Abstract
Type 1 diabetes is an autoimmune disease in which the immune system attacks insulin-producing beta cells of pancreatic islets. Type 1 diabetes can be treated with islet transplantation; however, patients must be administered immunosuppressants to prevent immune rejection of the transplanted islets if they are not autologous or not engineered with immune protection/isolation. To overcome biological barriers of islet transplantation, encapsulation strategies have been developed and robustly investigated. While islet encapsulation can prevent the need for immunosuppressants, these approaches have not shown much success in clinical trials due to a lack of long-term insulin production. Multiple engineering strategies have been used to improve encapsulation and post-transplantation islet survival. In addition, more efficient islet cryopreservation methods have been designed to facilitate the scaling-up of islet transplantation. Other islet sources have been identified including porcine islets and stem cell-derived islet-like aggregates. Overall, islet-laden capsule transplantation has greatly improved over the past 30 years and is moving towards becoming a clinically feasible treatment for type 1 diabetes.
Keywords: Diabetes, islet, macroencapsulation, microencapsulation, transplantation, immunogenicity, cryopreservation, differentiation
Graphical Abstract
1. INTRODUCTION
Type 1 diabetes mellitus affects over 1.5 million Americans and 20 million people worldwide.1,2 Type 1 diabetes is an autoimmune disorder where the body’s adaptive immune system attacks insulin-producing beta cells located in pancreatic islets.3 T cells eventually kill a large percentage of the body’s beta cells, which means blood glucose levels will fall outside of healthy ranges without medical intervention. The most common type 1 diabetes treatment method today is monitoring of blood glucose followed by insulin injections when levels are too high. However, insulin injections are time-consuming and patient compliance can waver, which can cause potentially fatal hypoglycemic peaks. Years of hyperglycemia due to lack of insulin can lead to organ damage.4 Pancreatic transplantation is another treatment method, but it requires a major surgical procedure and the patient must then be placed on immunosuppressant drugs for the rest of their lives to prevent organ rejection, which is detrimental to health.5 Transplantation of pancreatic islets is another method that has been widely studied in recent years. This method is less invasive, has shown promising results in animals and clinical trials, and can lead to a great improvement in quality of life for type 1 diabetes patients. Unfortunately, immunosuppressants are still necessary to prevent rejection of islets if they are non-autologous or not engineered with immune protection. In addition, a fibrous capsule may form around non-protected islets without immunosuppressants, keeping insulin from being secreted and nutrients from being absorbed.6 The need for immunosuppressive drugs can be eliminated by encapsulating islets in biocompatible materials. These biocompatible materials allow small molecules like insulin to diffuse out of the islets and oxygen and nutrients to diffuse in, but block larger immune cells and antibodies from entering.7,8 Islets are sourced from deceased donors, so a high yield of viable islets after transplantation is essential. Stem cell derived beta cells have been examined as an alternative to isolated islets, which can increase the number of cells available for transplantation.9 Problems still facing encapsulated islet survival include immune rejection, death due to lack of nutrients, and death due to post transplantation hypoxia. Survival after transplantation can be increased by improving the encapsulation technologies. This review will focus on the technologies behind islet encapsulation, the methods and materials in which islets can be encapsulated, and methods to improve islets survival.
2. BACKGROUND
Islets are clusters of cells located in the pancreas that are responsible for endocrine functions. These clusters contain multiple cell types including alpha cells, beta cells, delta cells, epsilon cells, pancreatic polypeptide (PP) cells, and a capillary network.10 Alpha cells are responsible for releasing glucagon when blood sugar levels are too low. Beta cells are responsible for secreting insulin when blood glucose levels are too high. Epsilon cells produce ghrelin which causes hunger, and PP cells secrete somatostatin which regulates alpha and beta cells.11 Islets are located throughout the pancreas and vary in shape, size, and density.12 Each person has about one million islets in their pancreas. The pancreas also contains glands with acinar cells and ductal cells, which are responsible for exocrine function. The exocrine tissue in the pancreas is all connected to the pancreatic duct.13
Type 1 diabetes is an autoimmune disease in which the body’s immune system attacks its beta cells, causing deterioration over time until they no longer function.3 This disease can start in early childhood and progresses throughout life. Type 2 diabetes is usually acquired later in life and can be the result of both genetic and environmental factors. In type 2 diabetes, beta cells still function well, but other cells no longer accept glucose, resulting in dangerous spikes in blood sugar levels.13 People administer insulin in various ways to reduce their blood glucose levels. Standard treatment methods for diabetes consist of constant blood glucose level monitoring accompanied by insulin injections or insulin pumps that need to be constantly worn.
Transplantation for type 1 diabetes treatment is a largely researched area. Transplantation can eliminate the need for the patient to constantly monitor and administer insulin. One method of treating type 1 diabetes is a full pancreatic transplantation from a deceased donor. This is a major surgical procedure, and the patient must be put on immunosuppressant drugs for the rest of their life to avoid immunorejection.6 These drugs can have extremely detrimental health effects including increased infection susceptibility, organ damage, and even cancer, greatly lowering quality of life. Another transplantation approach involves the use of only pancreatic islets. Islets, instead of the entire pancreas, are harvested from donors and transplanted into patients to regulate the body’s glucose levels. Since insulin-producing cells represent only 2–3 percent of the cells in the pancreas, it is appealing to only transplant the necessary tissue (e.g., islets) containing the insulin-producing cells.14 This method is also much less invasive than whole pancreas transplantation because islets are implanted into the liver through the portal vein.6 Studies have shown that for encapsulated islet transplantation, approximately 10,000 islet equivalents (IEQ, an islet with equal mass to the average islet of 150 μm in diameter) per kilogram of body weight are required in order to provide sufficient insulin dose.15 Patients that receive an islet transplantation must still be put on immunosuppression so that the implanted islets are not rejected. Although whole pancreas transplantation has a higher overall success rate for long-term euglycemia, they are also associated with a higher morbidity rate than islet transplantations.16
Encapsulating islets in a biocompatible material can overcome the possible immunogenicity of the tissue, potentially eliminating the need for patients to take immunosuppressants and improving their overall quality of life. Due to the lower surgical and post-surgical risks of islet transplantation and the potential for increased islet survival and long-term insulin production through improvements in encapsulation technologies, transplantation of encapsulated islets has recently become an appealing area of study and is the focus of this review.
3. GRAFT TRANSPLANTATION
There are various types of tissue grafts used in transplantation for diabetes treatment: allografts, autografts, and xenografts. Allografts involve transplantation from the same species and are the most common type of pancreatic transplantations.17 Autografts involve re-grafting tissue from the same person.18 In the context of islet transplantation, this involves removing the pancreas, digesting the exocrine tissue to remove the islets, and re-implanting encapsulated to prevent further damage by the immune system. Xenograft transplantation involves transplanting tissue from one species into another. Autograft transplantations will have a minimal immune response but involve organ removal which can add surgical complication.19 Since allograft transplantation is between the same species, the immune response will be less pervasive than xenotransplantation, but worse than autografts. Xenograft transplantation will have the largest immune response, but in the case of islet transplantation means an increased number of islets available for transplantation.
The most popular xenograft used in clinical trials is isolated porcine islets. Xenotransplantation of porcine islets is a popular area of research because there are a limited number of human islet donors available.20 Pig insulin is very similar to human insulin, differing only by one amino acid, which makes pigs a very appealing islet source. Although xenograft transplantation raises concerns about immunorejection, pig islets can be encapsulated in a biocompatible material to isolate them from the host immune system and avoid rejection21. There has been some concern that transplanting islets from pigs into humans would cause the transfer of retroviruses, but studies have shown successful islet transplantation with high amounts of insulin release and no sign of virus transmission.22,21 In vivo porcine xenotransplants have been shown to result in sustained patient normoglycemia in clinical trials as well.23,24
4. CLINICAL TRIALS
Clinical trials on whole pancreas transplantation were first reported in the 1960s,25 but with high morbidity and mortality rates.26 Eventually, these outcomes improved, especially when simultaneous pancreas and kidney (SPK) transplantation is done in uremic diabetic patients.27 The first islet transplantation was performed in 1974,28 but this procedure did not produce successful outcomes until the Edmonton protocol for islet isolation was developed.29,30 Since then, there have been two major transplantation types for diabetes treatments, whole pancreas and islet-only, which have greatly improved outcomes over recent years. Whole pancreas transplantation helps patients to become insulin-independent, but often results in complications leading to morbidity and even mortality.31 Islet-only transplantation, on the other hand, involves a much less invasive procedure and rarely results in morbidity or mortality, but has not consistently maintained insulin-independence over a long period of time.27
Lehmann et al.32 compared clinical SPK with simultaneous islet and kidney (SIK) transplantation and found that post-transplantation groups had over a 90% improvement rate of hypoglycemia. After 5 years, insulin dependence in the SPK group was 73.6% and it was 9.3% for the SIK group. The study also concluded that whole pancreas transplantations had significantly more complications. Moassesfar et al.27 compared clinical transplantation outcomes between pancreas transplantation alone (PTA) and islet transplantation alone (ITA). This study found that ITA patients had a 90% insulin independence at 1 year, and 70% insulin dependence at 3 years. PTA patients had 93% insulin dependence at 1 year, and 64% insulin dependence at 3 years. While two islet transplantations were needed for ITA patients, PTA patients had longer hospitalization times and more complications. Although there is still a disparity between long-term insulin independence after whole pancreas transplantation and islet-only transplantation, the lower risk of post-surgical complications makes islet transplantation a very appealing area of investigation.
In recent years, due to advances in islet isolation procedures and encapsulation technologies, the outcome of islet transplantation islets has improved. There are many islet transplantation clinical trials in which patients achieved normoglycemia for extended periods. Placing unencapsulated islets in the portal vein of the liver is the most common transplant type examined in clinical trials, but there are many problems with this method. In one study, seven patients with type 1 diabetes were implanted with islets isolated from human donors. Over 15 months of monitoring, patients were able to maintain normoglycemia after a total implantation of ~11,000 IEQs per kilogram body weight.29 Since the islets were not encapsulated, the patients also had to take immunosuppressants to prevent tissue rejection. To allow patients to forgo immunosuppressants while preventing the rejection of islets, especially when performing xenotransplantation, clinical trials moved towards using encapsulated islets. In another study, a patient was implanted with a macrodevice consisting of an oxygenated chamber made of alginate and a poly-membrane material containing human islets. The patient was able to maintain glucose response for 10 months with no immunosuppression and no indication of graft rejection.15 This improvement over previous studies may be attributed to increased oxygenation causing increased cell survival.
Xenotransplantation of encapsulated porcine islets has been explored in multiple clinical trials with varying levels of success. One clinical trial involving a collagen-covered islet encapsulation device resulted in six out of twelve patients being able to decrease their insulin requirements for four years. These devices contained porcine islets combined with Sertoli cells to provide islet protection, and were transplanted subcutaneously into patients that were not administered with immunosuppressant drugs.21
In another long-term study, alginate-encapsulated porcine islets were transplanted into one patient without the use of immunosuppressants. The patient’s blood glucose levels returned to pre-transplant amounts after 49 weeks. However, the encapsulated islets were removed and were tested after 9.5 years and were still alive, producing small amounts of insulin.24 The patients in these studies were not administered with immunosuppressive drugs, however insulin production did not persist long enough to be feasible for treatments on a larger scale. In general, encapsulation technologies need to be improved to achieve sustained, long-term insulin release.
Several clinical trials involving encapsulated islets are currently in progress but have not achieved consistent responsive insulin production for long enough periods to be a feasible standard of treatment for type 1 diabetes. There is a great deal of ongoing research to improve transplantation methods, specifically in improving the ways the islets are encapsulated. This review focuses on the technologies that improve islet transplantation, which will hopefully result in easily implantable encapsulated islets with long-term glucose responsiveness that can treat type 1 diabetes without the need for immunosuppression or insulin supplementation.
5. ENCAPSULATION
Various types of biocompatible encapsulation methods have been researched for islets. Encapsulation and transplantation of islets is also known as a bioartificial pancreas (BAP).33 Both larger encapsulation devices known as macrodevices (Figure 1A),34 and smaller encapsulation devices known as microdevices (Figure 1B) have been designed and studied. In addition, islets have been encapsulated in conformal coating for their protection (Figure 1C). The materials with which the devices are made and the way with which they are encapsulated also can contribute to the success of the device in vivo.
Figure 1. A schematic illustration of different types of islet encapsulation.
(A) Macroencapsulation devices contain a large number of islets and are usually implanted subcutaneously. The TheraCyte device (sketched) is an example of a macroencapsulation device34. A few macroencapsulation devices can hold the number of islets necessary for implantation. These devices are on the centimeter scale. (B) Microencapsulated islets. Microcapsules are usually between 300 μm and 1000 μm. Islets are encapsulated in a semipermeable microcapsule, which is most often alginate-based. There are only one to two islets per capsule and 10,000 islets per kg body weight are necessary to maintain normoglycemia (i.e., a large number of capsules need to be implanted into the body). (C) Conformally coated islets. Islets are encapsulated in a semipermeable membrane that is usually less than 50 μm thick. (D) Semipermeable membranes in the aforementioned approaches have pores of an optimal submicron size to allow the diffusion of insulin and metabolic waste out of the device, while permitting gas exchange, nutrient intake, and keep out of immune cells and large immune factors (e.g., immunoglobulin G or IgG).
5.1. Materials.
There has been a great deal of research in various materials that can be used to encapsulate islets. The purpose of encapsulating islets is to prevent/minimize immune reaction. The pores of the materials must be an optimal size so that they allow for nutrient exchange but prevent elicitation of an immune response (Figure 1D). Since immune cells have a diameter between ~6 μm and 10 μm, devices with pores in the submicron range should prevent immune cell infiltration.35 Since organic metabolites have diameters between 0.05 nm and 1 nm, and globular proteins have diameters between 2 nm and 10 nm, pore sizes should be up to 10 nm to allow for the diffusion of small molecules and macromolecules, such as oxygen, nutrients and growth factors. Using a maximum pore size of 10 nm also prevents immune cell infiltration into capsules.36,37 Alginate was the first material used for microencapsulation and remains the most popular38.
Alginate is currently the most commonly used material for islets encapsulation because it can be formulated to be biocompatible and non-degradable with adjustable stiffness, and has appropriate pore sizes to prevent immune cell infiltration.39 The pore sizes of alginate can also be adjusted based on the alginate composition.40,41 Alginate is a natural biomaterial that is derived from brown seaweed. It is also recognized by the FDA as safe, making it desirable for use.42 Alginate can form hydrogel through ionic crosslinking using a divalent cation such as Ca2+ and Ba2+. Alginate consists of β‐d‐mannuronic acid (M) and α-l-guluronic acid (G) as blocks of M, G, or MG.43 Changing the ratios of these monomers will change the properties of the alginate. It has been found that increasing the number of G blocks causes an increased stiffness because G is responsible for the crosslinking with divalent cations. Purifying alginate has also been shown to decrease immune reaction during islet transplantation which can be attributed to a higher content of G post purification which decreases the amount of capsule breakage, as well as the removal of toxins or impurities44. Some research has also shown that the block structure of M and G affect binding, which can be useful when tuning stiffnesses of alginate gel for minimizing immune response and determining the appropriate porosity.45 Barium crosslinking has been shown to lead to strong gel, but since barium can be toxic, calcium is used more for gelling alginate due to a fear of barium leakage.46,47 Alginate modification is a popular method of improving various aspects of transplantation. In Vériter et al., high viscosity alginate containing high mannuronic acid faired best for immune response prevention compared with unmodified and low viscosity alginate.48 Alginate modified with triazole-thiomorpholine dioxide (TMTD) was also shown to have immunosuppressive properties when the TMTD capsules were 1.5 mm in diameter, which is larger than most islet microencapsulations.49
The most popular methods include gelling through light-based cross-linking, thermally induced crosslinking, and ion-based crosslinking. Thermal and photo crosslinking methods are disadvantageous, as they might harm the cells. Polyethylene glycol (PEG) is another material that is often used for islet encapsulation due to biocompatibility, tunable properties, and gelation ability using crosslinking and photopolymerization.50 Weber et al. tested PEGs of varying molecular weights and found that increased crosslinking density in PEG resulted in a delay of insulin release from islets.51 In the work, dual layer PEG capsules were created, with each layer serving a different function. The inside layer was functionalized with either laminin or laminin peptides, leading to increased insulin secretion, and the exterior layer was composed of unmodified PEG to prevent attachment of fibroblasts.52 To decrease immune response, Su et al. created a PEG containing hydrogel network made of 4-armed PEG cysteine (4A-PEG-Cys) and 4-armed PEG thioester (4A-PEG-ThE). An IL-1 surface receptor peptide inhibitor was covalently linked to the hydrogel, and the combination resulted in decreased cytokine infiltration due to the surface peptides.53
PEG has also been used to create thinner islet encapsulations. In one study, a thin layer-by-layer PEG approach was used to create an ultra-thin capsule around islets, allowing for greatly increased molecular exchange over the membrane compared with previous methods.54 Other materials have been used to make thinner capsules. In Kozlovskaya et al.55 Poly(N-vinylpyrrolidone) (PVPON) based encapsulation in combination with tannic acid (TA), which has antioxidant properties, was used to encapsulate islets in thin multilayers. To create the capsules, islets were suspended in a solution containing PVPON which can be adsorbed to the islet surface, followed by a TA solution that can be absorbed to the PVPON through hydrogen bonding. This was repeated to create many layers of PVPON and TA.56 The TA allowed for free radical scavenging and inhibition of some proinflammatory cytokines.57
Another materials approach to improve biocompatibility is immobilizing cells on the surface of capsules to create a cell-based microcapsule. In one study, HEK293 cells were successfully immobilized on the surface of islets by coating the capsules with an amphiphilic PEG-lipid-biotin layer. After culturing coated islets with streptavidin immobilized HEK293 cells for 3–5 days, a capsule was successfully formed. Although this method has the potential of increasing microcapsules biocompatibility, insulin release was decreased due to HEK293 cells.58 Silk, a natural biomaterial, has been used for islet encapsulation due to its low immunogenicity.59 In Cabric et al.,60 a silk scaffold containing heparin was created to improve islet vascularization. Heparin was used due to its ability to increase islet angiogenesis through growth factor stabilization. The silk scaffold was created using lyophilization which allowed for the incorporation of various active compounds, and resulted in an open scaffold design enabling vascularization when placed in the epididymal fat pad of mice.61 Silk has also been used to create a self-assembling scaffold containing laminin, collagen IV, and mesenchymal stromal cells (MSCs) to mimic the ECM that normally surrounds the islets in vivo. The scaffolds were created through vortexing silk fibroin followed by adding EMC components and combining with isolated mouse islets. Adding these EMC factors stimulated insulin release.59
Prosurvival factors can be incorporated into encapsulation materials to prevent immune response. The chemokine CXCL12 has been shown to deter effector T-cells while retaining Tregs, which suppress immune response as well as prevent inflammation, and help improve beta cell survival through pro-survival signals.62,63,64 In Chen et al.,65 islets were encapsulated in alginate containing CXCL12 using syringe-driven droplet formation and crosslinking. In vivo mouse studies showed a lower amount of mononuclear cell infiltration. FTY720 is a small molecule that has been shown to increase vascularization by attracting anti-inflammatory monocytes when released from scaffolds.66 A pocket shaped device made of PLGA nanofibers combined with FTY720 was shown to maintain normoglycemia in an allograft diabetic mouse model. The pocket containing FTY720 was also shown to induce increased vascularization over the control without FTY720.67 In Zhang et al.,68 vascular endothelial growth factor (VEGF) cDNA delivered to islets was shown to improve the vascularization of non-encapsulated mouse islets implanted in the renal capsule during in vivo mouse studies. VEGF could also be used in islet encapsulations to improve vascularization.
Using components found in the ECM in the encapsulation materials has also been shown to improve biocompatibility. Collagen type IV is found in the ECM surrounding islets in the pancreas and has been shown to enhance islet survival.69 In Llacua et al.,70 human islets were encapsulated in an alginate/collagen IV mixture with laminin sequences of either RGD, LRE, or PDSGR. In vitro co-culture with cytokines showed an overall decrease in danger‐associated molecular pattern release and decreased apoptotic cell death, indicating protection against toxicity of cytokines. Insulin secretion has also been shown to be affected by the ECM. In Weber et al.,71 when islets were encapsulated in PEG gels containing collagen type IV and laminin, islet secretion of insulin increased. ECM from porcine pancreases was used to create microcapsules by Chaimov et al.72 where porcine pancreases were decellularized and the liquified ECM was collected. The liquified ECM was combined with alginate and capsules were created by using a needle and droplet spray and gelling with calcium chloride. The capsules were coated with poly-l-lysine (PLL) and then the alginate in the core was liquified, leaving a capsule made of ECM coated with PLL. It was found that the ECM capsules did not stimulate macrophage response in a diabetic mouse model.
Another way to improve immuno-isolation is through coating microcapsules with biocompatible or bioinert materials. Capsules have been coated with PLL with varying degrees of success. Studies have shown that PLL is good for capsule stability but can cause an immune response.73,74 In Cui et al.,75 islets were encapsulated with 2% alginate using an electrostatic capsule generation device resulting in 600 to 800 μm capsules. The capsules were coated by two alternating layers of PLL and alginate. Then an amphiphilic polymer was added to form mono-acrylate lipids resulting in successful encapsulation of rat islets, and normoglycemia in diabetic mice after transplantation in vivo.75 In Nabavimanesh et al.,76 the surface of alginate capsules was coated with methoxy polyethylene glycol (mPEG) for immuno-camouflaging the capsules. Alginate was modified with poly-L-ornithine (PLO) PEGylated, and in vitro testing using a lymphocyte co-culture was performed. PEGylation of the microcapsules decreased interleukin-2 secretion, meaning a decreased immune reaction.76
Material properties and composition are also important for creating conformally encapsulated islets. Lou et al. designed a PEG conformal coating to bind covalently to the amine groups on the surface of islets.77 Heparin was added to the PEG, and this coating reduced immune response during an in vitro APTT assay while still having excellence insulin release.77 In Fukuda et al.,78 a gel scaffold was used to individually coat beta cells with fibronectin and gelatin to mimic the ECM using a layer by layer coating technique. The coated cells were then fabricated into spheroids of ~300 μm in diameter. These dense spheroids had higher insulin release than the control spheroids in vitro, and higher insulin release in vivo.78
5.2. Types of Encapsulation.
Macrodevices and microcapsules are the most widely used encapsulation methods in clinical islet transplantation trials. Microdevices fall in the micron to millimeter scale and usually encapsulate one or two islets per device (Figure 1A). Macrodevices range from millimeters to centimeters in size and can encapsulate thousands of islets per device (Figure 1B). Conformal encapsulation of islets in a coating layer that is not much thicker than islets themselves are recently being explored to protect islets, leading to increased diffusion (Figure 1C).79
5.2.1. Microencapsulation.
Microcapsules are the most researched type of encapsulation for islet transplantation. Islet encapsulation was first described by Lim and Sun where rat islets were placed in an alginate hydrogel, and microcapsules were created using a syringe drop method and gelled using calcium chloride.80 This remains the most popular type of microencapsulation, but there have been many improvements in encapsulation methods and materials. Microcapsules contain one to a few islets inside the spherical hydrogel. The capsules usually range over 300 μm to 1000 μm. The biomaterials used have pores that are large enough to allow small molecule diffusion yet small enough to prevent immune cells from infiltrating, as seen in Figure 1D. The biomaterials surround the islet to minimize immune response, while still allowing a large surface area of the islets to be exposed with a limited amount of space between the islet and the surrounding tissue, which makes the diffusion of small molecules much faster. Microencapsulation of islets also allows for other material modifications that can aid in cell survival and prevention of immune response. The major drawback of this method is the lack of direct vascularization between the islets and the surrounding tissues, due to the surrounding biomaterial. Examples of microencapsulation devices can be found in Table 1.
Table 1.
A list of capsules reported in the literature for islet encapsulation and transplantation
| Type | Materials | Encapsulation method | Design advantage | Reference(s) |
|---|---|---|---|---|
| Photo-crosslinked |
|
|
|
51 |
| Ultra-thin layers |
|
|
|
54 |
| Thin layers |
|
|
|
55,56,57 |
| Cell-based capsule |
|
|
|
58 |
| ECM-based microcapsule |
|
|
|
78 |
| Ion-crosslinked microcapsule |
|
|
|
172 |
| Ion-crosslinked microcapsule |
|
|
|
85 |
| Core-shell microcapsule |
|
|
|
86 |
| Conformal coating |
|
|
|
87, 88 |
| Crosslinked on-chip capsule |
|
|
|
91 |
| High-throughput ion-crosslinked microcapsules |
|
|
|
97 |
Islet encapsulation methods are varied, but also paramount for successful application. In general, islet-laden hydrogel microcapsule drops are formed then stabilized via gelling. In the first paper describing the microencapsulation of islets, capsules were formed using a syringe pump technique.80 Sodium alginate solution containing islets was pushed through a syringe to form droplets, which deposited into a calcium chloride solution for gelling. Methods for generating capsules have greatly improved since then.
Droplet generators are most commonly used for creating droplets. There are various types of droplet generation including air shearing, electrostatic droplet formation, and acoustic vibrations.81 A droplet generator is comprised of a needle or syringe to hold an aqueous solution, a nozzle, and a gelling bath as seen in Figure 2. The aqueous solution flows out of the nozzle, eventually forming a drop which freely falls after extrusion into a gelling solution (Figure 2A). These droplets usually range from 100 –1000 μm and can contain varying numbers of islets. The non-homogeneous sizes and islet number per capsule, in addition to the formation of empty capsules, are major drawbacks of this method. However, the size of the microcapsules can be adjusted by changing the nozzle size and the speed at which the solution is flowing. More advanced droplet generators have been developed to control the capsule size and decrease the size dispersity in the same batch.
Figure 2. A schematic illustration of the different types of droplet generators.
(A) Basic droplet generator. The generator contains an aqueous solution of alginate suspended with islets. Microcapsules are formed by pushing the solution suspended with alginate into a gelling bath with an aqueous solution of divalent cations (often calcium chloride). Droplet size can be tuned by adjusting the size of the nozzle, but it is still difficult to control and can result in a large range of capsule sizes. (B) Air droplet generator. Co-axial airflow is used to control the size of the capsules for producing capsules with increased homogeneity in size. (C) Electrostatic droplet generator. Applying an electrostatic potential between the nozzle and the gelling solution helps to create smaller and more homogeneous (in size) capsules. (D) Co-axial droplet generator. This generator consists of a core channel containing islets suspended in a biocompatible core fluid surrounded by a co-axial channel of alginate solution. It produces core-shell capsules with islets in the core and alginate in the shell. Core-shell capsules decrease immunogenicity because islets are localized in the center of the capsule with no direct exposure to the in vivo environment. The core-shell design also allows for the use of various core fluid (e.g., collagen) besides sodium alginate solution, which may help to improve islet survival.
Air droplet generators use co-axial airflow to create microcapsules via surface tension. In the case of islet encapsulation, islets are mixed into an alginate solution which is then contained in an air droplet generator (Figure 2B). Air droplet generators have demonstrated improved microcapsule formation in multiple studies.82–83 Electrostatic droplets are generated via an electrostatic potential between the tip of the nozzle and hydrogel solution (Figure 2C). This method has shown to decrease microcapsule sizes and increase capsules uniformity. One study used a method called electrohydrodynamic jetting (EHDJ) in which a high-voltage electric field was applied between the gelling solution and the jetting needle. No negative effects on cell proliferation were detected.84 Electrostatic droplet generation has also been used for islet encapsulation.85,86 In Safley et al.,74 electrostatic droplet formation was used to create a double coated capsule with a high mannuronic acid alginate core coated with PLL, and then coated again using strontium-gelled alginate. In vivo studies in an immune compromised macaque showed that double layered capsules prevented exposure of islets to the surrounding environment.74
Another group employed a nozzle method to create a “conformal coating” around islets to reduce the microcapsule sizes.87 The method was performed using a microfluidic chamber coaxial jetting system with oil and aqueous phases. Islets were first covered with a precursor coating made of vinyl sulfone functionalized PEG and crosslinked with dithiothreitol (DTT). Alginate was added to the gelling materials to optimize the coating. Coatings with thicknesses between 10 μm and 50 μm were achieved but did not readily protect islets from immune cells.88
The majority of studies that used the droplet generator method for microcapsule extrusion utilized only one material such as alginate to contain the islets. One major drawback of this method is that the islets may become partially exposed to the external physiological environment. This can trigger immune reaction, defeating the purpose of encapsulation. Capsules can be coated with materials such as PLL after extrusion, but this adds time to the process and could cause unwanted capsule damage. To prevent islet exposure to the external environment, a core-shell capsule design can be used (Figure 2D). In the core-shell encapsulation, two different materials can be used in the core and shell, respectively. This is performed by using a co-axial flow of an aqueous solution, usually of alginate, to surround an islet-laden core fluid.81 Ma et al.86 used electro-jetting to generate islet-laden core-shell capsules. An in vivo evaluation of core-shell versus solid capsules revealed that core-shell capsules had less fouling and maintained normoglycemia in mice for a longer period than solid capsules.
Cell encapsulation using microfluidic devices has become popular in recent years due to the wide availability of photolithography and 3D printing for creating masters or molds. Photolithography-based mold creation involves applying photoresist to a silicon wafer to create a master mold. The master mold is then used repeatedly to generate PDMS-based microfluidic devices by soft lithography.89 Microfluidic devices are very tunable and can be adjusted to create different types of flow paths for various functions. Microcapsules are formed at fluid shearing junctions such as T-junctions (Figure 3A) and flow focusing junctions (Figure 3B).90 Once the microcapsules are created, they are gelled using divalent ions or ultraviolet light exposure. Alginate is the most popular material to use for microfluidic cell encapsulation because it is easily crosslinked using calcium chloride. PEG is another material commonly used to create microcapsules because it can be photo-crosslinked as the capsules flow through the device channels.
Figure 3. Microfluidic encapsulation of islets.
(A) T junction-based microfluidic mechanism for microcapsule generation to encapsulate islets. The immiscible oil flow is used to shear the aqueous flow suspended with islets into droplets which are further crosslinked into hydrogel for islet encapsulation. (B) Flow focusing-based microcapsule generation with a similar mechanism to the T junction-based approach. (C) Core-shell flow focusing-based microcapsule generation. Two (core and shell) aqueous flows are used for generating core-shell microcapsules with islets being encapsulated in the core. The core-shell design prevents direct contact between islets and the surrounding environment and allows the use of different biomaterials for the core and shell, which may improve islet survival and function.
Microfluidics has also been used for islet encapsulation. Using microfluidics to encapsulate islets increases the control of capsule size and uniformity. Smaller capsules can be produced compared to nozzle-based droplet generating methods which have much less control over size. Weaver et al. encapsulated murine islets in 300 μm PEG-based capsules using microfluidics. In the same study, the smallest capsules produced using electrostatic droplet generation were 500 μm.91 In Headen et al.,92 human islets were encapsulated on-chip in a 4-arm PEG maleimide (PEG-4MAL) gel using dithiothreitol crosslinking. This resulted in a 99% loading efficiency with capsules between 300 μm and 800 μm, most of which contained multiple islet92.
Core-shell microcapsules can also be created using microfluidics (Figure 3C), and have been used to encapsulate different cell types.93 After the cell-laden core and shell fluid streams meet in the device, they flow into a flow focusing junction with another shearing fluid. To improve capsule formation, these devices are sometimes designed to be non-planar.94 Typically, the heights of the channels vary with the core channel being the shortest so that the shell fluid can completely encapsulate the core. Multiple studies show the advantages of using a core-shell method for cell encapsulation.95,96 This microfluidic core-shell method could easily be applied to islet encapsulation which can result in improved cell survival through adjusting the core material.94 Islets also have less chance of stick out of the side of the capsule due to the shell, mitigating immune response.86
Due to the infinite number of designs you can make on microfluidic devices, there are innumerable ways to optimize the encapsulation process. One facet that has been explored recently is the scaling-up of capsule production. In one study, a microfluidic device with eight outlets demonstrated increased rates of islet encapsulation compared to other methods.97 For transplantation in clinical trials, humans need about 10,000 IEQs per kilogram of body weight. The rate of islet encapsulation is also important for maintaining the viability of the islets. While islets are being encapsulated, the already encapsulated islets will either have to be cultured or preserved in the wait time. This is very inconvenient and can lead to islet death. Methods that use multiple needles or channels could increase the number of islets encapsulated in a reduced period of time to allow for scale-up, without increasing the flow speed or shear stress on islets.
The purity of microcapsules has not been discussed in current literature in detail. When encapsulating islets, many of the generated capsules contain no cells due to the low density of islets used in the aqueous fluid. Separating islet-laden and empty capsules by hand is extremely difficult, and on-chip microcapsule sorting could fix this problem.94 On-chip microcapsule sorting using dielectrophoresis is a label-free sorting method that may be applied for islet encapsulation.98,99
Islets can also be encapsulated using other coating methods, as discussed previously in the microencapsulation materials section. Coating methods have been used to create ultra-thin layers (Table 1). In Manzoli et al.,88 islets were coated with a conformal coating of PEG or Matrigel and were found to have high survival in vivo. Islets were suspended in the encapsulation material (PEG and Matrigel) for coating and then incubated in DTT to crosslink the coating.88
5.2.2. Macroencapsulation.
Macrodevices are usually in millimeters to centimeters in size, allowing for multiple islet encapsulation. Their large size makes macrodevices easily tunable, as membrane size, thickness, and pore size can all be precisely adjusted. One device has the potential to hold the large number of islets necessary for diabetes treatment. However, the thickness of the device makes small molecule exchange difficult, which can lead to cell death and decreased insulin release. Because many islets are enclosed in one device, the surface area available for small molecule exchange is reduced. Additionally, some macrodevices are made of materials that have been shown to increase the immune response and fibrotic build-up.100 Examples of macrodevices can be found in Table 2.
Table 2.
A list of macrodevices reported in the literature for islet encapsulation and transplantation
| Type/ Name | Description | Design advantage | In Vivo / Clinical trials | Reference(s) |
|---|---|---|---|---|
| β-Air |
|
|
|
15,101,100 |
| 3D Printed Vascularized Device |
|
|
|
102 |
| Bioplotted scaffold |
|
|
|
104 |
| Silicon Nanopore Membrane Device |
|
|
|
105 |
| Theracyte |
|
|
|
34,107,108 |
| PES/PVP device |
|
|
|
109, 110, 111 |
| Encaptra |
|
|
|
168,169 |
Since limited oxygen transport is a major flaw of large devices, many macrodevice designs focus on increasing the amount of oxygen that reaches the islets. Beta-O2 Technologies designed a bioartificial pancreas called β-Air to enhance oxygen supply to islets and improve immune protection.101 The device consists of an islet module that holds an alginate slab containing islets, and a gas chamber which increases the oxygenation of the islets. The β-Air device is implanted subcutaneously, and has been tested in vivo in Macaques using xenotransplanted porcine islets and later used in clinical trials.15,100 Both in vivo trials and clinical trials showed successful insulin release post implantation. A major downside of the device is the fibrous capsule that formed around it which will eventually prevent small molecule exchange. Various other material compositions and design techniques have been used to increase oxygenation. Farina et al.102 3D-printed a vascularized device to create micro-reservoirs. The micro-reservoirs were designed to increase vascularization. This along with growth factors embedded in the scaffold resulted in increased vascularization during in vivo mouse studies using encapsulated human islets. Other transplantation areas for unencapsulated islets have been examined, including prevascularized subcutaneous sites.103
Degradable polymers have also been examined to improve device vascularization after the polymers are degraded post-transplantation. Marchioli et. al. used a Bioplotter to create a 3D degradable scaffold out of 4% alginate and 5% gelatin.104 The scaffold was 2×2 cm, had a pore diameter of 1.88 ± 0.18 mm, and a strand thickness of 1.55 ± 0.13 mm. The goal of this scaffold was to increase vascularization of islets by allowing blood vessels to grow into the pores. The bioplotting technique used to print the scaffold allowed for manipulation of the scaffold’s shape and pore size. Bioplotting also decreases damage to the islets during encapsulation compared to other techniques that require loading through small ports that can deform the islets. The scaffold keeps the islets from clumping together, increasing the surface area for small molecule exchange. The drawbacks of this method include low insulin release until the scaffold has degraded, as well as lack of oxygen and nutrient diffusion through the scaffolding materials. Also, once this scaffold degrades, there will likely be an increased immune reaction to the transplanted tissue because immune cells will now have access to islets. Moreover, fibrotic build-up can accumulate directly on the islets, which could decrease insulin release by reducing its transport.6,15
Increasing nutrient and insulin diffusion through adjustment of material pore size is also a largely studied area. Song et al. focused on increasing hydraulic permeability using a silicon nanopore membrane to study the effect of pores size by comparing membranes with pores of 40 nm and 10 nm. The device was created using a silicon wafer nanofabrication method. The study found that the device with 10 nm pores showed good permeability results in vivo.105 Chang et al. studied pore sizes ranging from 30 nm to 100 nm using a polycaprolactone-based microdevice with nanorods fabricated using a silicon substrate.106 The device was created to hold embryonic stem cell (ESC)-derived beta cells. The mold for the device was constructed by hydrothermally growing zinc oxide nanorods on a silicon wafer, adding a PEG and PCL solution to create a film, then etching the rods out of the film using sulfuric acid solution. After dissolving the PEG, a membrane with pores ranging from 30 to 100 nm was created. To support the system, a microporous membrane was added to the other side of the device, and the two membranes were fused together using heat sealing. This device was created to allow nutrient diffusion while preventing the escape of encapsulated cells because escape of ESC-derived beta cells in vivo can lead to teratomas if not all the ESCs are differentiated.106 The advantage of these devices is tunable pore sizes and prevention of teratoma escape when using differentiating cells instead of islets.
Other devices have been designed to encapsulate ESC-derived cells. The TheraCyte device was created to contain ESC-derived beta-cells.34 The device has a pouch-based design, composed of a semipermeable inner membrane with a pore size of 0.4 μm, and an outer membrane with pores of 5 μm to promote angiogenesis. The device is implanted subcutaneously. In Lee et al.,107 human fetal beta-cell precursors known as “islet-like clusters” were loaded in the device through the loading port and implanted in mice. Results showed that the islet-like clusters survived well and were protected from autoimmune rejection. Interestingly, the control group of encapsulated isolated islets showed poor survival. Lee et al. hypothesized that the young beta cell precursors were more resistant to cell death post-transplantation, which allowed for more insulin release and proper function.108
Due to their large size, macrodevices are prone to fouling, which may lead to infection and graft rejection. To combat this, researchers have investigated incorporating biocompatible and bioinert materials in their designs. Papenburg et al. created a microencapsulation design using thin microwell porous membranes made of non-degradable poly (ether sulfone) (PES)/polyvinylpyrrolidone (PVP).109 This material is hydrophilic, inhibiting cell adhesion and fouling. The device master was fabricated using phase separation micro-molding which allows for a “one step” creation of the microstructures. The lid had 0.45 μm pores which prevented cells from penetrating into the device.110 During in vitro testing using human islets, the islets did not aggregate which allowed for an increase in islet exchange surface area. The islets were seeded in the separate wells in the device, which is one advantage over other microencapsulation devices.111 Yang at al., used a “sheet” shaped encapsulation device created using an ethylene-vinyl alcohol membrane.112 This material has low tissue adhesive properties, resulting in low tissue adhesion and fibroblast invasion after in vivo implantation.
Varying the shape of the macrodevices has been shown to improve various aspects of implantation. Microfibers allow for large amounts of islets to be encapsulated in a long fiber which can decrease the thickness of the biomaterial layer surrounding the islets compared with other encapsulation designs because islets are in a tube rather than clumped in a large sphere. Microfibers usually have a diameter on the micron scale and can be from centimeters to meters long. Microfibers can also improve the implant’s ability of being stationary, as opposed to microcapsules which are more mobile in some implantation sites. One popular technique used to create microfibers is co-axial flow microfluidics. In Onoe et al.,113 rat islets were encapsulated in a core-shell hydrogel microfiber made of an islet-laden atelocollagen core and an alginate agarose shell using a double co-axial microfluidic system. The 20 cm long fibers were implanted into the renal capsular space of mice, resulting in normoglycemia for 36 days. In Jun et al.,114 a microfluidic chip was used to create fibers composed of collagen and alginate with a diameter of approximately 250 μm, which prevented the islets from protruding out of the fibers. Fibers containing 1200 IEQ were then transplanted into the intraperitoneal (IP) cavity of mice, resulting in normoglycemia for the four-week study.
In Bowers et al., an encapsulation pocket made of polyhydroxybutyrate (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) nanofibers was created to house islets and maintain their spherical shape which is important for islet function.67 In An et al.,115 a fiber design called the TRAFFIC (thread reinforced alginate fiber for islets encapsulation) device was created for easy retrieval so that it could be implanted and replaced using minimally invasive procedures. The device consists of a surface modified nylon suture with a nonporous coating to allow for Ca2+ release to help with uniform crosslinking during fabrication. This rope was then coated with alginate containing islets. Rat islets were encapsulated in the TRAFFIC device, resulting in normoglycemia in an in vivo diabetic mouse model, as well as easy device removal.115 Knobeloch et al. created an injectable PEG based hydrogel for islet encapsulation.116 A total of 50 islets were mixed into the PEG formulation and then gelled. In vivo mouse studies using encapsulated rat islets resulted in minimal inflammatory response but short-lived islet function. The advantage of this type of encapsulation is that it can be injected into a transplantation site using a 27 gauge needle, making surgery unnecessary.116
Macrodevices have many advantages including a large cell capacity, removability, and tunability of pore sizes and compositions. Diffusion over the limited area of microdevices is still a problem that leads to necrosis of islets. By using a microencapsulation or conformal encapsulation method, these small molecules can diffuse more easily between the islets and the surrounding environment.48
5.2.3. Conformal Encapsulation.
Recently, researchers have shifted towards the creation of conformal coating of islets, which involve very thin layers of materials coated onto the islets or using other methods to create a coating that is very similar to the actual size of the islets. This allows for increased diffusion across the biomaterial membrane, while still minimizing immune reaction (Figure 1C).79
5.3. Islet Co-Encapsulation.
Encapsulating islets with other cells has been shown to decrease the body’s immune response, reduce pericapsular fibrotic overgrowth (PFO), improve islet survival, improve graft revascularization, and increase insulin production. Mesenchymal stem cells (MSCs) have immune-evasive properties and can improve tolerance to transplanted tissue.117,118 In Nasr et al.,119 the immunomodulatory effects of autologous MSCs co-transplanted with islets were tested, and MSCs were shown to improve graft survival during in vivo mouse studies. In Vaithilingam et al.,120 in vivo mouse studies showed that co-encapsulation of islets and MSCs in barium alginate microcapsules led to decreased PFO after retrieval and assessment of encapsulated islets. This study showed that co-encapsulation led to increased insulin production in vitro. In vivo, co-encapsulation with MSCs achieved normoglycemia in 71.4% of mice, while co-encapsulation with stimulated (using proinfammatory cytokines) MSCs resulted in 100% normoglycemia, compared with 9.1% for transplantation of islets alone.120 Co-encapsulation of islets with preconditioned adipose tissue-derived stem cells was shown to improve graft revascularization during in vivo rat studies in Cavallari et al.121 In Teramura et at.,58 HEK293 cells were used to create a microcapsule around islets. This was shown to prevent necrosis of the islets. Islet co-encapsulation with other cells has great potential to improve transplantation outcomes by decreasing PFO, increasing insulin production, enhancing revascularization, and augmenting islet viability.
6. ISLET ISOLATION AND TRANSPLANTATION
6.1. Islet Isolation.
To ensure successful and long-lasting transplantation, it is vital to start with healthy islets. The most popular way to isolate islets is through a perfusion process. Most protocols follow the Edmonton Protocol for Islet Transplantation.30 Islets are separated by digesting away the exocrine tissue, often utilizing the Ricordi Chamber (Figure 4A).122 Adding the digestion solution through the pancreatic duct allows spreading throughout the exocrine tissue while leaving the islets intact. Collagenase-based digestion enzymes that are most often used for pancreatic digestion include Liberase MTF, Collagenase IV, and Collagenase XI among others.123 The enzyme selection is also species dependent.124,125 Following digestion, islets are separated from other digested tissue using gradient separation. Ficoll-based gradients are most typically used.126 Ficoll-400 solutions of varying densities are layered on top of one another, and after centrifugation, the islets will localize to one layer of the Ficoll. The islets can then be collected and washed.126 Other types of gradients that have been utilized to improve separation include bovine serum albumin and Visipaque.127,128 A major limitation of using Ficoll and many other separation gradients is that they are toxic to the islets after long exposure, so the process must be performed quickly.129 Moreover, islets can get lost during separation, and even after separation the extracted islets might still contain small amounts of exocrine tissues. The exocrine tissues must be manually removed by hand which can lead to contamination. The islet isolation process must be further optimized to be less cytotoxic and more efficient.
Figure 4. A schematic illustration of islet isolation, encapsulation, and transplantation process.
(A) Types of islets sources. Islets can be isolated from a deceased donor’s pancreas via various digestion and separation procedures. A popular method of islet tissue separation is using a Ricordi Chamber (top). Beta cells and islet-like structures can also be derived from stem cells, either coming from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) derived from somatic cells. The stem cells are differentiated to beta cell progenitors which will mature and form beta cell aggregates (bottom). (B) Methods of islet preservation. After islets are isolated, their health must be maintained through different preservation methods. Islets can be isolated and immediately cultured in an appropriate medium (top) or cryopreserved (e.g., by vitrification in liquid nitrogen) for future use (middle). Administration of antioxidants can help to improve islet survival after isolation and during culture. Nanoparticles have been used to facilitate the delivery of antioxidants (bottom). (C) Islet encapsulation can be performed using many methods (see figure 2), the most common being microencapsulation in alginate using a droplet generator (top). (D) Islet implantation sites. Various types of encapsulations are made for different transplantation sites. Macrodevices, such as the TheraCyte device, are commonly implanted subcutaneously (right). Microcapsules are typically implanted in the peritoneal cavity (left). Other popular implantation sites include the omentum pouch and intramuscular implantations.
Once islets are isolated, it is best to transplant them as soon as possible. It is beneficial to culture or cryopreserve isolated islets in vitro to maintain their viability/function before encapsulation (top and middle, Figure 4B). After isolation, the capillary network in islets is severed without blood supply. This can make it difficult to get nutrients and oxygen into the islet core of large islets and they may develop necrotic cores. As will be discussed in following sections, many methods can help prevent islet core necrosis post isolation.
6.2. Transplantation Areas.
Traditionally, non-encapsulated islets are transplanted into the portal vein of the liver. This allows for direct contact with the circulatory system, which can enhance vascularization, increase nutrient and oxygen supply, and improve survival.5 One drawback of using non-encapsulated islets for transplantation is that patients are required to be on immunosuppressants. On the other hand, encapsulated islets take up a lot more space than non-encapsulated islets because encapsulation requires additional biomaterials (often hydrogels) around the islets and the transplantation of empty microcapsules (Figure 4C). In addition, placing capsules into the portal vein can be dangerous due to possible blockage. The transplantation location for an islet-laden device is very important and dependent on the type of encapsulation.130
For encapsulated islets, many different transplantation areas have been tested (Figure 4D). Subcutaneous implantation is a very popular route since it has easy access and is non-invasive. Many macrodevices are implanted subcutaneously. Some examples include the TheraCyte and β-Air devices.101,107 Subcutaneous implants can also be easily removed or replaced with new devices. A major drawback of subcutaneous implants is the possible lack of oxygen exchange and nutrients, which can stifle islet survival. Intramuscular implantation is another popular route.131 However, the intramuscular implantation procedure is more difficult compared to other implantation site procedures. It is also difficult for oxygen to reach the implants due to a lack of surrounding vasculature at rest state and limited early neovascularization.132
The most popular transplantation area for microencapsulated islets is the peritoneal cavity.24,133,134 Transplantations in the peritoneal cavity require minimal surgical procedures and can contain a large amount of implanted material. Although devices implanted into this region can be removed, the removal process is more invasive than subcutaneous removal. More vascularization is observed intraperitoneally compared to subcutaneously, however, vascularization is still limited in the peritoneal cavity unless the islets are implanted on the cavity wall. In addition, it has been shown that the diffusion of insulin and nutrients is also better intraperitoneally than subcutaneously.130 Some implant areas (e.g. epididymal fat pads) are selected to increase vascularization potential.88
The greater omentum also has good vascularization potential, as well as proximity to the portal vein for improved insulin delivery to the body. In Kriz et al.,135 a pouch device was designed for placement into the greater omentum to achieve increased vascularization. This macrodevice was placed into the abdominal cavity, wrapped in the omentum, and sutured. In vivo testing demonstrated extensive vasculature growth and lumen formation.
Intravascular transplantation has the benefit of being directly inside the blood stream but can also pose several dangers for the patient. This procedure requires vascular surgery for implantation and has the potential to easily clog vasculature. In one study, a novel tube-shaped device with a coiled membrane in an acrylic housing was created to mitigate issues with device insertion and immune response, however, thrombosis still occurred.81 Due to multiple limitations and risks, the field no longer pursues intravascular transplantation of encapsulated islets.
Overall, the design of the device and material selection determine the most optimal transplantation location. In Liu et al,131 the immune responses to PEG and PLL microcapsules were compared in various transplantation areas. At the subcutaneous implant site, PEG demonstrated the lowest immunogenicity, whereas at the intra-epididymis implantation site, PLL displayed even lower immunogenicity. At the intramuscular implantation site, there was no significant difference between PEG and PLL biocompatibility. It was also determined that intramuscular implantation led to the least amount of fibrous overgrowth.131
6.3. Improving Islet Viability.
One major problem that occurs during islet transplantation is islet hypoxia. Since islets are a dense cluster of cells, it is easy for the core of the islet to become hypoxic due to the diffusion limit of oxygen and nutrients (often only a few hundred microns).136,137 Also, severance of capillary network present in the islet during isolation further hinders the core cells from obtaining sufficient nutrients. Islets may experience undesired hypoxia during the first 7 days after transplantation. During this time, up to 70% of cells could die.138 Significant islet death could also occur right after transplantation. Furthermore, exposure to a new environment in vivo can lead to significant cell death, diminishing the amount of insulin production.139
Physiological concentrations of antioxidants have been shown to help to prevent cell death in hypoxic conditions. Bilirubin is one of the most well-studied antioxidants regarding islet viability. Bilirubin interferes with hypoxia-induced apoptotic pathways through the downregulation of apoptotic genes including TNF-α and upregulation of anti-apoptotic genes including HO-1 and bcl-2. Bilirubin is an effective therapeutic at approximately 10 to 20μM for organ perfusion.140,141,142 In Adin et al.,141 bilirubin was shown to downregulate the release of damage-associated molecular patterns (DAMPs) from islets (which are major contributors to the host immune response), and incubation of islets with a physiologic dose of 20 μM bilirubin was shown to significantly decrease cell death under hypoxic stress. Nanoparticle encapsulation technology has been used to increase the uptake of bilirubin into islets. In Fullagar et al.,138 bilirubin-loaded nanoparticles were shown to improve islet uptake of bilirubin. Islets were incubated with nanoparticles containing 0–20 μM bilirubin, and it was found that 5–10 μM bilirubin led to lower cell death levels of an insulinoma cell line (INS-R3) during hypoxia in vitro (bottom, Figure 4B). Physiological concentrations of antioxidants have been shown to increase islet viability under hypoxic conditions and reduce immune response.141
When islets are transplanted, significant amounts of reactive oxygen species (ROS) are generated which leads to immune cell infiltration and pro-inflammatory cytokine-mediated apoptosis.143 In Lee et al.,144 an oxygen carrier material, perfluorodecalin (PFD), was added to alginate at a 1:5 ratio of 1.908g/mL PFD to 2% alginate. Islets were then encapsulated in the PDF-alginate mixture, which resulted in reduced production of ROS, improving cell viability. Shalaly et al.145 used islet dissociation to increase cell viability during culturing. Islets were dissociated into single cells so that nutrients could reach individual cells, preventing a hypoxic core during culture. The cells were further cultured and then seeded on silk scaffolds, resulting in the formation of islet-like clusters.
Device designs have also been used to increase the amount of oxygen that reaches cells. The β-Air device was designed to increase the amount of oxygen that reaches cells by using an integrated oxygen gas chamber which can be replenished with oxygen through an access point.101 Implanting devices into pre-vascularized sites can also help mitigate initial hypoxia. In one study, a subcutaneous site was pre-vascularized before the implantation of islets.103 It is also important to make sure islets receive sufficient oxygen after the initial post-transplantation period. Devices have also been designed to increase vascularization using bioplotting techniques.104 The TheraCyte uses an outer membrane that specifically promotes neovascularization.34 Other cell-based methods, such as co-encapsulation of islets and preconditioned adipose tissue-derived stem cells, have been used to increase vascularization around encapsulated islets.121
6.4. Preventing Fibrotic Overgrowth.
Pericapsular fibrotic overgrowth (PFO) is one of the main reasons that encapsulated islet transplantation has not had success in clinical trials.146 PFO is a fibrotic build-up that occurs due to an immune response to implanted materials. In the case of islet transplantation, this build-up prevents the islets from receiving nutrients and secreting insulin. PFO is much more prevalent in large animals than small animals, so it is essential to study ways to prevent fibrotic build-up for clinical translation.147
One approach to reduce the immune response after islet transplantation is modifying the alginate used for encapsulation of islets. In Bochenek et al.,148 seven different chemically modified alginate formulations were used to encapsulate islets, and were then compared in vivo in macaques. The resulting capsules had varying surface charges, elastic moduli, and permeabilities. Some formulations resulted in lower amounts of PFO after intraperitoneal transplantation into macaques, with one formulation having no PFO after four months in vivo, and islets were still responsive to glucose stimulation. In Kyung et al.,149 a chitosan coating on the capsules was used to decrease fibrotic build-up on alginate encapsulated islets. In vivo mouse model results showed that the chitosan coating led to decreased fibrotic growth and increased insulin secretory function. Modifying the surface of encapsulations is another approach to preventing PFO. In Vaithilingam et al.,150 corline heparin conjugate (CHC) was bound to alginate capsules to prevent PFO due to the anticoagulant property of heparin. In vivo rat studies showed that CHC binding significantly decreased PFO on alginate encapsulated islets. Co-encapsulation of islets with MSCs in barium alginate microcapsules was shown to reduce PFO in Vaithilingam et al.120 For encapsulation of islets to be clinically transplantable, long-term prevention of PFO is essential and a combination of these strategies could be useful in decreasing fibrotic build-up.
6.5. Islet Preservation.
Islet transplantations usually require 10,000 IEQ per kilogram of body weight.151 For human donors to be a more feasible islet source, islets must be stored until enough are collected to lead to an effective transplant. The islet isolation process is also complicated, and in order for transplantation to become more feasible on a large scale, better methods for islet banking must be developed to maintain optimal cell survival and function between islet isolation and transplantation into a patient.
Vitrification using high amounts of cryoprotectants (CPA) to avoid ice formation altogether, has been used to cryopreserve islets.152,153,154 Vitrification is performed using high concentrations of CPAs and by fast cooling samples to prevent ice formation (middle, Figure 4B).155,156 Different aspects of vitrification have been examined to improve cell survival outcomes.157 For example, the CPA itself has a large effect on cryopreservation outcome. Trehalose is a non-toxic sugar that has become a popular material for cryopreservation and an alternative to toxic CPAs, such as dimethyl sulfoxide. It has been implemented in CPA formulations to improve islet viability.152,153 Chen et al. examined a formulation of ethylene glycol, DMSO, and trehalose, which showed improved viability over traditional CPAs.154
Devices used to hold islet-containing samples for vitrification have been optimized to improve cryopreservation efficiency. In Nagaya et al., mouse islets were vitrified using a hollow fiber vitrification method (HFV), which decreased the necessary concentration of CPAs for vitrification. The islets demonstrated excellent function well after vitrification.158
Vitrification has also been performed on microencapsulated islets. Agarose-encapsulated islets were vitrified using a solution made of DMSO, PEG, and PVP K10. The resulting islets retained 55% of normal insulin secretion ability, and showed ~46 days of insulin secretion, on average, in vivo compared with 53 days for fresh islets without cryopreservation.159,160 Alginate microcapsules have also been shown to improve cell viability after vitrification. Alginate microcapsules that are crosslinked using calcium cations maintain microcapsule integrity well during vitrification, permitting a lower amount of necessary CPAs.161 It has also been shown that alginate microcapsules improve cell viability by preventing intracellular ice formation as a result of devitrification or recrystallization during the warming-back step of a cryopreservation procedure.162 Moving forward, a combination of low-damaging CPAs such as trehalose, improved vitrification devices, and pre-encapsulating islets in a protective gel should improve the islet banking process, making islet transplantation more clinically feasible and widespread.
7. STEM CELL AS AN ISLET ALTERNATIVE
As aforementioned, limited availability of islets and possible rejection of non-autologous islets are two major problems associated with islet transplantation for treating diabetes. One way to combat these two problems is through the use of stem cell differentiation. Recent research focuses on the differentiation of stem cells into beta cells, or clusters that contain multiple cell types found in islets.9,107,163 Stem cell-derived beta cells can be readily available and mitigate the lack of islet donors and the chance of immune rejection (bottom, Figure 4A). The latter is because stem cells derived from a patient could be used to differentiate into insulin-secreting cells/islets for transplantation. Both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have been studied in the area of islet replacement.
7.1. Embryonic Stem Cells (ESCs).
ESC is the most studied type of stem cells for beta cell differentiation. ESCs are pluripotent cells derived from the inner cell mass of blastocysts.164 In Amour et al.,163 human embryonic stem cells (hESCs) were differentiated into endocrine cells that demonstrated secretion of multiple pancreatic cell hormones including insulin. In Kroon et al.,9 hESCs were differentiated into pancreatic progenitor cells which successfully matured into functional pancreatic cells in vivo. Scaling up production is essential for using hESCs in large scale diabetes treatment. Schulz et al. demonstrated methods for improving the production and cell banking of hESCs.165 They subsequently differentiated the banked hESCs into pancreatic progenitors which then matured into insulin-secreting cells in vivo.
Microencapsulation can also be useful for ESC differentiation. Richardson et al. encapsulated hESCs in alginate and then differentiated the cells into pancreatic cell types, resulting in improved cell viability than 2D culture.166 Using this method, cells could be differentiated and then directly transplanted in vivo. Stem cell derived precursors have also been shown to function after macrodevice encapsulation and implantation. Beta-cell precursors were put in a TheraCyte device and matured into insulin-secreting beta cells with glucose responsiveness in vivo.107 These cells showed greater survival post transplantation in comparison to isolated adult islets. It was hypothesized that younger tissue may be more resistant to transplantation stressors. Robert et al. demonstrated differentiation of hESCs into glucose-responsive cells in an Encaptra device after subcutaneous implantation in mice.167 Insulin-producing cells have also been derived from hESCs in vitro, which allows for better control of the differentiation process and can ensure insulin production before implantation in patients.168 Encaptra is also currently in clinical trials.169
7.2. Induced Pluripotent Stem Cells (iPSCs).
In the last decade, iPSCs have become a promising option for cell therapies. iPSCs are derived from adult somatic tissue, and there is no need for human embryonic tissue that is limited and associated with ethical concerns.164 Moreover, differentiating patient-specific iPSCs will prevent rejection of the resulting beta cells after implantation. In one study, iPSCs were differentiated into pancreatic progenitor cells which secreted insulin in response to glucose challenges in vitro.170 In vivo testing of these cells in mice also showed regulated blood glucose levels. As iPSC differentiation into beta cells is more robustly characterized preclinically, the potential for islet replacement using this method may expand. Other cell types have also been reprogrammed into insulin-producing cells. Adult human liver cells (AHLCs) have been reprogrammed into insulin-producing cells using pancreatic transcription factors (pTFs).171 In one study, AHLCs transduced with pTFs were combined with human mesenchymal stem cells and encapsulated. This resulted in successful glycemic control in diabetic mice when implanted in vivo.72
8. OUTLOOK AND CONCLUSIONS
Islet encapsulation has greatly advanced over the past few decades. Although clinical trials are being performed on microencapsulated islets, there has not been a trial that has shown well-controlled blood glucose levels in patients over several years. Non-encapsulated islet transplantation requires patients to take immunosuppressants long-term, which can be very detrimental to health. The goal of islet encapsulation is to prevent the body from rejecting implanted islets while allowing the islets to release insulin and receive the nutrients they need to survive for many years.
For islet transplantation to become a viable standard of care, it must provide a long-term therapeutic effect. Long-term insulin release in patients first requires isolation methods for obtaining healthy islets. In addition, the encapsulation processes must not reduce the health of the islets. Methods for mitigating post transplantation hypoxia can also be implemented to decrease cell death. The encapsulation needs to permit the exchange of oxygen and nutrients while preventing immune response and fibrotic build-up. Vascularization and optimal implant location can also decrease cell death and increase plasma insulin concentration.
Researchers need to continue to focus on improving encapsulation technologies to reduce immune response and increase cell viability. Conformal encapsulation of islets has also been explored. It was done by using thin polymeric layers to surround islets with minimal resistance to the transport of nutrients, oxygen, and wastes. However, the conformal encapsulation technology still needs significant improvement in terms of stability and durability of the thin layers. Device vascularization after implantation is both vital and detrimental to the resulting efficacy. Vascularization can increase the immune reaction to the device since there will be little to no barrier protection between the islets and immune cells in the blood. On the other hand, increased oxygen and antioxidant delivery via the vasculature can decrease islet death and enhance the therapeutic effect of implantation.
Materials are another way in which devices can be improved. By changing the materials, the encapsulation types, chemical properties, and pore sizes can all be adjusted with the goal of increasing small molecule exchange and decreasing immune response. In addition, materials can be added to the encapsulation to aid with immune protection and cell survival. There have also been experiments that included other cell types to improve cell survival and prevent immune response.58,72,120
In order for islet transplantation to become the standard of diabetes treatment, there must be a way to have a sustainable number of islets for transplantation. The current standard is to isolate islets from deceased donors. Islet encapsulation allows for xenotransplantation using porcine islets, which is an almost unlimited source of islets. Another method to increase the number of available islets is stem cell differentiation. Stem cells collected from embryonic tissue have shown increasing clinical feasibility in recent years. However, iPSCs have the potential to generate an unlimited supply of personalized beta cells for transplantation with no ethical issues.
As encapsulation technology advances and becomes more developed, there will hopefully be an increased amount of successful clinical trials using implanted islets for long-term glucose level regulation. If islets can be produced and encapsulated on a large scale, costs of islet transplantation would diminish, making this a financially feasible treatment for diabetes patients.
ACKNOWLEDGMENTS
This work was supported by grants from the US National Institute of Health (NIH R01EB023632).
Footnotes
The authors declare no competing financial interest.
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