Microencapsulated Pig Islet Xenotransplantation as an Alternative Treatment of Diabetes

Introduction

In comparison with exogenous insulin administration and whole pancreas transplantation, islet transplantation is considered as a more viable and prospective treatment for patients with type 1 diabetes mellitus (T1DM), ¹ but shortage of available islet grafts (inability of human donor programs to meet present demands) greatly restricts the further development of clinical islet transplantation. ² Xenotransplantation, using pig as a source of islet cells, offers an attractive and appropriate solution for this dilemma. ³

Pig is considered as a promising candidate for xenologous islets due to several advantages, including structural similarity of insulin between pig and human, lack of amyloid formation, resistance to recurrent autoimmunity, feasibility in genetic immunomodulation/engineering, and few ethical issues. ⁴ However, initiation of clinical application of pig islet still requires additional progress in two major hurdles: immune rejection due to xenogeneic incompatibility, and virus- or pathogen-based xenosis.^5,6 Several strategies are developed to protect transplanted pig islet grafts from immune attacks of recipients. Nowadays, the mainstay of immunomodulatory therapy depends on the usage of conventional (e.g., steroid, tacrolimus, and cyclosporine) and/or relatively specific (e.g., costimulatory blocking agents) immunosuppressants to achieve sustained immunosuppressive state. These immunodepressors, while showing favorable efficacy in extending the functional survival of pig islet xenografts,^7–12 have numerous severe side effects,^13–15 such as opportunistic infection, malnutrition, neuritis, thrombogenesis, and islet toxicity. Therefore, to safely and efficiently promote experimental pig islet xenotransplantation into clinical trials, problems concerning life-long and heavy immunodepression must be solved first.

One alternative strategy for protecting the grafts against xenorejection is immunoisolation. Immunoisolation, that is, encapsulation of islets in a membrane that is selectively permeable for oxygen, nutrients, and insulin, but not for humoral and cellular components of the recipient's immune system (Fig. 1), enables successful pig islet xenotransplantation in the reduction or absence of immunosuppressive medication.^16–20

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FIG. 1.

Illustration of the concept of islet microencapsulation and the principle of immunoisolation. The semipermeable membranes inhibit the entry of immune cells and antibodies, meanwhile, they allow the diffusion of insulin, glucose, oxygen, nutrients, and metabolites. Color images available online at www.liebertpub.com/teb

There are several distinct approaches for the transplantation of immunoisolated pig islets as a bioartificial pancreas (BAP). Intravascular devices (encapsulation of pig islets in a perfusion chamber which is anastomosed to the vascular system of the host) require high-risk vascular surgery and intense anticoagulation therapy,^20,21 and therefore are unsuitable for clinical application currently. Extravascular approaches of immunoisolation are either macroencapsulation (encapsulation of a large mass of pig islets within a tubular diffusion chamber or planar chamber) or microencapsulation (encapsulation of individual or a small group of pig islets within a spherical chamber). Several considerations favor microcapsules over macrocapsules. Their spherical shape and low volume offers better diffusion profile due to higher surface-to-volume ratio. In addition, microcapsules require less complex manufacturing process, are mechanically durable, and can be simply injected without major surgery. A main drawback is the difficulty associated with the retrieval of transplanted capsules if necessary. However, fixation of pig islet microcapsules on a plasma-treated polydimethylsiloxane sheet, appears to be practicable for preventing the capsules from scattering in the body and perfectly retrieving them when required. ²² In the field of pig islet transplantation, there is a great deal more research and development with positive results for microdevices than for macrodevices,^6,23–28 and some encouraging achievements have been made in preclinical/clinical xenotransplantation of microencapsulated pig islets without immunodepression (Table 1).

Consequently, xenotransplantation of microencapsulated pig islets may provide a promising option of applicable treatment for insulin-dependent diabetes mellitus (IDDM) patients with minimal risks from permanent immunosuppression or complicated operation. This article reviews advances and challenges for xenotransplantation of microencapsulated pig islets, and discusses demands to successfully promote this therapy into widely clinical application.

Pig Islet Source

The most important element of BAP is the islet cell mass encapsulated within the semipermeable membrane. Pig islet with high-quality contributes a lot to the efficacy and functional duration of the BAP. Compared with other variable factors of gender and body weight, the age of the pig donor plays a much more significant impact on islet size, yield, and functionality.^4,29–31

Neonatal pig pancreatic cell clusters (NPCCs) offer additional advantages over adult pig islets (APIs), including apparent resistance to ischemia or inflammation, low sensitivity to collagenase variability, great ease of digestion and purification,^32,33 and low level of T-cell response. ³⁴ Usually, NPCCs obtained from piglets aged <5 days perform better viability and functionality than the older ones. ³⁵ Additionally, culture of NPCCs (from piglets aged 1–3 days) for a long time (12 days) offers the best balance in vivo functionality, evidenced by better diabetes reversal, lower levels of tissue factor (TF) expression, and higher gene expression for insulin, glucagon, and antiapoptotic Bcl-2. ³⁶ The NPCC represents an available and alternative source of islet grafts for human recipients,^4,37 and immunoisolation can further enhance the metabolic response of these islet xenografts in vivo.^38–40 In fact, microencapsulation process did not compromise the viability of NPCCs, and the encapsulated grafts could be held in culture for 5 weeks with maintenance of viability. ⁴¹ Also, a published study demonstrated that microencapsulation was able to maintain fine morphology/structure of cryopreserved NPCCs and effectively recover their endocrine function. ⁴² After in vivo implantation, the microencapsulated NPCCs showed considerable capacity for proliferation and further differentiation into β-cells, ⁴⁰ thus displaying the ability to restore normoglycemia or significantly reduce insulin dosage in both experimental^18,24,40 and clinical trials ¹⁶ (Table 1). Once the time lag between preparation and maturation of NPCCs was curtailed by preincubation with homologous Sertoli cells, the microencapsulated NPCCs could correct hyperglycemia more effectively in diabetic mice. ³⁵ Currently, commercial product of microencapsulated NPCCs manufactured by Living Cell Technology (LCT) are undergoing late-stage clinical trials. ⁴³

Taking into account the potential risk of zoonosis, islet xenografts should be derived from specific pathogen-free (SPF) or designated pathogen-free (DPF) pig herds. ⁴⁴ Donor pigs fulfilling DPF status are usually raised and kept in biosecure barrier facilities where all activities and operations are in compliance with Standard Operating Procedures and current Good Manufacturing Practices. To guarantee and maintain the DPF status, pigs are transferred into such facilities through Cesarean section and the source pigs are raised as a closed herd. Compared with the wild-type or market pig herds, the SPF Chicago Medical School (CMS) miniature pig, ⁴⁵ Wuzhishan (WZS) miniature pig, ⁴⁶ New Zealand Auckland Island pig, ⁴⁷ and the new conserved pig herd called XENO-1 ⁴⁸ may serve as more practicable and better islet sourcing for microencapsulation in clinical application.

In addition, genetically modified pigs are considered as alternative donor sources with potentially major benefits of reducing the early graft loss and protecting islets against immune rejection.^4,49 For example, humoral rejection can be fully suppressed by the crossbreeding of Gal gene knockout (GT-KO) pigs with transgenic pigs expressing human complement regulators such as CD46, CD59, and human decay-accelerating factor (hDAF, CD55)^50–53 ; transgenic expression of human heme oxygenase-1 (HO-1) and/or soluble human TNFRI-Fc (shTNFRI-Fc) can protect pig islets from oxidative and inflammatory injury^54,55; knockout of TF and overexpressing of human antithrombotic gene (CD39/thrombomodulin), may prevent the occurrence of instant blood-mediated inflammatory reaction (IBMIR) and coagulation dysfunction.^56–58 Also, genetic engineering of pigs (deletion of Gal epitope and/or knockin of human genes) results in few abnormalities in hematologic, biochemical, or coagulation parameters, and seems to modify these indicators closer to human values. ⁵⁹ More importantly, the genetic alterations do not display an overtly detrimental effect on islet function. ⁶⁰ Although significant variations exist between wild-type and transgenic pigs in terms of baseline metabolic data (e.g., insulin, blood glucose, C-peptide, glucagon levels) and responses to glucose and arginine, acceptable glucose metabolism can be achieved.^60,61

Materials for Microencapsulation

Polymers applied for microcapsules should satisfy the following requirements, including negative influence on viability and functional survival of encapsulated cells, be pliable, soft and mechanically stable, free exchange of molecules of interest (e.g., nutrients, oxygen, glucose, and insulin), and high biocompatibility with minor host response. ⁶²

Over the past two decades, a wide range of polymers, such as agarose,^63,64 polyethyleneglycol (PEG),^65,66 and sodium cellulose sulfate, ⁶⁷ have been used for microencapsulation of pig islets. Most of them have not been adequately and comprehensively studied in terms of biocompatibility with longevity with the enveloped islets and the recipients, and therefore do not qualify as safe and valid for human application for the moment.

To date, the most widely used and more preferred biomaterial for pig islet microencapsulation is alginate (Table 1),^{17,19,40,68–73} an unbranched anionic polysaccharide (comprised of β-D-mannuronic acid and α-L-guluronic acids) extracted from brown algae (Phaeophyta), whose physicochemical and gelling properties make it more efficient and promising in sequestering pig islets from the immune system with a long-term stability and no alteration of graft function. In general, islet-containing alginate solution is dropped through a nozzle into a divalent cation solution, either barium chloride or calcium chloride, to generate a rigid and durable microbead incorporating an individual or few islet grafts (Fig. 2). As alginate is a polysaccharide, it will be subject to a gradual enzymatic and hydrolytic degradation under physiological conditions, let alone inflammatory conditions. Undesired quick biodegradation of alginate inherently influences the physicomechanical properties of capsules owing to structural changes, resulting in poor stability and impaired semipermeability/immunoprotection and therefore failing to achieve a sustained viability/functionality of encapsulated islets after transplantation. Basically, higher molecular weight (MW) of alginate reduces the number of reactive positions available for hydrolysis degradation, which further promotes a slower degradation rate. By changing the concentration of the alginate, it is possible to control the rate of capsule degrading or breakdown over various periods of time (ranging 3 months [≤0.5–0.75% alginate] to 1 year [≥1.5% alginate]), which correspond to the functional longevity of encapsulated pig islets. ⁷⁴

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FIG. 2.

The principle, technologies, and apparatus for the production of alginate-based microbead. Nozzle modes: a. simple dripping mode; b. air flow driven mode; c. electrostatic mode; d. vibration-controlled mode. Color images available online at www.liebertpub.com/teb

In general, barium crosslinkage provides alginate gels with higher dimensional stability/strength, better biocompatibility, and weaker reaction than those found with calcium^75–78 ; however, the leakage of barium from alginate microcapsule and high risk of barium accumulation (chronic toxicity) largely limits its application in clinic. ⁷⁹ Using low concentrations of barium ions and intensive rinsing of barium beads may be useful strategies to eliminate the safety concerns.^79,80 Although different chemical formulations of alginates (e.g., high mannuronate or guluronate, high or low viscosity, with or without coupled peptide sequence) are selected for pig islet encapsulation; high mannuronic (≥50%) and high viscosity (≥100 mPa·s) alginate is considered as the most appropriate encapsulating material that possesses ideal biocompatible properties, with benefits including impermeability to natural/elicited antibodies (e.g., Ig G, 150 kDa), sufficient diffusion of nutrients/metabolites/insulin, suitable implant stability, low macrophage/lymphocyte recruitment and infiltration, as well as promotion of neoangiogenesis, and sufficient pO₂ delivery (approximately 40 mmHg). ⁸¹ The favorable stability referred to slight implant degradation (−27% of initial graft weight) and inapparent fibrosis/inflammatory process. By contrast, most of the other alginates degraded at 12 weeks as evidenced by ≤50% of initial implant. The rapid degradation seems to be associated with the strong inflammatory response. In a pig-to-primate model, this type of simple alginate microcapsule could significantly prolong API survival into monkeys for up to 6 months even in the presence of antibody response. ⁸² No graft fibrosis or cellular overgrowth was observed, and after explantation, the residual pig islets in capsules showed the capacity to secrete insulin after glucose exposure.

In most cases, several polycations are applied as positively charged coating on the simple alginate microbead to confer better permselectivity and mechanical stability. However, selection of optimal coating material remains controversial. Ponce et al. ⁸³ suggested that poly-l-lysine (PLL) was the best option available and that utilization of poly-l-ornithine (PLO) and poly-d-lysine (PDL) should be avoided due to ongoing inflammatory responses and high rates of capsular overgrowth. By contrast, Darrabie et al. ⁸⁴ demonstrated that PLO-coated alginate microcapsule (containing pig islets) was mechanically stronger and offered better permselectivity than PLL coating, suggesting that PLO is an alternative to PLL. Others preferred PEG-PLL diblock copolymers for preventing potential protein adsorption and improving biocompatible protection on the surface of capsules.^85,86 The commercial microcapsule containing pig islets, which is being tested in clinical trials, is composed of an alginate core coated with a layer of PLO and an outer coat of alginate.^87,88 But degradation of the outer layer of alginate will leave the underlying layer of PLO exposed, which eventually results in decreased biocompatibility in vivo. To address the problem, chemical crosslinking/modification of PLO layer using genipin, which has the properties of neutralizing immunogenic effect of PLO and reducing protein adsorption, offers a viable solution to improve the antidegradative, biocompatible, and hydrophilic properties of alginate/PLO/alginate capsules without any adverse effects on pig islets.^87,89 Lately, Hillberg el al. ⁹⁰ have shown that alginate microbead coating with glycol chitosan provides improved mechanical stability and biocompatibility over conventional alginate/PLO/alginate microcapsule. Within the novel microcapsule, comparable pig islet viability (>90%) and insulin release could be maintained over a month in vitro culture.

Methods for Fabrication of Microcapsule

In general, microcapsules used for immunoisolation of pig islets are generally fabricated by the following main steps (Fig. 3)^{40,82,91–94}: (1) Islet preparation. Adequate supply of pig islets with high viability/functionality is a prerequisite for obtaining high-grade microencapsulated islets. (2) Blending. Pig islets are suspended in the polymer solution (commonly alginate) which makes up the microbeads. (3) Droplet generation. This process disperses the mixture of polymer (alginate) and pig islets into small droplets containing few cells by different methods such as extrusion or emulsification. During this procedure, several main factors should be considered when choosing the droplet generation technique, including capability of maintaining high islet viability/functionality, good control of capsular size and shape, as well as size distribution/uniformity, and viscosity of polymer solution.^95,96 (4) Stabilization. The formed droplets are then further solidified and modified to improve the strength, permselectivity, and biocompatibility of microcapsules. All these procedures are also available for microencapsulation of islets isolated from other species, including rodents and humans.^97,98

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FIG. 3.

Flow chart for the fabrication of microencapsulated pig islets.

Isolation and purification of pig islets have been elaborated in published reviews.^5,33,99,100 Briefly, due to the lack of exocrine tissues and concomitant relative abundance of endocrine tissues, the immature pig islets, both fetal pig islet-like cell clusters (FPICCs) and NPCCs are easy to be obtained by simple enzymatic digestion and following pretransplantation culture (4–9 days). By contrast, it is relatively difficult to obtain APIs from adult pigs, despite many experiences in this field. Achieving successful API isolation needs complete blood exsanguination, extensive flushing of pancreas, reduced warm ischemia time (WIT, within 10 min), and appropriate cold ischemia time (CIT, within 2 h). Before storage of harvested pancreas in cold preservation solution (two-layer cold storage and controlled perfusion are advisable), morphological screening and rapid assessment of pancreas should be conducted, and only IEQs/cm² of splenic lobe biopsy is a reliable predictor of islet yield. In the subsequent digestion and purification processes, to improve pig islet yield and viability, Good Manufacturing Practices Grade Liberase MTF C/T with low endotoxin content (≤10 EU/mg) and iodixanol solution is recommended for pancreas digestion (usually in a digestion/filtration chamber) and islet purification (usually on COBE 2991 cell separator), respectively. Postisolation, in vitro culture of APIs (for 7 days, at 37°C) is also recommended for improving islet viability/secretory capacity (e.g., by increasing ATP content and oxygen consumption rate) and possibly reducing the immunogenicity, as well as stress-associated damage and cell apoptosis (e.g., by upregulating prosurvival JNK-3 expression and downregulating proapoptotic signaling of JNK1/2/c-fos).^101–103 In the process of bleeding, pig islets with high quality/purity and alginate with high purity are proposed for improving the biocompatibility and longevity of the subsequently formed capsulated grafts.^{17,82,104,105} The alginate/pig islet proportion (commonly used concentration range of ∼3000–10, 000 islet equivalents [IEQs]/mL alginate)^{17,25,40,82,91,105–107} should be properly adjusted so that one capsule will contain one or few islets, with fewer empty capsules.

For dispersion, with emulsion method, the aqueous solution (containing islet cells) is mixed and dispersed in an organic solvent (e.g., oil). When the dispersion reaches equilibrium, gel formation is triggered by cooling or by the addition of a gelling agent. Although this technique can be easily scaled-up, the shear stress during the procedure may induce islet injury and the process itself can result in heterogeneous dispersion. ⁹⁵ Compared with the less popular method of emulsification, extrusion is a much more practical and promising process to generate droplets by forcing the homogeneous suspension through a needle using either vibration, or coaxial airflow (called air-driven droplet generator), or electrostatic potential (called electrostatic droplet generator) (Fig. 2). Generally, greater mechanical strength and better diffusion efficiency of microcapsules are directly associated with smaller size. The vibrating nozzle technique can generate uniform-shaped microbeads with small diameter at a high production rate, but this method is limited to a relatively low viscosity of alginate (≤0.2 Pa/s). ¹⁰⁸ Mazzitelli et al. ¹⁰⁹ further discovered that the optimized parameters of vibrational encapsulation to obtain NPCCs containing monodisperse droplets were, 150 Hz of vibrational frequency, 1 mm of vibrational amplitude, 8.6 mL/min of alginate pumping rate, and 140 mm of distance between nozzle and surface of gelling bath, with no alternation of morphological and functional properties of the enveloped pig islets. Although electrostatic pulse system can produce smaller (250–350 μm vs. 400–880 μm in diameter) and more uniform microcapsules in comparison with air-jet technique,^{17,107,110,111} the high electrostatic voltage used probably interferes with pig islet viability. At present, coaxial airflow is the most commonly utilized method in producing droplets containing islet grafts, ⁹³ and smaller capsules (≤300 μm) with narrow size distribution can also be produced by a novel micro-airflow nozzle. ¹¹² Also, this method seems to be less sensitive for alginate with high viscosity.^108,113 To reduce the occurrence of fragmented droplets, the rate of air flow or polymer solution flow should be controlled within a certain range. ¹¹⁴ Koo et al. ²⁵ established an optimal condition for microencapsulation of pig islets using alginate and air-driven droplet generator. For alginate at different concentrations of 2% (wt/vol), 2.5%, and 3%, the appropriate CO₂ flow rate and alginate inflow rate were (2.0 L/min, 10 mL/h), (3.0 L/min, 20 mL/h), and (4.0 L/min, 40 mL/h), respectively. Park et al. ¹¹⁵ successfully laminated NPCCs within small microcapsules (<200 μm in diameter, also known as conformal encapsulation) using the air-driven encapsulator under the condition of alginate (2%) flow rate of 135 mL/h, and air pressure of 0.10 bar. With several advantages over large microcapsule (600–700 μm), including reduced capsular thickness, minimal dead space, and favorable metabolite transfer (e.g., oxygen and nutrients), the small capsule can acquire better selective permeability, tolerance, relatively low oxygen tensions without massive graft loss, result in a reasonable graft volume for implantation, and significantly enhance islet viability and insulin secretory response.^116–118

In the subsequent stabilization step, alginate-based droplets are converted into rigid microbeads by gelification in a divalent cation solution, mostly Ca²⁺. Ordinarily, the microbeads are then coated with a second layer (e.g., PLL, PLO, or PEG) and another outer layer of alginate (Fig. 1). This step may be the key tache for ensuring the mechanical stability, permeability, biocompatibility, and longevity of islet microcapsules. Also, the associated principles and some considerations have been described previously.

In addition, after microencapsulation, especially conformal encapsulation, separation of empty capsules appears to be essential since production of empty capsules results in enlargement of implant volume, as well as interference of nutrients and insulin transfer. In comparison with traditionally manual selection, the density gradient (e.g., Ficoll or Percoll)-based centrifugation is much more adaptable for large-scale processing to discard poorly encapsulated islets and generate sufficient uniformly sized encapsulated islets without any adverse effects on viability.^23,119

Cryopreservation of Microencapsulated Pig islet

Considering the effective protection of fragile islet from freezing-induced damage by microencapsulation,^{42,107,120,121} as well as advanced microcapsule design/fabrication technology and successful preclinical transplantation studies, a banking system (by cryopreservation) that allows for effective and long-term storage of the BAP, represents a critical prerequisite for facilitating donor pooling and promoting transplantation of microencapsulated islets as available cell-based medicine since graft demand is unpredictable and just-in-time processing of encapsulated islets is impossible.

Many improvements have been made to optimize cryopreservation technology for maintaining islet viability and functionality, as well as the biointegrity of microcapsule.^107,122 Zhou et al. ¹⁰⁷ reported acceptable cryopreservation of alginate-based microencapsulated pig islets by slow-cooling protocols. The freezing process neither induced morphological change and fragmentation of pig islet nor altered surface property of the microcapsule. After rapid thawing, the encapsulated pig islets preserved in liquid nitrogen could restore normoglycemia in diabetic mice for the duration of the study (90 days). It is notable that a tightly controlled freezing rate is essential in dealing with the delicate and sensitive pig islets. Moreover, the use of nucleation (ice seeding) seems to play an important role in preserving the physiological competence of the cryopreserved islet grafts. The major challenge of slow freezing is the relatively high risk of islet injury due to ice formation.

Another approach of cryopreservation is vitrification, which is less complicated and time-consuming than the slow-freezing method. Recently, Agudelo et al. ¹²² developed a novel vitrification solution KYO-1 for cryopreserving encapsulated islets, which showed more superior ability to maintain morphological integrity and mechanical/physicochemical property of the agarose microcapsules in comparison with dimethylsulfoxide-based conventional freezing solution. Subsequently, in a xenogeneic transplantation model, the cryopreserved agarose-encapsulated hamster islets (storage for at least 2 weeks) could reverse hyperglycemia in diabetic mice for a long period, up to 56 days. ¹²³ The feasibility studies serve as solid foundations for conducting available and practicable cryopreservation of microencapsulated pig islets.

As a note, the polymers (e.g., alginate, agarose) utilized for islet microencapsulation appear to be not very stable in the frozen–thaw cycle. The cryoprotectant (e.g., ethylene glycol) can directly influence the volume of alginate-based microcapsules during cryopreservation. ¹²⁴ Also, the cryopreservation process probably cause changes of network structures of agarose gel as evidenced by differences in diffusion coefficients for substances with intermediate MWs before and after freezing. ¹²⁵ Thus, more suitable protocol for cryopreservation needs to be developed in terms of achieving prolonged storage of encapsulated islets without any adverse effects on islet viability/functionality and capsular stability.

Other Factors Influencing Survivability and Functionality of Microencapsulated Pig islets

Biosafety and Clinical Trials

Conclusions

The BAP comprised of microencapsulated pig islets may overcome the major obstacles to islet transplantation: shortage of donor supply and extensive use of immunosuppressants. Although pilot preclinical/clinical trials demonstrate the safety, practicability, efficacy, and promise of microencapsulated pig islet xenotransplantation, the currently available therapy is far away from the final possible cure of T1DM. Several major obstacles remain to be overcome: reinforced immune protection, enhanced oxygen/nutrients supply and transfer for capsulated islets, reduced activation of destructive host immune reaction, and improved survival and functionality of islet xenografts when implanted. It is expected that sufficient supplies of pig islets or renewable insulin-producing cells with high quality and low xenoantigenicity, coupled with optimization of capsule design/size/preparation, advances in biomimetic materials, and immunomodulatory strategies, as well as better selection and bioengineering of the capsules' site of implantation may potentially lead to a reliable and sustained glycemic control. Furthermore, the achievements made in successful xenotransplantation of microencapsulated pig islets will provide potential guidance for the application of other bioartificial organs.