Islet transplantation is a valid treatment option for patients suffering from type 1 diabetes mellitus. To assure optimal islet cell quality, specialized islet isolation facilities have been developed. Utilization of such facilities necessitates transportation of islet cells to distant institutions for transplantation. Despite its importance, a clinically feasible solution for the transport of islets has still not been established. We here compare the functionality of isolated islets from C57BL/6 mice directly after the isolation procedure as well as after two simulated transport conditions, static versus rotation. Islet cell quality was assessed using real-time live confocal microscopy.
Human pancreatic islet transplantation has emerged as a potentially curative therapy for selected patients suffering from type 1 diabetes mellitus, especially those with inadequate glucose control despite intensive insulinotherapy [
Due to the complexity of islet isolation, networks have been established between specialized islet isolation facilities and distant islet transplantation centers. The use of such isolation facilities have proven successful in recent years, both in the United States and Europe; as they ensure optimized utilization of donated pancreata and guarantee supply of islets with consistently high quality. However, such facilities involve the necessity to ship islets from the isolation to the transplantation facility, thereby negatively influencing islet cell quality. Despite its importance there is no consensus on a clinically feasible solution for the transport of islets [
Recently, we were able to show that the use of a perfused rotary transport device (ROTi) allows high cell viability and quality of human islets, even after a simulated transport of 24 h [
We have previously shown that human pancreatic islets can be maintained in rotating wall vessels for up to one week without a significant loss of viability [
In the present study, we assessed murine islet quality and function
Eight- to ten-week-old male C57BL/6 mice obtained from Harlan-Winkelmann Co. (Borchen, Germany) were donor and recipient pairs. Animals were housed under standard conditions at the animal center of Innsbruck Medical University. All animals received humane care in compliance with the “Principles of Laboratory Animal Care” formulated by the National Society for Medical Research and the “Guide for the Care and Use of Laboratory Animals” prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH Publication number 86-23, revised 1985). All experiments were approved by the Austrian Federal Ministry for Education, Arts and Culture. Recipient mice were treated with 175 mg/kg body weight of streptozotocin (STZ, Sigma-Aldrich, St. Louis, MO, USA) to induce diabetes. Blood glucose was measured with a blood glucose monitor (SureStep; Lifescan, Milpitas, CA, USA); only animals with blood glucose levels over 350 mg/dL were included in the study.
Murine islets were isolated according to the method described by Ricordi et al. in 1988 with slight modifications as described earlier [
The first part of the study consisted of live confocal microscopy-based cell viability measurements conducted after isolation of the murine islets.
In the first group, cells were analyzed after 15 h simulated transport under standard sedimentation conditions in a 50 mL tube. The second group consisted of cells analyzed after 15 h simulated transport in a rotation chamber using the Synthecon RCCS-4D Rotation System (Synthecon, Houston, TX, USA) placed in an incubator (5% CO2, 37°C). Rotation speed was 8 rpm. Islets assessed immediately after isolation served as controls.
In the second part of the study 250 islets from C57BL/6 mice were transplanted into syngeneic recipients directly after isolation. In a second group the islets that underwent simulated transport of 15 h in the Synthecon device were transplanted likewise. Due to the low cell viability observed after simulated transport under static conditions, these islet cells were not transplanted.
The
In both transported groups viability of the islet cells was assessed by “real-time” confocal analysis after isolation and before transplantation. Confocal microscopy was performed with a microlens-enhanced Nipkow disk-based UltraVIEW RS confocal scanner (Perkin Elmer, Wellesley, MA, USA) mounted on an Olympus IX-70 inverse microscope (Olympus, Vienna, Austria). Cell morphology was visualized with fluorescein- (FITC-) labeled wheat germ agglutinin (WGA, 10
100 days after transplanting the islets under the kidney capsule, microcirculation was assessed by intravital confocal fluorescence microscopy. In order to enhance the contrast of the microvessels, 0.3 mL of a 0.4% fluorescein isothiocyanate (FITC)-labeled dextran (MW 150.000; Sigma Aldrich) was injected via the penile vein. For confocal microscopy we used the above-mentioned system. Each image consists of a z stack of 20 planes acquired with a 20x objective at a wavelength of 488 nm.
Blood glucose levels of islet recipients were measured on the morning of the day of transplantation and on postoperative days 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, and 130. Normoglycemia was defined as blood glucose below 150 mg/dL on at least two consecutive days.
Intraperitoneal glucose tolerance test (IPGTT) was performed in transplanted mice 30 days after transplantation. After 12 hours of fasting, mice were injected with 2.0 g/kg body weight of 20% glucose solution. Blood was sampled from the tail vein before and 30, 60, 90, and 120 min after intraperitoneal injection.
At 120 days after islet transplantation, nephrectomy of the islet-containing kidney was performed. For this purpose the left renal artery and vein as well as the ureter were ligated and the kidneys resected.
Immunohistochemistry of islet grafts was performed on day 130 after transplantation as follows: islet grafts were retrieved from individual animals by nephrectomy. After fixation in 10% phosphate-buffered formalin overnight, kidneys were embedded in paraffin. Consecutive sections (4
For histological examination, kidneys bearing islets were harvested and fixed in 4% formaldehyde for 24 h and embedded in paraffin. Sections of 4
Results are expressed as mean ± standard error of the mean (SEM). Statistical analysis was performed using GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA). For comparison of multiple groups the Kruskal-Wallis test was applied. If statistical significance was achieved, all pairs were compared among each other using the Mann-Whitney
Immediately after the isolation procedure, cell morphology and mitochondrial potentials were well preserved, as documented by WGA and TMRM staining (Figure
Islet cell viability and cell stress assessed by live confocal microscopy. (a) Cells imaged immediately after isolation and stained with WGA to assess cell morphology and with TMRM to detect cell viability. (b) Cells imaged after 15 h simulated transport under perfused rotary conditions; WGA and TMRM were used as dyes. (c) Cell morphology (WGA) and viability of islet cells after 15 h simulated transport under static conditions. (d) Islets analyzed immediately after isolation and stained with WGA for cell morphology and with Rhod-2 for the assessment of cell stress. (e), (f) Cell morphology and islet stress assessed after 15 h simulated transport under rotary versus static conditions.
Similar results were obtained from the analysis of intracellular calcium content using Rhod-2 as a marker for cell stress. Islet cells analyzed after 15 h of simulated transport under rotary conditions as well as immediately after isolation showed only minimal signs of stress (Figures
After induction of diabetes in C57BL/6 mice with a single injection of streptozotocin, we transplanted 250 either freshly isolated islets or islets cultured for 15 h under rotary conditions.
Islet cell transplantation immediately after isolation resulted in mean blood glucose values of 230 mg/dL
Functional assessment of islets cultured under the two different conditions following transplantation into syngeneic recipients. (a) Blood glucose levels of recipients following transplantation of 250 islets after simulated transport under rotary conditions or immediately after isolation. (b) Postoperative days to normoglycemia, showing a significant difference between islets transplanted after incubation under rotary conditions and islets transplanted immediately after isolation (
Interestingly, transplantation of freshly isolated islets into syngeneic recipients resulted in normoglycemia on day
Nephrectomy of the left kidney bearing the islet grafts was performed on day 100 after grafting and induced a diabetic state in all animals, proving that animals relied on islet graft function for physiological glucose homeostasis.
Figure
A glucose tolerance test performed on day 30 after grafting elicited no statistically significant difference in islet cell function between the two transplanted groups. Almost normal blood glucose values were observed as early as 60 min after intraperitoneal injection of a 20% glucose solution 0.3 mL (Figure
Figure
Murine islets 120 days after grafting under the kidney capsule of syngeneic recipients. (a) H&E; (b) immunohistochemical detection of insulin-producing cells. (c) Immunohistochemistry with CD31 was performed to assess neoangiogenesis at the implantation site. (d) and (e) Islet capillary system shown by confocal microscopy. (f) Graft-bearing kidney.
During the last decade progress has been made in the field of clinical islet cell transplantation. It has been shown that the expertise of an islet isolation center is crucial for success in clinical islet cell transplantation. As a consequence networks have been established between specialized islet isolation facilities and distant islet transplantation centers, thus, guaranteeing a supply of islets of a consistently high quality. However, no consensus has been reached on the best-suited modality for the transport of isolated islets to a transplantation center [
Damage associated with nonspecific inflammatory events occurs not only after the transplantation of islets but, more importantly, during the isolation process and during the transportation of isolated islets. Apoptosis of human islets during isolation has been discussed as an important pathomechanism [
A promising option is to incubate islets under rotary, microgravity conditions using rotating wall vessels. Rotary, microgravity conditions promote islet remodeling, which potentially results in formation of channels with external openings. Such openings have been shown to allow nutrients and oxygen to shift into the cells and thus facilitate angiogenesis and engraftment following transplantation. Furthermore, brief incubation under rotary conditions reduces the immunogenicity of allogeneic islets by depleting passenger dendritic cells [
Recently, using a rotary cell culture system combining microgravity, low shear force and high mass transfer with a perfused system of disposable tubes and a breeding chamber, we were able to demonstrate that human islets can sustain their functional properties, such as insulin secretion, for up to one week [
In the current study we assessed murine islet cell viability via real time live confocal microscopy directly after the isolation procedure as well as after two simulated transport conditions, static versus rotation. Due to the low cell viability observed after simulated transport under static conditions, we decided not to transplant these islets
Major findings of this study are that (1) all viability parameters of the islets cultured in the rotating chamber for 15 h were comparable to those of freshly isolated islets, whereas a simulated transport of 15 h under standard conditions had a devastating impact on assessed viability parameters and (2) islet function after transplantation under the kidney capsule was comparable for the freshly isolated islets and the islets kept under rotary conditions for 15 h.
We chose a 15 h incubation time because, firstly, this would allow us to demonstrate increased viability of murine islet cells cultured under microgravity conditions, even after prolonged simulated transport and, secondly, a time window of 15 h would make it possible to reach most transplant centers within Europe or the USA.
Clinical islet cell transplantation is faced with the problem that viability test and efficacy assays, which characterize islet cell preparation prior to transplantation, are unable to predict posttransplant outcome. Several different strategies for assessing islet cell viability have been described, including fluorescence microscopy, standard light microscopy, FACS, and the nude mouse bioassay [
Visualizing cell stress and predicting its consequences with regard to functional outcome after transplantation are of utmost importance. We previously described an approach for assessing islet viability by visualizing a range of stressed cells to dead cells using a combination of live stains and real-time live confocal imaging [
In line with the results of our cell viability analysis we were able to demonstrate that cell function of islets cultured under microgravity conditions following syngeneic transplantation is comparable to that of islets transplanted immediately after isolation. This was underscored by analogous IPGT test results as well as postoperative changes in body weight and blood glucose levels. Interestingly, the time until normoglycemia achieved was shorter in recipients of cultured islets. This underlines the functional equality, if not superiority, of islets cultured under microgravity conditions as compared with freshly isolated islets. Furthermore, in line with the results reported by Rutzky et al. we were able to show that the cultivation of murine islets under microgravity conditions can drastically improve cell function following syngeneic transplantation [
The rotating wall chamber tested in this study combines an excellent method for the preservation of islet cell function and viability and the practical advantage of good transportability. We therefore propose it as a mobile system for the transport of islet cells from the isolation to the transplantation center.
The authors declare that there is no conflict of interests regarding the publication of this paper.
Rupert Oberhuber and Christof Mittermair contributed equally to this work.
This study was supported by Grant no. 207 from the Medizinischer Forschungsfond Tirol and by Grant no. 2010022010 from the MUI-START Förderprogramm of Innsbruck Medical University.