Optimal Time to Ship Human Islets Post Tissue Culture to Maximize Islet

Access to functional high-quality pancreatic human islets is critical to advance diabetes research. The Integrated Islet Distribution Program (IIDP), a major source for human islet distribution for over 15 years, conducted a study to evaluate the most advantageous times to ship islets postisolation to maximize islet recovery. For the evaluation, three experienced IIDP Islet Isolation Centers each provided samples from five human islet isolations, shipping 10,000 islet equivalents (IEQ) at four different time periods postislet isolation (no 37°C culture and shipped within 0 to 18 hours; or held in 37°C culture for 18 to 42, 48 to 96, or 144 to 192 hours). A central evaluation center compared samples for islet quantity, quality, and viability for each experimental condition preshipment and postshipment, as well as post 37°C culture 18 to 24 hours after shipment receipt. Additional evaluations included measures of functional potency by static glucose-stimulated insulin release (GSIR), represented as a stimulation index. Comparing the results of the four preshipment holding periods, the greatest IEQ loss postshipment occurred with the shortest preshipment times. Similar patterns emerged when comparing preshipment to postculture losses. In vitro islet function (GSIR) was not adversely impacted by increased tissue culture time. These data indicate that allowing time for islet recovery postisolation, prior to shipping, yields less islet loss during shipment without decreasing islet function.

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Research-Focused Isolation of Human Islets From Donors With and Without Diabetes at the Alberta Diabetes Institute IsletCore

Endocrinology ◽

10.1210/en.2015-1562 ◽

2015 ◽

Vol 157 (2) ◽

pp. 560-569 ◽

Cited By ~ 38

Author(s):

James Lyon ◽

Jocelyn E. Manning Fox ◽

Aliya F. Spigelman ◽

Ryekjang Kim ◽

Nancy Smith ◽

...

Keyword(s):

Type 2 Diabetes ◽

Glycated Hemoglobin ◽

Organ Procurement ◽

Human Islets ◽

Human Islet ◽

Islet Function ◽

Small Contribution ◽

Islet Isolation ◽

Diabetes Status

Abstract Recent years have seen an increased focus on human islet biology, and exciting findings in the stem cell and genomic arenas highlight the need to define the key features of mature human islets and β-cells. Donor and organ procurement parameters impact human islet yield, although for research purposes islet yield may be secondary in importance to islet function. We examined the feasibility of a research-only human islet isolation, distribution, and biobanking program and whether key criteria such as cold ischemia time (CIT) and metabolic status may be relaxed and still allow successful research-focused isolations, including from donors with type 1 diabetes and type 2 diabetes. Through 142 isolations over approximately 5 years, we confirm that CIT and glycated hemoglobin each have a weak negative impacts on isolation purity and yield, and extending CIT beyond the typical clinical isolation cutoff of 12 hours (to ≥ 18 h) had only a modest impact on islet function. Age and glycated hemoglobin/type 2 diabetes status negatively impacted secretory function; however, these and other biological (sex, body mass index) and procurement/isolation variables (CIT, time in culture) appear to make only a small contribution to the heterogeneity of human islet function. This work demonstrates the feasibility of extending acceptable CIT for research-focused human islet isolation and highlights the biological variation in function of human islets from donors with and without diabetes.

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Interleukin-22 reverses human islet dysfunction and apoptosis triggered by hyperglycemia and LIGHT

Journal of Molecular Endocrinology ◽

10.1530/jme-17-0182 ◽

2018 ◽

Vol 60 (3) ◽

pp. 171-183

Author(s):

Shadab Abadpour ◽

Bente Halvorsen ◽

Afaf Sahraoui ◽

Olle Korsgren ◽

Pål Aukrust ◽

...

Keyword(s):

Islet Cells ◽

Harmful Effect ◽

Human Islets ◽

Protective Role ◽

Human Islet ◽

Islet Function ◽

Post Transplantation ◽

Interleukin 22 ◽

Glucose Stimulated Insulin Secretion

Interleukin (IL)-22 has recently been suggested as an anti-inflammatory cytokine that could protect the islet cells from inflammation- and glucose-induced toxicity. We have previously shown that the tumor necrosis factor family member, LIGHT, can impair human islet function at least partly via pro-apoptotic effects. Herein, we aimed to investigate the protective role of IL-22 on human islets exposed to the combination of hyperglycemia and LIGHT. First, we found upregulation of LIGHT receptors (LTβR and HVEM) in engrafted human islets exposed to hyperglycemia (>11 mM) for 17 days post transplantation by using a double islet transplantation mouse model as well as in human islets cultured with high glucose (HG) (20 mM glucose) + LIGHT in vitro, and this latter effect was attenuated by IL-22. The effect of HG + LIGHT impairing glucose-stimulated insulin secretion was reversed by IL-22. The harmful effect of HG + LIGHT on human islet function seemed to involve enhanced endoplasmic reticulum stress evidenced by upregulation of p-IRE1α and BiP, elevated secretion of pro-inflammatory cytokines (IL-6, IL-8, IP-10 and MCP-1) and the pro-coagulant mediator tissue factor (TF) release and apoptosis in human islets, whereas all these effects were at least partly reversed by IL-22. Our findings suggest that IL-22 could counteract the harmful effects of LIGHT/hyperglycemia on human islet cells and potentially support the strong protective effect of IL-22 on impaired islet function and survival.

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Comparison of Purification Solutions with Different Osmolality for Porcine Islet Purification

Cell Medicine ◽

10.3727/215517916x693140 ◽

2017 ◽

Vol 9 (1-2) ◽

pp. 53-59 ◽

Cited By ~ 9

Author(s):

Chika Miyagi-Shiohira ◽

Naoya Kobayashi ◽

Issei Saitoh ◽

Masami Watanabe ◽

Yasufumi Noguchi ◽

...

Keyword(s):

Salt Solution ◽

Stimulation Index ◽

Human Islets ◽

Human Islet ◽

Low Density ◽

Islet Isolation ◽

Continuous Density ◽

Yield Rate ◽

Purification Efficiency ◽

Porcine Islet

The osmolality of the purification solution is one of the most critical variables in human islet purification during islet isolation. We previously reported the effectiveness of a combined continuous density/osmolality gradient for the supplemental purification of human islets. We herein applied a combined continuous density/osmolality gradient for regular purification. The islets were purified with a continuous density gradient without osmolality preparation [continuous density/normal osmolality (CD/NO)] or continuous density/osmolality solution with osmolality preparation by 10× Hank's balanced salt solution (HBSS) [continuous density/continuous osmolality (CD/CO)]. The osmolality of the low-density solution was 400 mOsm/kg in both groups and that of the high-density solution was 410 mOsm/kg in the CD/NO group and 500 mOsm/kg in the CD/CO group. Unexpectedly, we noted no significant differences between the two solutions in terms of the islet yield, rate of viability and purity, score, stimulation index, or the attainability and suitability of posttransplantation normoglycemia. Despite reports that the endocrine and exocrine tissues of pancreata have distinct osmotic sensitivities and that high-osmolality solutions result in greater purification efficiency, the isolation and transplant outcomes did not markedly differ between the two purification solutions with different osmolalities in this study.

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Comparison of Clostripain and Neutral Protease as Supplementary Enzymes for Human Islet Isolation

Cell Transplantation ◽

10.1177/0963689718811614 ◽

2018 ◽

Vol 28 (2) ◽

pp. 176-184 ◽

Cited By ~ 2

Author(s):

Heide Brandhorst ◽

Paul R. Johnson ◽

Johanna Mönch ◽

Manfred Kurfürst ◽

Olle Korsgren ◽

...

Keyword(s):

Islet Transplantation ◽

Stimulation Index ◽

Effective Means ◽

Insulin Response ◽

Human Islets ◽

Human Islet ◽

Neutral Protease ◽

Islet Isolation ◽

Clinical Islet Transplantation ◽

Glucose Challenge

Although human islet transplantation has been established as valid and safe treatment for patients with type 1 diabetes, the utilization rates of human pancreases for clinical islet transplantation are still limited and substantially determined by the quality and composition of collagenase blends. While function and integrity of collagenase has been extensively investigated, information is still lacking about the most suitable supplementary neutral proteases. The present study compared islet isolation outcome after pancreas digestion by means of collagenase used alone or supplemented with either neutral protease (NP), clostripain (CP), or both proteases. Decent amounts of islet equivalents (IEQ) were isolated using collagenase alone (3090 ± 550 IEQ/g), or in combination with NP (2340 ± 450 IEQ/g) or CP (2740 ± 280 IEQ/g). Nevertheless, the proportion of undigested tissue was higher after using collagenase alone (21.1 ± 1.1%, P < 0.05) compared with addition of NP (13.3 ± 2.2%) or CP plus NP (13.7 ± 2.6%). Likewise, the percentage of embedded islets was highest using collagenase only (13 ± 2%) and lowest adding NP plus CP (4 ± 1%, P < 0.01). The latter combination resulted in lowest post-culture overall survival (42.7 ± 3.9%), while highest survival was observed after supplementation with CP (74.5 ± 4.8%, P < 0.01). An insulin response toward glucose challenge was present in all experimental groups, but the stimulation index was significantly decreased using collagenase plus NP (2.0 ± 0.12) compared with supplementation with CP (3.16 ± 0.4, P < 0.001). This study demonstrates for the first time that it is possible to isolate significant numbers of human islets combining collagenase only with CP. The supplementation with CP is an effective means to substantially reduce NP activity, which significantly decreases survival and viability after culture. This will facilitate the manufacturing of enzyme blends with less harmful characteristics.

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Dose-dependent effects of necrostatin-1 supplementation to tissue culture media of young porcine islets

PLoS ONE ◽

10.1371/journal.pone.0243506 ◽

2020 ◽

Vol 15 (12) ◽

pp. e0243506

Author(s):

Hien Lau ◽

Nicole Corrales ◽

Samuel Rodriguez ◽

Colleen Luong ◽

Mohammadreza Mohammadi ◽

...

Keyword(s):

Tissue Culture ◽

Insulin Secretion ◽

Beta Cells ◽

Culture Media ◽

Stimulation Index ◽

Optimal Dose ◽

Human Islets ◽

Porcine Islets ◽

Different Doses

Previous studies have shown that necrostatin-1 (Nec-1) supplementation improved the viability of murine islets following exposure to nitric oxide, increased the survival of human islets during hypoxic culture, and augmented the maturation of pre-weaned porcine islets (PPIs) after 7 days of tissue culture. A limitation of these studies is that only one concentration of Nec-1 was used, and no studies have determined the optimal dose of Nec-1 for PPIs. Thus, the present study examined the effects of Nec-1 on PPIs at four different doses—0, 25, 50, 100, and 200 μM—after 7 days of tissue culture when supplemented on day 3. PPIs were isolated from pancreata of pre-weaned Yorkshire piglets (8–15 days old) and cultured in a specific islet maturation media added with Nec-1 on day 3 of tissue culture at 4 different doses—0, 25, 50, 100, and 200 μM (n = 6 for each dose). After 7 days of tissue culture, islets were assessed for recovery, viability, endocrine cellular content, GLUT2 expression in beta cells, and insulin secretion after glucose challenge. Nec-1 did not affect the viability of both intact islets and dissociated islets cells during tissue culture regardless of doses. Islets cultured in media supplemented with Nec-1 at 100 μM, but not 25, 50, or 200 μM, had a significantly higher recovery, composition of endocrine cells, GLUT2 expression in beta cells, and insulin secretion capacity than control islets cultured in media without Nec-1 supplementation. Moreover, culturing islets in 200 μM Nec-1 supplemented media not only failed to improve the insulin release but resulted in a lower glucose-induced insulin stimulation index compared to islets cultured in media added with 100 μM Nec-1. Xenotransplantation using porcine islets continues to demonstrate scientific advances to justify this area of research. Our findings indicate that Nec-1 supplementation at 100 μM was most effective to enhance the in vitro maturation of PPIs during tissue culture.

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Mesenchymal stromal cells improve human islet function through released products and extracellular matrix

Clinical Science ◽

10.1042/cs20171251 ◽

2017 ◽

Vol 131 (23) ◽

pp. 2835-2845 ◽

Cited By ~ 36

Author(s):

Ahmed A. Arzouni ◽

Andreia Vargas-Seymour ◽

Chloe L. Rackham ◽

Paramjeet Dhadda ◽

Guo-Cai Huang ◽

...

Keyword(s):

Extracellular Matrix ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Human Islets ◽

Human Islet ◽

Human Mscs ◽

Islet Function ◽

Secretory Function ◽

Beneficial Effects

Aims: The aims of the present study were (i) to determine whether the reported beneficial effects of mesenchymal stromal cells (MSCs) on mouse islet function extend to clinically relevant human tissues (islets and MSCs), enabling translation into improved protocols for clinical human islet transplantation; and (ii) to identify possible mechanisms through which human MSCs influence human islet function. Materials and methods: Human islets were co-cultured with human adipose tissue-derived MSCs (hASCs) or pre-treated with its products – extracellular matrix (ECM) and annexin A1 (ANXA1). Mouse islets were pre-treated with mouse MSC-derived ECM. Islet insulin secretory function was assessed in vitro by radioimmunoassay. Quantitative RT-PCR was used to screen human adipMSCs for potential ligands of human islet G-protein-coupled receptors. Results: We show that co-culture with hASCs improves human islet secretory function in vitro, as measured by glucose-stimulated insulin secretion, confirming previous reports using rodent tissues. Furthermore, we demonstrate that these beneficial effects on islet function can be partly attributed to the MSC-derived products ECM and ANXA1. Conclusions: Our results suggest that hASCs have the potential to improve the quality of human islets isolated for transplantation therapy of Type 1 diabetes. Furthermore, it may be possible to achieve improvements in human islet quality in a cell-free culture system by using the MSC-derived products ANXA1 and ECM.

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Pseudoislets for diabetes research: Purifying and selective re-aggregating human islet cells v1

10.17504/protocols.io.bwsgpebw ◽

2021 ◽

Author(s):

Wei Liu ◽

Craig Dorrell ◽

Xiaojuan Chen

Keyword(s):

Islet Cells ◽

Human Islet ◽

Diabetes Research ◽

In Vitro Modeling

In vitro modeling of human islet cells for diabetes research utilizing purified and then selectively re-aggregated various combinations of human islet cells.

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Proteomic Profiling Reveals the Ambivalent Character of the Mesenchymal Stem Cell Secretome: Assessing the Effect of Preconditioned Media on Isolated Human Islets

Cell Transplantation ◽

10.1177/0963689720952332 ◽

2020 ◽

Vol 29 ◽

pp. 096368972095233

Author(s):

Heide Brandhorst ◽

Daniel Brandhorst ◽

Anju Abraham ◽

Samuel Acreman ◽

Simen W. Schive ◽

...

Keyword(s):

Culture Media ◽

Human Islets ◽

Human Islet ◽

Physical Contact ◽

Proteomic Profiling ◽

Protective Capacity ◽

Islet Culture ◽

Proinflammatory Mediators ◽

Related Proteins

Previous studies in rodents have indicated that function and survival of transplanted islets can be substantially improved by mesenchymal stem cells (MSC). The few human islet studies to date have confirmed these findings but have not determined whether physical contact between MSC and islets is required or whether the benefit to islets results from MSC-secreted proteins. This study aimed to investigate the protective capacity of MSC-preconditioned media for human islets. MSC were cultured for 2 or 5 days in normoxia or hypoxia before harvesting the cell-depleted media for human islet culture in normoxia or hypoxia for 6–8 or 3–4 days, respectively. To characterize MSC-preconditioned media, proteomic secretome profiling was performed to identify angiogenesis- and inflammation-related proteins. A protective effect of MSC-preconditioned media on survival and in vitro function of hypoxic human islets was observed irrespective of the atmosphere used for MSC preconditioning. Islet morphology changed markedly when media from hypoxic MSC were used for culture. However, PDX-1 and insulin gene expression did not confirm a change in the genetic phenotype of these islets. Proteomic profiling of preconditioned media revealed the heterogenicity of the secretome comprising angiogenic and antiapoptotic as well as angiostatic or proinflammatory mediators released at an identical pattern regardless whether MSC had been cultured in normoxic or hypoxic atmosphere. These findings do not allow a clear discrimination between normoxia and hypoxia as stimulus for protective MSC capabilities but indicate an ambivalent character of the MSC angiogenesis- and inflammation-related secretome. Nevertheless, culture of human islets in acellular MSC-preconditioned media resulted in improved morphological and functional islet integrity suggesting a disbalance in favor of protective factors. Further approaches should aim to eliminate potentially detrimental factors to enable the production of advanced clinical grade islet culture media with higher protective qualities.

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Bulk Cryopreservation of Isolated Islets of Langerhans

Cell Transplantation ◽

10.1177/096368979600500306 ◽

1996 ◽

Vol 5 (3) ◽

pp. 395-404 ◽

Cited By ~ 8

Author(s):

Jonathan R.T. Lakey ◽

Garth L. Warnock ◽

Ziliang Ao ◽

Ray V. Rajotte

Keyword(s):

Islets Of Langerhans ◽

Stimulation Index ◽

Glass Tube ◽

Human Islets ◽

Isolated Islets ◽

Freezing And Thawing ◽

Slow Cooling ◽

Glass Tubes ◽

Multiple Donors

Current methods to isolate human islets of Langerhans are limited and multiple donors are required for successful reversal of longstanding Type 1 diabetes mellitus. Cryopreservation of isolated islets is an effective method of storing and pooling islets. Current cryopreservation protocols are cumbersome due to current practices of placing small aliquots of islets per individual freezer tube. In the present study, we examined the application of a blood freezer bag for the cryopreservation of isolated islets by slow cooling and rapid thawing. Freezing and thawing profiles generated using thermocouples placed inside a 500 mL Cryocyte (Baxter) blood freezer bag showed that a longer equilibration period at −7.4°C was necessary to consistently achieve nucleation and cooling profiles similar to those observed in glass tubes. When known numbers of rat islets were placed in the freezer bag and the cryoprotectant dimethyl sulfoxide (DMSO) was added in a stepwise fashion and removed using a sucrose dilution, the islet recovery compared with glass tubes was 92 ± 4.8 vs. 90 ± 2.3% (n = 4, p = ns, Mann-Whitney U-test). When purified canine islets were cryopreserved in a single freezer bag or in multiple glass tubes, the recovery was similar (78.8 ± 12.5% recovery for freezer bag vs. 82.3 ± 5.3% for glass tubes; n = 6, p = ns). In vitro function was equivalent for both groups. The stimulation index of insulin release during glucose perifusion (stimulated over basal insulin secretion) for canine islets cryopreserved in a freezer bag vs. glass tubes was 3.2 ± 1.0 and 2.3 ± 1.3, respectively (n = 6, p = ns). These values were significantly lower than the nonfrozen control islets (6.9 ± 2.4, p < 0.05). When 2000 canine islets cryopreserved in either a freezer bag, or glass tubes were transplanted into diabetic nude mice, the animals became and remained normoglycemic posttransplant. We conclude that the survival of freshly isolated canine islets cryopreserved in a single freezer bag is equivalent to the glass tube method. Bulk cryopreservation of islets in a single freezer bag will facilitate effective low temperature tissue banking to support ongoing clinical trials of islet transplantation.

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