Recent advances in the design of implantable insulin secreting heterocellular islet organoids

Abstract

Islet transplantation has proved one of the most remarkable transmissions from an experimental curiosity into a routine clinical application for the treatment of type I diabetes (T1D). Current efforts for taking this technology one-step further are now focusing on overcoming islet donor shortage, engraftment, prolonged islet availability, post-transplant vascularization, and coming up with new strategies to eliminate lifelong immunosuppression. To this end, insulin secreting 3D cell clusters composed of different types of cells, also referred as heterocellular islet organoids, spheroids, or pseudoislets, have been engineered to overcome the challenges encountered by the current islet transplantation protocols. β-cells or native islets are accompanied by helper cells, also referred to as accessory cells, to generate a cell cluster that is not only able to accurately secrete insulin in response to glucose, but also superior in terms of other key features (e.g. maintaining a vasculature, longer durability in vivo and not necessitating immunosuppression after transplantation). Over the past decade, numerous 3D cell culture techniques have been integrated to create an engineered heterocellular islet organoid that addresses current obstacles. Here, we first discuss the different cell types used to prepare heterocellular organoids for islet transplantation and their contribution to the organoids design. We then introduce various cell culture techniques that are incorporated to prepare a fully functional and insulin secreting organoids with select features. Finally, we discuss the challenges and present a future outlook for improving clinical outcomes of islet transplantation.

Introduction

Type-1 diabetes (T1D) is an autoimmune disease characterized by severe destruction of insulin-secreting pancreatic β cells, resulting in insulin deficiency and high blood glucose levels. The first and most available treatment option in the clinic for T1D is insulin therapy, which provides exogenous insulin uptake either through daily multiple injections or insulin pumps depending on the severity of disease. It was shown that insulin pumps may provide more precise insulin delivery as well as adjusting basal insulin levels [1]. However, there are several systemic side effects of insulin therapy, such as hypoglycemia, weight gain and pain at injection site [2]. Additionally, recent advances have been made in the development of insulin analogues which are fast and long acting compared to insulin [3]. Since T1D is an autoimmune disease, immune therapy was also proposed as alternative treatment option. The main purpose of current effort is based on regulating autoimmune response such as using immunomodulatory agents to induce immune tolerance through activation of regulatory T cells (Tregs) [4]. Additionally, late-stage insulin-dependent T1D patients’ subgroups that exhibit severe hypoglycaemic episodes, hypoglycaemia unawareness and glycaemic lability whose symptoms cannot be reverted by exogenous insulin pumps and/or glucose monitorization therapies, are referred to islet allotransplantation [5]. The other treatment option to achieve normoglycemia without the requirement of insulin injection is allogeneic pancreas transplantation. Although this operation may allow for long-term insulin independence, it is a heavily invasive procedure and causes massive immune attack to the graft. Islet transplantation is a better alternative to whole-organ pancreas transplantation as the procedure itself does not require major surgery. Islet transplantation technique was improved with the “Edmonton Protocol” for isolation of islets from brain-dead donors and immediate transplantation through the portal vein of recipient patients along with steroid-free immunosuppressive drug regimen [6]. However, there are several challenges associated with the islet transplantation such as instant blood-mediated inflammatory reactions (IBMIR) after implantation followed by immune rejection of the graft, requirement of lifelong use of immunosuppressive drugs and scarcity of donor pancreas [7]. Additionally, after islet transplantation, vascularization around the graft is important for the maintenance of viability and functionality of islets. Another concept to facilitate transplantation of islets is by physically protecting from immune attack using an implantable immunoisolation device with different encapsulation approaches. Extensive research has been focused on optimization of these implantable devices. Some of these implants are commercially available and under preclinical and clinical investigation. One example is Encaptra device by Viacyte Inc. This device consists of stem cell–derived pancreatic progenitor cells (PEC-01) which are encapsulated in a cell delivery system (Clinical Trial: NCT02239354) [8,9]. Alternatively, recent progress has been made to promote vascularization and graft function by engineering cells in in vitro models which holds a great potential for clinical use of islets in future [10]. Current clinical strategies for the treatment of T1D is summarized elsewhere and we refer the interested readers to the literature [5,9,11,12].

Human and mice pancreatic islets share many common features which enables the use of mice islets for research purposes. On the other hand, one of the most distinct differences between mice and human pancreatic islet is the ordered cellular architecture of mice islets. Pancreatic islets of mice comprise of multiple endocrine cell types, including insulin-secreting β-cells at the core surrounded by glucagon-secreting α cells and somatostatin-secreting δ cells at the periphery, whereas in human pancreatic islets, endocrine cells are rather randomly distributed [13]. They have complex microstructure where different cell types are interconnected through extracellular matrix (ECM), cell-cell adhesion and cell-matrix adhesion molecules. The complex interactions between cells have direct effect on islet function, β-cell survival and insulin secretion capacity [14]. Morphology and β-cell to β-cell contact are critical for a pancreatic islet as intact islets have enhanced insulin secretion function compared to that of dissociated islet cells [10]. As alternative to pancreatic islets, insulin-secreting cell lines such as Mouse Insulinoma 6 (MIN6), Rat Insulinoma (RINm), INS-1 and β-TC6 cells are considered as promising cell sources in islet engineering studies [15,16]. These cell lines have been utilized to prepare engineered islet organoids, or pseudoislets, by co-culturing with accessory cells to improve the function and provide immunoisolation or vascularization. Rather than monolayer cell culture, insulin secreting cells are clustered into aggregates to form 3D spheroid structures. It is important to closely mimic the 3D architecture of native islets for in vitro preparation of organoids and to achieve appropriate insulin secretory responses [17]. However, these cell lines are engineered to explore further design parameters for functional pseudoislets mainly in the in vitro setup and currently, they have not transitioned to clinical setup. With new technologies being developed, engineered pseudoislets consisting of pancreatic cell lines could be translated into clinical studies.

Until now many attempts have been made to understand the interaction of β-cells with accessory cells such as stem cells, fibroblasts, hepatic cells, neural cells and endothelial cells [18]. These co-culture strategies have great importance as it may be possible to promote protection from immune attack through a cell-based biological barrier function and promote graft survival and function. Additionally, apart from formation of heterospheroids, native islets have also been co-cultured with these accessory cells to prevent graft loss and improve islet function after transplantation. Alternatively, native islets are indirectly co-cultured with other cell types where cell signaling occurs through paracrine signaling as a result of secretion of some soluble factors [19]. There are several 3D cell culture methods to form heterocellular islet organoids with spherical shape and uniform size. The important parameters that should be considered for the selection of appropriate 3D culture method include homogeneous cell distribution, improved cell activity and efficient production of insulin.

In the following sections, we discuss about the design of implantable heterocellular islet organoids in terms of accessory cell type and co-culture method (Fig. 1). We present recent findings about co-culture of insulin secreting β-cells or native islets with different accessory cells to promote post-transplant graft acceptance and function (Table 1). In addition, this review provides a description of various co-culture methods used in islet organoid preparation. Finally, we discuss the potential and the challenges associated with the co-culture systems and describe the contributions brought by the accessory cells.