Re-engineering Islet Cell Transplantation

Abstract

We are living exciting times in the field of beta cell replacement therapies for the treatment of diabetes. While steady progress has been recorded thus far in clinical islet transplantation, novel approaches are needed to make cell-based therapies more reproducible and leading to long-lasting success. The multiple facets of diabetes impose the need for a transdisciplinary approach to attain this goal, by targeting immunity, promoting engraftment and sustained functional potency. We discuss herein the emerging technologies applied to beta cell replacement therapies.

1. Introduction

1.1. Diabetes Mellitus

Diabetes is a metabolic disorder characterized by elevated glucose levels in the blood (hyperglycemia). Type 1 diabetes mellitus (T1DM) is caused by an autoimmune-mediated destruction of the insulin-producing β-cells in the pancreatic islets [1]. Type 2 diabetes mellitus (T2DM) is associated with dyslipidemia, obesity, initial hyperinsulinemia and insulin resistance in target tissues (fat, liver and muscle), resulting in progressive islet cell dysfunction and ultimately in insulinopenia and need for exogenous insulin therapy [2]. Glycemic metabolism can be controlled, at least to a certain extent, by daily administration of exogenous insulin, frequent monitoring of blood sugar levels combined with diet and exercise. Achieving tight glycemic control is desirable in patients with diabetes [3]. Unfortunately, even with a careful insulin treatment based on the use of improved insulin formulations, infusion systems and continuous glucose monitoring systems, daily glycemic excursions are difficult to keep tightly in the normal range. Thus, chronic and degenerative complications, such as retinopathy, nephropathy, neuropathy, and atherosclerosis, still occur in a considerable fraction of patients with diabetes, contributing to the poor quality of life, reduced life expectancy and to the elevated medical costs associated with diabetes.

1.2. Restoration of Physiologic Metabolic Control

Restoration of physiologic glucose metabolic control is highly desirable in patients with diabetes. Replacement of islet β-cells can be performed either by whole pancreas or isolated pancreatic islet transplantation. The experience of the last three decades supports the positive impact on metabolic control of the biologic replacement of β-cells via allogeneic islet and whole pancreas transplantation. Notably, islet transplantation requires less risky implantation approaches than invasive surgery. Moreover, the possibility of engineering the islet transplant to promote its engraftment and long-term function makes of islet transplantation an appealing therapeutic approach to restore β-cell function.

1.2.1. Islet Transplantation

The islet transplantation procedure is currently performed with a minimally invasive approach consisting of a percutaneous cannulation of the portal vein, through which islets are infused into the recipient’s liver [4–7]. This technique has been utilized since the 1970’s mainly to prevent or ameliorate metabolic control in patients with chronic pancreatitis requiring pancreatectomy (autologous islet transplantation) [8,9], and to restore metabolic control in patients with unstable T1DM associated with frequent severe hypoglycemic episodes [7,10]. Recently, autologous islet transplantation has also been proposed for patients with resectable neoplastic lesions of the pancreas [11–14]. Clinical islet allogeneic transplantation trials performed in patients with brittle T1DM demonstrated restoration of metabolic control with complete independence from (when adequate islets are implanted) or dramatic reduction of exogenous insulin requirements (i.e., transplantation of suboptimal islet numbers or development of graft dysfunction), as well as prevention of severe hypoglycemic episodes paralleled by improved quality of life [10,15,16]. Interestingly, preliminary data suggest improvement of diabetes complications following islet transplantation [10,17–19].

1.2.2. Current Challenges of Islet Transplantation

Even though islet transplantation has become a promising clinical therapeutic option in recent years, several challenges currently limit its application to the most severe cases of unstable diabetes characterized by hypoglycemia unawareness and frequent debilitating, severe hypoglycemia, at times life-threatening. Amongst the key hurdles recognized to the widespread application of islet transplantation is the variable long-term success of intra-hepatic implantation, which may result from a combination of variables likely secondary to lack of vasculature in the early peri-transplant period and to nonspecific inflammation triggered by islet isolation and transplantation procedures, collectively resulting in reduced islet engraftment (β-cell death and functional impairment), as well as in triggering of adaptive immunity affecting graft survival.

Cadaveric human donor pancreata represent an unsustainable source of transplantable islets since variables related to donor (i.e., age, sex, cause of death and duration of intensive care) and organ characteristics (i.e., warm and cold ischemia, preservation technique utilized, presence of intra-parenchymal fat infiltration, etc.), islet isolation (i.e., enzyme and purification methods) and culture conditions may yield quite disparate results in terms of islet integrity, numbers, potency and immunogenicity, all ultimately determining graft outcome after transplantation [20].

Development of reliable tests and algorithms able to predict the success of islet isolation and transplantation based on donor variables [20–22] or final cell product assessment [23–31] may be of assistance in achieving higher success rates after islet transplantation more reproducibly. However, even by refining and expanding donor selection criteria (i.e., use of organ donation after cardiac arrest and marginal donors) and improving the efficiency of organ recovery, the number of pancreata suitable for transplant may fall short of the needs of the large patient population potentially benefiting of a biologic replacement of β-cell function.

Another challenge is the immunosuppression utilized in islet transplant recipients, which relies on agents that may impair tissue remodeling and neovascularization (e.g., mTOR inhibitors) as well as affect β-cell function over time (e.g., calcineurin inhibitors, amongst other). Moreover, the immunosuppression required to prevent rejection may not adequately target autoreactive immune responses, in turn allowing progressive loss of graft function to autoimmunity recurrence in patients with T1DM. Development of novel approaches to promote and enhance islet engraftment and long-term function, as well as to modulate immunity are needed to make islet transplantation a more reproducible therapeutic option in the near future.

1.3. Alternative Sources for Transplantable Islet Cells

Undeniably, there is an urgent need for unlimited source of transplantable ‘islets’, which may come from xenogeneic donors (e.g., porcine islet cells), conversion of adult or embryonic stem cells into endocrine pancreatic cells. Islet transplantation of islets obtained from xenogeneic donors is appealing. Porcine islets may represent a readily available source, and pilot human clinical trials have been attempted, with demonstration of transient function of implanted islets without adventitious effects related to zoonotic diseases (i.e., porcine endogenous retrovirus infections, PERV) [32–35]. Moreover, remarkable progress has been documented with the development of specific pathogen-free herds, as well as of genetically modified strains that aim at overcoming the immunological barriers [36,37], which may enable achieving long-term restoration of metabolic control in combination of safe immunotherapies and bioengineering of the transplant microenvironment (i.e., immunoisolation).

Embryonic stem cells (ESCs) are a promising alternative cell source for treating diabetes. They are pluripotent stem cells capable of unlimited replicative capacity and the potential to differentiate into different cell phenotypes. Differentiation of insulin-producing cells from mouse and human ESCs has been demonstrated. A milestone in the field was the work by D’Amour et al. demonstrating the differentiation of human ESCs into endocrine cells able to produce insulin, glucagon, somatostatin, pancreatic polypeptide and ghrelin using a five step protocol mimicking the pancreatic development in vitro through a series of endoderm intermediates [38]. However, the release of C-peptide by these cells in response to glucose in vitro was marginal. Interestingly, these immature cells can subsequently differentiate in vivo into endocrine cells capable to support metabolic function in chemically-induced diabetic mice [39]. These studies have stimulated the field and led to a phase 1/2 clinical trial in patients with T1DM currently underway (Table 1).

Table 1.

Islet Clinical Trials (clinicaltrials.gov, accessed Jan 31, 2015)

Sponsor	Title of Trial	Phase	Registry No.*
Viacyte, San Diego, CA, USA	ESC in Macrodevice	I/II	NCT02239354
University of Miami/Diabetes Research Institute, Miami, FL, USA	Allogeneic Islet Transplant into the Omentum	I/II	NCT 02213003
Vrije Universiteit Brussel, Brussels, Belgium	Long Term Function of Beta Cell Allografts in Non-Uremic T1D Patients	I/II	NCT00798785
Ospedale San Raffaele, Milan, Italy	Bone Marrow as an Alternative Site for Islet Transplantation	I	NCT01345227
Ospedale San Raffaele, Milan, Italy	Bone Marrow vs Liver as Site for Islet Transplantation (IsletBOM 2)	I/II	NCT01722682
The Nordic Network For Clinical Islet Transplantation, Sweden and Norway	Intraportal or Intramuscular Site for Islets in Simultaneous Islet and Kidney Transplantation	II	NCT01967186
Uppsala University Hospital, Sweden	Autologous Mesenchymal Stem Cell Transplantation	II	NCT02057211
Uppsala University Hospital, Sweden	Beta/Air Device for Encapsulation of Transplanted Human islets	I/II	NCT02064309
Cliniques Universitaires Saint-Luc-Université Catholique de Louvain, Leuven, Belgium	Encapsulated Human Islets in a "Monolayer Cellular Device"	I	NCT00790257
Novocell, Irvine, CA, USA	Allogeneic Cultured Islet Cells (Human); Encapsulated	I/II	NCT00260234
Living Cell Technologies, Auckland, New Zealand	Alginate-Encapsulated Porcine Islets for Xenotransplantation	I/II	NCT00940173 NCT01736228 NCT01739829
Vrije Universiteit Brussel, Brussels, Belgium	Transplantation of Encapsulated Beta Cells	II	NCT01379729
Fuzhou General Hospital, Xiamen University, China	Cotransplantation of Islet and Mesenchymal Stem Cell	I/II	NCT00646724
University of Alberta, Edmonton, BC, Canada	Sernova Cell Pouch	I/II	NCT01652911
University of Perugia, Italy	Transplantation of Immunoprotected Pancreatic Islets for the Therapy of Type 1 Diabetes Mellitus (T1DM)	I	ISRCTN43557935
Technische Universität Dresden, Germany	Beta O2 Immunoisolation Device	“individual treatment attempt”	PMID24167261

^*NCT: clincialtrials.gov; ISRCTN: isrctn.com; PMID: ncbi.nlm.nih.gov/PubMed

Conversion of pancreatic exocrine cells into insulin-producing cells in adult mouse pancreas has been achieved by specific combination of transcription factors (namely, Ngn3, Pdx1, and Mafa) [40]. Moreover, pancreatic acinar cells can be converted into somatostatin and glucagon cells by Ngn3 and Ngn3+Mafa respectively [41]. It has also been demonstrated that pancreatic ductal structure may contain precursor cells that can yield to insulin-producing cells [42–45]. Collectively, these studies point to the potential of developing protocols for the large scale production of pancreatic endocrine cells for transplantation in vitro from tissue that is currently considered waste product of islet isolation processing. Additionally, it may lead to optimization of approaches to promote endocrine cell differentiation and/or expansion in vivo targeting impure (e.g., containing acinar and ductal structures) islet preparations transplanted in engineered sites that can be targeted selectively with the appropriate treatment for controlled (time and amounts) delivery.

1.4. Cell-Based Immunomodulation

Immunity represents one of the critical hurdles for islet grafts, particularly since their fate depends on the combination of allogeneic and autoreactive immune responses in patients with T1DM [46,47]. Restoration of self-tolerance and/or donor-specific hyporesponsiveness/tolerance is the desirable goal of diabetes and transplant immunobiology. Different approaches have been proposed that may be of assistance in achieving this ambitious objective, likely by combining immunotherapy and cell-based treatments. A growing body of literature supports the potential beneficial impact of the use of hematopoietic stem cells (HSC) and of regulatory immune cell subsets (i.e., T regulatory cells, Tregs; Mesenchymal Stromal Cells, MSC; Dendritic Cells, DC; amongst others) to achieve such goal in patients with T1D [48–52] and in organ transplant recipients [53–60]. In the context of clinical islet transplantation, the use of donor-specific HSC was shown to associate with the achievement of graft function and transient hematopoietic chimerism, though the discontinuation of immunosuppression by protocol (in the absence of tests predictive of immunomodulation) invariably resulted in loss of graft function [4,54,61]. Combination of islets with MSC treatment was shown to improve islet engraftment in rodents [62–65] and large animal models [66], and is currently under evaluation in the clinical settings (Table 1).

The initial reports on the use of cell-based immunotherapies are quite encouraging, though current challenges include the lack of reliable immune markers predictive of patient response, as well as the fact that different trials rely on quite different protocols for cell isolation and concomitant immunotherapy utilized, rendering cumbersome the comparison of outcomes. A coordinated effort amongst Centers is desirable, along with the use of standardized approaches and readouts is paramount towards the development of successful cell-based therapies for the treatment of diabetes and their widespread clinical application.

1.5. Bioengineering Approaches for Islet Grafts

The critical importance of creating a microenvironment suitable for islet cell has prompted many tissue engineering approaches for the development of a bioartificial pancreas, which can ensure an optimal site of implantation to favor both engraftment and long-term function of islet cells. Ultimate goal of engineering the transplant microenvironment is to enhance islet cell viability, to promote prompt neo-angiogenesis, and to modulate immunity.

1.5.1. Extra-Hepatic Islet Transplantation

The choice of the site of implant for islet grafts may ultimately influence clinical outcomes. For the last three decades, clinical islet transplantation has relied on their embolization in the hepatic sinusoids of the portal system (intra-hepatic islet transplantation) [67]. It has been recognized that the liver microenvironment may not be ideal for islet cells. The portal system is exposed to high concentrations of immunosuppressive drug levels that may result in impaired islet cell potency [68]. Moreover, glycemic levels are higher than in the liver than in the pancreas, which may contribute to increase endocrine cell basal activity leading to exhaustion. Contact of islets with blood induces an instant blood-mediated inflammatory reaction (IBMIR) that results from the activation of coagulation and complement cascades, neutrophils and leukocyte recruitment [69,70] that contributes to amplifying the activation of transplant microenvironment via endothelial cell and macrophage activation associated with islet embolization in the hepatic sinusoids [71] and hypoxia negatively affecting islet cell viability and engraftment. Notably, prompt neovascularization of islet grafts is essential to achieve functional competence after transplantation. The isolation process results in damage of intrainsular vascular structures [72] that may contribute to immunogenicity and delayed neovascularization of transplanted islets, which may require weeks to be fully re-established [73,74]. It is conceivable also that the microvascular complications of diabetes may altered endothelial repair capabilities in the transplant microenvironment [75]. Progressive loss of functional islet mass has been reported also in large animal models of intrahepatic autologous islet transplantation in the absence of chronic rejection and autoimmunity recurrence or toxicity of immunosuppressive drugs [76], further supporting the need for the development of alternative, extra-hepatic sites for islet grafts.

An ideal site for islet transplantation may include: sufficient capacity to host large volumes of tissue containing impure (non-endocrine) fractions, portal drainage, ease of access using minimally invasive techniques, potential of microenvironment manipulation (tissue engineering), and accessible for noninvasive imaging and biopsy [77]. In recent years, different studies have explored extrahepatic implantation sites for islet cells in experimental models [77,78]. Implantation sites such as the omentum may meet the requirements of ideal portal venous drainage and space pliable for tissue engineering approaches [79–84]. Also, the intestinal subserosa [85–87], excluded vascular structures [88], amongst other sites, were shown to represent an interesting implantation site for islet grafts (Figure 1).

Figure 1. Implantation sites for islet cells.

Several implantation sites have been proposed for islet cells, besides the intrahepatic portal system. An ideal site may be spacious, easily accessible for minimally invasive implantation and explant, have portal venous drainage, modifiable to enhance engraftment and modulate immunity, and allow for graft monitoring via noninvasive or minimally invasive approaches. The sites in the boxes have been tested in humans.

Evaluation of the feasibility and efficacy of extrahepatic sites in the clinical setting is also underway. Allogeneic and xenogeneic islets incorporated in immunoisolating microcapsules have been implanted in the peritoneal cavity of patients with diabetes, but the achievement of sustained function has been difficult to attain so far in the clinical settings, which may require better characterization of the suitability of the site for islet cells [89,90]. The subcutaneous space is easily accessible with minimally invasive surgery, and initial attempts with implantable devices have been reported [34,91,92]. Engraftment and functional competence of autologous islets implanted in the intramuscular site (forearm) has also been reported in human subjects and may represent a viable approach [93–96]. More recently, the feasibility of intra-bone marrow islet transplantation has been demonstrated in a pilot series of human islet autografts in patients with contraindication for intrahepatic route, in which successful engraftment without adverse events were reported [77,97]. Collectively, it is likely that future clinical trials will increasingly rely on extrahepatic implantation sites to enhance the long-term success of β-cell replacement therapies (Table 1).

1.5.2. Scaffolds

Given the high metabolic demand of islets, it would be desirable to design a device that allows for both intra-device vascularization and to achieve spatial distribution of the cells within the device to avoid clumping and generation of hypoxic conditions. The use of a biomaterial to spatially distribute the cells would allow for a more desirable three-dimensional arrangement of the cells and result in a more efficient delivery of nutrients. Moreover, devices may be engineered to incorporate drugs, generate oxygen and several biomaterials have been used, both for in-vitro and in-vivo studies, to generate macroporous scaffolds, including collagen, chitosan, hydroxyapatite, poly(α- hydroxy esters) like poly(glycolic acid) (PGA), poly(lactic acid) (PLA), and poly(lacticco- glycolic acid) (PLGA).

In general, natural materials have the benefit of high cellular recognition and tissue compatibility, but are costly, variable, biodegradable, and require high levels of purification [98]. Synthetic materials, on the other hand, can be fabricated with uniformity between batches, precise control over rates of degradation, and modified chemical properties.

Progressive nonimmunologic loss of functioning islets may be related to the lack of a functional islet microenvironment after isolation from the whole pancreas. Developing technologies that provide a better microenvironment for islet grafts may represent a viable strategy to extend islet longevity as well as reduce the islet mass required to achieve insulin independence [99]. Problems associated with the hepatic transplantation of islets may preclude the broad application of islet transplantation. Thus, new approaches to the extrahepatic transplantation of islets using a synthetic biodegradable polymer scaffold have been developed.

Several basic requirements for cell transplantation on macro- and micro-porous scaffolds have been identified, including biocompatibility, a high surface area/volume ratio with sufficient mechanical integrity, and a suitable environment for new tissue formation that can integrate with the surrounding tissue [100]. Porous polymer-based scaffolds are and not intended to serve as an immune barrier, like macro- and micro-encapsulation. Rather, they were specifically designed to provide a solid support for islets that would allow cellular infiltration and formation of a vascular network within the transplant graft. Scaffolds with a high surface area/volume ratio not only have sufficient surface area to support cell adhesion, but can also support nutrient transport by diffusion from surrounding tissue. Moreover, they can be fabricated from material that has sufficient mechanical properties to resist collapse while maintaining an interconnected pore structure that allows for cell infiltration from the surrounding tissue. This is important not only for integration of the engineered tissue with the host, but also for development of a vascular network throughout the tissue to supply the needed metabolites once the transplanted cells are engrafted.

Reversal of diabetes in diabetic rodents was shown after seeding islets onto a highly porous Poly(D,L14 lactide-co-glycolide) (PLG) scaffold implanted in the intraperitoneal fat [101]. Similar results were obtained using porous scaffolds fabricated in biocompatible poly(dimethylsiloxane) (PDMS) that supported adequate neovascularization and functional potency of syngeneic islets in rodents [84,102]. These findings indicate that a synthetic polymer scaffold can serve as a platform for islet transplantation. Notably, the scaffold may also be useful to deliver bioactive molecules such as oxygen, trophic factors (i.e., proangiogenic molecules) or immunomodulatory agents to modify the microenvironment surrounding the transplanted islets and, thus, enhance islet survival and function [102–104].

Synthetic biodegradable polymers have been extensively utilized in tissue engineering [100], as their degradation kinetics and mechanical properties can be tailored to meet the needs of a specific application [105]. Successful engraftment of islets loaded on a synthetic (VICRYL (Polyglactin 910) and PDS (poly-P-Dioxanon) biodegradable scaffold implanted an omental pouch of diabetic monkeys supported the feasibility in preclinical settings [81].

In recent years, there has been a growing interest in developing biocompatible scaffolds containing extracellular matrix (ECM) components to improve islet culture and transplantation [106]. Cultured on collagen, fibrin or surrogate matrices, islets show a decrease in apoptosis, better survival in culture and improved and prolonged function in vitro [107]. A key factor in choosing this compound is that fibrin is already available as a clinical grade tissue sealant. Alternatively, sufficient quantities of fibrinogen-rich plasma can potentially be obtained from islet recipients prior to transplantation [82]. In addition, fibrin scaffolds have been shown to be beneficial for islets in culture: they increase human islet cell mass in vitro [108], stimulate endothelial cell proliferation [109], and have been used successfully in experimental islet transplantation [110]. An additional and appealing source for extracellular matrices applicable to islet grafts are decellularized pancreata (human or from other species) that may be used as 3-D structures for implantation or as lyophilized matrices for co-implantation with islet cells [111– 113].

1.5.3. Macrodevices

Macrodevices may be designed to support engraftment and physical containment of the cellular grafts. Prevascularization of the islet implantation site may be achieved by using spacers (e.g., Polytetrafluoroethylene, PTFE) containing or not pro-angiogenic factors to prime the environment and that are replaced before islet transplantation. While this approach does not confer immune isolation, it may help mitigating the local inflammation and allow adequate engraftment of the cellular implants [34,91,114–123].

Amongst devices that provide a physical barrier to the graft is the TheraCyte™ macroencapsulation system, a planar pouch featuring a bilaminar PTFE semipermeable membrane. The outer layer promotes tissue integration, whereas an inner, cell impermeable, membrane has a 0.4µm pore size. Its subcutaneous placement allows cells to be transplanted in a minimally-invasive manner and retrieved if necessary. The device is biologically inert and when transplanted into human patients for a year, there were no adverse effects reported [124]. This type of device has been utilized to protect cells from immune reaction, but also to prevent dissemination of the cell implant in the recipient body. Feasibility of this approach was demonstrated for the implantation of genetically engineered cells specially designed for the in vivo delivery of therapeutic proteins, such as endostatin, which circumvents the problem of limited half-life and variation in circulating levels [125]. This device limits direct contact of host immune cells with the implanted cells, which could reduce or delay rejection while allowing the exchange of subcellular materials. The use of TheraCyte device has yielded some degree of successes reported with stem cell derived endocrine cells [126], as well as with allogeneic and xenogeneic pancreatic islet transplantation in experimental animals [127], including in the presence of autoimmunity in NOD mice [128]. Immunoisolation of pancreatic islets using the TheraCyte device has shown to protect against allograft rejection in non-immunized recipients [129]. A similar device, aimed at both confining and immunoprotecting human embryonic stem cell-derived endocrine cells is under evaluation for clinical testing (Table 1).

1.5.4. Islet Encapsulation

Islet encapsulation offers a means to create a physical barrier around islet clusters to shield them from the immune system, while allowing free exchange of insulin and nutrients throughout the cells [comprehensive review [130]]. Amongst the challenges in this field is the selection of materials that protect islets from the immune system. The encapsulation material must perform two vital functions: permit diffusion of insulin and waste products, and isolate the encapsulated islets from the immune system (i.e., macrophage and lymphocyte infiltration)[131]. Different approaches have been proposed that rely on macrodevices and microdevices containing multiple islets or thin-layer (conformal) coating of individual islets [132,133]. One of the materials widely used for islets encapsulation is alginate, an anionic polysaccharide produced by seaweed, whose biocompatibility and gelling properties make it useful for macro- and micro-encapsulation [126,134]. To maximize biocompatibility and elimination of potential pyrogenic contaminants (e.g., endotoxin), rigorous purification of the naturally occurring compound is required [135]. In addition to alginate, polysulphone (PSU) has also been proposed as a possible encapsulation material because it does not limit the secretion and diffusion of insulin in macroencapsulated islets [136,137]. Other hydrogels, such as poly-ethylene glycol (PEG) [138] or methacrylate copolymers, have been proposed for islets encapsulation [131,139].

Many materials in different configurations have been tested in experimental models of islet transplantation in recent years, but only few have been evaluated in humans. Thus, little is known on their effective biocompatibility and suitability for long-term implantation as part of islet transplantation approaches in the clinical settings. New approaches are focused on the conjugation or incorporation of biomolecules, such as extracellular matrices that may reduce anoikis and stabilize cell membranes, to the biomaterials for encapsulation [140,141].

Intravascular macrocapsules consist of a synthetic hollow fiber semipermeable membrane that passes through a compartment seeded with pancreatic islets [142,143], where blood flows through the lumen of the hollow fibers allowing islets to promptly sense changes in glucose homeostasis and timely release of insulin, while they are protected from immunity by the membrane. This kind of device provides transport of nutrients and oxygen to the islets, but with a trade-off increased risk of damaging a blood vessel during surgery.

Extravascular macrocapsules are referred to macroencapsulated cells that are implanted outside the vasculature (e.g., in the peritoneal cavity as well as subcutaneously). The easy implant and explant relying on minimally invasive techniques reduce the risk of the procedure. The major drawback is the limitation of oxygen diffusion and nutrients transport related to the thickness of the device and tissue reaction around the foreign material. Encouraging results have been reported on the use of immunoisolation macrodevices in preclinical models of diabetes, supporting the ability to immune protect xenogeneic islets and improve metabolic control for extended periods of times [144–146]. Another approach consisting of the incorporation of an oxygen delivery system to the immunoisolation macrodevice to support the graft and help overcoming potential diffusion limitation from surrounding tissue, showed sustained human islet function in a pilot clinical case [92,147].

In microcapsules, islets are immobilized inside microspheres of a biocompatible material, usually alginate, coated with a semipermeable membrane, and implanted in the recipient – generally, as free intraperitoneal grafts. This kind of device has been the most studied thus far because of the simplicity of manufaturing and flexibility for modifying key components, which allowed researchers to play with key parameters like wall thickness or pore size to overcome significant nutrient and oxygen diffusion limitations seen in all extravascular implantation sites. Drawbacks of microencapsulation are mainly related to their size (500 – >1000um) that limits oxygen diffusion to the core of the capsule and in turn favors the survival of islets close to its surface. Also, the large volume of the capsules requires a large implantation site such as the peritoneal cavity, where generally the capsules are injected and tend to pellet in the pelvis by gravity expoosing the islets to unfavorable hypoxic conditions. Also, removal of the implants from the peritoneal cavity is cumbersome.

The concept of microencapsulated islets was first introduced in 1980 by Lim and Sun [148], who encapsulated pancreatic islets inside calcium-gelated alginate microcapsules: alginate microcapsules were cross-linked with polycationic poly-L-lysine (PLL) and an outer layer of alginate. However, these capsules were found to cause fibrosis when engrafted inside an animal. Islet encapsulation based on Ba²⁺ ions as the gelating cation combined with protamine sulfate (PS) residues as alginate cross linker have been tested. Ba²⁺ ions have a higher affinity than Ca²⁺ ions, so the mixing barium and alginate produces a stronger barium-alginate hydrogel and it supported a higher cell viability as compared to conventional alginate caspules [149]. Interestingly, unlike calcium–gelled capsules, barium capsules are intrinsically radiopaque, which could be easily imaged in vivo with micro-computed tomography. Improved survival of xenogeneic islets was demonstrated in the stringent NOD mouse model by combining bariumgelled alginate microcapsules with co-stimulation blockade [150,151].

Drawbacks of macrocapsules include loss of islet cells to hypoxia due to poor diffusion of vital molecules such as oxygen and nutrients into the central core of the constructs, and the unfavorable transplants volumes for the clinically preferred intraportal route of transplantation [152]. Incorporation of emulsions with oxygen carrying moieties, such as perfluorinated chemicals has been proposed to help overcoming oxygen diffusion within macro- and microcapsules, [153,154].

As an alternative approach that may overcome the limitations of macro- and microencapsulation, conformal coating of pancreatic islets may represent a viable strategy to allow for a more physiologic release of insulin into the bloodstream, while improving oxygen and nutrient diffusion supporting their survival and function after implantation. The use of polyhetylene glycol (PEG) coating obtained by photopolymerization for islet immunoisolation showed great promise in rodents [155] and nonhuman primate models of diabetes [130]. Multilayered nanofilms can be applied directly to the surface of the islet clusters to confer immunoislation after transplantation [135,156]. Improved islet allograft survival has been reported for conformally-coated islets in experimental animal models in recent years [157].

Novel techniques are needed in order to improve the efficiency of conformal coating of pancreatic islets, which are challenging due to the variability of size of the clusters comprised in the final preparation. The use of fluid dynamics approach may be of assistance to address such limitations, and encouraging initial results have been recently reported using PEG hydrogels [138]. Translation of these approaches to the clinical setting is appealing, and hopefully will allow attaining sustained graft function with lower or no requirement for lifelong immunosuppression.

1.6. Conclusions

We are living exciting times in the field of beta cell replacement therapies for the treatment of diabetes. While steady progress has been recorded thus far in clinical islet transplantation, novel approaches are needed to make cell-based therapies more reproducible and leading to long-lasting success. The multiple facets of diabetes impose the need for a transdisciplinary approach to attain this goal, by targeting immunity, promoting engraftment and sustained functional potency.

For stem cell based therapies to become a definitive solution to the shortage of transplantable endocrine cells, it may be desirable to fully recapitulate the complex structure and function leading to the tight regulation of human islets, which are in fact micro-organs. This goal should parallel the long-term safety of stem cell-based therapies (namely, no tumor formation).

Exploring novel sites for islet implantation that are accessible and pliable to modification of the microenvironment may allow for targeted interventions to enhance engraftment, reduce immune cell activation and islet cell loss at the time of transplant. The choice of the ideal site for islet implantation should take into account safety concerns and sustainability of implanted cell function. With the emerging evidence that non-endocrine pancreatic tissue represents a potential source for precursor cells able to maintain functional beta cell mass, it may be desirable to transplant impure islet cell preparations. This would require the availability of implantation sites that can accommodate relatively large volumes of tissues while avoiding competition for nutrients and oxygen.

The use of scaffolds or macrodevices is appealing for the potential to modify the local transplant microenvironment. The choice for biodegradable or non-biodegradable materials should be weighted, since the immune reaction for degradation and persistence of foreign material, respectively, may affect the fate of the graft long-term.

Physical modifications of the islet surface using biologics, gene therapy or polymers may be of assistance in also preventing immune mediated destruction, and allow lowering the immunotherapy needs to sustain graft function, and even enable the success of protocols aimed at immune tolerance. The use of transient coating of islet surface with polymers and/or ECM products may contribute to stabilize the graft (i.e., addressing ainoikis) and possibly protect it during the early post-transplant period, resulting in reduced attrition and microenvironment activation ultimately leading to improved outcome. Thus far, immunoisolation approaches have not met the desirable goal of a long-term function of transplanted islets. This may be the result of attrition over time due to inadequate adaptation of the graft to the implant conditions (i.e., lack of vascularization, inadequate nutrients and oxygen, waste products accumulation inside the constructs, amongst others).

Encouraging experimental data supports the potential value of strategies that combine different cellular types (i.e., mesenchymal stromal cells, endothelial cell precursors, or others) to promote engraftment and modulate immunity, which may synergize with the bioengineering approaches discussed herein. In fact combination products (e.g., comprising biomaterials, cell products and drugs) along with rationale systemic immunotherapy may lead to a permanent restoration of beta cell function in people with insulin-requiring diabetes. Only through a sequential, integrated approach, and a collective effort of the research community this ambitious goal can be achieved.

Figure 2. Schematic representation of immunoisolation approaches.

Macrodevices may house large amount of islet cell products and may be implanted subcutaneously or intraabdominally (i.e., intraomental pouch). Macro- and Micro-encapsulation devices are 400–1000um in diameter and may accommodate different numbers of islet clusters within each capsule. The diameter and overall volume of the graft, limits the possibility of implantation to the intraperitoneal cavity. Conformal (thin) coating consists of a thin polymer layering (e.g., hydrogel) on the islet clusters’ surface to confer immunoprotection.

Figure 3. Differences between conformal coating and microencapsulation.

Effective immunoisolation should rely on biocompatible materials to protect the islets from immune cells and humoral factors (i.e., antibodies), while allowing adequate exchange of oxygen and nutrients from the surrounding microenvironment, elimination of cellular waste, and timely inflow of glucose and outflow of insulin. In conformal coating immunoisolation (Left panel), a thin layer of polymer is applied on each islet cluster. The thickness of the coating is considered to represent an advantage when compared to micro- and macro- encapsulation where the distance between islet surface and the outer environment may hamper or delay inflow diffusion of oxygen and nutrients, as well as the outflow of cellular waste that may result in death or impairment of islet cell function (Right panel). Novel formulations of polymers able to enhance intracapsular oxygen generation or diffusion, and incorporation of oxidative scavenging molecules may be of assistance in overcoming the current limitations of larger constructs.

Abbreviations

DC

Dendritic Cells

ECM

Extracellular matrix

ESC

Embryonic Stem Cells

HSC

Hematopoietic Stem Cells

MSC

Mesenchymal Stromal Cells

PDMS

Poly(dimethylsiloxane)

PEG

Poly-ethylene glycol

PERV

Porcine Endogenous Retrovirus

PGA

Poly(glycolic acid)

PLA

Poly(lactic acid)

PLGA

Poly(lacticco- glycolic acid)

PLL

Polycationic poly-L-Lysine

PS

Protamine sulfate

PSU

Polysulphone

PTFE

Polytetrafluoroethylene

T1DM

Type 1 Diabetes Mellitus

T2DM

Type 2 Diabetes Mellitus

Tregs

T regulatory cells