ABSTRACT
Type 2 diabetes mellitus (T2D) affects 463 million individuals worldwide. β-cell dysfunction and relatively inadequate β-cell mass has been implicated in the pathogenesis of T2D. Primary human islets from T2D patients can reveal the islet dysfunction and the underlying mechanisms and thus have become valued resources for diabetes research. Our center (Human Islet Resource Center, China) has prepared a number of batches of human islets from T2D organ donors. The present study aims to characterize islet isolation processes, islet yields, and qualities of T2D pancreases by comparing with non-diabetic (ND) ones. Overall, 24 T2D and 80 ND pancreases were obtained with informed research consents. The digestion time, islet purity, yield, size distribution, islet morphology score, viability, and function in each islet preparation were analyzed. We found that at digestion stage, T2D pancreases need significantly longer digestion duration and have worse digestion rates and lower gross islet yields. At purification stage, T2D pancreases have poorer purity, purification rate, morphology score, and islet yields after purification. Functional evaluation by GSI assay showed that the human T2D islets have significantly lower glucose stimulated insulin secretion ability. In conclusion, the features of longer digestion duration, lower yields and quality, and impaired insulin secretion in T2D group are consistent with the pathological condition of this disease. Both islet yields and islet function evaluation results did not support human T2D islets as clinical transplantation resources. However, they could serve as good research models for T2D disease studies and promote the advancement of diabetes research.
KEYWORDS: Human islet preparation, islet morphology, islet of Langerhans, islet purity, islet quality, islet yield, Type 2 diabetes mellitus
GRAPHICAL ABSTRACT
Introduction
Islet failure either by mass loss or dysfunction underlies all forms of diabetes, including Type 2 diabetes mellitus (T2D), but the underlying mechanism remains obscure.1 Human islet research is crucial to understand the cellular biology and pathology of human islets.2 Our knowledge on this field is expanding, benefitting from the foundation of human distribution programs around the world, such as Integrated Islet Distribution Program (IIDP), Alberta Diabetes Institute IsletCore, and The Human Pancreas Analysis Program.3–5 Over the past two decades, these programs and commercial sources have supplied millions of human islets to hundreds of scientists, leading to a dramatic increase in the number of scientific publications describing research with human islets. However, study on human T2D islets is relatively lacking but necessary for a better understanding of the cellular biology of the pancreas with T2D. In addition, human islets from diabetic donors are important research materials for new therapies development aiming to improve islet function in T2D. Therefore, our center has begun to isolate human T2D islets for scientific research usage since 2017. Here we made a retrospective comparison of the islet isolation procedure and outcomes between the human T2D pancreases and non-diabetic (ND) ones. The digestion and purification processes, as well as the islet yields, size distributions, morphology scores, purity, viability, and functionality were compared.
Materials and methods
Donor information
Human pancreases were obtained from organ donors with informed research consents in the Tianjin First Central Hospital. The organ donation procedure follows the regulations in China. The donor information is shown in Table 1.
Table 1.
Donor characteristics.
| ND | T2D | P Value | ||
|---|---|---|---|---|
| Age (years) | 45.26 ± 7.83 | 49.21 ± 10.12 | 0.0503 | |
| Gender | Male (%) | 85 | 75 | 0.2561 |
| Female (%) | 15 | 25 | ||
| BMI (kg/m2) | 25.18 ± 4.06 | 26.59 ± 3.90 | 0.1529 | |
| HbA1c (%) | 5.33 ± 0.36 | 7.13 ± 1.22 | <0.0001*** | |
| C-Peptide (ng/ml) | 8.52 ± 7.07 | 7.64 ± 6.64 | 0.6783 | |
| CIT (h) | 5.70 ± 3.89 | 4.71 ± 3.05 | 0.2838 | |
| Pancreas mass (g) | 86.86 ± 24.52 | 82.48 ± 21.14 | 0.3213 | |
Data were shown as mean±SD. ND (n = 80); T2D (n = 24).
BMI, body mass index; CIT, cold ischemia time; ND, non-diabetic; T2D, Type 2 diabetes.
Pancreas procurement
The pancreas perfusion with University of Wisconsin (UW) solution was conducted via the abdominal aorta in situ under low temperature and low pressure. Then the pancreas was dissected with the pancreatic capsule retained. Being preserved in cold UW solution, the pancreas was transferred to the Current Good Manufacturing Practice (cGMP) facility, Human islet resource center, Department of cell transplantation, Tianjin First Central Hospital.
Islet isolation
The pancreas was further finely trimmed and cannulated prior to the enzyme solution perfusion via the head and body/tail using an automated perfusion apparatus (BioRep Technologies, Miami, FL, USA). The enzymes of Liberase MTF C/F (Roche Diagnostics, Roche Applied Science, Indianapolis, IN, USA) collagenase were perfused to the pancreas. The pancreas was put in an isolation chamber with continuous enzyme solution circulation and a gentle mechanical dissociation, as described by Ricordi et al. (1988).6 Tissue collection was initiated when ＞50% of islets were released. The digested tissues were resuspended with 150 mL UW solution for 30 min. Free islets were separated from exocrine tissues by continuous density gradient centrifugation of Biocoll separating solution (Biochrom AG, Ontario, Canada) in an apheresis system (model 2991, Cobe Laboratories). Purified islets were cultured in CMRL-1066 medium (Corning, NY, USA), supplemented with 10% Human Serum Albumin (Baxter, Shanghai, China), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C in 5% CO2.
Islet yields assessment
Islet yields were calculated both before and after purification. A total of 100 µL resuspended islets are diluted to dithizone-containing PBS (3.12 mmol/L). Particles stained with a red color is identified as islets and counted under the microscope. Islets at different sizes were counted by a calibrated grid under microscope. The islet particle number (IPN) was calculated by the sum of total counts of all islets, and the islet equivalent number was calculated using a published conversion table.7–9 The islet yields per gram of pancreas tissue were calculated via dividing the total islet yields (IEQ or IPN) by pancreas weight (g).
Islet purity assessment
Islet purity after purification in each tube was determined by the proportion of dithizone-stained tissue volume versus total tissue volume.
Islet morphology score
Islet morphology parameters including size, degree of fragmentation, density of islet stained by dithizone (Sigma-Aldrich), border, and shape were scored after islet purification. The five individual subscores ranged from 0 to 2; the total islet score was the sum of the five subscores and ranged from 0 to 10.10 Meanwhile, the fragmentation rate and acinar trapped rate were estimated.
Viability assay
The islets viability was analyzed via fluorosceindiacetate/propidium iodide (FDA/PI) staining followed by fluorescence imaging. FDA produces bright green fluorescence (viable cells) cells, while PI produces red fluorescence (dead or dying cells). The viability of 50 islet particles was evaluated. The ratio of green fluorescence area to the total islet area was calculated as the viable rate.
Insulin secretion assay
Ten islets were pretreated in 1 mL low glucose (1.67 mM) Krebs–Ringer bicarbonate buffer (KRB; supplemented with 0.5% BSA, pH 7.4) for 1 hour in 12-well plate, followed by sequential 1-hour treatment with 1 mL low-glucose KRB solution (1.67 mM) and high-glucose KRB solution (16.7 mM) for 1 hour. Insulin concentration at low and high glucose were measured by ELISA (Human Insulin Elisa Kit; Mercodia, Uppsala, Sweden). Insulin secretion of islets was expressed as the glucose stimulated index (GSI; insulin content in the high glucose media/insulin content in the low glucose media).
Statistical analysis
All data were analyzed by two-tailed student’s t test using GraphPad Prism (GraphPad Software 7.0, La Jolla, CA, USA). p < 0.05 were considered statistically significant.
Results
Donor characteristics
The key characteristics of pancreas donors are shown in Table 1. Overall, 24 T2D pancreases from 18 male and 6 female organ donors and 80 ND pancreases from 68 male and 12 female organ donors were surveyed in this study. There were no statistical differences in age, BMI, and gender composition, as well as serum c-peptide, cold ischemia time, and pancreas mass. The mean HbA1c in the T2D organ donors was significantly higher HbA1c (p < 0.001), consistent with their disease conditions.
Digestion
The digestion duration of the T2D pancreases is 11.95 ± 3.98 min by average, while that of the ND counterparts is 10.13 ± 2.67 min. It takes 1.82 minutes longer to digest a T2D diseased pancreas than an ND pancreas. Despite this, the digestion efficiency is still not as efficient as that of an ND pancreas (61.65 ± 20.16 vs 73.32 ± 14.26, p < 0.01). The longer digestion duration but lower digestion efficiency means that there is more digestion resistance for T2D pancreases, possibly due to the fibrosis, fat infiltration, inflammation, etc., which frequently occurs in T2D pancreases.
After digestion, the islet yields were determined, with STZ staining as an indicator (Figure 1a). The average total islet yield for ND pancreases was 471163 ± 217286 IEQ per pancreas. Yet, that of T2D pancreases was 256,238 ± 184662 IEQ per pancreas, which is only a half of its counterparts. To rule out the influence of pancreas sizes, we normalized the islet yields to each corresponding pancreas mass. The islet yield for each gram of pancreas tissue is 5565 ± 2691 IEQ in ND group and 2930 ± 2428 in T2D group, and the latter is still roughly a half of its counterpart. In accordance with the difference between IEQ, the total islet particle numbers (IPNs) and the normalized IPNs per gram of pancreas tissue also each showed a 46% reduction.
Figure 1.
Representative images of islet purity and viability. (a) Representative images of DTZ-stained human islets before or after purification from both T2D group and ND group. (b) Islet purity distribution between ND and T2D groups. High: purity ≥ 70%; Medium: 40% ≤ purity < 70%; Low: purity < 40%.
Purification
The digested tissues were resuspended with 150 mL UW solution and then went through a continuous linear density gradient centrifugation. The purified islets were collected into six tubes, coding tube 1 to tube 6 according to the order that they were collected. Representative images of DTZ-stained islets in each group post purification are shown in Figure 1a. It can be seen that the islet isolations from pancreatic of donors with T2D were generally successful, with the uniform islet morphology and an appreciable amount of islets trapped in acinar tissue. The obvious difference was that the purity of islet from T2D was lower than that of ND group (Table 2). In the ND group, the purity in the first three tubes maintained at around 70–80%, and the purity in the following three tubes began to descend to 53% in tube 4, 33% in tube 5, and 19% in tube 6. In the T2D group, only the first two tubes maintained over 70% purity and that in tubes 3 and 4 in T2D group was significantly lower than their counterparts in ND group. The islets after purification were divided into three groups with high purity (≥70%), medium purity (40%-69%), and low purity (<40%). The distribution of islets in three groups has no significant difference (Figure 1b).
Table 2.
Digestion and purification.
| ND | T2D | P Value | |
|---|---|---|---|
| Digestion procedure | |||
| Digestion duration | 10.13 ± 2.76 | 11.95 ± 3.98 | 0.0143* |
| Digestion efficiency | 73.32 ± 14.26 | 61.65 ± 20.16 | 0.0023** |
| Islet yields | |||
| IEQ | 471163 ± 217286 | 256238 ± 184662 | <0.0001*** |
| IEQ/g pancreas | 5565 ± 2691 | 2930 ± 2428 | 0.0001*** |
| IPN | 514296 ± 259240 | 279855 ± 179746 | 0.0001*** |
| IPN/g pancreas | 6214 ± 3362 | 3267 ± 2643 | 0.0003*** |
| IEQ/IPN | 0.33 ± 0.24 | 0.25 ± 0.32 | 0.3772 |
| Islet yields after purification | |||
| IEQ | 229199 ± 142663 | 96817 ± 70888 | 0.0001*** |
| IEQ/g pancreas | 2719 ± 1683 | 1085 ± 687 | <0.0001*** |
| IPN | 273381 ± 180514 | 106186 ± 73301 | 0.0004*** |
| IPN/g pancreas | 3234 ± 2245 | 1199 ± 756 | 0.0002*** |
| Purification efficacy | |||
| IEQ | 0.49 ± 0.24 | 0.35 ± 0.29 | 0.0349* |
| IPN | 0.56 ± 0.24 | 0.38 ± 0.25 | 0.0098** |
| Purity | |||
| Tube 1 | 80.31 ± 20.33 | 77.65 ± 24.37 | 0.6463 |
| Tube 2 | 82.43 ± 18.24 | 74.75 ± 22.09 | 0.1123 |
| Tube 3 | 70.96 ± 21.49 | 57.38 ± 25.91 | 0.0157* |
| Tube 4 | 52.75 ± 24.94 | 36.25 ± 26.64 | 0.0106* |
| Tube 5 | 33.18 ± 21.13 | 26.44 ± 22.67 | 0.2327 |
| Tube 6 | 18.81 ± 14.93 | 17.61 ± 18.93 | 0.7737 |
Data were shown as mean ± SD. IEQ: islet equivalents; IEQ/g: islet equivalents/gram pancreas tissue; IPN: islet particle number; IPN/g: islet particle number/gram pancreas tissue.
The total recovery rate is also significantly lower in T2D group. The islet yields in ND group are 229199 ± 142663 IEQ and 273381 ± 180514 IPNs per preparation and 2719 ± 1683 IEQ and 3234 ± 2245 IPNs by per gram pancreas tissue. However, these numbers in T2D sharply decreased to 96817 ± 70888 IEQ and 106186 ± 73301 IPNs per preparation and 1085 ± 687 IEQ and 1199 ± 756 IPNs by per gram pancreas tissue, significantly lower than those in ND group (Table 2).
The average recovery rate in ND group is 49% by IEQ and 56% by IPN, which is comparable to the reports by other groups. Yet that in T2D group is only 35% by IEQ and 38% by IPN (Table 2).
Islet quality
Islet quality was assessed using the previously reported islet morphology score system.10,11 The islet morphology scores were significantly lower in T2D donors group comparing to the ND group (Table 3). The subscores of size, border, and shape were similar between the two groups, but those of degree of fragmentation and density of islet stained by dithizone were significantly lower in the T2D groups than that in ND group (Table 3).
Table 3.
Islet morphology.
| ND | T2D | P Value | |
|---|---|---|---|
| Total morphology score | 8.16 ± 1.01 | 7.26 ± 1.18 | 0.0036** |
| Size | 1.68 ± 0.28 | 1.55 ± 0.29 | 0.1082 |
| Degree of fragmentation | 1.59 ± 0.30 | 1.30 ± 0.42 | 0.0020** |
| Density of islet stained by dithizone | 1.67 ± 0.31 | 1.48 ± 0.21 | 0.0289* |
| Border | 1.58 ± 0.34 | 1.46 ± 0.37 | 0.2243 |
| Shape | 1.63 ± 0.30 | 1.46 ± 0.24 | 0.0628 |
| Trapped rate | 23.77 ± 16.55 | 25.71 ± 18.38 | 0.6912 |
| Fragmentation rate | 6.43 ± 4.02 | 12.5 ± 10.33 | 0.0002*** |
There is no significant difference in the trapped islet rate between the two groups, but the islet fragment rate from T2D donors was significantly higher comparing to that from ND donors (Table 3).
FDA/PI staining results demonstrated that the isolated islets from T2D donors maintained similar viability as the ND donors (Figure S).
Islet size distribution
We then compared the islet size distribution in each group. The islets were divided into six groups according to the islet diameter: 50–100 µm, 101–150 µm, 151–200 µm, 201–250 µm, 251–300 µm, and ≥301 µm. The islets with a diameter between 50 and 100 μm accounted for the largest proportion in both T2D and ND groups, followed by the islets between 101–150 μm and 151–200 μm. Islets over 200 μm only accounted for a small percentage (Figure 2a). There was no significant difference in the islet distribution between the two groups (Figure 2a). Regarding the islet number reduction in each group of T2D islets, we found that islets with diameters under 300 µm were all significantly reduced in T2D (Figure 2b), which is consistent with overall shrinkage of islets yields in T2D (Table 2). The number of super large islets (>300 µm) has no statistical difference, possibly due to the extremely low proportions in this group (Figure 2b). Together, these results suggested that the decrease in islet yields in T2D is rather an overall reduction in islets with different sizes than a reduction of islets with special size ranges.
Figure 2.
Islet size distribution. Islets were grouped by diameters, 50–100 µm, 101–150 µm, 151–200 µm, 201–250 µm, 251–300 µm, and ≥300 µm. (a) Proportions of each group of islets in ND and T2D. (b) Numbers (IPNs) of each group of islets with different diameters in ND and T2D.
Islet function
The glucose stimulated insulin secretion assay showed that the glucose stimulation index (GSI), which is calculated by the ratio of insulin secretion in high-glucose condition to that in low-glucose condition, was significantly lower in T2D group than that in ND group (p < 0.05) (Figure 3a). The basal insulin secretion at low-glucose condition had no significant difference between the ND group and the T2D group, yet the insulin secretion at high-glucose condition was significantly lower in T2D group (n = 12) than that in ND group (n = 50) (p < 0.05, Figure 3b). GSI of islets was negatively correlated with HbA1c (p < 0.05) (Figure 3c). These results showed that the islet function has been significantly impaired in T2D islets.
Figure 3.
Glucose stimulated index of human T2D and ND islets. (a) GSI was significantly decreased in human T2D islets compared to ND ones. t-test. (b) Insulin contents in the culture medium of 10 human islets for 1 hour at low (1.67 mM) and high (16.7 mM) glucose concentrations, respectively. t-test. (c) GSI of islets was negatively correlated with HbA1c. Simple linear regression. *p < 0.05, ***p < 0.001.
Discussion
In this study, we systematically analyzed the manufacturing procedure of human islets from organ donors with T2D and evaluated the quantity and quality characteristics of the produced islets, in comparison with that for ND donors.
The digestion duration was generally longer on average but still with lower digestion efficacy in T2D pancreases, indicating that the pancreases with T2D is more difficult to digest than that in non-diabetic condition. The physiological and morphological features are more complicated in T2D pancreases, such as the prevalence of pancreas atrophy,12 fibrosis,13 fat deposition,14 amyloid deposition,15 etc., which may influence the working efficiency of enzymes or increase the digestion resistance physically.
The islet yields were lower in T2D pancreases than that for ND pancreases. This is consistent with previous studies that islet yield and purity were impacted by donor HbA1c and diabetes status5,16. Reduced islet mass itself in T2D subjects in Chinese is a possible reason. Our previous study has suggested that there is a decreasing trend of islet density in human T2D pancreas sections.17
The recovery rate of islets during purification in T2D islet isolation was lower than that in group, and the final islet purity is also significantly lower in T2D. The islet size distribution, acinar trapped rate, and islet viability did not show significant difference between the two group, denying the contribution of these factors on the significantly lower islet purity in T2D group. However, the morphology scores were significantly lower and the fragmentation rate was significantly higher in human T2D islets, suggesting that these two factors might be mainly accounted for the difficulty of purification.
In addition, glucose stimulated index is a regular test of islet function in our center. Here, we also compared the GSI of the two groups and confirmed that the GSI of human T2D islets are significantly lower that of ND ones, which is consistent with the disease condition.
The limitations of this study include the following: (1) The purity calculations is dependent on the DTZ staining of islets, which may lead to a higher estimation of the islets purity when compared with the more rigorous approach of the whole islet volume fraction based on EM determination of cell composition18. However, given its advantage of repaid assessment, this DTZ-based method is still frequently used for clinical islet transplantation. (2) The glucose stimulated insulin secretion assay in this study was normalized to islet number, which is not as accurate as being normalized by DNA contents or protein contents. (3) The intracellular insulin content and glucagon secretions are also an important function of islets, yet it was not accessed in the islet quality evaluation for clinical islet transplantation. These above assays should be involved in future studies.
In summary, we described the features of islet manufacturing from human T2D organ donors in comparison with that from non-diabetic ones in Chinese populations. The T2D phenotype did result in the otherness in islet isolation process, islet yields, and quality. At the enzyme digestion stage, the T2D pancreases have longer average digestion duration, worse digestion rate, and significantly lower gross islet yields. At the purification stage, the T2D pancreases have poorer purity, purification rate, and islet yields after purification. Functional evaluation by GSI assay showed that human T2D islets have significantly lower glucose stimulated insulin secretion ability. These features are consistent with the pathological condition of this disease. Both islet yields and islet function evaluation results did not support human T2D islets as clinical transplantation resources. However, they could serve as good research models for T2D disease studies and promote the advancement of diabetes research. A systematic comparison of islet manufacturing process and outcomes between human T2D pancreases and their non-diabetic counterparts is crucial for the refinement of islet isolation, banking, and distribution procedures for human T2D islets.
Funding Statement
This work was supported by National Key Research and Development Program of China (2020YFA0803704), National Natural Science Foundation of China (82070805, 82100841, 82100840, 82200890), Tianjin Municipal Science and Technology Bureau (20JCQNJC01720, 21JCQNJC01850), Tianjin Municipal Human Resources and Social Security Bureau (XB202011), Tianjin Municipal Health Commission (ZC20222), Tianjin First Central Hospital (2020CL01, Youth Talent Cultivation Program to R. Liang.).
Abbreviations
- BMI
body mass index
- CIT
cold ischemia time
- GSIS
glucose-stimulated insulin secretion
- HbA1c
glycated hemoglobin A1c
- IEQ
islet equivalent
- T1DM
type 1 diabetes mellitus
- T2D
type 2 diabetes mellitus
Disclosure statement
No potential conflict of interest was reported by the author(s).
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