Alginate has long been the material of choice for immunoprotection of islets due to its low cost and ability to easily form microspheres. Unfortunately, this seaweed-derived material is notoriously prone to fibrotic overgrowth in vivo, resulting in premature graft failure. The purpose of this study was to test an alternative, hyaluronic acid (HA-COL), for in vitro function, viability, and allogeneic islet transplant outcomes in diabetic rats. In vitro studies indicated that the HA-COL gel had diffusion characteristics that would allow small molecules such as glucose and insulin to enter and exit the gel, whereas larger molecules (70 and 500 kDa dextrans) were impeded from diffusing past the gel edge in 24 h. Islets encapsulated in HA-COL hydrogel showed significantly improved in vitro viability over unencapsulated islets and retained their morphology and glucose sensitivity for 28 days. When unencapsulated allogeneic islet transplants were administered to the omentum of outbred rats, they initially were normoglycemic, but by 11 days returned to hyperglycemia. Immunohistological examination of the grafts and surrounding tissue indicated strong graft rejection. By comparison, when using the same outbred strain of rats, allogeneic transplantation of islets within the HA-COL gel reversed long-term diabetes and prevented graft rejection in all animals. Animals were sacrificed at 40, 52, 64, and 80 weeks for evaluation, and all were non-diabetic at sacrifice. Explanted grafts revealed viable islets in the transplant site as well as intact hydrogel, with little or no evidence of fibrotic overgrowth or cellular rejection. The results of these studies demonstrate great potential for HA-COL hydrogel as an alternative to sodium alginate for long-term immunoprotected islet transplantation.

When working with isolated islet preparations, measuring the volume of tissue is not a trivial matter. Islets come in a large range of sizes and are often contaminated with exocrine tissue. Many factors complicate the procedure, and yet knowledge of the islet volume is essential for predicting the success of an islet transplant or comparing experimental groups in the laboratory. In 1990, Ricordi presented the islet equivalency (IEQ), defined as one IEQ equaling a single spherical islet of 150 μm in diameter. The method for estimating IEQ was developed by visualizing islets in a microscope, estimating their diameter in 50 μm categories and calculating a total volume for the preparation. Shortly after its introduction, the IEQ was adopted as the standard method for islet volume measurements. It has helped to advance research in the field by providing a useful tool improving the reproducibility of islet research and eventually the success of clinical islet transplants. However, the accuracy of the IEQ method has been questioned for years and many alternatives have been proposed, but none have been able to replace the widespread use of the IEQ. This article reviews the history of the IEQ, and discusses the benefits and failings of the measurement. A thorough evaluation of alternatives for estimating islet volume is provided along with the steps needed to uniformly move to an improved method of islet volume estimation. The lessons learned from islet researchers may serve as a guide for other fields of regenerative medicine as cell clusters become a more attractive therapeutic option.

Cell microencapsulation is a rapidly expanding field with broad potential for stem cell therapies and tissue engineering research. Traditional alginate microspheres suffer from poor biocompatibility, and microencapsulation of more advanced hydrogels is challenging due to their slower gelation rates. We have developed a novel, noncytotoxic, nonemulsion-based method to produce hydrogel microspheres compatible with a wide variety of materials, called core-shell spherification (CSS). Fabrication of microspheres by CSS derived from two slow-hardening hydrogels, hyaluronic acid (HA) and polyethylene glycol diacrylate (PEGDA), was characterized. HA microspheres were manufactured with two different crosslinking methods: thiolation and methacrylation. Microspheres of methacrylated HA (MeHA) had the greatest swelling ratio, the largest average diameter, and the lowest diffusion barrier. In contrast, PEGDA microspheres had the smallest diameters, the lowest swelling ratio, and the highest diffusion barrier, while microspheres of thiolated HA had characteristics that were in between the other two groups. To test the ability of the hydrogels to protect cells, while promoting function, diabetic NOD mice received intraperitoneal injections of PEGDA or MeHA microencapsulated canine islets. PEGDA microspheres reversed diabetes for the length of the study (up to 16 weeks). In contrast, islets encapsulated in MeHA microspheres at the same dose restored normoglycemia, but only transiently (3-4 weeks). Nonencapsulated canine islet transplanted at the same dose did not restore normoglycemia for any length of time. In conclusion, CSS provides a nontoxic microencapsulation procedure compatible with various hydrogel types.

Cell therapies are hampered by a lack of available delivery systems, resulting in inconsistent outcomes in animal studies and human clinical trials. Hydrogel encapsulants offer a broad range of tunable characteristics in the design of cell delivery vehicles. The focus of the hydrogel field has been on durable encapsulants that provide long-term paracrine function of the cells. However, some cell therapies require cell-to-cell contact in order to elicit their effect. Controlled release microencapsulants would be beneficial in these situations, but appropriate polymers have not been adaptable to microsphere manufacturing because they harden too slowly. We developed and tested a novel microencapsulant formulation (acrylated hyaluronic acid: AHA) with degradation characteristics as a controlled release cell delivery vehicle. The properties of AHA microspheres were evaluated and compared to those of poly­(ethylene glycol) diacrylate (PEGDA), a durable hydrogel. AHA microspheres possessed a higher swelling ratio, lower diffusion barrier, faster degradation rate, a lower storage modulus, and a larger average diameter than microspheres composed of PEGDA. Additionally, in vitro cell viability and release and short-term in vivo biocompatibility in immune competent Sprague–Dawley rats was assessed for each microsphere type. Compared to PEGDA, microspheres composed of AHA resulted in significantly less foreign body response in vivo as measured by a lack of cellularity or fibrotic ring in the surrounding tissue and no cellular infiltration into the microsphere. This study illustrates the potential of AHA microspheres as a degradable cell delivery system with superior encapsulated cell viability and biocompatibility with the surrounding tissue.

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BackgroundCanine diabetes is a strikingly prevalent and growing disease, and yet the standard treatment of a twice-daily insulin injection is both cumbersome to pet owners and only moderately effective. Islet transplantation has been performed with repeated success in canine research models, but has unfortunately not been made available to companion animals. Standard protocols for islet isolation, developed primarily for human islet transplantation, include beating-heart organ donation, vascular perfusion of preservation solutions, specialized equipment. Unfortunately, these processes are prohibitively complex and expensive for veterinary use. The aim of the study was to develop a simplified approach for isolating canine islets that is compatible with the financial and logistical restrictions inherent to veterinary medicine for the purpose of translating islet transplantation to a clinical treatment for canine diabetes.ResultsHere, we describe simplified strategies for isolating quality islets from deceased canine donors without vascular preservation and with up to 90 min of cold ischemia time. An average of more than 1500 islet equivalents per kg of donor bodyweight was obtained with a purity of 70% (N = 6 animals). Islets were 95% viable and responsive to glucose stimulation for a week. We found that processing only the body and tail of the pancreas increased isolation efficiency without sacrificing islet total yield. Islet yield per gram of tissue increased from 773 to 1868 islet equivalents when the head of the pancreas was discarded (N = 3/group).ConclusionsIn summary, this study resulted in the development of an efficient and readily accessible method for obtaining viable and functional canine islets from deceased donors. These strategies provide an ethical means for obtaining donor islets.

Background Protection of islets without systemic immunosuppression has been a long-sought goal in the islet transplant field. We conducted a pilot biocompatibility/safety study in healthy dogs followed by a dose-finding efficacy study in diabetic dogs using polyethylene glycol diacrylate (PEGDA) microencapsulated allogeneic canine islets. Methods Prior to the transplants, characterization of the canine islets included the calculations determining the average cell number/islet equivalent. Following measurements of purity, insulin secretion, and insulin, DNA and ATP content, the islets were encapsulated and transplanted interperitoneally into dogs via a catheter, which predominantly attached to the omentum. In the healthy dogs, half of the microspheres injected contained canine islets, the other half of the omentum received empty PEGDA microspheres. Results In the biocompatibility study, healthy dogs received increasing doses of cells up to 1.7 M cells/kg body weight, yet no hypoglycemic events were recorded and the dogs presented with no adverse events. At necropsy the microspheres were identified and described as clear with attachment to the omentum. Several of the blood chemistry values that were abnormal prior to the transplants normalized after the transplant. The same observation was made for the diabetic dogs that received higher doses of canine islets. In all diabetic dogs, the insulin required to attempt to control blood glucose was cut by 50–100% after the transplant, down to no required insulin for the course of the 60-day study. The dogs had no adverse events and behavioral monitoring suggested normal activity after recovery from the transplant. Conclusions and implications The study provides evidence that PEGDA microencapsulated canine islets reversed the signs of diabetes without immunosuppression and led to states of insulin-independence or significantly lowered insulin requirements in the recipients.

FDA approved Clinical Trials are already under way using stem cell-derived insulin-producing cells (IPCs) for the treatment of type 1 diabetes in humans. Differentiation methods used to derive beta-cell like IPCs have greatly improved over the last few years, resulting in highly efficacious treatment of high blood glucose in diabetic animals models. However, there still remains major challenges of developing an effective cure in humans due to challenges of delivery and long term survival and function of grafts. Like humans, spontaneous canine diabetes is a commonly diagnosed, multifactorial endocrine disorder in dogs which clinically presents as a type 1 diabetes disorder. In order to develop successful human diabetes therapies using stem cells, spontaneous canine diabetes offers a very attractive translational model. Dogs have major similarities to humans including the clinical signs, pathophysiology and long-term clinical management of the disease by use of exogenous insulin injections. Using cadaveric canine islets and our Core-Shell Spherification (CSS) microencapsulation methods, we have successfully reversed hyperglycemia in dogs for varying time periods. Our data confirms that similar to humans, islet transplantation is a viable option for diabetes treatment in dogs. To further develop canine diabetes as a translational model for human diabetes, while developing stem cell therapies for dogs, we have successfully generated IPCs from human pluripotent stem cells. The IPC clusters express key beta-cell biomarkers including PDX1, NK6.1, NeuroD1, and insulin. The majority of endocrine cells in clusters are single hormonal where separate cells express ARX and glucagon. These cell clusters corrected induced diabetes in mouse models when transplanted under the kidney capsule. Our studies will further allow us to further test the use of the canine model, both in allo- and xenogeneic transplant environments, for further pre-clinical development in humans. Disclosure A. Wildey: None. S.J. Williams: None. M. Hamilton: None. S. Harrington: None. L. Ott: Employee; Self; Likarda, LLC. F. Karanu: Employee; Self; Likarda, LLC.