High Seeding Density Induces Local Hypoxia and Triggers a Proinflammatory Response in Isolated Human Islets

Abstract

Hypoxia is the main threat to morphological and functional integrity of isolated pancreatic islets. Lack of oxygen seems to be of particular importance for functionality of encapsulated islets. The present study was initiated as an experimental model for the environment experienced by human islets in a confined space present during culture, shipment, and in an implanted macrodevice. Quadruplicate aliquots of isolated human islets (n = 12) were cultured for 24 h at 37°C under normoxic conditions using 24-well plates equipped with 8-μm pore size filter inserts and filled with islet aliquots adjusted to obtain a seeding density of 75, 150, 300, or 600 IEQ/cm². After culture viability, glucose-stimulated insulin release, DNA content as well as Bax and Bcl-2 gene expression were measured. Culture supernatants were collected to determine production of VEGF and MCP-1. Viability correlated inversely with IEQ seeding density (r = −0.71, p < 0.001), while the correlation of VEGF and MCP-1 secretion with seeding density was positive (r = 0.78, p < 0.001; r = 0.54, p < 0.001). Decreased viability corresponded with a significant increase in the Bax/Bcl-2 mRNA ratio at 300 and 600 IEQ/cm² and with a sigificantly reduced glucose-stimulated insulin secretion and insulin content compared to 75 or 150 IEQ/cm² (p < 0.01). The present study demonstrates that the seeding density is inversely correlated with islet viability and in vitro function. This is associated with a significant increase in VEGF and MCP-1 release suggesting a hypoxic and proinflammatory islet microenvironment.

Keywords

Human islet culture Islet macroencapsulation Seeding density Hypoxia Inflammation

Introduction

Pancreatic islet transplantation is a highly successful treatment for reversing life-threatening hypoglycemic unawareness and restoring normal glucose homeostasis in people with type 1 diabetes mellitus (T1DM)^1,2. It has the advantage of being a minimally invasive procedure, and it also has the potential to stabilize or reverse the secondary complications of T1DM including nephropathy, retinopathy, and cardiovascular disorders^3–5.

Although the procedures for pancreas preservation and islet isolation have been improved over the last decade^6–8 inadequate oxygenation is still the main negative determinant of the morphological and functional integrity of pancreatic islets isolated for subsequent transplantation. Due to the specific preference of islets for the respiratory pathway of glucose breakdown⁹, any ischemic or hypoxic situation has dramatic and immediate effects on energy generation of islets¹⁰. In view of the knowledge that even normoxic culture conditions can induce hypoxia-related cell death in the core of islets^11,12, the supply of oxygen by diffusion appears to be a determining factor of islet function after transplantation. While the lack of oxygen affects the postransplant function of free islets¹³ it seems to be even more important for survival of encapsulated islets^14,15.

One of the main determinants of survival of islets maintained in an artificial environment is the seeding density¹⁶. Culture conditions can be optimized by an adequate number of culture vessels filled according to islet yield and purity with a thin layer of culture medium to reduce the diffusion distance for oxygen. In contrast, the capacity of an immunoprotective macrodevice to host a certain amount of islet tissue is predetermined by its design and limited size. So far, mathematical modeling has been used as the main tool to try to define the ideal physical requirements to preserve islet viability in an artificial environment^16–18. Our study is one of the first experimental attempts to quantify the effects of seeding density on the viability and in vitro function of isolated human islets.

Materials and Methods

Islet Isolation

With appropriate informed consent and ethical approval by the NHS Research Committee, human pancreases were retrieved from 12 human multiorgan brain-dead donors. The male-to-female ratio was 6 to 6, and the mean donor age was 47.6 ± 3.0 years. The mean body mass index of the donors was 30.4 ± 1.4. All pancreases were perfused with University of Wisconsin solution (UW; Bridge to Life, London, UK) and shipped in UW to the Oxford DRWF Human Islet Isolation Facility within 6.8 ± 1.0 h of cold ischemia, defined as the interval between pancreas placement in the ice box and injection of the collagenase blend into the pancreatic duct. Enzymatic pancreas digestion¹⁹ and islet purification²⁰ were performed using standard techniques. Isolated and purified islets were suspended in cold UW and stored for 24 to 48 h at 6°C in tissue culture flasks equipped with filter caps (Greiner Bio-One, Stonehouse, UK). Average purity was 70.4 ± 1.4%.

Islet Culture

After storage in UW recovered human islets were washed and collected in CMRL 1066 supplemented with 1 mmol/L L-glutamine, 25 mmol/L HEPES (PAA, Pasching, Austria), 5% fetal calf serum (FCS), and 100 U/μg/ml penicillin–streptomycin (Life Technologies, Paisley, UK). Islet yield was determined using a standardized procedure converting islet number to islet equivalents (IEQ) with an average diameter of 150 μm²¹. After counting, quadruplicate aliquots of islets were transferred into 24-well plates equipped with 8-μm pore size filter inserts (Greiner). Aliquot volume was adjusted to obtain an islet seeding density of 75, 150, 300, or 600 IEQ/cm². The final incubation volume was 500 μl per well resulting in a diffusion distance of 1.6 mm if measured from the islets in the insert to the surface of the medium. Subsequently, 24-well plates were incubated at 37°C in normal atmosphere enriched with 5% carbon dioxide. After culture, islets were recovered and processed for characterization. Conditioned culture medium was collected and assessed for islet chemokine release.

Islet Characterization

After 24 h of culture at different seeding densities, islet viability was assessed as membrane integrity utilizing 0.67 μmol/L of fluorescein diacetate (FDA; Sigma-Aldrich, Dorset, UK) and 4.0 μmol/L of propidium iodide (PI; Sigma-Aldrich) for staining of viable and dead cells, respectively²². The fluorescence of FDA-PI was quantified utilizing a fluorometric plate reader.

In vitro islet function was assessed in duplicate during static glucose incubation. Filter inserts were transferred into 24-well plates and sequentially incubated for 45 min in 1 ml of bicarbonate-free CMRL 1066 (Applichem GmbH, Darmstadt, Germany) supplemented with 2 mmol/L glucose followed by 45 min at 20 mmol/L finally followed by a second period of 45 min at 2 mmo/L glucose. After stimulation, islets were recovered and sonified in distilled water for subsequent determination of intracellular insulin and DNA content⁷. Intracellular and secreted insulin was determined utilizing an enzyme immunoassay specific for human insulin (Mercodia, Uppsala, Sweden) and normalized to islet DNA content measured by a Pico Green assay (Life Technologies). The glucose stimulation index was calculated by dividing the insulin release at 20 mmol/L glucose by the mean of the two basal periods.

The ATP content in islets cultured at different seeding densities was evaluated utilizing a commercially available high sensitivity kit (Biothema, Handen, Sweden). Briefly, triplicate aliquots of cultured islets were washed and placed in a white 96-well plate. Prior to disintegration by means of a kit-provided lysis reagent, islets were incubated for 10 min with an ATP-eliminating reagent to remove extracellular ATP. Afterward, luciferase was added and the light emission quantified by a luminometric plate reader. Subsequently, a reference sample was added and measured to calculate the intraislet ATP content. ATP was normalized to DNA assessed as described above.

Islet Chemokine Release

After 24 h of culture at different seeding densities, islet-preconditioned supernatants were collected and assessed for production and secretion of hypoxia- and inflammation-related chemokines. The production and secretion of vascular endothelial growth factor (VEGF) and monocyte chemotactic protein 1 (MCP-1) by cultured islets was detected utilizing enzyme immunoassays specific for these human chemokines (Life Technologies).

Quantitative Real-Time Polymerase Chain Reaction

Gene expression within cultured islets was measured using Taqman-based quantitative real-time polymerase chain reaction (qRT-PCR). Cultured islets were collected in phosphate-buffered saline (PBS) and lysed for 15 min at 72°C utilizing the CellsDirect One-Step qRT-PCR kit (Invitrogen, Paisley, UK) for cell lysis and all subsequent steps. Lysates were stored at −80°C until further processing of the samples. Gene-specific PCR products were continuously measured in triplicate during 35 cycles of a singleplex reaction performed on a QuantStudio 7 cycler (Life Technologies). The target genes Bax (Hs00180269_m1 FAM-MGB) and Bcl-2 (Hs00608023_m1 FAM-MGB) were normalized to β-actin (Hs01060665_g1 VIC-MGB). All primers were provided by Applied Biosystems (Paisley, UK).

Data Analysis

All statistical analysis was performed using Prism 6.0d for MacIntosh (GraphPad, La Jolla, CA, USA). Comparisons of data were carried out by Friedman test followed by Dunn's test for multiple comparisons. Insulin release at low- and high-glucose concentrations within an experimental group was compared by the Wilcoxon test. Correlations were analyzed using Spearman's rank correlation. Differences were considered significant at p < 0.05. Values of p > 0.05 were defined as nonsignificant (NS). For clarity, results are expressed as mean ± standard error (SEM) rather than the correct nonparametric measures of median and quartiles.

Results

Islet Characterization

Isolated islets were aliquoted to obtain an islet seeding density of 75, 150, 300, or 600 IEQ/cm². Considering the purity of each preparation the number of islets plus non-endocrine particles per cm² for each category was 107 ± 2.3, 214 ± 4.6, 428 ± 9.3, and 856 ± 18.5, respectively. The accuracy of seeding was validated measuring the DNA content in the wells (Table 1), which significantly correlated with the number of IEQ (r = 0.58, p < 0.001) and the number of particles (r = 0.57, p < 0.001) considering also non-endocrine tissue. As demonstrated in Table 1, the seeding density had a significant impact on islet viability, which dropped at 300 (p < 0.001 vs. 75 IEQ/cm²) and 600 IEQ/cm² (p < 0.001 vs. 75 IEQ/cm²; p < 0.01 vs. 150 IEQ/cm²). No significant effect was noted when correlating islet purity with viability (r = −0.27, NS). Islet viability corresponded with the expression of pro- and antiapoptotic genes determined as Bax-to-Bcl-2 ratio. A significant increase in this ratio was measured in islets cultured at a seeding density of 600 IEQ/cm² when compared to 75 IEQ/cm² (p < 0.05) or 150 IEQ/cm² (p < 0.001) (Table 1). Although ATP correlated inversely with the DNA content (r = −0.36, p < 0.05) the differences between the experimental groups were not significant (Table 1).

Table 1.

Islet Characterization

Seeding Density (IEQ/cm²)	DNA (ng/cm²)	Viability (%)	ATP (pM/ng DNA)	Bax/Bcl-2 Ratio (%)
75	307 ± 101	61.0 ± 2.9	5.7 ± 3.2	100
150	877 ± 397	56.9 ± 2.6	5.5 ± 3.1	72.8 ± 12.8
300	1,743 ± 680^*	47.6 ± 2.5^†	3.7 ± 1.6	147.5 ± 18.3
600	4,220 ± 1,958^†^‡	41.6 ± 2.6^†^‡	3.6 ± 1.8	296.5 ± 47.2^§^¶

p < 0.01 versus 75 IEQ/cm².

†

p < 0.001 versus 75 IEQ/cm².

‡

p < 0.01 versus 150 IEQ/cm².

p < 0.05 versus 75 IEQ/cm².

p < 0.001 versus 150 IEQ/cm².

Islet In vitro Function

After 24 h of culture at different seeding densities, islet in vitro function was assessed during sequential incubation at 2 and 20 mmol/L glucose. Islets were stimulated as recovered aliquots and not manually selected. Basal (r = −0.67, p < 0.001) as well as stimulated insulin release (r = −0.75, p < 0.001) inversely correlated with the amount of DNA recovered after culture. As shown in Table 2, a seeding density of 300 and 600 IEQ/cm² significantly decreased basal and stimulated insulin release when compared to 75 or 150 IEQ/cm². As a result, islets cultured at a density of ≥300 IEQ/cm² did not respond adequately toward a glucose challenge when compared to 75 and 150 IEQ/cm². The lower secretory capacity of islets cultured at higher seeding densities is reflected by the glucose stimulation index, which was significantly reduced in islets cultured at 300 (2.20 ± 0.70) and 600 IEQ/cm² (1.47 ± 0.21) when opposed to islets cultured at 75 (3.48 ± 1.21, p < 0.01 vs. 300 IEQ/cm², p < 0.001 vs. 600 IEQ/cm²) and 150 IEQ/cm² (2.95 ± 0.98, p < 0.05 vs. 300 IEQ/cm², p < 0.01 vs. 600 IEQ/cm²). Although islet purity did not affect the glucose stimulation index (r = 0.08, NS), basal (r = 0.56, p < 0.001) as well as stimulated (r = 0.50, p <0.001) insulin secretion was influenced by this parameter.

Table 2.

Insulin Secretion and Intracellular Insulin Content

	Insulin Secretion
Seeding Density (IEQ/cm²)	2.0 mM (μU/ng)	20 mM (μU/ng)	2.0 mM (μU/ng)	Stimulation Index (20 mM/2.0 mM)	Insulin Content (μU/ng)
75	18.5 ± 9.2	52.7 ± 29.3	9.7 ± 4.8	3.48 ± 1.21	309.6 ± 77.1
150	20.4 ± 11.0	41.9 ± 27.4	10.0 ± 5.5	2.95 ± 0.98	217.0 ± 61.7
300	15.2 ± 11.5^*	20.9 ± 14.4^†	8.3 ± 5.0	2.20 ± 0.70^†^‡	145.5 ± 43.8^†
600	4.7 ± 2.2^‡^§	6.6 ± 4.1^§^¶	4.1 ± 1.7^*	1.47 ± 0.21^§^¶	128.1 ± 32.3^†

p < 0.05 versus 75 IEQ/cm².

†

p < 0.01 versus 75 IEQ/cm².

‡

p < 0.05 versus 150 IEQ/cm².

p < 0.001 versus 75 IEQ/cm².

p < 0.01 versus 150 IEQ/cm².

Significant differences were also present between experimental groups regarding the intracellular insulin content. Islets cultured at a seeding density of 75 IEQ/cm² had a significantly higher intracellular insulin content when compared to 300 and 600 IEQ/cm² (p < 0.05). As observed for insulin release, an inverse correlation was found between the amount of DNA and the intracellular insulin content (r = −0.66, p < 0.001). In agreement, islet purity had a positive correlation with the intracellular insulin content (r = 0.44, p < 0.01).

Islet Chemokine Release

VEGF and MCP-1 production of human islets was investigated in culture supernatants collected after 24 h of incubation. Although VEGF production varied in a wide range between different donors, an incremental increase in VEGF secretion was always found in the supernatants of higher seeding densities (Table 3). In comparison with a seeding density of 75 (3.3 ± 3.3 pg/pg DNA) or 150 IEQ/cm² (82.4 ± 67.0 pg/pg), 300 (467 ± 158 pg/pg, p < 0.05 vs. 75 IEQ/cm²) and 600 IEQ/cm² (1,014 ± 253 pg/pg, p < 0.001 vs. 75 and 150 IEQ/cm²) induced a stronger VEGF release in islets.

Table 3.

Islet Chemokine Release

Seeding Density (IEQ/cm²)	VEGF (pg/pg DNA)	MCP-1 (pg/pg DNA)
75	3.3 ± 3.3^*^†	471 ± 232^†^‡
150	82.4 ± 67.0^†	812 ± 391^§
300	467 ± 158	2,113 ± 771
600	1,014 ± 253	2,767 ± 813

p < 0.05 versus 300 IEQ/cm².

†

p < 0.001 versus 600 IEQ/cm².

‡

p < 0.01 versus 300 IEQ/cm².

p < 0.01 versus 600 IEQ/cm².

Compared to VEGF, MCP-1 production was of higher magnitude and variability, reaching its maxima at 300 IEQ/cm² (2,113 ± 771 pg/pg, p < 0.01 vs. 75 IEQ/cm²) and 600 IEQ/cm² (2,767 ± 813 pg/pg, p < 0.001 vs. 75 IEQ/cm², p < 0.01 vs. 150 IEQ/cm²).

No relevant effect of islet purity on MCP-1 (r = 0.11, NS) release was found. In contrast, VEGF correlated slightly but significantly with islet purity (r = 0.31, p < 0.05), indicating that mainly islet tissue contributed to hypoxia-induced signaling.

Discussion

In contrast to immortalized cell lines or tumor-derived cells, primary human islets isolated from adult donors are not at the risk of suffering from nutrient depletion within the medium if maintained in culture for a limited period of time until transplantation²³. This does not exclude that both survival and function of cultured human islets are deteriorated when growth or survival factors are not provided by supplements or serum^24,25. Moreover, it has been shown that hypoxia synergistically enhances islet cell death when islets are exposed to serum deprivation during culture²⁶. The cellular response toward hypoxia is mainly determined by the activation of numerous HIF-1α-controlled genes that are related to protective or proapoptotic mechanisms such as VEGF or caspase 3 formation^27–29. However, even when culture conditions are characterized by normoxic atmosphere and serum supplementation, local hypoxia can occur in the microenvironment of cultured islets as demonstrated by the coexpression of VEGF and central necrosis in rat islets^11,30.

Mathematical models indicate that this local hypoxia is mainly related to the ellipsoid structure of islets composed of several hundred cells³¹, which reduces the passive diffusion of oxygen into the core of the islets^12,18. The diffusion rate of oxygen is also defined by the media volume used for islet suspension. The amount of culture medium determines the distance between islets at the bottom of a culture vessel and the upper surface of the fluid forming the interface between the islets and the gaseous phase^32,33. In the present experimental setting the incubation volume was minimized to guarantee a maximum diffusion distance of 1.6 mm between the islets and the atmosphere, which corresponds to clinically established culture protocols³⁴.

Considering the specifically high respiratory rate of islets^9,35,36 it seems to be obvious that higher seeding densities (defined as IEQ per cm² of culture vessel area) will additionally reduce the local oxygen concentrations in the islet microenvironment. In accordance with Papas et al.'s calculations¹⁶, the data of the present study demonstrate that the seeding density of human islets inversely correlates with viability of islets even when islets are cultured under conditions that meet the physical requirements of human islet culture for subsequent clinical islet transplantation³³. While Papas et al.'s model calculated a threshold for anoxia-induced cell death at a seeding density ranging from 110 to 150 IEQ/cm², our experimental study indicates that the critical seeding density for human islets is between 150 IEQ/cm² and 300 IEQ/cm². The deviation between our experimental findings and the expected values may be explained by idealized conditions assumed for a mathematical model on the one hand and the variability observed in human pancreas donors and islet counting on the other³⁷. In addition, the viability in our preparations, as measured by unbiased plate reader technique, would result in lower respiratory rates³⁸, which seems to facilitate islet culture at higher seeding densities as expected¹⁶.

The measurement of the VEGF production of human islets cultured at different seeding densities revealed that high numbers of large cell clusters concentrated in small areas is an important contributor to local hypoxia. Seeding densities ≥300 IEQ/cm² induced a VEGF secretion that reached a level approximately fivefold higher than the potential threshold of 150 IEQ/cm². In addition, we found that any change in VEGF secretion is associated with a similar adaption of MCP-1 production. This finding has practical implications for posttransplant function, particularly for encapsulated islets. Previous studies have clearly demonstrated that the reduction of exogenous insulin requirements in islet-transplanted type 1 diabetic patients inversely correlates with the MCP-1 secretion of human islets during pretransplant culture³⁹. Thus, our study is in agreement with previous observations indicating that human islets produce increased amounts of VEGF and MCP-1 and exhibit signs of apoptosis when exposed to hypoxia^40,41. Moreover, we could also detect significant differences in islet viability using a normoxic atmosphere. Consistent with the reduction of islet viability we observed a significant increase in the Bax-to-Bcl-2 mRNA ratio, suggesting a shift toward proapoptotic mechanisms within hypoxic human islets and thereby confirming previous studies^27,28,40.

Although the differences in the ATP content of the different experimental groups were not significant, the inverse correlation between DNA and ATP content in cultured islets indicates that the oxygen supply at higher seeding densities is insufficient for oxidative glucose metabolism as the preferred pathway for glucose breakdown in human islets^42,43. As a consequence, glucose-stimulated insulin secretion is coupled with oxidative glucose metabolization and correlates with the increase in oxygen consumption rate in stimulated islets^38,44. Vice versa, hypoxia has not only an inhibitory effect on glucose oxidation and glucose-stimulated insulin release^28,45–47 but also on insulin biosynthesis and intracellular insulin content^48,49. In agreement with these previous observations and calculations of Papas et al.¹⁶, revealing that higher seeding densities increase the anoxic volume within islets, we found that higher seeding densities are characterized by a decreased insulin secretory capacity and reduced insulin content. The overall importance of sufficient oxygen supply for β-cell-specific pathways is additionally demonstrated by the finding that β cells are much more sensitive toward hypoxia-induced apoptosis than a cells⁵⁰.

Another possible explanation for the decrease in insulin secretion at higher seeding densities is the existence of a potential negative feedback loop for insulin release. Previous studies indicated that insulin inhibits its own glucose-stimulated secretion when a static incubation system is used^51,52. The extent of inhibition seems to be related to the number of islets incubated⁵³. In contrast, islets that were loaded into a dynamic perifusion system do not show a secretory downregulation through ambient insulin⁵⁴ even when seeded at a density of 4,000 islets per cm²⁽⁵⁵⁾.

As with any other organ, the metabolic demand of islets is clearly related to the environmental temperature. According to the Q10 temperature coefficient any reduction of the environmental temperature by 10°C reduces the metabolic rate of organisms and cells by a factor of approximately 3 or more⁵⁶. The reduced metabolic activity of islets cultured at a temperature of 22°C or 24°C is reflected by a lower insulin release at basal conditions and by a decreased insulin stimulatory capacity when compared to islets cultured at 37°C^57,58. Because hypothermia mainly affects the mitochondrial pathways of glucose breakdown the demand for oxygen is also strongly reduced⁵⁹. As a consequence, the extent of central necrosis is significantly decreased and correlates with higher survival at lower temperatures^58,60. Nevertheless, while hypothermia reduces the metabolic demand of islets in culture, particularly when seeded at higher densities, this benefit does not apply when islets are encapsulated in immunoprotective macrodevices implanted in diabetic recipients.

These considerations, combined with the findings of our study, may have implications for optimal human islet culture and for islet shipment between isolation facility and islet transplant center³³ particularly when no efficient oxygenation devices are available¹⁶. The relevance of our findings may be even more significant for islets encapsulated in immunoprotective extravascular macrodevices or seeded on scaffolds and then implanted within the peritoneal cavity, the omental pouch, or at the subcutaneous site. The nutritive support of islets loaded into an extravascular macrodevice solely depends on passive diffusion of nutrients such as glucose, amino acids, and, most important, oxygen⁶¹. It can be assumed that the already poor nutritive support of macroencapsulated islets is further deteriorated at a higher seeding density as shown by our data. Particularly, when encapsulated islets are contaminated by significant proportions of exocrine tissue the functional potency and viability of implanted islets are significantly decreased⁶².

To avoid any cluster formation within the device, islet immobilization using an extracellular matrix protein or a porous structure seems to be an essential step to support islet function⁶³. The only study that has quantified the effect of seeding density on the survival of transplanted islets, so far, demonstrated that the survival of pig islets seeded on porous scaffolds is substantially reduced by 55% when the density was increased from 750 to 3,000 IEQ/cm². However, no direct correlation between seeding density and islet functional capacity was analyzed in this study⁶⁴.

A simple enlargement of an extravascular macrodevice may not provide the final solution as it is limited not only by anatomical restrictions but also by any extension of the oxygen diffusion distance, which determines the rate of islet necrosis. Conversely, increased cell death requires a higher tissue load to compensate for fewer functional islets⁶⁵. In addition, increasing the dimensions of a macrodevice has the added significant disadvantage of deteriorating the kinetics of insulin efflux⁶⁶.

In summary, this study clearly demonstrates that the seeding density at normoxic atmospheric conditions is inversely correlated with islet viability and islet in vitro function. This is associated with a significant increase in VEGF and MCP-1 production, suggesting the involvement of hypoxia and proinflammatory mechanisms. Optimization of islet seeding densities and the development of novel strategies for improving oxygenation are both essential if islet encapsulation strategies are to be successfully translated into clinical practice.

Footnotes

Acknowledgments

This study is performed as part of a collaborative project to develop a bioartificial pancreas to treat type 1 diabetes (BIOSID). BIOSID is supported at the level of 5,469,603 € through the Cooperation Program of the European Community's FP7. The grant agreement No. is HEALTH-F2-2012-305746. Daniel Brandhorst and Heide Brandhorst participated in the research design, in the performance of research, in the data analysis, and in the writing of the manuscript. Niamh Mullooly and Samuel Acreman participated in the performance of research and in the data analysis. Paul R. V. Johnson participated in the writing of the manuscript. The authors would like to thank all members of the Oxford Human Islet Isolation team for providing the isolated human islets. Isolation of human islets for research was supported by the Oxford NIHR Biomedical Research Centre and a Juvenile Diabetes Research Foundation (JDRF) award (31-2008-617) to Paul R. V. Johnson. The authors declare no conflicts of interest.

References

Meyer

, Hering

B.J.

, Grossmann

, Brandhorst

, Gerich

, Federlin

, Bretzel

R.G.

Improved glucose counterregulation and autonomic symptoms after intraportal islet transplants alone in patients with long-standing type I diabetes mellitus. Transplantation 1998; 66(2): 233–40.

Ryan

E.A.

, Shandro

, Green

, Paty

B.W.

, Senior

P.A.

, Bigam

, Shapiro

A.M.

, Vantyghem

M.C.

Assessment of the severity of hypoglycemia and glycemic lability in type 1 diabetic subjects undergoing islet transplantation. Diabetes 2004; 53(4): 955–62.

Fiorina

, Folli

, Maffi

, Placidi

, Venturini

, Finzi

, Bertuzzi

, Davalli

, D'Angelo

, Socci

, Gremizzi

, Orsenigo

, La Rosa

, Ponzoni

, Cardillo

, Scalamogna

, Del Maschio

, Capella

, Di Carlo

, Secchi

Islet transplantation improves vascular diabetic complications in patients with diabetes who underwent kidney transplantation: A comparison between kidney-pancreas and kidney-alone transplantation. Transplantation 2003; 75(8): 1296–301.

Warnock

G.L.

, Thompson

D.M.

, Meloche

R.M.

, Shapiro

R.J.

, Ao

, Keown

, Johnson

J.D.

, Verchere

C.B.

, Partovi

, Begg

I.S.

, Fung

, Kozak

S.E.

, Tong

S.O.

, Alghofaili

K.M.

, Harris

A Multi-Year Analysis of islet transplantation compared with intensive medical therapy on progression of complications in type 1 diabetes. Transplantation 2008; 86(12): 1762–1766.

Fiorina

, Folli

, Zerbini

, Maffi

, Gremizzi

, Di Carlo

, Socci

, Bertuzzi

, Kashgarian

, Secchi

Islet transplantation Is associated with improvement of renal function among uremic patients with type I diabetes mellitus and kidney transplants. J Am Soc Nephrol. 2003; 14: 2150–58.

Brandhorst

, Brandhorst

, Hesse

, Ambrosius

, Brendel

, Kawakami

, Bretzel

R.G.

Successful human islet isolation utilizing recombinant collagenase. Diabetes 2003; 52(5): 1143–6.

Brandhorst

, Asif

, Andersson

, Theisinger

, Andersson

H.H.

, Felldin

, Foss

, Salmela

, Tibell

, Tufveson

, Korsgren

, Brandhorst

A new oxygen carrier for improved long-term storage of human pancreata before islet isolation. Transplantation 2010; 89(2): 155–60.

Friberg

A.S.

, Stahle

, Brandhorst

, Korsgren

, Brandhorst

Human islet separation utilizing a closed automated purification system. Cell Transplant. 2008; 17(12): 1305–13.

Erecinska

, Bryla

, Michalik

, Meglasson

M.D.

, Nelson

Energy metabolism in islets of Langerhans. Biochim Biophys Acta 1992; 1101(3): 273–95.

10.

Hellman

, Idahl

L.A.

, Danielsson

Adenosine triphosphate levels of mammalian pancreatic B cells after stimulation with glucose and hypoglycemic sulfonylureas. Diabetes 1969; 18(8): 509–16.

11.

Vasir

, Aiello

L.P.

, Yoon

K.H.

, Quickel

R.R.

, Bonner-Weir

, Weir

G.C.

Hypoxia induces vascular endothelial growth factor gene and protein expression in cultured rat islet cells. Diabetes 1998; 47(12): 1894–903.

12.

Williams

S.J.

, Huang

H.H.

, Kover

, Moore

, Berkland

, Singh

, Smirnova

I.V.

, MacGregor

, Stehno-Bittel

Reduction of diffusion barriers in isolated rat islets improves survival, but not insulin secretion or transplantation outcome. Organogenesis 2010; 6(2): 115–24.

13.

Lau

, Mattsson

, Carlsson

, Nyqvist

, Kohler

, Berggren

P.O.

, Jansson

, Carlsson

P.O.

Implantation site-dependent dysfunction of transplanted pancreatic islets. Diabetes 2007; 56(6): 1544–50.

14.

Jia

, Sharma

, Kumagai-Braesch

, Wernerson

A.M.

, Sorenby

A.K.

, Yamamoto

, Wang

, Tibell

A.B.

Exendin-4 increases the expression of hypoxia-inducible factor-1alpha in rat islets and preserves the endocrine cell volume of both free and macroencapsulated islet grafts. Cell Transplant. 2012; 21(6): 1269–83.

15.

Barkai

, Weir

G.C.

, Colton

C.K.

, Ludwig

, Bornstein

S.R.

, Brendel

M.D.

, Neufeld

, Bremer

, Leon

, Evron

, Yavriyants

, Azarov

, Zimmermann

, Maimon

, Shabtay

, Balyura

, Rozenshtein

, Vardi

, Bloch

, de Vos

, Rotem

Enhanced oxygen supply improves islet viability in a new bioartificial pancreas. Cell Transplant. 2013; 22(8): 1463–76.

16.

Papas

K.K.

, Avgoustiniatos

E.S.

, Tempelman

L.A.

, Weir

G.C.

, Colton

C.K.

, Pisania

, Rappel

M.J.

, Friberg

A.S.

, Bauer

A.C.

, Hering

B.J.

High-density culture of human islets on top of silicone rubber membranes. Transplant Proc. 2005; 37(8): 3412–4.

17.

Avgoustiniatos

E.S.

, Colton

C.K.

Effect of external oxygen mass transfer resistances on viability of immunoisolated tissue. Ann NY Acad Sci. 1997; 831: 145–67.

18.

Buchwald

FEM-based oxygen consumption and cell viability models for avascular pancreatic islets. Theor Biol Med Model. 2009; 6: 5.

19.

Cross

S.E.

, Hughes

S.J.

, Clark

, Gray

D.W.

, Johnson

P.R.

Collagenase does not persist in human islets following isolation. Cell Transplant. 2012; 21(11): 2531–5.

20.

Barbaro

, Salehi

, Wang

, Qi

, Gangemi

, Kuechle

, Hansen

M.A.

, Romagnoli

, Avila

, Benedetti

, Mage

, Oberholzer

Improved human pancreatic islet purification with the refined UIC-UB density gradient. Transplantation 2007; 84(9): 1200–3.

21.

Ricordi

, Gray

D.W.R.

, Hering

B.J.

, Kaufman

D.B.

, Warnock

G.L.

, Kneteman

N.M.

, Lake

S.P.

, London

N.J.M.

, Socci

, Alejandro

, Zeng

, Scharp

D.W.

, Viviani

, Falqui

, Tzakis

, Bretzel

R.G.

, Federlin

, Pozza

, James

R.F.L.

, Rajotte

R.V.

, Di Carlo

, Morris

P.J.

, Sutherland

D.E.R.

, Starzl

T.E.

, Mintz

D.H.

, Lacy

P.E.

Islet isolation assessment in man and large animals. Acta Diabetol Lat. 1990; 27(3): 185–95.

22.

London

N.J.

, Contractor

, Lake

S.P.

, Aucott

G.C.

, Bell

P.R.

, James

R.F.

A fluorometric viability assay for single human and rat islets. Horm Metab Res Suppl. 1990; 25: 82–7.

23.

Freshney

R.I.

Defined media and supplements. In: Freshney

R.I.

, editor. Culture of animal cells: A manual of basic technique. Hoboken (NJ): John Wiley & Sons, Inc.; 2005. p. 99–114.

24.

Kerr-Conte

, Vandewalle

, Moerman

, Lukowiak

, Gmyr

, Arnalsteen

, Caiazzo

, Sterkers

, Hubert

, Vantyghem

M.C.

, Pattou

Upgrading pretransplant human islet culture technology requires human serum combined with media renewal. Transplantation 2010; 89(9): 1154–60.

25.

Avgoustiniatos

E.S.

, Scott

W.E.

, Suszynski

T.M.

, Schuurman

H.J.

, Nelson

R.A.

, Rozak

P.R.

, Mueller

K.R.

, Balamurugan

A.N.

, Ansite

J.D.

, Fraga

D.W.

, Friberg

A.S.

, Wildey

G.M.

, Tanaka

, Lyons

C.A.

, Sutherland

D.E.

, Hering

B.J.

, Papas

K.K.

Supplements in human islet culture: Human serum albumin is inferior to fetal bovine serum. Cell Transplant. 2012; 21(12): 2805–14.

26.

Giuliani

, Moritz

, Bodmer

, Dindo

, Kugelmeier

, Lehmann

, Gassmann

, Groscurth

, Weber

Central necrosis in isolated hypoxic human pancreatic islets: Evidence for postisolation ischemia. Cell Transplant. 2005; 14(1): 67–76.

27.

Moritz

, Meier

, Stroka

D.M.

, Giuliani

, Kugelmeier

, Nett

P.C.

, Lehmann

, Candinas

, Gassmann

, Weber

Apoptosis in hypoxic human pancreatic islets correlates with HIF-1alpha expression. Faseb J. 2002; 16(7): 745–7.

28.

Cantley

, Walters

S.N.

, Jung

M.H.

, Weinberg

, Cowley

M.J.

, Whitworth

T.P.

, Kaplan

, Hawthorne

W.J.

, O'Connell

P.J.

, Weir

, Grey

S.T.

A preexistent hypoxic gene signature predicts impaired islet graft function and glucose homeostasis. Cell Transplant. 2013; 22(11): 2147–59.

29.

Cantley

, Grey

S.T.

, Maxwell

P.H.

, Withers

D.J.

The hypoxia response pathway and beta-cell function. Diabetes Obes Metab. 2010; 12(Suppl. 2): 159–67.

30.

Gorden

D.L.

, Mandriota

S.J.

, Montesano

, Orci

, Pepper

M.S.

Vascular endothelial growth factor is increased in devascularized rat islets of Langerhans in vitro. Transplantation 1997; 63(3): 436–43.

31.

Morini

, Braun

, Onori

, Cicalese

, Elias

, Gaudio

, Rastellini

Morphological changes of isolated rat pancreatic islets: A structural, ultrastructural and morphometric study. J Anat. 2006; 209(3): 381–92.

32.

Murdoch

T.B.

, McGhee-Wilson

, Shapiro

A.M.

, Lakey

J.R.

Methods of human islet culture for transplantation. Cell Transplant. 2004; 13(6): 605–17.

33.

Ichii

, Sakuma

, Pileggi

, Fraker

, Alvarez

, Montelongo

, Szust

, Khan

, Inverardi

, Naziruddin

, Levy

M.F.

, Klintmalm

G.B.

, Goss

J.A.

, Alejandro

, Ricordi

Shipment of human islets for transplantation. Am J Transplant. 2007; 7(4): 1010–20.

34.

Fraker

, Montelongo

, Szust

, Khan

, Ricordi

The use of multiparametric monitoring during islet cell isolation and culture: A potential tool for in-process corrections of critical physiological factors. Cell Transplant. 2004; 13(5): 497–502.

35.

Malaisse

W.J.

, Sener

Hexose metabolism in pancreatic islets. Feedback control of D-glucose oxidation by functional events. Biochim Biophys Acta 1988; 971(3): 246–54.

36.

Sener

, Malaisse

W.J.

Nutrient metabolism in islet cells. Experientia 1984; 40(10): 1026–35.

37.

Friberg

A.S.

, Brandhorst

, Buchwald

, Goto

, Ricordi

, Brandhorst

, Korsgren

Quantification of the islet product: Presentation of a standardized current good manufacturing practices compliant system with minimal variability. Transplantation 2011; 91(6): 677–83.

38.

Wang

, Upshaw

, Strong

D.M.

, Robertson

R.P.

, Reems

Increased oxygen consumption rates in response to high glucose detected by a novel oxygen biosensor system in non-human primate and human islets. J Endocrinol. 2005; 185(3): 445–55.

39.

Piemonti

, Leone

B.E.

, Nano

, Saccani

, Monti

, Maffi

, Bianchi

, Sica

, Peri

, Melzi

, Aldrighetti

, Secchi

, Di Carlo

, Allavena

, Bertuzzi

Human pancreatic islets produce and secrete MCP-1/CCL2: Relevance in human islet transplantation. Diabetes 2002; 51(1): 55–65.

40.

Lai

, Brandhorst

, Hossain

, Bierhaus

, Chen

, Bretzel

R.G.

, Linn

Activation of NFkappaB dependent apoptotic pathway in pancreatic islet cells by hypoxia. Islets 2010; 1(1): 19–25.

41.

Hals

I.K.

, Rokstad

A.M.

, Strand

B.L.

, Oberholzer

, Grill

Alginate microencapsulation of human islets does not increase susceptibility to acute hypoxia. J Diabetes Res. 2013; 2013: 374925.

42.

Tamarit-Rodriguez

, Idahl

L.A.

, Gine

, Alcazar

, Sehlin

Lactate production in pancreatic islets. Diabetes 1998; 47(8): 1219–23.

43.

Malaisse

W.J.

, Rasschaert

, Conget

, Sener

Hexose metabolism in pancreatic islets. Regulation of aerobic glycolysis and pyruvate decarboxylation. Int J Biochem. 1991; 23(9): 955–9.

44.

Sweet

I.R.

, Gilbert

, Jensen

, Sabek

, Fraga

D.W.

, Gaber

A.O.

, Reems

Glucose stimulation of cytochrome C reduction and oxygen consumption as assessment of human islet quality. Transplantation 2005; 80(8): 1003–11.

45.

Hellman

, Idahl

L.A.

, Sehlin

, Taljedal

I.B.

Influence of anoxia on glucose metabolism in pancreatic islets: Lack of correlation between fructose-1,6-diphosphate and apparent glycolytic flux. Diabetologia 1975; 11(6): 495–500.

46.

Dionne

K.E.

, Colton

C.K.

, Yarmush

M.L.

Effect of hypoxia on insulin secretion by isolated rat and canine islets of Langerhans. Diabetes 1993; 42(1): 12–21.

47.

Malaisse

W.J.

, Rasschaert

, Zahner

, Sener

Hexose metabolism in pancreatic islets: The Pasteur effect. Diabetes Res. 1988; 7(2): 53–8.

48.

, Moruzzi

, Catrina

S.B.

, Hals

, Oberholzer

, Grill

, Bjorklund

Preconditioning with associated blocking of Ca2+ inflow alleviates hypoxia-induced damage to pancreatic beta-cells. PLoS One 2013; 8(7): e67498.

49.

Wang

, Khan

S.A.

, Prabhakar

N.R.

, Nanduri

Impairment of pancreatic beta-cell function by chronic intermittent hypoxia. Exp Physiol. 2013; 98(9): 1376–85.

50.

Bloch

, Vennang

, Lazard

, Vardi

Different susceptibility of rat pancreatic alpha and beta cells to hypoxia. Histochem Cell Biol. 2012; 137(6): 801–10.

51.

Sodoyez

J.C.

, Sodoyez-Goffaux

, Foa

P.P.

Evidence for an insulin-induced inhibition of insulin release by isolated islets of Langerhans. Proc Soc Exp Biol Med. 1969; 130(2): 568–71.

52.

Loreti

, Dunbar

J.C.

, Chen

, Foa

P.P.

The autoregulation of insulin secretion in the isolated pancreatic islets of lean (obOb) and obese-hyperglycemic (obob) mice. Diabetologia 1974; 10(4): 309–15.

53.

Hahn

H.J.

, Michael

Inhibition of insulin release by endogeneous insulin in vitro. Horm Metab Res. 1970; 2(2): 119–20.

54.

Marincola

, Frank

, Clark

, Douglas

, Merrell

The independence of insulin release and ambient insulin in vitro. Diabetes 1983; 32(12): 1162–7.

55.

Sweet

I.R.

, Khalil

, Wallen

A.R.

, Steedman

, Schenkman

K.A.

, Reems

J.A.

, Kahn

S.E.

, Callis

J.B.

Continuous measurement of oxygen consumption by pancreatic islets. Diabetes Tech Ther. 2002; 4(5): 661–72.

56.

Heldmaier

, Ruf

Body temperature and metabolic rate during natural hypothermia in endotherms. J Comp Physiol B 1992; 162(8): 696–706.

57.

Ono

, Lacy

P.E.

, Michael

H.E.

, Greider

M.H.

Studies of the functional and morphologic status of islets maintained at 24 C for four weeks in vitro. Am J Pathol. 1979; 97(3): 489–503.

58.

Brandhorst

, Brandhorst

, Hering

B.J.

, Bretzel

R.G.

Long-term survival, morphology and in vitro function of isolated pig islets under different culture conditions. Transplantation 1999; 67(12): 1533–41.

59.

Escolar

J.C.

, Hoo-Paris

, Castex

, Sutter

B.C.

Effect of low temperatures on glucose-induced insulin secretion and glucose metabolism in isolated pancreatic islets of the rat. J Endocrinol. 1990; 125(1): 45–51.

60.

Noguchi

, Naziruddin

, Jackson

, Shimoda

, Ikemoto

, Fujita

, Chujo

, Takita

, Kobayashi

, Onaca

, Levy

M.F.

, Matsumoto

Low-temperature preservation of isolated islets is superior to conventional islet culture before islet transplantation. Transplantation 2010; 89(1): 47–54.

61.

Avgoustiniatos

E.S.

, Colton

C.K.

Effect of external oxygen mass transfer resistances on viability of immunoisolated tissue. Ann NY Acad Sci. 1997; 831: 145–67.

62.

Kessler

, Jesser

, Belcourt

, Pinget

Influence of acinar tissue contamination on encapsulated pancreatic islets: Morphological and functional studies. Transplantation 1997; 63(10): 1537–40.

63.

Y.J.

, Inoue

, Shinohara

, Doi

, Kogire

, Aung

, Sumi

, Imamura

, Fujisato

, Maetani

Xenotransplantation of bioartificial pancreas using a mesh-reinforced polyvinyl alcohol bag. Cell Transplant. 1994; 3(Suppl. 1): S19–21.

64.

Gibly

R.F.

, Zhang

, Graham

M.L.

, Hering

B.J.

, Kaufman

D.B.

, Lowe

W.L.

Jr Shea

L.D.

Extrahepatic islet transplantation with microporous polymer scaffolds in syngeneic mouse and allogeneic porcine models. Biomaterials 2011; 32(36): 9677–84.

65.

Dulong

J.L.

, Legallais

A theoretical study of oxygen transfer including cell necrosis for the design of a bioartificial pancreas. Biotechnol Bioeng. 2007; 96(5): 990–8.

66.

Scharp

D.W.

, Marchetti

Encapsulated islets for diabetes therapy: History, current progress, and critical issues requiring solution. Adv Drug Deliv Rev. 2014; 67–68: 35–73.