Optimizing Temperature and Oxygen Supports Long-term Culture of Human Islets

Komatsu, Hirotake; Rawson, Jeffrey; Medrano, Leonard; Cook, Colin A.; Barriga, Alyssa; Gonzalez, Nelson; Salgado, Mayra; Omori, Keiko; Kandeel, Fouad; Tai, Yu‐Chong; Mullen, Yoko

doi:10.1097/tp.0000000000002280

Cited by 18 publications

(21 citation statements)

References 44 publications

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“…It is also associated with a loss of beta cells but not with a reduction in their percentage [22], and it can be used to combine preparations if quantitative criteria are not immediately met, while preserving the desired metabolic effect in patients [8]. However, since beta cell recovery after culture may be influenced by many confounders [22,25,26], the measurement of beta cells after purification was used to examine if DCD III organs are suitable for the preparation of islet cell grafts. Islet mass serves since long as a criterium to select pancreatic islet isolates for clinical transplantation [1,6,15].…”

Section: Discussionmentioning

confidence: 99%

Lower beta cell yield from donor pancreases after controlled circulatory death prevented by shortening acirculatory warm ischemia time and by using IGL-1 cold preservation solution

et al. 2021

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Organs from donors after controlled circulatory death (DCD III) exhibit a higher risk for graft dysfunction due to an initial period of warm ischemia. This procurement condition can also affect the yield of beta cells in islet isolates from donor pancreases, and hence their use for transplantation. The present study uses data collected and generated by our Beta Cell Bank to compare the number of beta cells in isolates from DCD III (n = 141) with that from donors after brain death (DBD, n = 609), before and after culture, and examines the influence of donor and procurement variables. Beta cell number per DCD III-organ was significantly lower (58 x 106 versus 84 x 106 beta cells per DBD-organ; p < 0.001) but their purity (24% insulin positive cells) and insulin content (17 μg / 106 beta cells in DCD III-organs versus 19 μg / 106 beta cells in DBD-organs) were similar. Beta cell number correlated negatively with duration of acirculatory warm ischemia time above 10 min; for shorter acirculatory warm ischemia time, DCD III-organs did not exhibit a lower beta cell yield (74 x 106 beta cells). Use of Institut Georges Lopez-1 cold preservation solution instead of University of Wisconsin solution or histidine-tryptophan-ketoglutarate also protected against the loss in beta cell yield from DCD III-organs (86 x 106 for IGL-1 versus 54 x 106 and 65 x 106 beta cells respectively, p = 0.042). Multivariate analysis indicates that both limitation of acirculatory warm ischemia time and use of IGL-1 prevent the reduced beta cell yield in islet cell isolates from DCD III-organs.

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Section: Discussionmentioning

confidence: 99%

Lower beta cell yield from donor pancreases after controlled circulatory death prevented by shortening acirculatory warm ischemia time and by using IGL-1 cold preservation solution

et al. 2021

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“…This explains why viability-conventional is overestimated compared to viability-semi-automated in islet batches containing large islets. Automated viability determination has recently been applied with success in various studies 10 , 16 . In addition to being a resourceful tool for assessing islet viability, a similar automated method has been developed to evaluate islet purity using zinc-chelating DTZ staining 24 , 25 .…”

Section: Discussionmentioning

confidence: 99%

“…Nonobese diabetic, severe combined immunodeficiency mice (Charles River Laboratories, Wilmington, MA, USA) were used as recipients for islet transplantations, as previously described 16 . Mice were rendered diabetic by streptozotocin injections (50 mg/kg, intraperitoneal injections in consecutive 3 d).…”

Section: Methodsmentioning

confidence: 99%

Semi-Automated Assessment of Human Islet Viability Predicts Transplantation Outcomes in a Diabetic Mouse Model

et al. 2020

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In clinical and experimental human pancreatic islet transplantations, establishing pretransplant assessments that accurately predict transplantation outcomes is crucial. Conventional in vitro viability assessment that relies on manual counting of viable islets is a routine pretransplant assessment. However, this method does not correlate with transplantation outcomes; to improve the method, we recently introduced a semi-automated method using imaging software to objectively determine area-based viability. The goal of the present study was to correlate semi-automated viability assessment with posttransplantation outcomes of human islet transplantations in diabetic immunodeficient mice, the gold standard for in vivo functional assessment of isolated human islets. We collected data from 61 human islet isolations and 188 subsequent in vivo mouse transplantations. We assessed islet viability by fluorescein diacetate and propidium iodide staining using both the conventional and semi-automated method. Transplantations of 1,200 islet equivalents under the kidney capsule were performed in streptozotocin-induced diabetic immunodeficient mice. Among the pretransplant variables, including donor factors and post-isolation assessments, viability measured using the semi-automated method demonstrated a strong influence on in vivo islet transplantation outcomes in multivariate analysis. We calculated an optimized cutoff value (96.1%) for viability measured using the semi-automated method and showed a significant difference in diabetes reversal rate for islets with viability above this cutoff (77% reversal) vs. below this cutoff (49% reversal). We performed a detailed analysis to show that both the objective measurement and the improved area-based scoring system, which distinguished between small and large islets, were key features of the semi-automated method that allowed for precise evaluation of viability. Taken together, our results suggest that semi-automated viability assessment offers a promising alternative pretransplant assessment over conventional manual assessment to predict human islet transplantation outcomes.

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“…However, isolated islets were stressed, and islet function was impaired during the in vitro cultivation period, which may result in unsatisfactory transplant outcomes. Various efforts have been made to preserve islet mass and function before transplantation, including supplementation of additives like green tea extract ( 48 ) and liraglutide ( 49 ); use of scaffolds or extracellular matrix ( 50 – 52 ), and modulation of the culture temperature and gas environment ( 53 , 54 ). To the best of our knowledge, this is the first study that demonstrated how using human amniotic membrane extract as medium supplementation to improve islet viability and function.…”

Section: Discussionmentioning

confidence: 99%

Amniotic Membrane Extract Protects Islets From Serum-Deprivation Induced Impairments and Improves Islet Transplantation Outcome

Yang

Zhang

et al. 2020

Front. Endocrinol.

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Islet culture prior to transplantation is a standard practice in many transplantation centers. Nevertheless, the abundant islet mass loss and function impairment during this serum-deprivation culture period restrain the success of islet transplantation. In the present study, we used a natural biomaterial derived product, amniotic membrane extract (AME), as medium supplementation of islet pretransplant cultivation to investigate its protective effect on islet survival and function and its underlying mechanisms, as well as the engraftment outcome of islets following AME treatment. Results showed that AME supplementation improved islet viability and function, and decreased islet apoptosis and islet loss during serum-deprived culture. This was associated with the increased phosphorylation of PI3K/Akt and MAPK/ERK signaling pathway. Moreover, transplantation of serum-deprivation stressed islets that were pre-treated with AME into diabetic mice revealed better blood glucose control and improved islet graft survival. In conclusion, AME could improve islet survival and function in vivo and in vitro, and was at least partially through increasing phosphorylation of PI3K/Akt and MAPK/ERK signaling pathway.

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Optimizing Temperature and Oxygen Supports Long-term Culture of Human Islets

Abstract: By optimizing temperature and oxygen concentration, we cultured human islets for 2 weeks with minimal loss of volume and function.

Cited by 18 publications

References 44 publications

Lower beta cell yield from donor pancreases after controlled circulatory death prevented by shortening acirculatory warm ischemia time and by using IGL-1 cold preservation solution

Lower beta cell yield from donor pancreases after controlled circulatory death prevented by shortening acirculatory warm ischemia time and by using IGL-1 cold preservation solution

Semi-Automated Assessment of Human Islet Viability Predicts Transplantation Outcomes in a Diabetic Mouse Model

Amniotic Membrane Extract Protects Islets From Serum-Deprivation Induced Impairments and Improves Islet Transplantation Outcome

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