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Original Clinical Science—General

Use of Culture to Reach Metabolically Adequate Beta-cell Dose by Combining Donor Islet Cell Isolates for Transplantation in Type 1 Diabetes Patients

Lee, DaHae MD1,2; Gillard, Pieter MD, PhD1,2; Hilbrands, Robert MD, PhD2; Ling, Zhidong MD, PhD2; Van de Velde, Ursule MSCN2; Jacobs-Tulleneers-Thevissen, Daniel MD, PhD2; Maleux, Geert MD, PhD3; Lapauw, Bruno MD, PhD4; Crenier, Laurent MD, PhD5; De Block, Christophe MD, PhD6; Mathieu, Chantal MD, PhD1; Pipeleers, Daniel MD, PhD2; Keymeulen, Bart MD, PhD2

Author Information
doi: 10.1097/TP.0000000000003321

Abstract

INTRODUCTION

Islet beta-cell transplantation has improved considerably in recent years, but the duration of graft function remains heterogeneous. Both donor and recipient factors were identified as important factors determining long-term results.1-3 Concerning graft characteristics, a minimum number of islet equivalents (IEQ)/kg (or beta cells per/kg) in the graft, is considered to be the most determinant factor of successful outcome.2-4 Most centers will proceed with transplantation only when the procurement of the donor pancreas and subsequent isolation procedure lead to a satisfactory islet yield after which islets from this single donor are infused immediately or after overnight culture.5-9

In most transplant centers, the duration of islet culture preparations is limited to 24–72 hours,2,5,7,9-13 because of the progressive reduction in the absolute number of beta cells during culture, and due to the well-established relationship between graft function and the number of IEQ that are infused intraportally.14 In these centers, the islets are not transplanted if a predefined number of IEQ is not reached after isolation. In regions with less than optimal islet donors (those of more advanced age and with lower body mass index [BMI] and longer mean stay in intensive care), such inferior isolation outcomes are common. Another option is to keep the preparations under culture for a longer duration.

In our islet cell program, we combine cell preparations derived from various islet yields so that at least 2 million beta cells per kilogram are transplanted.4 This strategy requires a longer culture time of the cells resulting in the possible disadvantage of losing functional beta cells during the culture or after transplantation.15 Prolonged culture, on the other hand, allows the facilitation of planning of the infusion procedure and thus reducing the stress of patients in the pretransplant period. It is also possible to reduce the cytokine release symptoms related to antithymocyte globulin (ATG) administration at the time of islet infusion, since the first administration of ATG can be done 24–48 hours before. Maintenance immune suppression target levels are reached most easily during the first days posttransplantation (PT).

In this retrospective study, we investigated if longer culture time (≥96 h) of the first combined graft (defined as the graft at initiation of immune suppression) led to the longer transplant survival.

MATERIALS AND METHODS

Graft Recipients

Data were collected from 40 islet beta-cell graft recipients with type 1 diabetes mellitus and hypoglycemia unawareness, who were transplanted beween December 2001 and January 2011 (Figure 1). These recipients were consecutively transplanted based on their waiting time and body weight since we aimed at transplanting at least 2 × 106 beta cells per kilogram of recipient body weight. No changes occurred in recipient selection during the study period. All recipients had type 1 diabetes for >5 years, were plasma C-peptide negative (<0.09 ng/mL), showed a large variation in blood glucose levels (glycemic variability [GV] >25%), HbA1c >7% and had 1 or more chronic diabetes complications. Before the transplantation, autoantibodies for ICA, GAD, and IA2,16 T-cell autoreactivity to GAD and IA2,17 HLA class I and II antibodies, and lymphocyte subsets18,19 were determined as previously described.4 All patients were complement-dependent cytotoxicity (CDC)-HLA class I negative and crossmatch before transplantation was negative in all patients.

FIGURE 1.
FIGURE 1.:
Design of the study. Number of cells and composition were determined from a combined sample before transplantation. The immune suppression regimen consisted of induction therapy with ATG-Fresenius (Fresenius, HemoCare, Redmond, WA) and maintenance therapy with MMF (Cellcept, Roche, Basel, Switzerland) or mycophenolic acid (Myfortic, Novartis, Basel, Switzerland) and tacrolimus (Prograft, Astellas Pharma Europe, Staines, United Kingdom). ATG, antithymocyte globulin; IQR, interquartile range; MMF, mycophenolate mofetil.

The Ethics Committees of the Belgian Diabetes Registry and the participating university hospitals approved the protocol. This study was performed according to the Declaration of Helsinki, and institution review board approval was obtained (institutional review board protocol BK/3 and BK/136; clinicaltrials.gov NCT00623610 and NCT00798785). A written informed consent was obtained from every patient. The local ethical committee approved the protocols (CME 2005/118 2010/193).

Preparation of Islet Beta-cell Grafts

Donor organs were procured after brain death as well as circulatory death through the Eurotransplant Foundation (Leiden, The Netherlands) and processed at the Beta-cell Bank in Brussels as previously described.20 No changes occurred in donor selection criteria during the study period.

Islet cell–enriched fractions were cultured as described previously.21,22 In short, fractions were suspended in T175 culture flasks with 47-mL culture medium (approximate 1 million cells/mL) in CO2 incubator at 37°C. Culture medium was Ham’s F10 containing 0.5% human albumin, 7.5 mmol/L glucose, 2 mmol/L glutamine, and 2 mmol/L nicotinamide. During 2000–2007, 2% human AB serum was added during the first 1 or 2 days culture. Preparations used for 9 recipients were cultured at room temperature for 24 hours before implantation.

During culture, the preparations were analyzed for their beta-cell number and purity.4,20,22 Data were used to select preparations that, after combination, would constitute a graft with minimal 2 × 106 beta cells per kilogram of recipient body weight (Figure 1).4 The final cellular composition and beta-cell mass of each graft were determined on samples that were taken after the combination of different preparations just before implantation.4 This final result was therefore obtained after infusion. Total cell number was determined with DNA assay (6.5 pg per cell)4 before year 2007 and with nuclei count assay after 2007. The cell composition was determined by electron microscopy and immunocytochemistry. Beta-cell number was derived from a total cell count and the percentage of insulin-positive cells in the preparations.4,22 This method achieves a direct and specific assessment of beta-cell mass but takes more time than the classically used IEQ method. We did not simultaneously measured IEQ values as these are questionable in cultured islet cell preparations, because their dithizone staining for beta cells becomes less intense due to degranulation and loss of zinc, and thus it becomes more difficult to distinguish from nonspecific staining. Additionally, changes in the intercellular adhesion and extracellular volume in the cell aggregates interfere also with the correct comparison with measurements from freshly isolated preparations.

The final multidonor grafts were composed from cell preparations with each different culture times ranging from 16 to 511 hours for the first graft and 19 to 821 hours for the second graft. Ten patient received first grafts containing cells that were all cultured for at least 96 hours compared with 30 patients who received grafts containing cells cultured for <96 hours (Table S1, SDC, http://links.lww.com/TP/B946). All second grafts except 1 contained cells cultured for <96 hours.

Transplantation Protocol and Immune Suppression

Patients received in total 4.7 × 106 beta cells/kg body weight (interquartile range [IQR], 3.8–5.7), most frequently via 2 intraportal injections with a median interval of 2.7 months (IQR, 2.3–3.1) (Figure 1). None of the patients received >2 implants. The grafts were infused into the portal vein over 5–6 minutes via a catheter inserted in the umbilical vein (n = 18)23 or through subcutaneous transhepatic puncture under ultrasound guidance (n = 22).24 The immune suppression regimen was the same during the study period and consisted of induction therapy with ATG-Fresenius (Fresenius, HemoCare, Redmond, WA) and maintenance therapy with mycophenolate mofetil (Cellcept, Roche, Basel, Switzerland) or mycophenolic acid (Myfortic, Novartis, Basel, Switzerland) and tacrolimus (Prograf(t)), Astellas Pharma Europe, Staines, United Kingdom).4 A single dose of steroids (500 mg methylprednisolone) was administered 2 hours before each infusion. Antiplatelet and anticoagulant therapy was started 1 day after infusion and consisted of acetylsalicylic acid 100 mg once daily in all patients and additional low-molecular weight heparin was administered at a preventive dosage (0.4 mL Fraxiparin once daily). In 3 patients undergoing laparoscopic implantation, heparin was added to the transplant medium at 70 U/kg with continuous infusion of heparin containing saline (10 U/mL) at 500 U/h (50 mL/h) up to 24 hours PT. Doppler ultrasonography of the portal vein and liver was performed within 24 hours PT to rule out bleeding and thrombosis.

Measurement of Implant Function

Graft recipients were followed in our outpatient clinic weekly until PT week 6, and every 2 weeks between PT week 6 and 12 and monthly thereafter. All patients performed 4- to 7-point glycemia profiles of home blood glucose monitoring. GV was defined as the coefficient of variation of prebreakfast glycemia measured from home blood glucose monitoring during a 2-month period. Plasma C-peptide, glycemia, and HbA1c concentrations were measured in the central laboratory of the Belgian Diabetes Registry in Brussels.4

The main outcome parameter was the duration of islet graft function, defined as plasma C-peptide positivity ≥0.5 ng/mL. Secondary outcome parameters were duration of plasma C-peptide levels ≥1 ng/mL associated with low GV (<25%)25 and duration of insulin independence. The duration for each endpoint was calculated by subtracting the start day of the endpoint from the day when it no longer fulfilled the criteria after 2 consecutive measurements.

Statistical Methods

All values are expressed as median and IQR or mean ± SD (SE of mean) when indicated. Factors that are based on previous publications2,3,16,17,26-35 related to graft function (ie, patient characteristics, graft characteristics, as well as immune parameters including pretransplant cellular autoreactivity, presence of pretransplant autoantibodies, and HLA antibody status pretransplantation) were selected for analysis. For the comparison of the baseline patient characteristics, Pearson’s Chi-squared test was used for categorical data.

To evaluate the impact of culture time ≥96 hours on graft survival, results were compared between patients that received first grafts with all cells cultured ≥96 hours with those receiving first grafts with cells cultured <96 hours. To evaluate the impact of age on graft survival the total population was divided in groups based on age quartiles: quartile 1 (<39 y), quartile 2 and 3 (39–52 y), and quartile 4 (>52 y).

Differences between varying points in time during the follow-up were calculated using Fisher’s exact test for categorical data and Mann-Whitney U test for continuous data. All analysis was performed using SPSS (version 25.0) and the graphics were computed by using Graph Pad Prism (version 5.0). Analysis of correlations was performed using the Pearson’s rank correlation test. Multiple linear regression including all parameters with P < 0.1 was used for multivariate analyses. Kaplan-Meier estimates for outcome measures were made for the overall data and for strata-defined variables and were compared by means of the log-rank chi-squared test. All reported P are 2-sided, and P < 0.05 was considered statistically significant.

RESULTS

Baseline Recipient and Graft Characteristics

Graft recipients (17 females/23 males) had type 1 diabetes for >20 years (IQR, 21–33) with a median age of 46 years and BMI of 24 kg/m2. They were all C-peptide negative (<0.09 ng/mL), had suboptimal glucose control before transplantation (median HbA1c of 7.7% [IQR, 7.0–8.3]; 61 mmol/L [53–67]) with large GV (46% [40–51]), and were using insulin at 0.57 units/kg/day (0.49–0.76).

The median donor and graft composition are shown in Table S2, SDC, http://links.lww.com/TP/B946. Mean age, BMI, and cold ischemia time were 48 years, 25 kg/m2, and 7.8 hours, respectively. The final grafts contained around 40% endocrine cells and a low percentage of damaged cells when observed under electron microscopy.

Duration of Graft Function Correlates to Culture Time of the First Graft and Recipient Age

All patients achieved plasma C-peptide ≥0.5 ng/mL PT for a median of 51 months although duration of graft function varied considerably between recipients, illustrated by IQR between 14 and 90 months. A longer duration of C-peptide ≥0.5 ng/mL was noted in recipients (n = 10) who received a first graft that was composed from cell preparations that were all cultured for ≥96 hours (Figure 2A). Kaplan-Meier survival analysis shows that grafts of these 10 recipients had a half-life that was >80 months longer with respect to the presence of C-peptide ≥0.5 ng/mL. In those 10 patients, mean duration of C-peptide ≥0.5 ng/mL was 103 months, compared with 48 months when not all cells were cultured for ≥96 hours (P = 0.005).

FIGURE 2.
FIGURE 2.:
Graft survival curves. Time to graft failure (panel A), to loss of C-peptide ≥1.0 ng/mL and low glycemic variability (panel B) and to loss of insulin independence (panel C) in recipients who received islet cells that were all cultured for at least 96 h (blue line) or not (red line). The horizontal dotted line indicates 50% survival. The points on X-axis that intersect with the vertical dotted line indicate the duration that the endpoint was reached. Log-rank test was used for statistical significance. GV, glycemic variability.

In univariate and multivariate analysis, not only culture time of ≥96 hours but also recipient age at transplantation correlated significantly with longer duration of C-peptide ≥0.5 ng/mL (Table 1). The youngest recipients (age <39 y) had a significantly shorter duration of graft function and insulin independence (Table 2).

TABLE 1. - Factors correlated with duration of C-peptide ≥0.5 ng/mL
Parameters Univariate Multivariate
Estimate P Estimate P
Graft characteristics
 Beta 1
  Beta-cell number (×106) 0.022 0.891
  Beta-cell number/kg body weight (×106) 0.022 0.866
 Beta 2
  Beta-cell number (×106) 0.013 0.994
  Beta-cell number/kg BW (×106) 0.092 0.630
 Beta 1 + beta 2
  Beta-cell number (×106) –0.068 0.679
  Beta-cell number/kg BW (×106) –0.034 0.834
Culture time
 Beta 1
  All cells in culture ≥96 h (yes/no) 0.486 0.001 0.422 0.005
 Patient characteristics
  Age at transplantation 0.313 0.049 0.307 0.036
  BMI (kg/m2) –0.273 0.088
  HbA1c (%) –0.292– 0.071
  B-lymphocytes (CD19) 0.083 0.611
  T lymphocytes (CD3) –0.053 0.733
  Donor-specific HLA antibodies –0.038 0.815
  Presence of autoantibodies (yes/no) –0.225 0.162
  T-cell autoreactivity for GAD (yes/no) –0.232 0.166
  T-cell autoreactivity for IA2 (yes/no) –0.107 0.530
  HLA A24 (yes/no) –0.250 0.120
Analysis of correlations was performed using the Pearson’s rank correlation test. Multiple linear regression including all parameters with P < 0.1 (in bold) was used for multivariate analyses.
BMI, body mass index; GAD, glutamate decarboxylase; IA2, islet antigen 2.

TABLE 2. - Median duration of 3 different outcomes according to age at transplantation
Median age (y) n Duration of C-peptide ≥0.5 ng/mL (mo) Duration of C-peptide ≥1 ng/mL and GV <25% (mo) Duration of insulin independence (mo)
<39 9 36 (2–70) 5 (1–10) 1 (0–2)
39–52 22 68 (46–92) 24 (15–33)** 12 (7–17)*
>52 9 88 (51–125)* 18 (7–30)* 15 (0–31)
Data are mean (95% confidence interval). Statistical significance *P < 0.05 and **P < 0.005 compared with age <39 y.
GV, glycemic variability.

Beta-cell number (both per graft and total) and other baseline patient characteristics, such as BMI, HbA1c, and immune parameters had no correlation to the duration of graft function.

Culture Time of First Graft and Recipient Age Correlate With the Duration of C-peptide ≥1.0 ng/mL and Low Glycemic Variability and Insulin Independence

In total, 34 patients achieved C-peptide ≥1.0 ng/mL with low GV for a median duration of 22 months (IQR, 7–27) and 24 patients achieved insulin independence for a median duration of 15 months (IQR, 3.5–23).

Grafts following ≥96 hours culture resulted in more patients with low GV and plasma C-peptide ≥1.0 ng/mL, both at PT month 12 (9/10 versus 12/30; P = 0.005) and PT month 24 (7/10 versus 6/30; P = 0.007). In this subgroup, 9/10 patients became insulin-independent versus 15/30 in the other subgroup (P = 0.03). The duration of C-peptide ≥1.0 ng/mL with low GV was >12 months longer in recipients of the ≥96 hours grafts (Figure 2B) while no difference was measured in duration of insulin independence (Figure 2C).

Recipients age <39 years showed shortest duration of C-peptide ≥1.0 ng/mL with low GV and insulin independence compared with recipients of 39–52 years and >52 years (Table 2).

Graft Composition Relates to Culture Time

To further understand the influence of culture time on the duration of graft function, actual graft composition and baseline characteristics were studied in function of culture times longer or shorter than 96 hours.

As expected, grafts composed from cells that were all cultured for ≧96 hours contained a lower total number of cells. The number of beta cells was also reduced but to a lesser extent. However, endocrine purity was significantly higher in long-term cultured grafts (P = 0.028), illustrating that longer culture time results in grafts enriched with endocrine cells (Table 3). They also contained significantly less exocrine (P = 0.03) and duct cells.

TABLE 3. - Cell number and composition of grafts that contained cells that were all cultured for 96 h or not
First graft P
≧96 h in culture <96 h
Number of recipients 10 30
Beta cell per kg body weight (106/kg BW) 2.6 (2.1–3.1) 3.1 (2.7–3.5) 0.187
Graft cell number (×106)
 Total 491 (392–591) 757 (665–850) 0.001
 Beta cells 167 (135–200) 208 (185–232) 0.043
 Exocrine cells 4 (0.2–7) 40 (18–62) <0.001
 Duct cells 205 (131–279) 392 (323–461) 0.001
 Damaged cells 49 (30–67) 69 (56–81) 0.095
Graft composition (%)
 Endocrine 49 (38–60) 36 (31–41) 0.028
 Exocrine 0.7 (0.2–1.4) 4.7 (2.4–7.0) 0.003
 Duct 41 (31–50) 51 (46–55) 0.058
 Damaged 10 (7–12) 9 (8–10) 0.590
Data are mean (95% confidence Interval). Bold numbers indicate statistical significance (P < 0.05).

Recipients of grafts with a longer culture time had lower body weight and BMI than recipients of grafts with shorter culture times (Table 4). No differences in age and pretransplant HbA1c, autoimmune, and alloimmune measurements were found.

TABLE 4. - Baseline characteristics of recipients in function of culture time of first grafts
All cells cultured ≥96 h <100% cells cultured <96 h
First graft (n = 40) 10 30
Body weight (kg) 62.4 ± 7.4 70.3 ± 8.4 a
BMI (kg/m2) 22.0 ± 1.9 24.3 ± 2.4 a
Age at transplantation (y) 46.5 ± 6.4 45.4 ± 9.4
HbA1c (%) 7.2 ± 0.8 7.8 ± 0.9
Total lymphocyte count 1890 ± 996 1822 ± 500
CD3+ count 1384 ± 627 1259 ± 272
CD19+ count 296 ± 155 236 ± 98
Leukocyte count before ATG 5993 ± 2764 5972 ± 1438
CD4/CD8 ratio 2.0 ± 0.7 2.0 ± 1.0
Autoantibodies (ICA/GAD/IA2) 0/3/4 8/14/7
Positivity of 2 or more antibodies 4 18
T-cell autoreactivity against
 IA2 and/or GAD (yes) 4/10 19/26
 IA2 (yes) 1/10 10/26
 GAD (yes) 2/10 16/26
Presence of donor-specific HLA antibodies 2/10 3/30
Second graft (n = 30) 6/10 24/30
Total lymphocyte count 449.6 ± 165 661 ± 324
CD3+ count 201 ± 71 287 ± 163
CD19+ count 124 ± 67 216 ± 147
Leukocyte count 4583 ± 2254 3029 ± 1002
T-cell autoreactivity against
 IA2 and/or GAD (yes) 4/6 14/24
 IA2 (yes) 2/6 11/24
 GAD (yes) 4/6 8/24
Presence of donor-specific HLA antibodies 3/6 8/23
aStatistical significance P < 0.05 between 2 groups, independent T test. Data are mean ± standard deviation.
ATG, antithymocyte globulin; BMI, body mass index; GAD, glutamate decarboxylase; IA2, islet antigen 2; ICA, islet cell antibodies.

DISCUSSION

In this retrospective study, culture of islet preparations for at least 96 hours in first multidonor grafts was associated with prolonged graft survival. It also increased the duration of low glucose variability and a higher chance of reaching insulin independence, which is compatible with obtaining a higher graft beta-cell mass in the first-month PT.

To the best of our knowledge, this article is the first to compare short (<96 h) with long (≥96 h) culture time of human islet cell preparations.

More patients transplanted with longer cultured cells achieved clinically relevant graft function and insulin independence and achieved better preserved graft function significantly longer than recipients of shorter cultured cells. Several mechanisms might explain these beneficial effects of prolonged culture.

First, prolonged culture resulted in a reduced percentage of duct cells in the preparations. Human pancreatic duct cells are known to exert tissue-factor dependent procoaglulant activity36 and to express CD40,37 which are both linked to instant blood–mediated inflammatory responses and cell death.38 In vitro human data from Ramnath et al39 suggest that short-term culture already reduces the instant blood–mediated inflammatory response after intraportal infusion, and prolonging culture time even more might further improve these conditions.

Second, the lower number of exocrine cells and higher endocrine purity seen in preparations cultured ≥96 hours might further reduce their immunogenicity. Data in animal models showed a better survival of cultured islet transplants possibly linked to a higher percentage of endocrine cells in the graft and reduced immunogenicity.31,40-43 Previous studies indicate that prolonged culture reduces the number of endothelial cells and white blood cells within graft,44-46 which may propagate islet transplant rejection.46,47 Whatever the mechanisms involved, changes induced by long-term culture may be especially important in the first days and weeks of immune suppression. This study does not report on HLA antibody formation but our group previously reported on HLA sensitisation in patients with type 1 diabetes receiving islet cell grafts from multiple donors under the same immunosuppression as recipients in our current analysis.48 HLA antibodies assessed by solid-phase flow-based Luminex method and CDC assay were low in patients on immunosuppression. Allosensitization by Luminex but not CDC assay increased after immunosuppression withdrawal and was correlated with lower beta-cell purity but not with number of donors.48

No human data exist comparing transplantation of noncultured “fresh” islets with islet cells that have been cultured for a prolonged time. In this study, all preparations were cultured for at least 16 hours, so that the comparison between fresh and prolonged culture in terms of transplant outcome was not possible. It cannot be excluded that the beneficial effects of higher beta-cell mass and function in fresh preparations49-52 outweighs the benefits of reduced procoagulant activity and immunogenicity of long-cultured islet preparations.

Our data confirm the relationship between recipient age and achievement of good graft survival and insulin independence3,13,53,54 and extend this finding to duration of these beneficial effects. It is well known in other organ transplantation settings that older age correlates with lower rates of rejection and improved graft survival.55-57 Another reason for worse graft function in younger patients might be less compliance with the immunosuppressive regimen.58,59 It is conceivable that combining younger recipient age with grafts that have higher immunogenicity maximizes the risk for acute rejection and therefore lower beta-cell mass, resulting in shorter duration of graft function and insulin independence observed in our cohort. Currently, there are no established methods to diagnose rejection after islet transplantation, making proof of this hypothesis not feasible at this moment.

Our data also provide insight in the long-term survival of 1–2 grafts transplanted within a 3 months period. We report a 10-year graft survival rate of 50% in recipients of long-cultured islet grafts, which is in line with other centers.60,61 However, without repeated infusions, no graft function is preserved longer than 15 years after the first 2 implants, in line with previously published registry data.13 For that reason, our center recently started with reinfusions of islet grafts in patients that lost graft function several years after the first implants. Future data will show if this approach results in restoring graft function with improvement of metabolic control beyond 15 years.

Our study has limitations, such as the small number of patients who received long-cultured preparations. It still remains to be determined, ideally in a prospective study, if a minimum culture time of 96 hours for the first graft is the most optimal when other culture conditions or methods are used. However, the scarcity of donor organs and the variability of isolation outcomes make such a prospective study very unlikely. Additional analysis in larger cohorts is also needed to determine the absolute of risk of allosensitization after exposure to multiple donors, especially after discontinuation of immune suppression.

In summary, human islet isolates with insufficient beta-cell mass for implantation within 72 hours can be cultured for 96 hours and longer to combine preparations that together reach required dose and result in a metabolic benefit. The better outcome of grafts with a longer culture history was correlated to their higher endocrine purity, which is in line with observations in animal models.

ACKNOWLEDGMENTS

The authors thank Brigitta Swennen, Jolien Vincent en Mieke Verdeyen (University Hospitals Leuven); Soniya Thomas, Valerie Van Damme (Diabetes kliniek UZ Brussel) for their work in the clinical follow-up of the patients. The authors thank Cindy Tettelin and Veerle Kemels for data entry and coordination of the logistics. The authors also thank the Eurotransplant International Foundation and its transplant surgeons and coordinators for organ procurement. Finally, the authors thank the staff of the Beta cell Bank, the Belgian Diabetes Registry, the Diabetes Research Center of the Vrije Universiteit Brussel, and the Clinical Biology Department of UZ Brussel.

REFERENCES

1. CITR Research Group. 2007 update on allogeneic islet transplantation from the Collaborative Islet Transplant Registry (CITR). Cell Transplant. 2009; 18:753–767
2. Barton FB, Rickels MR, Alejandro R, et al. Improvement in outcomes of clinical islet transplantation: 1999-2010. Diabetes Care. 2012; 35:1436–1445
3. Balamurugan AN, Naziruddin B, Lockridge A, et al. Islet product characteristics and factors related to successful human islet transplantation from the Collaborative Islet Transplant Registry (CITR) 1999-2010. Am J Transplant. 2014; 14:2595–2606
4. Keymeulen B, Gillard P, Mathieu C, et al. Correlation between beta cell mass and glycemic control in type 1 diabetic recipients of islet cell graft. Proc Natl Acad Sci U S A. 2006; 103:17444–17449
5. Lablanche S, Borot S, Wojtusciszyn A, et al.; GRAGIL Network. Five-year metabolic, functional, and safety results of patients with type 1 diabetes transplanted with allogenic islets within the Swiss-French GRAGIL network. Diabetes Care. 2015; 38:1714–1722
6. Ludwig B, Reichel A, Kruppa A, et al. Islet transplantation at the Dresden diabetes center: five years’ experience. Horm Metab Res. 2015; 47:4–8
7. O’Connell PJ, Holmes-Walker DJ, Goodman D, et al.; Australian Islet Transplant Consortium. Multicenter Australian trial of islet transplantation: improving accessibility and outcomes. Am J Transplant. 2013; 13:1850–1858
8. Koh A, Senior P, Salam A, et al. Insulin-heparin infusions peritransplant substantially improve single-donor clinical islet transplant success. Transplantation. 2010; 89:465–471
9. Hering BJ, Kandaswamy R, Ansite JD, et al. Single-donor, marginal-dose islet transplantation in patients with type 1 diabetes. JAMA. 2005; 293:830–835
10. Hering BJ, Clarke WR, Bridges ND, et al.; Clinical Islet Transplantation Consortium. Phase 3 trial of transplantation of human islets in type 1 diabetes complicated by severe hypoglycemia. Diabetes Care. 2016; 39:1230–1240
11. Tekin Z, Garfinkel MR, Chon WJ, et al. Outcomes of pancreatic islet allotransplantation using the Edmonton protocol at the University of Chicago. Transplantation Direct. 2016; 2:e105–e112
12. Vantyghem MC, de Koning EJP, Pattou F, et al. Advances in β-cell replacement therapy for the treatment of type 1 diabetes. Lancet. 2019; 394:1274–1285
13. CITR Coordinating Center. Collaborative Islet Transplant Registry (CITR) 2015 (Tenth) Annual Report. 2018:Rockville, MD: The Emmes Corporation; 1–328
14. Ryan EA, Lakey JR, Paty BW, et al. Successful islet transplantation: continued insulin reserve provides long-term glycemic control. Diabetes. 2002; 51:2148–2157
15. Holmes MA, Clayton HA, Chadwick DR, et al. Functional studies of rat, porcine, and human pancreatic islets cultured in ten commercially available media. Transplantation. 1995; 60:854–860
16. Huurman VA, Hilbrands R, Pinkse GG, et al. Cellular islet autoimmunity associates with clinical outcome of islet cell transplantation. PLoS One. 2008; 3:e2435
17. Hilbrands R, Huurman VA, Gillard P, et al. Differences in baseline lymphocyte counts and autoreactivity are associated with differences in outcome of islet cell transplantation in type 1 diabetic patients. Diabetes. 2009; 58:2267–2276
18. Piemonti L, Everly MJ, Maffi P, et al. Alloantibody and autoantibody monitoring predicts islet transplantation outcome in human type 1 diabetes. Diabetes. 2013; 92:1656–1664
19. Naziruddin B, Matsumoto S, Noguchi H, et al. Improved pancreatic islet isolation outcome in autologous transplantation for chronic pancreatitis. Cell Transplant. 2012; 21:553–558
20. Keymeulen B, Ling Z, Gorus FK, et al. Implantation of standardized beta-cell grafts in a liver segment of IDDM patients: graft and recipients characteristics in two cases of insulin-independence under maintenance immunosuppression for prior kidney graft. Diabetologia. 1998; 41:452–459
21. Scharp DW, Lacy PE, Santiago JV, et al. Insulin independence after islet transplantation into type I diabetic patient. Diabetes. 1990; 39:515–518
22. Ling Z, Pipeleers DG. Prolonged exposure of human beta cells to elevated glucose levels results in sustained cellular activation leading to a loss of glucose regulation. J Clin Invest. 1996; 98:2805–2812
23. Movahedi B, Keymeulen B, Lauwers MH, et al. Laparoscopic approach for human islet transplantation into a defined liver segment in type-1 diabetic patients. Transpl Int. 2003; 16:186–190
24. Maleux G, Gillard P, Keymeulen B, et al. Feasibility, safety, and efficacy of percutaneous transhepatic injection of beta-cell grafts. J Vasc Interv Radiol. 2005; 16:1693–1697
25. Gillard P, Hilbrands R, Van de Velde U, et al. Minimal functional β-cell mass in intraportal implants that reduces glycemic variability in type 1 diabetic recipients. Diabetes Care. 2013; 36:3483–3488
26. Deters NA, Stokes RA, Gunton JE. Islet transplantation: factors in short-term islet survival. Arch Immunol Ther Exp (Warsz). 2011; 59:421–429
27. Fiorina P, Vergani A, Petrelli A, et al. Metabolic and immunological features of the failing islet-transplanted patient. Diabetes Care. 2008; 31:436–438
28. Kin T, Senior P, O’Gorman D, et al. Risk factors for islet loss during culture prior to transplantation. Transpl Int. 2008; 21:1029–1035
29. Bertuzzi F, Ricordi C. Prediction of clinical outcome in islet allotransplantation. Diabetes Care. 2007; 30:410–417
30. Hirsch D, Odorico J, Danobeitia JS, et al. Early metabolic markers that anticipate loss of insulin independence in type 1 diabetic islet allograft recipients. Am J Transplant. 2012; 12:1275–1289
31. Ihm SH, Matsumoto I, Zhang HJ, et al. Effect of short-term culture on functional and stress-related parameters in isolated human islets. Transpl Int. 2009; 22:207–216
32. Ihm SH, Matsumoto I, Sawada T, et al. Effect of donor age on function of isolated human islets. Diabetes. 2006; 55:1361–1368
33. Nano R, Clissi B, Melzi R, et al. Islet isolation for allotransplantation: variables associated with successful islet yield and graft function. Diabetologia. 2005; 48:906–912
34. Street CN, Lakey JR, Shapiro AM, et al. Islet graft assessment in the Edmonton Protocol: implications for predicting long-term clinical outcome. Diabetes. 2004; 53:3107–3114
35. Demeester S, Balke EM, Van der Auwera BJ, et al. HLA-A*24 carrier status and autoantibody surges posttransplantation associate with poor functional outcome in recipients of an islet allograft. Diabetes Care. 2016; 39:1060–1064
36. Beuneu C, Vosters O, Movahedi B, et al. Human pancreatic duct cells exert tissue factor-dependent procoagulant activity: relevance to islet transplantation. Diabetes. 2004; 53:1407–1411
37. Vosters O, Beuneu C, Nagy N, et al. CD40 expression on human pancreatic duct cells: role in nuclear factor-kappa B activation and production of pro-inflammatory cytokines. Diabetologia. 2004; 47:660–668
38. Moberg L, Johansson H, Lukinius A, et al. Production of tissue factor by pancreatic islet cells as a trigger of detrimental thrombotic reactions in clinical islet transplantation. Lancet. 2002; 360:2039–2045
39. Ramnath RD, Maillard E, Jones K, et al. In vitro assessment of human islet vulnerability to instant blood-mediated inflammatory reaction (IBMIR) and its use to demonstrate a beneficial effect of tissue culture. Cell Transplant. 2015; 24:2505–2512
40. Keymeulen B, Anselmo J, Pipeleers D. Length of metabolic normalization after rat islet cell transplantation depends on endocrine cell composition of graft and on donor age. Diabetologia. 1997; 40:1152–1158
41. Pipeleers DG, Pipeleers-Marichal M, Vanbrabandt B, et al. Transplantation of purified islet cells in diabetic rats. II. Immunogenicity of allografted islet beta-cells. Diabetes. 1991; 40:920–930
42. Jacobs-Tulleneers-Thevissen D, Bartholomeus K, Suenens K, et al. Human islet cell implants in a nude rat model of diabetes survive better in omentum than in liver with a positive influence of beta cell number and purity. Diabetologia. 2010; 53:1690–1699
43. Jacobs-Tulleneers-Thevissen D, Chintinne M, Ling Z, et al.; Beta Cell Therapy Consortium EU-FP7. Sustained function of alginate-encapsulated human islet cell implants in the peritoneal cavity of mice leading to a pilot study in a type 1 diabetic patient. Diabetologia. 2013; 56:1605–1614
44. Lacy PE, Davie JM, Finke EH. Effect of culture on islet rejection. Diabetes. 1980; 29Suppl 193–97
45. Kuttler B, Hartmann A, Wanka H. Long-term culture of islets abrogates cytokine-induced or lymphocyte-induced increase of antigen expression on beta cells. Transplantation. 2002; 74:440–445
46. Lafferty KJ, Prowse SJ, Simeonovic CJ, et al. Immunobiology of tissue transplantation: a return to the passenger leukocyte concept. Annu Rev Immunol. 1983; 1:143–173
47. Rose ML. Endothelial cells as antigen-presenting cells: role in human transplant rejection. Cell Mol Life Sci. 1998; 54:965–978
48. Hilbrands R, Gillard P, Van der Torren CR, et al. Predictive factors of allosensitization after immunosuppressant withdrawal in recipients of long-term cultured islet cell grafts. Transplantation. 2013; 96:162–169
49. Noguchi H, Naziruddin B, Jackson A, et al. Fresh islets are more effective for islet transplantation than cultured islets. Cell Transplant. 2012; 21:517–523
50. King A, Lock J, Xu G, et al. Islet transplantation outcomes in mice are better with fresh islets and exendin-4 treatment. Diabetologia. 2005; 48:2074–2079
51. Brandhorst D, Brandhorst H, Hering BJ, et al. ATP content of isolated islets: indication for species-dependent vulnerability for cell-mediated graft rejection? Transplant Proc. 1997; 29:2058
52. Fraga DW, Sabek O, Hathaway DK, et al. A comparison of media supplement methods for the extended culture of human islet tissue. Transplantation. 1998; 65:1060–1066
53. Al-Adra DP, Gill RS, Imes S, et al. Single-donor islet transplantation and long-term insulin independence in select patients with type 1 diabetes mellitus. Transplantation. 2014; 98:1007–1012
54. Lee D, Keymeulen B, Hilbrands R, et al. Age and early graft function relate with risk-benefit ratio of allogenic islet transplantation under antithymocyte globulin-mycophenolate mofetil-tacrolimus immune suppression. Transplantation. 2017; 101:2218–2227
55. Tullius SG, Tran H, Guleria I, et al. The combination of donor and recipient age is critical in determining host immunoresponsiveness and renal transplant outcome. Ann Surg. 2010; 252:662–674
56. Kidney Disease: Improving Global Outcomes (KDIGO) CKD-MBD Work Group. KDIGO clinical practice guideline for the diagnosis, evaluation, prevention, and treatment of chronic kidney disease-mineral and bone disorder (CKD-MBD). Kidney Int Suppl. 2009; 76:S1–S130
57. Pratschke J, Dragun D, Hauser IA, et al. Immunological risk assessment: the key to individualized immunosuppression after kidney transplantation. Transplant Rev (Orlando). 2016; 30:77–84
58. Gaynor JJ, Ciancio G, Guerra G, et al. Graft failure due to noncompliance among 628 kidney transplant recipients with long-term follow-up: a single-center observational study. Transplantation. 2014; 97:925–933
59. Bosma OH, Vermeulen KM, Verschuuren EA, et al. Adherence to immunosuppression in adult lung transplant recipients: prevalence and risk factors. J Heart Lung Transplant. 2011; 30:1275–1280
60. Vantyghem MC, Chetboun M, Gmyr V, et al.; Members of the Spanish Back Pain Research Network Task Force for the Improvement of Inter-Disciplinary Management of Spinal Metastasis. Ten-year outcome of islet alone or islet after kidney transplantation in type 1 diabetes: a prospective parallel-arm cohort study. Diabetes Care. 2019; 42:2042–2049
61. Lehmann R, Graziano J, Brockmann J, et al. Glycemic control in simultaneous islet-kidney versus pancreas-kidney transplantation in type 1 diabetes: a prospective 13-year follow-up. Diabetes Care. 2015; 38:752–759

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