Survival and Maturation of Microencapsulated Porcine Neonatal Pancreatic Cell Clusters Transplanted into Immunocompetent Diabetic Mice

Abstract

Differentiation and maturation of porcine neonatal pancreatic cell clusters (NPCCs) microencapsulated in barium alginate were assessed after transplantation into immunocompetent mice. Microencapsulated NPCCs were transplanted into the peritoneal cavity of streptozocin-induced diabetic B6AF1 mice (n ‫؍‬ 32). The microcapsules were removed at 2, 6, and 20 weeks and examined for cellular overgrowth, insulin content, and insulin secretory responses to glucose and glucose with theophylline. The differentiation, maturation, and proliferation of the ␤-cells in the NPCCs were assessed by immunohistochemistry. Blood glucose levels were normalized in 81% of the animals that received a transplant and remained normal until termination of the experiments at 20 weeks. Hyperglycemic blood glucose levels after explantation of the capsules confirmed the function of the encapsulated NPCCs. Insulin content of the encapsulated NPCCs was increased 10-fold at 20 weeks after transplantation compared with pretransplantation levels. A 3.2-fold increase of the ratio of the ␤-cell area to the total cellular area was observed at 20 weeks, demonstrating the maturation of NPCCs into ␤-cells. A s a result of recent progress (1), there is increased interest in islet transplantation as a potential therapy for type 1 diabetes. However, two major barriers must be overcome before islet transplantation can be provided for more patients: 1) the limited availability of human pancreatic tissue and 2) the need for permanent immunosuppression to prevent graft rejection and autoimmunity (2). Xenogeneic islets from pigs and cows (3-5) have been considered as potential sources of islets for transplantation. Many factors favor the use of pigs: the similar structure of porcine and human insulin, the comparable glucose levels, and that both pigs and humans are omnivores. Islet cells can be isolated in large numbers from adult (6 -9) or neonatal pigs (10,11). However, adult pig islets have proved to be difficult to isolate and tend to fare poorly in tissue culture, which has limited their use. Neonatal pancreatic cell clusters (NPCCs) contain a high proportion of islet precursor cells, can be maintained in culture, and differentiate into ␤-cells after transplantation (11). Naked (10,12) or microencapsulated NPCCs (13) have been shown to restore normoglycemia after transplantation into streptozotocin (STZ)-diabetic nude mice.The concept of a bioartificial pancreas, consisting of islets enclosed within immunobarrier membranes, provides a potential way to overcome the need for immunosuppression. Our group recently developed a promising encapsulation method that uses highly purified alginate cross-linked with BaCl 2 , without a separate permselective barrier, which protects islets against allorejection and autoimmunity (14). The aims of this study were to assess the protective capacity of simple barium-alginate capsules in a xenotransplantation model of NPCCs transplanted into STZ-induced diabetic immunocompetent mice and then to evaluate the growth, maturation, a...

Improved survival of microencapsulated islets during in vitro culture and enhanced metabolic function following transplantation

“…Nonetheless, an open pore alginate capsule is not so much an immune barrier as a support matrix to enhance islet viability, which is a critical essential step before a perm-selective membrane is placed on the islet. The ability of these capsules to so successfully restore glucose responsiveness may largely be a result of the high purity and low endotoxin content of the alginate [6,32]. Therefore, microencapsulating islets with an alginate of excellent biocompatibility can provide an environment that supports long-term islet graft survival and function, ultimately reducing beta cell loss and dysfunction in the immediate post-transplant period.…”

“…Since islets placed under the kidney capsule tend to fuse together forming large clumps, these grafts may be exposed to more hypoxic conditions resulting in necrosis and/or apoptosis [31]. Furthermore, encapsulation of islets, while not providing a perfect immune barrier, certainly may prolong graft survival [6,32]. While the exclusion of poly-L-lysine from our alginate capsule increases the porosity of the capsule [33], the capsule retains the ability to prevent contact of host cells such as macrophages with the islets, ultimately extending graft survival [34].…”

Preparation and in vitro studies of microencapsulated cells releasing human tissue inhibitor of metalloproteinase-2

“…A simple technique that consists of one step encapsulation with a highly purified alginate crosslinked with barium, without a traditional permselective layer such as PLL (poly-Llysine) can produce microcapsules working very well, showing even higher stability and biocompatibility. Porcine neonatal pancreatic cell clusters encapsulated with simple barium alginate can differentiate into β-cells and reverse high blood glucose levels in immunocompetent mice without immunosuppression for >20 weeks (Omer et al, 2003). By means of air-jet technique, we obtained barium alginate microcapsules having consistently the appearance of a sphere, inside which the encapsulated cells survived well at least 6 weeks after preparation in vitro, confirming the permeability of barium alginate microcapsules that allows the exchange of nutrients, oxygen and waste products.…”

Ectopic expression of neurogenin 3 in neonatal pig pancreatic precursor cells induces (trans)differentiation to functional alpha cells

“…Thus, inducing the differentiation of beta cells from precursors within the neonatal pancreas in vitro prior to transplantation would perhaps lead to enhanced in vivo function after transplantation. This has been demonstrated to be the case when neonatal pancreatic cells are provided an alginate extracellular matrix or cultured with nicotinamide [4,8].…”