isolation procedures from 126 category 3 DCD and 258 DBD pancreas were performed in a 9-year period. Islet yield after isolation was significantly lower for DCD compared to DBD pancreas (395 515 islet equivalents [IEQ] and 480 017 IEQ, respectively; p = .003). The decrease in IEQ during 2 days of culture was not different between the two groups. Warm ischemia time was not related to DCD islet yield. In vitro insulin secretion after a glucose challenge was similar between DCD and DBD islets. After islet transplantation, DCD islet graft recipients had similar graft function (AUC C-peptide) during mixed meal tolerance tests and Igls score compared to DBD graft recipients. In conclusion, DCD islets can be considered for clinical islet transplantation.Diabetes mellitus: pathophysiological changes and therap

Metabolic needs of the kidney graft undergoing normothermic machine perfusion

Normothermic machine perfusion (NMP) is emerging as a novel preservation strategy. During NMP, the organ is maintained in a metabolically active state that may not only provide superior organ preservation, but that also facilitates viability testing before transplantation, and ex situ resuscitation of marginal kidney grafts. Although the prevailing perfusion protocols for renal NMP are refined from initial pioneering studies concerning short periods of NMP, it could be argued that these protocols are not optimally tailored to address the putatively compromised metabolic plasticity of marginal donor grafts (i.e., in the context of viability testing and/or preservation), or to meet the metabolic prerequisites associated with prolonged perfusions and the required anabolic state in the context of organ regeneration. Herein, we provide a theoretical framework for the metabolic requirements for renal NMP. Aspects are discussed along the lines of carbohydrates, fatty acids, amino acids, and micronutrients required for optimal NMP of an isolated kidney. In addition, considerations for monitoring aspects of metabolic status during NMP are discussed.Transplant surger

Novel explanted human liver model to assess hepatic extraction, biliary excretion, and transporter function

Realistic models predicting hepatobiliary processes in health and disease are lacking. We therefore aimed to develop a physiologically relevant human liver model consisting of normothermic machine perfusion (NMP) of explanted diseased human livers that can assess hepatic extraction, clearance, biliary excretion, and drug–drug interaction (DDI). Eleven livers were included in the study, seven with a cirrhotic and four with a noncirrhotic disease background. After explantation of the diseased liver, NMP was initiated. After 120 minutes of perfusion, a drug cocktail (rosuvastatin, digoxin, metformin, and furosemide; OATP1B1/1B3, P-gp, BCRP, and OCT1 model compounds) was administered to the portal vein and 120 minutes later, a second bolus of the drug cocktail was co-administered with perpetrator drugs to study relevant DDIs. The explanted livers showed good viability and functionality during 360 minutes of NMP. Hepatic extraction ratios close to in vivo reported values were measured. Hepatic clearance of rosuvastatin and digoxin showed to be the most affected by cirrhosis with an increase in maximum plasma concentration (Cmax) of 11.50 and 2.89 times, respectively, compared with noncirrhotic livers. No major differences were observed for metformin and furosemide. Interaction of rosuvastatin or digoxin with perpetrator drugs were more pronounced in noncirrhotic livers compared with cirrhotic livers. Our results demonstrated that NMP of human diseased explanted livers is an excellent model to assess hepatic extraction, clearance, biliary excretion, and DDI. Gaining insight into pharmacokinetic profiles of OATP1B1/1B3, P-gp, BCRP, and OCT1 model compounds is a first step toward studying transporter functions in diseased livers. Cellular mechanisms in basic and clinical gastroenterology and hepatolog

Functional islets and where to find them

This thesis explores methods and strategies to most optimally isolate viable, functional pancreatic islets from donor pancreases. After analyzing the current challenges within the field, the manuscript examines the applicability of donation after circulatory death pancreases for islet isolation. The results of these isolations are discussed in the next chapter. A novel strategy to judge suitability of all donor pancreases is presented hereafter. The Islet Isolation Outcome Prediction Score is a multifactorial donor risk index in predicting obtained islet equivalents after isolation. After an organ is allocated and accepted, it must be preserved before it arrives at the isolation facility. To optimize this process, the use of hypothermic machine perfusion is explored in a feasibility study. Next, a novel islet isolation technique, the PancReatic Islet Separation Method is presented. This method can vastly improve isolation duration, efficiency, and standardization. Lastly, the results of the thesis are discussed.</p

Functional islets and where to find them

This thesis explores methods and strategies to most optimally isolate viable, functional pancreatic islets from donor pancreases. After analyzing the current challenges within the field, the manuscript examines the applicability of donation after circulatory death pancreases for islet isolation. The results of these isolations are discussed in the next chapter. A novel strategy to judge suitability of all donor pancreases is presented hereafter. The Islet Isolation Outcome Prediction Score is a multifactorial donor risk index in predicting obtained islet equivalents after isolation. After an organ is allocated and accepted, it must be preserved before it arrives at the isolation facility. To optimize this process, the use of hypothermic machine perfusion is explored in a feasibility study. Next, a novel islet isolation technique, the PancReatic Islet Separation Method is presented. This method can vastly improve isolation duration, efficiency, and standardization. Lastly, the results of the thesis are discussed.</p

PRISM: A Novel Human Islet Isolation Technique

Background. Successful pancreatic islet isolations are a key requirement for islet transplantation in selected patients with type 1 diabetes. However, islet isolation is a technically complex, time-consuming, and manual process. Optimization and simplification of the islet isolation procedure could increase islet yield and quality, require fewer operators, and thus reduce cost. Methods. We developed a new, closed system of tissue collection, washing, buffer change, and islet purification termed PancReatic Islet Separation Method (PRISM). In the developmental phase, pump and centrifuge speed was tested using microspheres with a similar size, shape, and density as digested pancreatic tissue. After optimization, PRISM was used to isolate islets from 10 human pancreases. Results. Islet equivalents viability (fluorescein diacetate/propidium iodide), morphology, and dynamic glucose-stimulated insulin secretion were evaluated. PRISM could be performed by 1 operator in 1 flow cabinet. A similar islet yield was obtained using PRISM compared to the traditional islet isolation method (431 234 +/- 292 833 versus 285 276 +/- 197 392 islet equivalents, P = 0.105). PRISM islets had similar morphology and functionality. Conclusions. PRISM is a novel islet isolation technique that can significantly improve islet isolation efficiency using fewer operators.Nephrolog

PRISM: A Novel Human Islet Isolation Technique

Background. Successful pancreatic islet isolations are a key requirement for islet transplantation in selected patients with type 1 diabetes. However, islet isolation is a technically complex, time-consuming, and manual process. Optimization and simplification of the islet isolation procedure could increase islet yield and quality, require fewer operators, and thus reduce cost. Methods. We developed a new, closed system of tissue collection, washing, buffer change, and islet purification termed PancReatic Islet Separation Method (PRISM). In the developmental phase, pump and centrifuge speed was tested using microspheres with a similar size, shape, and density as digested pancreatic tissue. After optimization, PRISM was used to isolate islets from 10 human pancreases. Results. Islet equivalents viability (fluorescein diacetate/propidium iodide), morphology, and dynamic glucose-stimulated insulin secretion were evaluated. PRISM could be performed by 1 operator in 1 flow cabinet. A similar islet yield was obtained using PRISM compared to the traditional islet isolation method (431 234 +/- 292 833 versus 285 276 +/- 197 392 islet equivalents, P = 0.105). PRISM islets had similar morphology and functionality. Conclusions. PRISM is a novel islet isolation technique that can significantly improve islet isolation efficiency using fewer operators

Good functionality but lower yield after islet isolation from donation after circulatory death pancreata

Diabetes mellitus: pathophysiological changes and therap

Hypothermic oxygenated machine perfusion of the human pancreas for clinical islet isolation: a prospective feasibility study

Due to an increasing scarcity of pancreases with optimal donor characteristics, islet isolation centers utilize pancreases from extended criteria donors, such as from donation after circulatory death (DCD) donors, which are particularly susceptible to prolonged cold ischemia time (CIT). We hypothesized that hypothermic machine perfusion (HMP) can safely increase CIT. Five human DCD pancreases were subjected to 6 h of oxygenated HMP. Perfusion parameters, apoptosis, and edema were measured prior to islet isolation. Five human DBD pancreases were evaluated after static cold storage (SCS). Islet viability, and in vitro and in vivo functionality in diabetic mice were analyzed. Islets were isolated from HMP pancreases after 13.4 h [12.9-14.5] CIT and after 9.2 h [6.5-12.5] CIT from SCS pancreases. Histological analysis of the pancreatic tissue showed that HMP did not induce edema nor apoptosis. Islets maintained >90% viable during culture, and an appropriate in vitro and in vivo function in mice was demonstrated after HMP. The current study design does not permit to demonstrate that oxygenated HMP allows for cold ischemia extension; however, the successful isolation of functional islets from discarded human DCD pancreases after performing 6 h of oxygenated HMP indicates that oxygenated HMP may be a useful technology for better preservation of pancreases