Effect of short-term culture on functional and stress-related parameters in isolated human islets

Abstract

Summary The Edmonton protocol for islet transplantation utilizes fresh islet grafts but other protocols increasingly transplant short‐term cultured grafts mainly for practical reasons. To improve our understanding of the impact of culture pretreatment of human islets, we assessed post‐transplant function by nude mouse bioassay, islet ATP, activity of stress‐activated MAP kinases, and expression of stress‐related genes by focused cDNA array in freshly isolated and cultured islets. Mean blood glucose levels over 4 weeks after transplantation (2000 IE) of (i) freshly isolated, (ii) cultured and preculture counted (recovery rate; 78 ± 6%), and (iii) cultured and postculture counted islets in diabetic mice were 330 ± 40, 277 ± 65, and 256 ± 52 mg/dl (i versus ii, P = 0.004; i versus iii, P = 0.002). During culture, islet ATP/DNA and ATP/ADP increased; JNK and p38 MAPK activities decreased. Among 96 genes studied, mRNA expression of heat shock protein 70 genes decreased >twofold during culture in all four pairs; expression of cyclooxygenase‐2, superoxide dismutase‐2, interleukin‐6 and cytochromes P450 1A1 genes increased. Our results show that culturing human islets before transplantation is not disadvantageous in regard of functional recovery from changes induced by nonphysiologic stimuli during islet isolation. The increase in expression of several stress‐related genes during culture also shows that improving culture conditions may further enhance post‐transplant islet function.

Quantitative Assessment of Islets of Langerhans Encapsulated in Alginate

“…15,16 Examination of viability by assessment of membrane integrity with vital stains such as fluorescein diacetate/propidium iodide does not reflect true viability because it may not account for cells undergoing early cell death processes, during which cells have not yet developed damage to their cell membrane. 8,9,17,18 Measures of cellular energetic state, including ATP content [19][20][21][22][23][24] and ATP/ADP and related ratios, [25][26][27] have been investigated with modest success. ATP content varies enormously between islets from different species, between human islet preparations, [20][21][22] with time in culture, 20,21 and with donor age.…”

Endoplasmic Reticulum Stress: An Unrecognized Actor in Solid Organ Transplantation

“…Isolated human pancreatic islets are susceptible to many insults that occur during donorbrain death, organ preservation, and islet isolation resulting in ischemic, mechanical, osmotic, and oxidative stress for islets. The islet grafts are further exposed to a local inflammatory and hypoxic microenvironment in the early stage posttransplant (54). Most of these extracellular insults can result in ER stress.…”

Pathogen Inactivation of Human Serum Facilitates its Clinical Use for Islet Cell Culture and Subsequent Transplantation

“…A period of culture has benefits not only for islet recovtries (50), has been reported to be safe in preclinical studies (9)(10)(11) and to be well tolerated in a wide range ery, as it replenishes islet ATP stores depleted during both cold ischemia period and the isolation procedure, of patients (22, 41,42,45,52,56). It has even been used to prevent transfusion transmission of Chikungunya virus but also increases posttransplant function of human islets transplanted into diabetic nude mice (2,5,23,26).…”