Pancreatic islet (PI) transplantation (Tx) to the portal vein is an established therapeutic modality for selected type 1 diabetic patients. However, a comprehensive review considering the effects of PIs on surrounding liver tissue is lacking. Typical interactions can be detected in the early and delayed phases. This review summarizes known side effects of PI transplantation. In early phase the interaction occurs immediately upon contact of the PI into portal vein blood. Mechanical obstruction, exacerbated by thrombosis as part of the instant blood-mediated inflammatory reaction (IBMIR), leads to ischemic injury to adjacent liver tissue. Delayed changes, such as focal steatosis and glycogen accumulation appear days to weeks after Tx and are caused by local overstimulation of hepatocytes by insulin in supraphysiological concentrations. In animal models these lesions could progress over months to cystic cholangiomas or hepatocellular carcinomas. Such neoplastic changes have been observed in experimental animals; they have not been reported in human patients. In conclusion, while PITx into the liver is not an optimal procedure, it currently represents the site offering the best functional integration of the graft. The adverse effects discussed are pronounced but generally not severe, nor do they appear to compromise the overall health status of the recipients.

Introduction: The correct islet graft dosing helps predict the transplantation outcome. The current quantification methods are based on the estimation of volumes of individual islets. The islet sizes and shapes are heterogeneous. The spherical islet model [1,2] presently used as the surrogate marker of the content of insulin secreting cells was challenged as grossly inaccurate [3]. Our long-term purpose is to establish an automated tool for islet graft quantification. To this end we are developing more accurate models for predicting the volumes as well as the cell content of individual islets from 2D-microscopic projections. Methods: Our models are based on detailed observations of individual islets using the coupled 2D-and 3D-microscopy. High quality 2D-images of unstained islets (Fig. 1A) were acquired using Apotome microscope and manually segmented (Fig. 1B) and analyzed using the My-Segmentation tool in IsletNet.com. The Optical Projection Tomography (OPT) was used to collect 3D-images for the islet shapes and volume measurements. The Light Sheet Microscopy on cleared individual DAPI-stained islets (Fig. 1C) was used for the cell content quantification (Fig.1D). 3D-images were analyzed using Fakir (volume measurement) and Fiji macros (nuclear counting, Fig.1D) [4]. The islet cell content was validated by the determination of total DNA content in islet lysates using a fluorescence-based physical method (Qubit) and the genome size for cell number calculation.Results: Our spinacle model for predicting the islet volume from 2D-projection was developed using 68 islets. The spinacle model was validated on 97 islets (size range: 83-546 µm, 8 donors), the volume of which was determined using OPT and Fakir software. The accuracy improvement using the spinacle model in comparison to the current standard method (spherical model) is shown in Fig. 2. Direct 3D-observation of the islet nuclei (49 islets, 4 donors, in blue) was in a good agreement with the islet DNA content (44 islets, 2 donors, in red), Fig.3. The results revealed that the current cell dissociation-based model [3] grossly underestimates the real islet cell content. The validation of our new proposed 2D-cell-count model on additional donors is underway.Conclusion: Our proposed models predict the islet volume and islet cell content more accurately than the current models. Their implementation into the web-service IsletNet is a step towards the cell-based graft quantification. Our findings are relevant for a fair comparison of the isolated vs. re-aggregated and stem cell-derived islets in the transplantation context. This research was supported by grant NU22-01-00141 from Czech Health Research Council. References: 1. Ricordi C, Pancreas. 1991; 2, 6: 242-244. 2. Niclaus N, et al. Transplantation. 2008; 11, 86: 1603-1609. 3. Ramachandran K, et al. Cell Transplantation. 2015; 7, 24: 1183-1194. 4. Stringer C et al. Nature Methods. 2021; 1, 18: 100-106.

Background. Transplantation of pancreatic islets into subcutaneous cavities in diabetic rats may be as or even more effective than transplantation into the portal vein. Identifying the optimal timing of the individual steps in this procedure is critical. Methods. Macroporous scaffolds were placed in the subcutaneous tissue of diabetic male Lewis rats for 7 or 28 d and the healing of the tissue inside the scaffolds was monitored. A marginal syngeneic graft comprising 4 islets/g of recipient body weight was transplanted at the best timing focusing mainly on vascularization. Recipients were monitored for blood glucose levels and tolerance tests. Histological examination was performed in all implanted scaffolds. The presence of individual endocrine cells was analyzed in detail. Results. Blood glucose levels remained within the physiological range in all recipients until the end of experiment as well as body weight increase. Coefficients of glucose assimilation were normal or slightly reduced with no statistically significant differences between the groups 40 and 80 d after transplantation. Histological analysis revealed round viable islets in the liver similar to those in pancreas, but alpha cells practically disappeared, whereas islets in the scaffolds formed clusters of cells surrounded by rich vascular network and the alpha cells remained partially preserved. Conclusions. Subcutaneous transplantation of pancreatic islets is considerably less invasive but comparably efficient as commonly used islet transplantation into the portal vein. In consideration of alpha and beta cell ratio, the artificial subcutaneous cavities represent a promising site for future islet transplantation therapy.

Subcutaneously implanted polymeric scaffolds represent an alternative transplantation site for pancreatic islets (PIs) with the option of vascularisation enhancement by mesenchymal stem cells (MSC). Nevertheless, a proper timing of the transplantation steps is crucial. In this study, scaffolds supplemented with plastic rods were implanted into diabetic rats and two timing schemes for subsequent transplantation of bioluminescent PIs (4 or 7 days after rod removal) were examined by multimodal imaging. The cavities were left to heal spontaneously or with 10 million injected MSCs. Morphological and vascularisation changes were examined by MRI, while the localisation and viability of transplanted islets were monitored by bioluminescence imaging. The results show that PIs transplanted 4 days after rod removal showed the higher optical signal and vascularisation compared to transplantation after 7 days. MSCs slightly improved vascularisation of the graft but hindered therapeutic efficiency of PIs. Long-term glycaemia normalisation (4 months) was attained in 80% of animals. In summary, multimodal imaging confirmed the long-term survival and function of transplanted PIs in the devices. The best outcome was reached with PIs transplanted on day 4 after rod removal and therefore the suggested protocol holds a potential for further applications.

BACKGROUND:Pancreas transplantation (PTx) represents the method of choice in type 1 diabetic patients with conservatively intractable hypoglycemia unawareness syndrome. In 2005, the Institute for Clinical and Experimental Medicine (IKEM) launched a program to investigate the safety potential of islet transplantation (ITx) in comparison to PTx.AIM:This study aims to compare the results of PTx and ITx regarding severe hypoglycemia elimination, metabolic control, and complication rate.METHODS:We analyzed the results of 30 patients undergoing ITx and 49 patients treated with PTx. All patients were C-peptide-negative and suffered from hypoglycemia unawareness syndrome. Patients in the ITx group received a mean number of 12,349 (6,387-15,331) IEQ/kg/person administered percutaneously into the portal vein under local anesthesia and radiological control. The islet number was reached by 1-3 applications, as needed. In both groups, we evaluated glycated hemoglobin, insulin dose, fasting and stimulated C-peptide, frequency of severe hypoglycemia, and complications. We used the Mann Whitney test, Wilcoxon signed-rank test, and paired t-test for analysis. We also individually assessed the ITx outcomes for each patient according to recently suggested criteria established at the EPITA meeting in Igls.RESULTS:Most of the recipients showed a significant improvement in metabolic control one and two years after ITx, with a significant decrease in HbA1c, significant elevation of fasting and stimulated C-peptide, and a markedly significant reduction in insulin dose and the frequency of severe hypoglycemia. Seventeen percent of ITx recipients were temporarily insulin-independent. The results in the PTx group were comparable to those in the ITx group, with 73% graft survival and insulin independence in year 1, 68% 2 years and 55% 5 years after transplantation. There was a higher rate of complications related to the procedure in the PTx group. Severe hypoglycemia was eliminated in the majority of both ITx and PTx recipients.CONCLUSION:This report proves the successful initiation of pancreatic islet transplantation in a center with a well-established PTx program. ITx has been shown to be the method of choice for hypoglycemia unawareness syndrome, and may be considered for application in clinical practice if conservative options are exhausted.

Clinical islet transplantation programs rely on the capacities of individual centers to quantify isolated islets. Current computer-assisted methods require input from human operators. Here we describe two machine learning algorithms for islet quantification: the trainable islet algorithm (TIA) and the nontrainable purity algorithm (NPA). These algorithms automatically segment pancreatic islets and exocrine tissue on microscopic images in order to count individual islets and calculate islet volume and purity. References for islet counts and volumes were generated by the fully manual segmentation (FMS) method, which was validated against the internal DNA standard. References for islet purity were generated via the expert visual assessment (EVA) method, which was validated against the FMS method. The TIA is intended to automatically evaluate micrographs of isolated islets from future donors after being trained on micrographs from a limited number of past donors. Its training ability was first evaluated on 46 images from four donors. The pixel-to-pixel comparison, binary statistics, and islet DNA concentration indicated that the TIA was successfully trained, regardless of the color differences of the original images. Next, the TIA trained on the four donors was validated on an additional 36 images from nine independent donors. The TIA was fast (67 s/image), correlated very well with the FMS method ( R 2 = 1.00 and 0.92 for islet volume and islet count, respectively), and had small REs (0.06 and 0.07 for islet volume and islet count, respectively). Validation of the NPA against the EVA method using 70 images from 12 donors revealed that the NPA had a reasonable speed (69 s/image), had an acceptable RE (0.14), and correlated well with the EVA method ( R 2 = 0.88). Our results demonstrate that a fully automated analysis of clinical-grade micrographs of isolated pancreatic islets is feasible. The algorithms described herein will be freely available as a Fiji platform plugin.

Variability of pancreatic donors may significantly impact the success of islet isolation. The aim of this study was to evaluate donor factors associated with isolation failure and to investigate whether immunohistology could contribute to organ selection. Donor characteristics were evaluated for both successful (n=61) and failed (n=98) islet isolations. Samples of donor pancreatic tissue (n=78) were taken for immunohistochemical examination. Islet isolations with 250000 islet equivalents were considered successful. We confirmed that BMI of less than 25 kg/m2 (P<0.001), cold ischemia time more than 8 hours (P<0.01), hospitalization longer than 96 hours (P<0.05), higher catecholamine doses (P<0.05), and edematous pancreases (P<0.01) all unfavorably affected isolation outcome. Subsequent immunohistochemical examination of donor pancreases confirmed significant differences in insulin-positive areas (P<0.001). ROC analyses then established that the insulin-positive area in the pancreas could be used to predict the likely success of islet isolation (P<0.001). At the optimal cutoff point (>1.02%), sensitivity and specificity were 89% and 76%, respectively. To conclude, while the insulin-positive area, determined preislet isolation, as a single variable, is sufficient to predict isolation outcome and helps to improve the success of this procedure, its combination with the established donor scoring system might further improve organ selection.

The use of RT-qPCR provides a powerful tool for gene expression studies; however, the proper interpretation of the obtained data is crucially dependent on accurate normalization based on stable reference genes. Recently, strong evidence has been shown indicating that the expression of many commonly used reference genes may vary significantly due to diverse experimental conditions. The isolation of pancreatic islets is a complicated procedure which creates severe mechanical and metabolic stress leading possibly to cellular damage and alteration of gene expression. Despite of this, freshly isolated islets frequently serve as a control in various gene expression and intervention studies. The aim of our study was to determine expression of 16 candidate reference genes and one gene of interest (F3) in isolated rat pancreatic islets during short-term cultivation in order to find a suitable endogenous control for gene expression studies. We compared the expression stability of the most commonly used reference genes and evaluated the reliability of relative and absolute quantification using RT-qPCR during 0-120 hrs after isolation. In freshly isolated islets, the expression of all tested genes was markedly depressed and it increased several times throughout the first 48 hrs of cultivation. We observed significant variability among samples at 0 and 24 hrs but substantial stabilization from 48 hrs onwards. During the first 48 hrs, relative quantification failed to reflect the real changes in respective mRNA concentrations while in the interval 48-120 hrs, the relative expression generally paralleled the results determined by absolute quantification. Thus, our data call into question the suitability of relative quantification for gene expression analysis in pancreatic islets during the first 48 hrs of cultivation, as the results may be significantly affected by unstable expression of reference genes. However, this method could provide reliable information from 48 hrs onwards.

Islet transplantation (ITx) started in 2005 in IKEM as a potentially safer alternative to pancreas transplantation (PTx), which so far had represented the method of choice in type-1 diabetic patients with conservatively intractable hypoglycemia unawareness syndrome. The aim of the study was to compare these two methods with regard to severe hypoglycemia elimination and to frequency of complications.Up to November 2015 a total number of 48 patients underwent ITx. The results from 22 patients with hypoglycemia unawareness were statistically analyzed. The mean number of transplanted islet equivalents was 12,096 (6,93316,705) IEQ/kg administered percutaneously in local anesthesia under radiological control to the portal vein. 44 patients underwent PTx from 1996. We evaluated glycated hemoglobin(HbA1c), insulin dose, fasting and stimulated C-peptide, frequency of severe hypoglycemia and complications. Medians (interquartile range) were analyzed using the Wilcoxon signed-rank test.One and two years after ITx, HbA1c decreased, C-peptide became significantly positive, insulin dose and frequency of severe hypoglycemia decreased and 18 % of ITx recipients were temporarily insulin-independent. Bleeding was present in 41 % of patients. One year after PTx, 73 % of patients were insulin and hypoglycemia-free, after two years 68 % of patients were insulin and hypoglycemia-free; graftectomy occurred in 20 % of recipients.Both methods led to restoration of insulin secretion and severe hypoglycemia elimination. PTx made more recipients insulin-independent at the cost of serious complications.

An artificial site for cell or pancreatic islet transplantation can be created using a polymeric scaffold, even though it suffers subcutaneously from improper vascularisation. A sufficient blood supply is crucial for graft survival and function and can be enhanced by transplantation of mesenchymal stem cells (MSCs). The purpose of this study was to assess the effect of syngeneic MSCs on neoangiogenesis and cell engraftment in an artificial site by multimodal imaging.MSCs expressing a gene for luciferase were injected into the artificial subcutaneous site 7 days after scaffold implantation. MRI experiments (anatomical and dynamic contrast-enhanced images) were performed on a 4.7-T scanner using gradient echo sequences. Bioluminescent images were acquired on an IVIS Lumina optical imager. Longitudinal examination was performed for 2 months, and one animal was monitored for 16 months.We confirmed the long-term presence (lasting more than 16 months) of viable donor cells inside the scaffolds using bioluminescence imaging with an optical signal peak appearing on day 3 after MSC implantation. When compared to controls, the tissue perfusion and vessel permeability in the scaffolds were significantly improved at the site with MSCs with a maximal peak on day 9 after MSC transplantation.Our data suggest that the maximal signal obtained by bioluminescence and magnetic resonance imaging from an artificially created site between 3 and 9 days after MSC transplantation can predict the optimal time range for subsequent cellular or tissue transplantation, including pancreatic islets.

The porous polymer foams act as a template for neotissuegenesis in tissue engineering, and, as a reservoir for cell transplants such as pancreatic islets while simultaneously providing a functional interface with the host body. The fabrication of foams with the controlled shape, size and pore structure is of prime importance in various bioengineering applications. To this end, here we demonstrate a thermally induced phase separation (TIPS) based facile process for the fabrication of polymer foams with a controlled architecture. The setup comprises of a metallic template bar (T), a metallic conducting block (C) and a non-metallic reservoir tube (R), connected in sequence T-C-R. The process hereinafter termed as Dip TIPS, involves the dipping of the T-bar into a polymer solution, followed by filling of the R-tube with a freezing mixture to induce the phase separation of a polymer solution in the immediate vicinity of T-bar; Subsequent free-drying or freeze-extraction steps produced the polymer foams. An easy exchange of the T-bar of a spherical or rectangular shape allowed the fabrication of tubular, open- capsular and flat-sheet shaped foams. A mere change in the quenching time produced the foams with a thickness ranging from hundreds of microns to several millimeters. And, the pore size was conveniently controlled by varying either the polymer concentration or the quenching temperature. Subsequent in vivo studies in brown Norway rats for 4-weeks demonstrated the guided cell infiltration and homogenous cell distribution through the polymer matrix, without any fibrous capsule and necrotic core. In conclusion, the results show the “Dip TIPS” as a facile and adaptable process for the fabrication of anisotropic channeled porous polymer foams of various shapes and sizes for potential applications in tissue engineering, cell transplantation and other related fields.