Method For Isolation Of Islets Research Articles

Advancing Diabetes Research: A Novel Islet Isolation Method from Living Donors.

Pancreatic islet isolation is critical for type 2 diabetes research. Although -omics approaches have shed light on islet molecular profiles, inconsistencies persist; on the other hand, functional studies are essential, but they require reliable and standardized isolation methods. Here, we propose a simplified protocol applied to very small-sized samples collected from partially pancreatectomized living donors. Islet isolation was performed by digesting tissue specimens collected during surgery within a collagenase P solution, followed by a Lympholyte density gradient separation; finally, functional assays and staining with dithizone were carried out. Isolated pancreatic islets exhibited functional responses to glucose and arginine stimulation mirroring donors' metabolic profiles, with insulin secretion significantly decreasing in diabetic islets compared to non-diabetic islets; conversely, proinsulin secretion showed an increasing trend from non-diabetic to diabetic islets. This novel islet isolation method from living patients undergoing partial pancreatectomy offers a valuable opportunity for targeted study of islet physiology, with the primary advantage of being time-effective and successfully preserving islet viability and functionality. It enables the generation of islet preparations that closely reflect donors' clinical profiles, simplifying the isolation process and eliminating the need for a Ricordi chamber. Thus, this method holds promises for advancing our understanding of diabetes and for new personalized pharmacological approaches.

Isolated bovine pancreatic islets as an alternate in vitro model for diabetes research.

Background & objectives Isolation of functional pancreatic islets for diabetes research and clinical islet transplantation stands as a big challenge despite the advancements in the field. In this context, the non-availability of human/animal tissues is one of the major impediments to islet-based research, which has tremendous scope for translation. The current study explores the feasibility of using the bovine pancreas as an alternative source to isolate pancreatic islets and assess its functionality for in vitro studies. Methods The bovine pancreas was collected from a registered slaughterhouse and transported in an ice-cold medium - Hank's Balanced Salt Solution (HBSS) to the laboratory. Islets were isolated by sequential collagenase digestion followed by a two-step filtration and purification by density gradient separation method. After isolation, islets were identified with dithizone staining and the islet function was assayed in vitro for assessing the dynamic insulin secretory function by monitoring the glucose-stimulated insulin secretion (GSIS), in response to low and high glucose. Staining techniques were also used to understand the cytoarchitecture of the bovine pancreas. Results The islet yield was 157±23 islets per gram of pancreas and was viable. The cold ischaemia time was reduced to 60-75 min. The islets released insulin with glucose stimulation. The insulin release was observed more with high glucose (28 mM) than with low glucose (2.8 mM). Dithizone staining confirmed the presence of islets after isolation and the size of islets ranged from 50 to 600 µm size. The mantled islets (islets with acinar tissue) were also noted with the pure islets in culture. Hematoxylin and eosin (H&E) and aldehyde- fuchsin showed islets interspersed in the acinar tissue of the bovine pancreas. Special stain defined the islets better than regular staining. Fluorescent and diaminobenzidine (DAB) staining with insulin, glucagon and somatostatin revealed the arrangement of the cells in each islet. The beta cells were majorly found in the islet core with alpha cells interspersed with the delta cells in the periphery. Interpretation & conclusions The isolation procedure described in this study yielded viable islets for in vitro studies which showed a differential response to glucose challenge, confirming their viability. We provide a simple and reproducible method for small-scale isolation of functional islets from the bovine pancreas. This model proffers the beginner a hands-on in islet experiments and helps to re-iterate the process that could be extrapolated to other pancreatic tissues as well as to expand on diabetes research.

Isolation of Human Pancreatic Islets from the Indian Population - Unveiling the Synergistic Interplay between different blends of Collagenases and Neutral Proteases

Innovative and effective therapeutic approaches for diabetes such as islet cell transplantation are much needed as diabetes increases the risk of heart failure. In India, the success rate of human clinical islet transplantation (allo-or auto-) mandates sufficiency of functional islets from the pancreas of either brain-dead or chronic pancreatitis donors. Human islet transplantation outcome is significantly affected by collagenases, which decides the yield and quality of isolated pancreatic islet cells. Comparison of collagenase blends carried out in a few islet transplantation centers yields conflicting results for islet quantity and quality. Therefore, we have researched on the right blend of enzymes to achieve an efficient pancreatic islet release, first-ever in Indian population. Our prospective approach evaluated the outcome of islet isolations performed in humans with three different collagenases, namely Serva Collagenase NB1, Roche Liberase and Vitacyte supplemented with Neutral Protease NB. Eight brain-dead humans served as organ donors for pancreatic islet isolation. We modified the islet isolation method to test these three new enzymes using the traditional split pancreas methodology. The islet isolation outcomes were compared for morphological differences, yield, and viability. This was correlated statistically with the donor characteristics using graph-pad prism analysis. Islet yield was substantial in all the three enzyme blends. But post-purification of islets in the Indian population, we found that the yield was better in Serva collagenase NB1 compared to Vitacyte and Roche liberase. For every gram of pancreas, the islet yield, proportion and morphology of islet cells (both free and intact) were higher in Serva collagenase compared to the other two enzyme blends.

PRISM: A Novel Human Islet Isolation Technique.

Successful pancreatic islet isolations are a key requirement for islet transplantation in selected patients with type 1 diabetes. However, islet isolation is a technically complex, time-consuming, and manual process. Optimization and simplification of the islet isolation procedure could increase islet yield and quality, require fewer operators, and thus reduce cost. We developed a new, closed system of tissue collection, washing, buffer change, and islet purification termed PancReatic Islet Separation Method (PRISM). In the developmental phase, pump and centrifuge speed was tested using microspheres with a similar size, shape, and density as digested pancreatic tissue. After optimization, PRISM was used to isolate islets from 10 human pancreases. Islet equivalents viability (fluorescein diacetate/propidium iodide), morphology, and dynamic glucose-stimulated insulin secretion were evaluated. PRISM could be performed by 1 operator in 1 flow cabinet. A similar islet yield was obtained using PRISM compared to the traditional islet isolation method (431 234 ± 292 833 versus 285 276 ± 197 392 islet equivalents, P = 0.105). PRISM islets had similar morphology and functionality. PRISM is a novel islet isolation technique that can significantly improve islet isolation efficiency using fewer operators.

A Preliminary Characterization of a Novel Recombinant Clostridial Collagenase Blend.

Clostridial collagenases are essential biotechnological tissue dissociation agents owing to their ability to cleave different types of collagen. Standardization of collagenase-based protocols has been hampered by impurities in products manufactured from Clostridium histolyticum. To enhance the purification process, we produced recombinant collagenase classes G and H, taking advantage of the Escherichia coli expression system. The respective gene sequences were derived from C. histolyticum and modified by addition of a C-terminal polyhistidine tag. Harvested bacteria were lysed and the collagenase protein was affinity purified using a His-tag column. The purity, identity, integrity of the eluted collagenases G and H were determined by SDS electrophoresis and Western blot. The proteolytic activity of the collagenase G and H blend (rColGH) was determined by the standard FALGPA assay. The tissue dissociation activity was verified using a standardized method for isolation of rat pancreatic islets. Biocompatibility of the blend was validated by a standardized viability assay on the isolated islets. Two batches of rColGH were produced and compared to a commercially available collagenase. Based on our results, we conclude that rColGH is a functional and non-toxic novel recombinant collagenase worth further characterization and blend optimization in order to make it a competitive commercial product.

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other important biological functions. The islets primarily consist of five types of hormone-secreting cells: α cells secrete glucagon, β cells secrete insulin, δ cells secrete somatostatin, ε cells secrete ghrelin, and PP cells secrete pancreatic polypeptide. Sixty to 80% of the cells in the islets are β cells, which are the most important cell population to study insulin secretion. Pancreatic islets are a crucial model system to study ex vivo insulin secretion. Acquiring high quality islets is of great importance for diabetes research. Most islet isolation procedures require technically difficult to access site of collagenase injection, harsh and complex digestion procedures, and multiple density gradient purification steps. This paper features a simple high yield mouse islet isolation method with detailed descriptions and realistic demonstrations, showing the following specific steps: 1) injection of collagenase P at the ampulla of Vater, a small area joining the pancreatic duct and the common bile duct, 2) enzymatic digestion and mechanical separation of the exocrine pancreas, and 3) a single gradient purification step. The advantages of this method are the injection of digestive enzyme using the more accessible ampulla of Vater, more complete digestion using combination of enzymatic and mechanical approaches, and a simpler single gradient purification step. This protocol produces approximately 250—350 islets per mouse; and islets are suitable for various ex vivo studies. Possible caveats of this procedure are potentially damaged islets due to enzymatic digestion and/or prolonged gradient incubation, all of which can be largely avoided by careful ad justification of incubation time.

Improved recovery of human islets from young donor pancreases utilizing increased protease dose to collagenase for digesting peri-islet extracellular matrix.

Human islet isolation from young donor pancreases (YDP) utilizing the current purified standard dose of collagenase-protease enzyme mixtures often results in the release of a high percentage of mantled islets. Mantled islets are those surrounded by exocrine tissue and are difficult to purify by density gradient centrifugation, leading to poor islet recovery. Based on difference in extracellular matrix, and total collagen content between YDP and old donor pancreas (ODP, > 35 Y) led us to compare results from islet isolation using increased collagenase combination (ICC) or increased protease combination (IPC), to the standard enzyme combination (SEC) in a "trisected" pancreas model to overcome the donor-to-donor variability. These results showed a reduced percentage of mantled islets (17%±7.5%) and higher postpurification islet recovery (83.8%±5.6%) with IPC. Furthermore, these results were confirmed in 13 consecutive whole pancreas islet isolations utilizing IPC from VitaCyte, Roche, or SERVA collagenase-protease enzyme mixtures. Results obtained from in vitro and in vivo islet functional assessment indicated that islets isolated using IPC retained normal islet morphology, insulin secretion, and the ability to reverse diabetes after transplantation in diabetic nude mice. This is the first report utilizing trisected pancreas to assess the effectiveness of different enzyme combinations to improve islet recovery from young donor pancreases.

Protective Effect of NecroX-7 on Islets against Oxidative Stress and Inflammation

Oxidative stress generated during islet isolation, culture, and transplantation causes islet cell damage mediated by hypoxia, reactive oxygen species (ROS) and inflammatory responses. We investigated the protective effects of NecroX-7, a novel ROS scavenger, during isolation and/or culture of islets. For ex vivo studies, islets from heterozygote human islet amyloid polypeptide (hIAPP+/-) mice and C57BL/6J mice were isolated by collagenase with and without supplementation with 20 uM of NecroX-7. Supplementation with NecroX-7 provided markedly increased islet viability and ATP contents, and attenuated ROS, transcription of c-Jun N-terminal kinases, high mobility group box-1 (HMGB1) and proinflammatory cytokines including interleukin-1beta (IL-1b), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-a). Transplantation of islets, which is derived from islet isolation method using supplementation with NecroX-7, into syngeneic subrenal subcapsular of hIAPP+/- mice also improved post-transplant blood glucose levels. Also, NecroX-7 protected RINm5F cells from t-BHP-induced and serum deprivation induced toxicity in in vitro culture by dose-dependent manner from 0.1 uM to 20 uM. Supplementation of medium with NecroX-7 during serum-deprived culture condition also improved impaired viability and serum deprivation-induced ROS in islets from hIAPP+/- mice by dose-dependent manner from 0.1 uM to 20 uM. These findings suggest that NecroX-7 supplementation during the islet isolation and/or culture process suppressed inflammatory responses and ROS induced by serum deprivation and provides a potential benefit to improve post-transplantation outcomes. Disclosure Y. Kwon: None. H. Lee: None. H. Kim: None. G. Kim: None. S. Jin: None. M. Lee: None. J. Kim: None.

A rapid, efficient, and economic device and method for the isolation and purification of mouse islet cells.

Rapid, efficient, and economic method for the isolation and purification of islets has been pursued by numerous islet-related researchers. In this study, we compared the advantages and disadvantages of our developed patented method with those of commonly used conventional methods (Ficoll-400, 1077, and handpicking methods). Cell viability was assayed using Trypan blue, cell purity and yield were assayed using diphenylthiocarbazone, and islet function was assayed using acridine orange/ethidium bromide staining and enzyme-linked immunosorbent assay-glucose stimulation testing 4 days after cultivation. The results showed that our islet isolation and purification method required 12 ± 3 min, which was significantly shorter than the time required in Ficoll-400, 1077, and HPU groups (34 ± 3, 41 ± 4, and 30 ± 4 min, respectively; P < 0.05). There was no significant difference in islet viability among the four groups. The islet purity, function, yield, and cost of our method were superior to those of the Ficoll-400 and 1077 methods, but inferior to the handpicking method. However, the handpicking method may cause wrist injury and visual impairment in researchers during large-scale islet isolation (>1000 islets). In summary, the MCT method is a rapid, efficient, and economic method for isolating and purifying murine islet cell clumps. This method overcomes some of the shortcomings of conventional methods, showing a relatively higher quality and yield of islets within a shorter duration at a lower cost. Therefore, the current method provides researchers with an alternative option for islet isolation and should be widely generalized.

Matrix metalloproteinases as reagents for cell isolation.

Cell isolation methods for therapeutic purposes have seen little advancement over the years. The original methods of stem cell and islet isolation using bacterial collagenases were developed in the early 1980s and are still used today. Bacterial collagenases are subject to autodegradation, and isolates obtained with these enzymes may be contaminated with endotoxins, reducing cell viability and contributing to toxicity in downstream applications. Here we describe a novel method for isolation of mesenchymal stem cells from adipose tissue (ADSC) utilizing recombinantly produced matrix metalloproteases (MMPs). The ADSCs isolated by MMPs displayed essentially identical morphological and phenotypical characteristics to cells isolated by bacterially-derived collagenase I and Liberase™. Samples isolated with MMPs and Liberase™ had comparable levels of CD73, CD90, and CD105. The adipogenic and osteogenic potential of the ADSCs isolated by MMPs was retained as compared to cells isolated with Liberase™. However, ADSCs isolated by Liberase™ displayed 6% contamination with other cells as per negative markers revealed by PE staining, as opposed to<1% for all MMP-treated samples. MMP-based cell isolation may contribute to optimization of transplantation technology.

The Different Faces of the Pancreatic Islet.

Type 1 diabetes (T1D) patients who receive pancreatic islet transplant experience significant improvement in their quality-of-life. This comes primarily through improved control of blood sugar levels, restored awareness of hypoglycemia, and prevention of serious and potentially life-threatening diabetes-associated complications, such as kidney failure, heart and vascular disease, stroke, nerve damage, and blindness. Therefore, beta cell replacement through transplantation of isolated islets is an important option in the treatment of T1D. However, lasting success of this promising therapy depends on durable survival and efficacy of the transplanted islets, which are directly influenced by the islet isolation procedures. Thus, isolating pancreatic islets with consistent and reliable quality is critical in the clinical application of islet transplantation.Quality of isolated islets is important in pre-clinical studies as well, as efforts to advance and improve clinical outcomes of islet transplant therapy have relied heavily on animal models ranging from rodents, to pigs, to nonhuman primates. As a result, pancreatic islets have been isolated from these and other species and used in a variety of in vitro or in vivo applications for this and other research purposes. Protocols for islet isolation have been somewhat similar across species, especially, in mammals. However, given the increasing evidence about the distinct structural and functional features of human and mouse islets, using similar methods of islet isolation may contribute to inconsistencies in the islet quality, immunogenicity, and experimental outcomes. This may also contribute to the discrepancies commonly observed between pre-clinical findings and clinical outcomes. Therefore, it is prudent to consider the particular features of pancreatic islets from different species when optimizing islet isolation protocols.In this chapter, we explore the structural and functional features of pancreatic islets from mice, pigs, nonhuman primates, and humans because of their prevalent use in nonclinical, preclinical, and clinical applications.

The Choice of Enzyme for Human Pancreas Digestion is a Critical Factor for Increasing the Success of Islet Isolation.

We evaluated three commercially available enzymes for pancreatic digestion by comparing key parameters during the islet isolation process, as well as islet quality post-isolation. Retrospectively compared and analyzed islet isolations from pancreata using three different enzyme groups: Liberase HI (n=63), Collagenase NB1/Neutral Protease (NP) (n=43), and Liberase Mammalian Tissue Free Collagenase/Thermolysin (MTF C/T) (n=115). A standardized islet isolation and purification method was used. Islet quality assessment was carried out using islet count, viability, in vitro glucose-stimulated insulin secretion (GSIS), glucose-stimulated oxygen consumption rate (ΔOCR), and in vivo transplantation model in mice. Donor characteristics were not significantly different among the three enzyme groups used in terms of age, sex, hospital stay duration, cause of death, body mass index (BMI), hemoglobin A1c (HbA1c), cold ischemia time (CIT), and pancreas weight. Digestion efficacy (percentage of digested tissue by weight) was significantly higher in the Liberase MTF C/T group (73.5 ± 1.5 %) when compared to the Liberase HI group (63.6 ± 2.3 %) (p<0.001) and the Collagenase NB1/NP group (61.7 ± 2.9%) (p<0.001). The stimulation index for GSIS was significantly higher in the Liberase MTF C/T group (5.3 ± 0.5) as compared to the Liberase HI (2.9 ± 0.2) (p<0.0001) and the Collagenase NB1/NP (3.6 ± 2.9) (p=0.012) groups. Furthermore, the Liberase MTF C/T enzymes showed the highest success rate of transplantation in diabetic NOD Scid mice (65%), which was significantly higher than the Liberase HI (42%, p=0.001) and the Collagenase NB1/NP enzymes (41%, p<0.001). Liberase MTF C/T is superior to Liberase HI and Collagenase NB1/NP in terms of digestion efficacy and glucose-stimulated insulin secretion in vitro. Moreover, Liberase MTF C/T had a significantly higher success rate of transplantation in diabetic NOD Scid mice compared to Liberase HI and Collagenase NB1/NP enzymes.

Modification of human islet preparation: an effective approach to improve graft outcome after islet transplantation?

Revascularisation of transplanted islets is an essential prerequisite for graft survival and function. However, current islet isolation procedures deprive the islets of endothelial tubulets. This may have a detrimental effect on the revascularisation process of islets following transplantation. We hypothesise that modification of the isolation procedure that preserves islet endothelial vessels may improve the islet revascularisation process following transplantation. Here, we present a modified islet isolation method by which a substantial amount of endothelial cells still attached to the islets could be preserved. The islets with preserved endothelial cells isolated by this method were revascularised within 3 days, not observed in islets isolated by standard methods. Further, we observed that grafts of islets isolated by standard methods had more patches of dead tissue than islet grafts obtained by the modified method, indicating that attached endothelial cells may play an important role in the islet revascularisation process and potentially help to improve the transplantation outcome.

Hypothermic machine perfusion of the pancreas; a promising preservation method for islet isolation

Hypothermic machine perfusion of the pancreas; a promising preservation method for islet isolation

A method for mouse pancreatic islet isolation and intracellular cAMP determination.

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing prevalence of diabetes, both immune-mediated type 1 and obesity-linked type 2, studies aimed at delineating diabetes pathophysiology and therapeutic mechanisms are of critical importance. The β-cells of the pancreatic islets of Langerhans are responsible for appropriately secreting insulin in response to elevated blood glucose concentrations. In addition to glucose and other nutrients, the β-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on β-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased β-cell function, mass, and incretin responsiveness are well-understood to contribute to the pathophysiology of type 2 diabetes, and are also being increasingly linked with type 1 diabetes. The present mouse islet isolation and cAMP determination protocol can be a tool to help delineate mechanisms promoting disease progression and therapeutic interventions, particularly those that are mediated by the incretin receptors or related receptors that act through modulation of intracellular cAMP production. While only cAMP measurements will be described, the described islet isolation protocol creates a clean preparation that also allows for many other downstream applications, including glucose stimulated insulin secretion, [3(H)]-thymidine incorporation, protein abundance, and mRNA expression.

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing prevalence of diabetes, both immune-mediated type 1 and obesity-linked type 2, studies aimed at delineating diabetes pathophysiology and therapeutic mechanisms are of critical importance. The β-cells of the pancreatic islets of Langerhans are responsible for appropriately secreting insulin in response to elevated blood glucose concentrations. In addition to glucose and other nutrients, the β-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on β-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased β-cell function, mass, and incretin responsiveness are well-understood to contribute to the pathophysiology of type 2 diabetes, and are also being increasingly linked with type 1 diabetes. The present mouse islet isolation and cAMP determination protocol can be a tool to help delineate mechanisms promoting disease progression and therapeutic interventions, particularly those that are mediated by the incretin receptors or related receptors that act through modulation of intracellular cAMP production. While only cAMP measurements will be described, the described islet isolation protocol creates a clean preparation that also allows for many other downstream applications, including glucose stimulated insulin secretion, [3H]-thymidine incorporation, protein abundance, and mRNA expression.

Pancreatic islet transplantation: from dogs to humans and back again.

Pancreatic islet transplantation is a cell-based therapy that provides a potential cure for type 1 diabetes mellitus. After the introduction of an automated method for islet isolation and steroid-free immunosuppressive protocols, reversal of diabetes by islet transplantation is now performed at major human medical centers around the world. Despite extensive use of animal models in islet transplantation research, practical concerns have slowed the introduction of the technique into clinical veterinary practice and only a small number of studies have reported results of transplantation in dogs with spontaneously occurring diabetes mellitus; however, recent advances in islet isolation and encapsulation may make it possible to perform islet transplantation without immunosuppression in companion animals. This review summarizes experimental and clinical studies of pancreatic islet transplantation in dogs, including future directions for cell therapy in animals with naturally occurring disease.

Implementation of a Simplified Method of Islet Isolation for Allogeneic Islet Transplantation in Cynomolgus Monkeys

The present study describes a simple and cost-effective islet isolation procedure. Using this method, allogeneic islets reverse diabetes in cynomolgus monkeys. Pancreatic tissue from 11 cynomolgus monkeys were digested, collected, and purified using a simplified method. Islet quantification, purity, viability, and glucose static incubation were conducted immediately after isolation. Five streptozotocin-induced monkeys with diabetes were transplanted intrahepatically, and liver biopsies from 3 of these monkeys were taken at different time points for histologic study. The mean (SD) of viability, purity, and static glucose incubation stimulation index were 94.4% (2.3%), 91.8% (3.4%), and 2.6 (1.7), respectively. Monkeys who received a mean (SD) dose of 19,968 (2273) islet equivalent per kilogram (n = 4) from 2 to 3 donors who achieved prolonged normoglycemia (57-232 days), whereas the single monkey who received an islet dose of 8000 islet equivalent per kilogram did not experience diabetes reversal. Immunohistochemical assessment of the liver biopsies taken from the monkeys with normoglycemia revealed an insulin- and glucagon-positive islet graft for up to 6 months with minimal peri-islet inflammatory infiltration. This study demonstrates that cynomolgus monkey islets can be successfully and efficiently harvested using a simple isolation method, and these islets can restore normoglycemia in monkeys with diabetes.

097 Non-enzymatic cryo-isolation of viable Islets from juvenile porcine pancreas after storage for >6 months at −135 °C

Cryo-isolation of therapeutic cells such as pancreatic islets has been proposed as a novel new alternative to conventional enzymatic digestion of the pancreas (Taylor, Baicu., Transpl Proc. 43 (2011) 3181–3183. And Cell Transpl. 2013 (In Press)). Apart from avoiding the expense, inconsistencies and toxicity associated with the collagenase-digestion process, cryo-isolation embodies several inherent advantages including the convenience of biobanking for storage and shipping. This study investigates the feasibility of obtaining viable islets from juvenile porcine pancreas after storage at −135 °C for >6 months using the cryo-isolation technique. Pancreases were procured from juvenile pigs using approved procedures and processed using the cryo-isolation protocol. This involved: (1) Infiltrating islets in situ preferentially with 2M dimethyl sulfoxide as the cryoprotectant (CPA) via antegrade perfusion of the major arteries; (2) retrograde ductal infusion of water, and then (3) the entire pancreas was frozen solid to −160 °C and stored in the vapor phase of liquid nitrogen. After storage at −135 °C for 1 day to 8 months, pancreases were further processed as follows: The frozen pancreas was mechanically crushed and pulverized into small fragments which were then thawed, filtered and washed with RPMI 1640 culture medium to remove the CPA. Finally, the filtered effluent (cryo-isolate) was stained with dithizone for identification of intact islets, and with Syto13/Propidium iodide (PI) for fluorescence viability testing and assessed for function using a conventional glucose-stimulated insulin secretion (GSIS) assay. The cryo-isolate from pancreata stored for >6 months contained pancreatic fragments comprising discrete dithizone-positive islets embedded in a shell of dead amorphous material. The cryo-isolate product was similar to that obtained from frozen pancreases stored for 1 day in that there was an abundance of largely intact and viable (>90%) islets. Moreover, the cryo-isolated islets were typically larger (range 50–500 μm) than their counterparts isolated from juvenile pigs using conventional collagenase digestion techniques and demonstrated GSIS indices (>3), which was not significantly different from controls (fresh islets from collagenase digestion). An enzyme-free method of islet isolation relying on in situ cryopreservation of islets with simultaneous freeze-destruction of acinar tissue is feasible and proposed as a new and novel method that avoids the problems associated with conventional collagenase digestion methods and permits long-term biobanking of pancreases as a ready source of islets. Source of funding: This work was funded in part by a grant from NIH/NIDDK#1R43DK096773. Conflict of interest: The authors are, or have been, employed by Cell and Tissue Systems. mtaylor@celltissuesystems.com

Serial Viability and Flow Cytometry Analysis of Maturing Young Porcine Islet Tissue

Introduction: Porcine pancreatic islets have been explored as source of islets for xenotransplantation into patients with Type 1 diabetes. Methods of islet isolation from market weight pigs is both labor intensive, costly and islets tend to fragment resulting in suboptimal yields for transplantation. Methods of islet isolation and maturation of young piglet islets, yield viable function islets and protocols that are scalable for clinical trials. The aim of this study was to further characterize young porcine islets while tracking changes in islet cellular characteristics using flow cytometry and standard isolation endpoints. Methods: Pancreases from Yorkshire pigs (age range 14 to 20 days) were rapidly recovered and partially digested using low dose purified collagenase MA/BP protease (Vitacyte, LLC). Pancreatic tissue was then matured in tissue culture (37°C, 5%CO2) for up to 14 days with media changes every 48 hrs. During in vitro maturation tissue samples were analyzed for quality by islet yield (IEQ), purity (dithizone) and cell viability (FDA/PI). In vitro function was assessed using a static glucose stimulated insulin release (GSIR) assay. Samples were characterized for islet cellular composition by dissociating islets, using Acutase, and analyzing by immunohistochemistry for antibodies targeted for glucagon (α-cells), insulin (β-cells), and amylase (acinar tissue), using flow cytometery (BD FACS Aria II, FACS Diva software). Results: The proportion of dithizone positive tissue increased during tissue culture (12.6x103±183 IEQ (mean±sem) to 33.3x103±136 after 7 days of culture (n=6), p< 0.05)) with a majority of islets between 50-150 um in diameter (94.52±11% after 7 days). Islet function (GSIR) improved during time in culture (SI 1.3±0.1 at day 0, to 2.6±0.2 after 7 days of culture). During islet maturation in tissue culture, using flow cytometry amylase proportions decreased from 64.7±4% to 25.6±11%, while beta cell proportion within the islet increased from 42±9% to 70±5% after 2 and 7 days in tissue culture (p< 0.05). We observed no difference in glucagon positive cells during in vitro culture (37.3±17.3% at day 2 vs 43.6±18.6% at day 7, p=ns). Conclusion: During maturation in tissue culture, young porcine islets increase their beta cell proportion while decreasing exocrine portions. Viable functional young piglet islets are achieved using this protocol and these cells are being considered for future transplant studies.