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Microencapsulation Technology
Chapter 43
Microencapsulation Technology Rajesh A. Pareta, John P. McQuilling, Alan C. Farney, and Emmanuel C. Opara Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem, NC
Chapter Outline 43.1 Introduction 43.2 Islet Isolation 43.3 Alginate-Based Microencapsulation of Islets 43.3.1 Semipermeable Membrane Coating Techniques for Alginate 43.3.2 Studies of Microencapsulated Islet Grafts in Large Animals and Humans 43.3.3 Microencapsulation Devices
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43.1 INTRODUCTION The hallmark of type 1 diabetes is destruction of the insulin-producing β-cells of the pancreas by an autoimmune disease [1] resulting in the obligatory need for exogenous insulin to regulate blood glucose in the afflicted individuals. The bioartificial pancreas (BAP) involves a bioengineering approach to treating diabetes with islet transplants, which will secrete pancreatic hormones in response to the host blood glucose like a real pancreas. Presently, islet transplantation is limited by a shortage of pancreas for human islets isolation and allogenic islet recipients also require long-term immunosuppression to prevent rejection and recovery of autoimmunity. Design of BAP should take this into account. In type 1 diabetes, the patients have preexisting antibodies and immune cells primed against β-cell surface markers and insulin [2], and hence a simple islet transplantation without immunosuppression is not viable. In the BAP construct illustrated in Figure 43.1, islets require a protective semipermeable coating to immunoisolate them and preserve their viability and functionality upon transplantation. This approach opens up possibilities for allo- and xenotransplants, and thus has the potential to overcome the shortage of islets while addressing the issue of transplant rejection. To summarize briefly, the BAP construct is not only a viable option to address the human islet shortage but also offers tremendous benefits to the
Regenerative Medicine Applications in Organ Transplantation. © 2014 Elsevier Inc. All rights reserved.
43.3.4 Oxygen Requirements of Islets and the Effect of Transplantation Site on BAP Function 43.4 Potential Role of ECM-Based Technology in the Development of the BAP 43.5 Conclusions Acknowledgment References
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transplant recipient such as relief from long-term use of immunosuppressant drugs. In this review, we will focus on two technologies which may enhance the development of a viable BAP. The first is the use of alginate to microencapsulate islets. Alginate is a widely used naturally occurring biopolymer for islet microencapsulation [3]. The second technology, which is currently in its infancy, is the use of extracellular matrix (ECM) to influence the viability and function of islets in BAP constructs. ECM is a three-dimensional meshwork of proteins and polysaccharides that impart structure and mechanical stability to tissues [3], which can be obtained after decellularization of tissue and organs. Alginate has been extensively used because it has unique properties and advantages for islet microencapsulation such as its mechanical strength, hydrophilic nature, and ability to cross-link at physiological conditions, ECM is a relatively new and upcoming technology where the tissue’s own natural scaffold can be used to promote the interactions between the islets and a construct matrix.
43.2 ISLET ISOLATION In fabricating a BAP, a critical starting point is the procurement of pancreatic islets from an abundant source using reliable techniques to preserve β-cell function. Thus, isolation of whole islets without inflicting any
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FIGURE 43.1 Concept of a BAP. A protective semipermeable coating to immunoisolate islets (iso-, allo-, or xenosource) and preserve their viability and functionality upon transplantation [1].
significant damage to the cells is a key component of developing a viable BAP. A critical balance of composition, process, and duration of collagenase digestion is required for isolating islets with integrity, viability, and high purity with a significant yield. This overall process has tremendous impact on the clinical outcome of islet transplants [4]. The pancreas is digested with combined collagenase and protease action, which disintegrates the intercellular matrix of collagen, releasing islets. These islets are isolated, purified, tested for viability, and sometimes cultured before being transplanted in the patient. Collagenase digestion disrupts islet exocrine tissue adhesive contacts [5]. Thus, shorter duration or lower concentration of collagenase would lead to incomplete digestion of islets from exocrine tissue, leading to reduced yield on purification. On the other hand, extended duration of incubation or higher concentration of collagenase would adversely affect the islet cell cell adhesion, leading to loss of islet integrity and viability. Intra-islet cell cell adhesion is protease sensitive, while extra-islet cell matrix adhesion is collagenase sensitive. In the pig pancreas, very little periinsular capsule is present, and the structural integration of the porcine islet in the exocrine pancreas is almost exclusively cell cell adhesion. In canine, the islets are almost exclusively encapsulated with very little exocrine endocrine cell cell contact. In rodent and human, the situation is intermediate with a tendency toward predominance of cell matrix adhesion. The presence of protease in the collagenase preparations has been reported to reduce the yield and quality of isolated islets from rats [6], however, it is more efficient for the isolation of pig islets [7].
43.3 ALGINATE-BASED MICROENCAPSULATION OF ISLETS It is crucial that the biomaterial used to encapsulate islets must be biocompatible and permeable (for hormonal, nutrient, and oxygen exchange). Hydrogels are such
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semisolid materials, which not only are soft but remain stable under mechanical stress. Hydrogels are very attractive for making microcapsules. Hydrogels also provide higher permeability for low-molecular weight nutrients and metabolites. Furthermore, the soft and pliable features of the gel reduce the mechanical or frictional irritations to surrounding tissue [8]. Alginate is one such hydrogel which has been widely used owing to its many excellent properties conducive to islet transplants. Alginate molecules are linear block copolymers of β-D-mannuronic (M) and α-L-guluronic acids (G). It forms a gel in the presence of divalent ions like Ca21 and Ba21. Recent studies have shown that divalent ions cross-link not only G blocks but also blocks of alternating M and G (M G blocks) [9]. Mainly calcium is used for gelling, as barium is known to be toxic and concerns have been raised about patients’ safety if it is used as the crosslinking agent. Alginate is one of the few biopolymers that allow cell encapsulation at physiological conditions. The encapsulation can be done at room or body temperature, at physiological pH, and in isotonic solutions. Alginatebased capsules have been shown to be stable for years in both animals and humans [10 12]. Also, since alginate materials are negatively charged, the attachment of immune cells to the microcapsule is limited due to the negative charge on the cell surface thus making alginate very highly biocompatible [13]. In most tissues it has been shown that maximum diffusion distance for effective oxygen and nutrient diffusion from blood capillary to cells is about 200 μm. Absence of this convection inside a capsule induces a nutrient gradient from the capsule surface to center of cells. Present insights suggest microcapsules as a preferable system over macrocapsules due to their high surface to volume ratio for fast exchange of hormones and nutrients. Microencapsulation uses the interfacial precipitation predominantly, where a polyanionic polymer (alginate) gels with a divalent cation (Ca21, Ba21). Cells are suspended in an alginate solution and its droplets are generated by air jet spray method [14], electrostatic generators [15,16], submerged oscillating coaxial extrusion nozzles [17], conformal coatings [18], and spinning disk atomization [19]. Of these methods, the air jet spray method, which uses a two-channel air droplet microencapsulator, is most commonly used. Two-channel air droplet microencapsulators operate by allowing the alginate cell suspension to drip through an inner channel of the device while the outer channel uses an air jacket to shear off the alginate droplet. Using this method, the diameters of the inner and outer channels, the flow rate of the alginate, and air pressure of the outer channel can be adjusted to vary the microcapsule size [14]. In order to prevent hypoxic damage to cells, microencapsulation must be done relatively quickly even at lower temperature such as 4 C.
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A reduction in capsule size would benefit the cells and also exponentially decrease the total transplant volume. Therefore, much work has been done with various new technologies to make beads as small as 185 μm (diameter) which is about four times smaller than conventional beads (800 μm). The smaller the diameter of the capsules the better the diffusion of nutrients and oxygen to the cells, and it has been shown that microcapsules with a diameter of 600 6 100 μm had improved stability in vivo compared to larger capsules with diameters of 1000 6 100 μm [20]. Uncoated non-permselective alginate microbeads have been reported to have a high permeability (.600 kD). Uptake studies with IgG (150 kD) and thyroglobulin (669 kD) suggested that they permeated uncoated alginate microbeads. Similarly, uncoated alginate microbeads implanted in peritoneum were positive for both IgG and C3 components after only 1 week [21]. Therefore, various immunoreactive molecules from macrophages and T-cells to smaller cytokine molecules such as IL-1β, TNF-α, and IFN- can easily penetrate into the microcapsules and can damage or destroy the encapsulated islets [22]. The role of permselective coating of alginate microcapsules cannot be overemphasized, and this issue can be illustrated with studies of encapsulated islet xenografts in the spontaneously diabetic nonobese diabetic (NOD) mouse. While studies performed with pig islet xenografts encapsulated with permselective alginate microcapsules showed prolonged reversal of hyperglycemia in immunocompetent diabetic NOD mice [23,24], another study using uncoated alginate microcapsules to encapsulate fish islets showed rapid destruction of the xenografts in NOD mice [25], thus highlighting the need to provide immunoisolation for islets within alginate microcapsules designated for transplant studies. Encapsulated islets may incite a host inflammatory response. There are two general targets of the hostderived responses: 1. Inflammatory reaction against the capsule material: With the present technology these reactions can be successfully prevented by applying purification steps to the materials to be used [8]. 2. Host response against the allogenic or xenogenic cellderived bioactive factors or antigens that leak out of the capsules. It results in overgrowth by macrophages and lymphocytes on a small portion (B10%) of the capsules and in a humoral immune response against the encapsulated tissue. It has long been known that islets secrete cytokines upon stress [26]. Encapsulated islets have been shown to produce the cytokines MCP-1, MIP, nitric oxide (NO), and IL-6 under stress (stress induced by adding IL-1β and TNF-α), and these cytokines are well known to contribute to the recruitment
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and activation of inflammatory cells [27]. Also, it has been demonstrated that activated macrophages on the 2 10% microcapsules with overgrowth do secrete the cytokines IL-1β and TNF-α when cultured with encapsulated islets but not with empty capsules [28]. This activation of inflammatory cells results in the production of cytokines, which are deleterious not only to the islet cells in the overgrown capsules but also the islets in the vast majority of transplanted, clean, and nonovergrown capsules.
43.3.1 Semipermeable Membrane Coating Techniques for Alginate To provide immunoisolation for the microcapsules, it is essential to apply a permeability barrier between the encapsulated cells and the host immune system. Applying a polyamino acid layer, followed by an additional outer coating of alginate, creates an adequate barrier from the host system. The positively charged polyamino molecules will readily bind to the negatively charged alginate molecules forming a complex membrane [29,30], which significantly reduces the pore size of the microcapsule and prevents immune cells from entering into it [31 33]. In order to prevent interactions of nonbound polyamines to host tissue, a thin second layer of alginate is added. This polyamino acid barrier also acts as a shell, providing mechanical stability to the microcapsule, allowing for the liquefaction of the inner alginate core [34]. The thickness and pore size of this barrier can be varied through adjustments in incubation time and concentration of the polymer used [35]. The most researched permselective biomaterial is poly-L-lysine (PLL) which was the first material used to generate this barrier [36]; however, more recent research has shown that poly-L-ornithine (PLO) has markedly reduced immune response and provides more mechanical support to the microcapsules. Like PLL, PLO is a positively charged polyamine which, when applied to alginate microcapsules, forms a semipermeable membrane which significantly reduces the porosity of the microcapsules, allowing for immunoisolation without impairing oxygen and nutrient diffusion. PLO has been shown to evoke less of an immune response as well as to have improved mechanical properties in comparison to PLL [37 41]. When compared to alginate PLL microcapsules, alginate PLO microcapsules have been shown to better resist swelling and bursting under osmotic stress [37]. Bead swelling is an important factor to take into consideration because it can cause increases in pore size and permeability, as well as in shear stress, leading to decreased islet viability [42,43]. It has been hypothesized that the improved mechanical properties of alginate PLO
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microcapsules over alginate PLL microcapsules are due to the improved bonding of PLO to alginate owing to the shorter monomer structure of PLO [37,44]. Also, while PLL seems to bind to M G sequences, PLO has been shown to prefer M M sequences [45]. Long-term studies, in which empty alginate PLO microcapsules were injected intraperitoneally in rodents, dogs, or pigs have always resulted in retrieval of intact and overgrowth-free microcapsules up to 1-year postimplant [46]. Usually after the coating with a cationic poly(amino acid), e.g., PLL or PLO is followed by a surface coating of low viscosity alginate, resulting in a microcapsule morphology that presents encapsulated cells in a sol layer of alginate, followed by PLL/PLO coating and gel layer of alginate on exterior, thus creating an alginate PLL/PLO alginate construct known as APA microcapsules.
43.3.2 Studies of Microencapsulated Islet Grafts in Large Animals and Humans The technique of microencapsulation of islets prior to transplantation has shown promise in both large animal trials and pilot clinical trials. Multiple canine and primate studies have been conducted and have demonstrated the ability of encapsulated islets to maintain insulin independence [47 49]. A study conducted by Sun et al. demonstrated the ability of encapsulated islet xenografts to reverse diabetes for periods of time greater than 800 days [11]. A more recent study by Dufrane et al. demonstrated the ability of encapsulated islets to survive and produce insulin in the kidney capsule of Cynomolgus macacus for up to 6 months [51]. Several pilot clinical studies [10,12,45,50] have been conducted in humans. While these trials have failed to establish long-term insulin independence in any of the subjects, they have shown that the implantation of viable encapsulated islets can stabilize the blood glucose levels and reduce the required amount of exogenous insulin required. In a study by Soon-Shiong et al., a long-term type 1 diabetic patient was implanted with 15,000 encapsulated islet equivalents per kilogram body weight and evaluated for up to 9 months posttransplantation. In this study, average blood glucose levels were maintained at 135 mg/dL, and daily insulin requirements decreased from 0.69 6 0.01 U/kg to 0 U/kg, and hyperglycemic episodes (.200 mg/dL) decreased from 11.7% to 6.14% at 9 months. Furthermore, the patient’s quality of life was evaluated and shown to have greatly improved over the duration of the study [11]. The study by Calafiore et al. evaluated two individuals 60 days after receiving the encapsulated allografts. Although insulin independence was not attained, there was a significant reduction in the daily insulin requirements as well as a significant
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reduction in the number of hypoglycemic events [45]. A third human study by Elliot et al. evaluated the effectiveness of porcine xenograft encapsulated islets up to 9.5 years after implantation. In this study, immediately after implantation, the daily insulin dosage was reduced by 30%, and C-peptide was present in urine samples up to 14 months posttransplantation. Retrieval of the capsules 9.5 years later revealed that the islets were still capable of producing insulin, however, the levels of insulin were significantly reduced and C-peptide could not be measured [12]. Tuch et al. studied the safety and viability of human islets microencapsulated in non-permselective alginate microbeads and found that allografts of these encapsulated islets were safe but had no efficacy in diabetic patients [50]. In addition to these small pilot trials, larger clinical trials are underway in New Zealand and Russia by Living Cell Technologies Limited (LCT). LCT is currently performing phase I and II clinical trials with DIABECELLs which are encapsulated neonatal porcine islets that are injected into the peritoneal cavity via laparoscopy at doses of 10,000 20,000 islet equivalents/kg. Currently, the shortterm and long-term safety and effectiveness as well as proper dosage are being evaluated [51].
43.3.3 Microencapsulation Devices One major limitation to the development of the microencapsulated islet technology is the scarcity of highthroughput devices. Current available microencapsulation devices are incapable of efficiently encapsulating large numbers of islets in a reasonable amount of time. This may result in hypoxic stress and loss of islet viability or islet function [3]. A newly proposed alternative procedure for islet microencapsulation utilizes multichannel air jacket microfluidic devices. These devices have the advantage of rapidly encapsulating large numbers of islets into microcapsules at speeds in excess of eight times of those of conventional methods without affecting the functionality of the islets. Additionally, this microfluidic approach can be used to produce microcapsules in the size range of 300 500 μ in diameter and are easily scaled up to increase production rates and can be cost effectively produced using rapid prototyping technology [52]. Figure 43.2 is a picture of PLO-coated APA microcapsules made with a prototype microfluidic device in our laboratory. An important consideration in the evaluation of the devices is the ability to produce microcapsules with uniformity in shape and size as the morphology of the microcapsules used to encapsulate islets plays a critical role in the performance of the BAP construct. Spherical microcapsules are necessary for long-term functionality; irregularities or imperfections in the microcapsules can cause an immune response and result in loss of islet function [53].
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FIGURE 43.2 Alginate PLO alginate (APA) microcapsules made with a prototype microfluidic device. Islet stained with dithizone.
43.3.4 Oxygen Requirements of Islets and the Effect of Transplantation Site on BAP Function Although islets constitute approximately 1% of the pancreas, they receive about 6 10% of its blood flow [54], indicating a disproportionate level of perfusion in which islets receive and consume lots of oxygen. The usual high oxygen requirement of islets is interrupted during the process of islet isolation and processing when islets are used for transplantation, and studies have shown that hypoxia has significant deleterious effects on the survival and function of islets [55]. In the immediate posttransplant period, isolated islets are forced to depend upon diffusion of oxygen and nutrients through peripheral perfusion from the surrounding tissue within the site of transplantation [56], until revascularization by angiogenesis, a process that requires 7 10 days [57]. The peritoneal cavity is a commonly used site for implantation of microencapsulated islets. An advantage of the peritoneal site is the ease of transplantation, but the site has a number of disadvantages, including a low vascular density. Observations suggest that engraftment is slow and inefficient within the peritoneal cavity, probably because of the low vascular density and an extended period of hypoxia before engraftment occurs. Therefore, the death of most of the encapsulated islet grafts owing to severe hypoxia results in the need for large quantities of microencapsulated islets to achieve normoglycemia in studies performed in large animals and humans [3]. Considering the issue of adequate nutrient supply as discussed above, it is necessary to find a site where encapsulated islets are in close contact with the blood stream. Unfortunately, it is difficult to find such a site since it should combine the capacity to bear a large
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graft volume in the immediate vicinity of blood vessels. Transplantation of encapsulated islets is most commonly done intraperitoneally, as it offers the advantages of laparoscopic implantation or through injection and allows ample room to implant numerous microcapsules [58]. In addition to the problem of avascular supply discussed earlier, another major disadvantage is that microcapsules that are implanted intraperitoneally are vulnerable to an immune response from intraperitoneal T-cells and macrophages [59 61] and have less access to the vasculature. This results in an increased likelihood of fibrotic growth over encapsulated islets, a loss of graft functionality, and a delay in insulin uptake into the blood circulation [62]. Consequently, alternative transplantation sites have been investigated, including transplanting into liver [63], kidney capsule, subcutaneously, and into an omentum pouch [3,51,64 66]. In the study conducted by Toso et al., microcapsules were injected into the portal veins of rats; however, the results of the study showed that immunosuppressants were necessary to prevent fibrotic overgrowth, and the risk of hepatic thrombosis makes this approach impractical. The studies by Dufrane et al. that investigated implant sites such as subcutaneous and the kidney capsule showed that encapsulated islets implanted in these two sites had less cellular overgrowth compared to encapsulated islets implanted intraperitoneally. The studies by Dufrane et al. demonstrated the functionality of encapsulated islets implanted within the kidney capsule of primates [51]; however, clinical application would be difficult given the limited space within this site [8]. The attraction for the omentum pouch is that like the kidney capsule, it offers a well-vascularized site for transplantation but has more space for microcapsules and is easier to access [67]. In addition, microencapsulated islets transplanted in the omentum pouch are easily retrievable for posttransplant evaluation [3]. Taking the oxygen requirements of islets into consideration, a recent study has described a promising approach that involves enclosure of microencapsulated islets in a macrochamber specifically engineered for islet transplantation. The subcutaneous implantable device allows for controlled and adequate oxygen supply and provides immunological protection of donor islets against the host immune system. This minimally invasive implantable BAP was shown to normalize blood sugar in streptozotocin-induced diabetic rodents for up to 3 months after subcutaneous transplantation [68]. In another study, investigators showed that encapsulation of solid calcium peroxide within hydrophobic polydimethylsiloxane resulted in sustained oxygen generation that lasted for more than 6 weeks and was enough to prevent hypoxiainduced cell dysfunction and death in insulin-producing cells [69].
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43.4 POTENTIAL ROLE OF ECM-BASED TECHNOLOGY IN THE DEVELOPMENT OF THE BAP In organ bioengineering, seeding of cells on supporting scaffolding material offers an exciting opportunity to enhance the clinical application of bioartificial organs [70 73]. In the last decade, more than 50 patients have received an organ manufactured from autologous cells which were seeded on supporting scaffolding material, with no use of immunosuppression at any time after the implantation. With this groundbreaking, history-making achievement tissue engineering has shown the potential to dramatically impact solid organ transplantation by successfully addressing the two major barriers to solid organ transplantation, namely the need for a new, ideally inexhaustible source of organs and for immunosuppressionfree status posttransplantation. Scaffolds may be synthetic or natural. Natural scaffolds consist of the innate ECM of animal or human organs and can now consistently be produced by perfusion of detergent solutions through the organ’s vasculature, a process called decellularization [74]. Innate ECM represents a biochemically, geometrically, and spatially ideal platform for bioengineering investigations, because it is biocompatible [75], it has both basic components (proteins and polysaccharides) and matrix-bound growth factors and cytokines preserved [76]. It retains an intact and patent vasculature which—when implanted in vivo— sustains the physiologic blood pressure [75], and it is able to drive differentiation of progenitor cells into an organspecific phenotype [77]. Importantly, when cells are seeded on ECM samples, they attach and expand well; when cells are seeded within whole, intact ECM scaffolds and allowed to mature into bioreactors, cells proliferate and show signs of active metabolism and effective function [78 81]. Pancreatic islet transplantation seems to be an ideal ground for the implementation of ECM-based bioengineering technology, because the role of matrix integrin interactions on beta-cell survival and function is well known. This was demonstrated more than a decade ago by Wang and Rosenberg. In their classic study [82], the ability of canine islets in culture to attach to a collagen matrix was shown to decline progressively over 6 days. This decline was accompanied by a decrease in integrin expression and beta-cell function and an increase in apoptosis; yet it could be prevented or delayed by exposure of islets to matrix proteins. This observation provided evidence that the disruption of the cell matrix relationship following pancreatic islets isolation can be prevented by restoration of a culture microenvironment that includes matrix proteins. Later, several investigations were focused on either increasing the survival of islets in vitro through
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support in a solid matrix or on restoring the ECM environment and determining the effect of cell matrix and cell cell interaction on survival. In one study, Daoud et al. tried to identify the factors responsible for postisolation islet survival and promotion of function in vitro [83]. By investigating the effects of collagen I and IV, fibronectin, and laminin on human islet adhesion, survival, and functionality, the authors observed that collagen I/IV and fibronectin are essential for cell adhesion, while fibronectin is the only ECM protein capable of maintaining islet structural integrity and insulin content distribution. Some groups are currently using pancreas ECM to support and enhance the islet viability in vivo in small animal models. Rat pancreata were minced and decellularized to obtain acellular ECM. When seeded on ECM patches, islets adhered well to the pancreatic matrix, maintained their long-term viability and function, and showed a constant glucose-induced insulin release during long-term in vitro incubation. In contrast, islets cultured on plastic or on non-pancreatic matrix showed a progressive reduction [84]. Moreover, when acellular matrix/islet cultures were inserted into poly(vinyl alcohol)/poly(ethylene glycol) tubes to obtain implantable devices, an in vitro constant insulin release could be detected. When the devices were implanted into diabetic rats, a reduced insulin requirement was noted suggesting insulin secretory activity of islets contained in the device. Later, immunofluorescence confirmed the presence of insulinand glucagon-producing cells in the explanted devices. From the foregoing illustration of the important role of ECM on islet function, we envisage a scenario where coencapsulation of micro/nanoparticles of ECM with islets in alginate microcapsules would result in significant enhancement of the function and longevity of encapsulated islet grafts. Indeed, it has recently been shown that the coencapsulation of islets with ECM proteins and mesenchymal stromal cells in a silk hydrogel resulted in enhanced function of the islets in in vitro studies [85]. Overall, ECM-based bioengineering technology holds a great promise for pancreatic islet transplantation research because of the essential role played by ECM islet interactions for islet integrity. Nevertheless, knowledge of pancreas ECM biology and of the interactions with pancreatic islets remains inadequate and represents the biggest hurdle to overcome before it can be used in the development of a BAP for the treatment of diabetes. Acellular pancreas ECM can be produced effectively and consistently; however we still do not know how much damage is caused, and whether, in doing so, we destroy molecular domains that are essential for cells/islet to attach and grow. Also, in other scenarios, innate ECM has been used to drive differentiation of progenitor cells toward an organ-specific phenotype. Therefore, in the
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future, pancreas ECM may be used also to obtain insulinproducing cells from different strains of progenitor cells, a situation which would enhance the availability of islets for BAP constructs.
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[9]
43.5 CONCLUSIONS Alginate-based and ECM-based materials offer complementary technologies, which working together have the potential to advance the development of a viable BAP and provide metabolic function and a cure for type 1 diabetes. At present, we know more about the encapsulation biomaterials and semipermeable membrane materials. We can transplant more volume (smaller microcapsules size) as well as purified alginates/polymers which are biocompatible. With recent developments in technologies for the BAP as discussed in this chapter, there is tremendous hope that routine use of BAP constructs will become a clinical reality in the not so distant future.
[10]
[11]
[12]
[13] [14]
ACKNOWLEDGMENT The authors would like to acknowledge financial support from the National Institutes of Health (RO1 DK080897) and the Vila Rosenfeld Estate, Greenville, NC for the work in Dr. Opara’s laboratory at the Wake Forest Institute for Regenerative Medicine.
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LIST OF ABBREVIATIONS BAP bioartificial pancreas ECM extracellular matrix
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[18]
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nonvascularized microencapsulated adult porcine islets. Transplantation 2009;88:160 9. Xu BY, Yang H, Serreze DV, MacIntosh R, Yu W, Wright Jr JR. Rapid destruction of encapsulated islet xenografts by NOD mice is CD4-dependent and facilitated by B-cells: innate immunity and autoimmunity do not play significant roles. Transplantation 2005;80:402 9. Cardozo AK, Proost P, Gysemans C, Chen MC, Mathieu C, Eizirik DL. IL-1β and IFN-γ induce the expression of diverse chemokines and IL-15 in human and rat pancreatic islet cells, and in islets from pre-diabetic NOD mice. Diabetologia 2003;46:255 66. De Groot CJ, Woodroofe MN. The role of chemokines and chemokine receptors in CNS inflammation. Prog Brain Res 2001;132:533 44. de Vos P, de Haan BJ, de Haan A, van Zanten J, Faas MM. Factors influencing functional survival of microencapsulated islet grafts. Cell Transplant 2004;13:515 24. Bystricky´ S, Malovı´kova´ A, Sticzay T. Interaction of alginates and pectins with cationic polypeptides. Carbohydr Polym 1990;13:283 94. Thu B, Bruheim P, Espevik T, Smidsrod O, Soon-Shiong P, Skjak-Braek G. Alginate polycation microcapsules. II. Some functional properties. Biomaterials 1996;17:1069 79. King GA, Daugulis AJ, Faulkner P, Goosen MFA. Alginate-polylysine microcapsules of controlled membrane molecular weight cutoff for mammalian cell culture engineering. Biotechnol Prog 1987;3:231 40. Halle JP, Leblond FA, Pariseau JF, Jutras P, Brabant MJ, Lepage Y. Studies on small ( , 300 microns) microcapsules: II– Parameters governing the production of alginate beads by high voltage electrostatic pulses. Cell Transplant 1994;3:365 72. Kulseng B, Thu B, Espevik T, Skjak-Braek G. Alginate polylysine microcapsules as immune barrier: permeability of cytokines and immunoglobulins over the capsule membrane. Cell Transplant 1997;6:387 94. Darrabie M, Freeman BK, Kendall Jr WF, Hobbs HA, Opara EC. Durability of sodium sulfate-treated polylysine-alginate microcapsules. J Biomed Mater Res 2001;54:396 9. Gugerli R, Cantana E, Heinzen C, von Stockar U, Marison IW. Quantitative study of the production and properties of alginate/ poly-L-lysine microcapsules. J Microencapsul 2002;19:571 90. Lim F, Sun AM. Microencapsulated islets as bioartificial endocrine pancreas. Science 1980;210:908 10. Darrabie MD, Kendall WF, Opara EC. Characteristics of poly-Lornithine-coated alginate microcapsules. Biomaterials 2005;26:6846 52. Brunetti P, Basta G, Faloerni A, Calcinaro F, Pietropaolo M, Calafiore R. Immunoprotection of pancreatic islet grafts within artificial microcapsules. Int J Artif Organs 1991;14:789 91. Calafiore R, Basta G, Boselli C, Bufalari A, Giustozzi GM, Luca G, et al. Effects of alginate/polyaminoacidic coherent microcapsule transplantation in adult pigs. Transplant Proc 1997;29:2126 7. Calafiore R, Basta G, Luca G, Calvitti M, Calabrese G, Racanicchi L, et al. Grafts of microencapsulated pancreatic islet cells for the therapy of diabetes mellitus in non-immunosuppressed animals. Biotechnol Appl Biochem 2004;39:159 64.
Endocrine Pancreas and Islets of Langerhans
[41] Kizilel S, Garfinkel M, Opara E. The bioartificial pancreas: progress and challenges. Diabetes Technol Ther 2005;7:968 85. [42] Thu B, Bruheim P, Espevik T, Smidsrod O, SoonShiong P, SkjakBraek G. Alginate polycation microcapsules .1. Interaction between alginate and polycation. Biomaterials 1996;17:1031 40. [43] Ching CD, Harland RC, Collins BH, Kendall W, Hobbs H, Opara EC. A reliable method for isolation of viable porcine islet cells. Arch Surg 2001;136:276 9. [44] Inaki Y, Tohnai N, Miyabayashi K, Miyata M. Isopoly-L-ornithine derivative as nucleic acid model. Nucleic Acids Symp Ser 1997;:25 6. [45] Calafiore R, Basta G, Luca G, Lemmi A, Montanucci MP, Calabrese G, et al. Microencapsulated pancreatic islet allografts into nonimmunosuppressed patients with type 1 diabetes: first two cases. Diabetes Care 2006;29:137 8. [46] de Vos P, Faas MM, Strand B, Calafiore R. Alginate-based microcapsules for immunoisolation of pancreatic islets. Biomaterials 2006;27:5603 17. [47] Kendall Jr WF, Collins BH, Opara EC. Islet cell transplantation for the treatment of diabetes mellitus. Expert Opin Biol Ther 2001;1:109 19. [48] Soon-Shiong P, Feldman E, Nelson R, Komtebedde J, Smidsrod O, Skjak-Braek G, et al. Successful reversal of spontaneous diabetes in dogs by intraperitoneal microencapsulated islets. Transplantation 1992;54:769 74. [49] Wang T, Adcock J, Kuhtreiber W, Qiang D, Salleng KJ, Trenary I, et al. Successful allotransplantation of encapsulated islets in pancreatectomized canines for diabetic management without the use of immunosuppression. Transplantation 2008;85:331 7. [50] Tuch BE, Keogh GW, Williams LJ, Wu W, Foster JL, Vaithilingam V, et al. Safety and viability of microencapsulated human islets transplanted into diabetic humans. Diabetes Care 2009;32:1887 9. [51] Zukerman W. ‘Pig Sushi’ diabetes trial brings xenotransplant hope. New Sci 2010;:. [52] Tendulkar S, McQuilling JP, Childers C, Pareta R, Opara EC, Ramasubramanian MK. A scalable microfluidic device for the mass production of microencapsulated islets. Transplant Proc 2011;43:3184 7. [53] Hobbs HA, Kendall Jr WF, Darrabie M, Opara EC. Prevention of morphological changes in alginate microcapsules for islet xenotransplantation. J Investig Med 2001;49:572 5. [54] Eddlestone GT, Oldham SB, Lipson LG, Premdas FH, Beigelman PM. Electrical activity, cAMP concentration, and insulin release in mouse islets of Langerhans. Am J Physiol 1985;248:C145 53. [55] Dionne KE, Colton CK, Yarmush ML. Effect of hypoxia on insulin secretion by isolated rat and canine islets of Langerhans. Diabetes 1993;42:12 21. [56] Davalli AM, Scaglia L, Zangen DH, Hollister J, BonnerWeir S, Weir GC. Vulnerability of islets in the immediate posttransplantation period - Dynamic changes in structure and function. Diabetes 1996;45:1161 7. [57] Menger MD, Jaeger S, Walter P, Feifel G, Hammersen F, Messmer K. Angiogenesis and hemodynamics of microvasculature of transplanted islets of Langerhans. Diabetes 1989;38(Suppl. 1):199 201. [58] Elliott RB, Escobar L, Garkavenko O, Croxson MC, Schroeder BA, McGregor M, et al. No evidence of infection with porcine
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endogenous retrovirus in recipients of encapsulated porcine islet xenografts. Cell Transplant 2000;9:895 901. De Vos P, Van Straaten JF, Nieuwenhuizen AG, de Groot M, Ploeg RJ, De Haan BJ, et al. Why do microencapsulated islet grafts fail in the absence of fibrotic overgrowth? Diabetes 1999;48:1381 8. De Vos P, Smedema I, van Goor H, Moes H, van Zanten J, Netters S, et al. Association between macrophage activation and function of micro-encapsulated rat islets. Diabetologia 2003;46:666 73. Safley SA, Kapp LM, Tucker-Burden C, Hering B, Kapp JA, Weber CJ. Inhibition of cellular immune responses to encapsulated porcine islet xenografts by simultaneous blockade of two different costimulatory pathways. Transplantation 2005;79:409 18. De Vos P, De Haan B, Pater J, Van Schilfgaarde R. Association between capsule diameter, adequacy of encapsulation, and survival of microencapsulated rat islet allografts. Transplantation 1996;62:893 9. Toso C, Mathe Z, Morel P, Oberholzer J, Bosco D, Sainz-Vidal D, et al. Effect of microcapsule composition and short-term immunosuppression on intraportal biocompatibility. Cell Transplant 2005;14:159 67. McQuilling JP, Arenas-Herrera J, Childers C, Pareta RA, Khanna O, Jiang B, et al. New alginate microcapsule system for angiogenic protein delivery and immunoisolation of islets for transplantation in the rat omentum pouch. Transplant Proc 2011;43:3262 4. Kobayashi T, Aomatsu Y, Iwata H, Kin T, Kanehiro H, Hisanga M, et al. Survival of microencapsulated islets at 400 days posttransplantation in the omental pouch of NOD mice. Cell Transplant 2006;15:359 65. Moya ML, Garfinkel MR, Liu X, Lucas S, Opara EC, Greisler HP, et al. Fibroblast growth factor-1 (FGF-1) loaded microbeads enhance local capillary neovascularization. J Surg Res 2010;160:208 12. Kin T, Korbutt GS, Rajotte RV. Survival and metabolic function of syngeneic rat islet grafts transplanted in the omental pouch. Am J Transplant 2003;3:281 5. Ludwig B, Rotem A, Schmid J, Weir GC, Colton CK, Brendel MD, et al. Improvement of islet function in a bioartificial pancreas by enhanced oxygen supply and growth hormone releasing hormone agonist. Proc Natl Acad Sci U S A 2012;109:5022 7. Pedraza E, Coronel MM, Fraker CA, Ricordi C, Stabler CL. Preventing hypoxia-induced cell death in beta cells and islets via hydrolytically activated, oxygen-generating biomaterials. Proc Natl Acad Sci U S A 2012;109:4245 50. Orlando G, Bendala JD, Shupe T, Bergman C, Bitar KN, Booth C, et al. Cell and organ bioengineering technology as applied to gastrointestinal diseases. Gut 2013;62:774 86. Orlando G, Wood KJ, De Coppi P, Baptista PM, Binder KW, Bitar KN, et al. Regenerative medicine as applied to general surgery. Ann Surg 2012;255:867 80.
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[72] Orlando G, Wood KJ, Stratta RJ, Yoo JJ, Atala A, Soker S. Regenerative medicine and organ transplantation: past, present, and future. Transplantation 2011;91:1310 7. [73] Orlando G, Baptista P, Birchall M, De Coppi P, Farney A, Guimaraes-Souza NK, et al. Regenerative medicine as applied to solid organ transplantation: current status and future challenges. Transpl Int 2011;24:223 32. [74] Badylak SF, Taylor D, Uygun K. Whole-organ tissue engineering: decellularization and recellularization of three-dimensional matrix scaffolds. Annu Rev Biomed Eng 2011;13:27 53. [75] Orlando G, Farney AC, Iskandar SS, Mirmalek-Sani SH, Sullivan DC, Moran E, et al. Production and implantation of renal extracellular matrix scaffolds from porcine kidneys as a platform for renal bioengineering investigations. Ann Surg 2012;256:363 70. [76] Wang Y, Cui CB, Yamauchi M, Miguez P, Roach M, Malavarca R, et al. Lineage restriction of human hepatic stem cells to mature fates is made efficient by tissue-specific biomatrix scaffolds. Hepatology 2011;53:293 305. [77] Ross EA, Williams MJ, Hamazaki T, Terada N, Clapp WL, Adin C, et al. Embryonic stem cells proliferate and differentiate when seeded into kidney scaffolds. J Am Soc Nephrol 2009;20:2338 47. [78] Ott HC, Clippinger B, Conrad C, Schuetz C, Pomerantseva I, Ikonomou L, et al. Regeneration and orthotopic transplantation of a bioartificial lung. Nat Med 2010;16:927 33. [79] Ott HC, Matthiesen TS, Goh SK, Black LD, Kren SM, Netoff TI, et al. Perfusion-decellularized matrix: using nature’s platform to engineer a bioartificial heart. Nat Med 2008;14:213 21. [80] Uygun BE, Soto-Gutierrez A, Yagi H, Izamis ML, Guzzardi MA, Shulman C, et al. Organ reengineering through development of a transplantable recellularized liver graft using decellularized liver matrix. Nat Med 2010;16:814 20. [81] Baptista PM, Siddiqui MM, Lozier G, Rodriguez SR, Atala A, Soker S. The use of whole organ decellularization for the generation of a vascularized liver organoid. Hepatology 2011;53:604 17. [82] Wang RN, Rosenberg L. Maintenance of beta-cell function and survival following islet isolation requires re-establishment of the islet-matrix relationship. J Endocrinol 1999;163:181 90. [83] Daoud J, Petropavlovskaia M, Rosenberg L, Tabrizian M. The effect of extracellular matrix components on the preservation of human islet function in vitro. Biomaterials 2010;31:1676 82. [84] De Carlo E, Baiguera S, Conconi MT, Vigolo S, Grandi C, Lora S, et al. Pancreatic acellular matrix supports islet survival and function in a synthetic tubular device: in vitro and in vivo studies. Int J Mol Med 2010;25:195 202. [85] Davis NE, Beenken-Rothkopf LN, Mirsoian A, Kojic N, Kaplan DL, Barron AE, et al. Enhanced function of pancreatic islets coencapsulated with ECM proteins and mesenchymal stromal cells in a silk hydrogel. Biomaterials 2012;33:6691 7.
Microencapsulation Technology Rajesh A. Pareta, John P. McQuilling, Alan C. Farney, and Emmanuel C. Opara Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem, NC
Chapter Outline 43.1 Introduction 43.2 Islet Isolation 43.3 Alginate-Based Microencapsulation of Islets 43.3.1 Semipermeable Membrane Coating Techniques for Alginate 43.3.2 Studies of Microencapsulated Islet Grafts in Large Animals and Humans 43.3.3 Microencapsulation Devices
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43.1 INTRODUCTION The hallmark of type 1 diabetes is destruction of the insulin-producing β-cells of the pancreas by an autoimmune disease [1] resulting in the obligatory need for exogenous insulin to regulate blood glucose in the afflicted individuals. The bioartificial pancreas (BAP) involves a bioengineering approach to treating diabetes with islet transplants, which will secrete pancreatic hormones in response to the host blood glucose like a real pancreas. Presently, islet transplantation is limited by a shortage of pancreas for human islets isolation and allogenic islet recipients also require long-term immunosuppression to prevent rejection and recovery of autoimmunity. Design of BAP should take this into account. In type 1 diabetes, the patients have preexisting antibodies and immune cells primed against β-cell surface markers and insulin [2], and hence a simple islet transplantation without immunosuppression is not viable. In the BAP construct illustrated in Figure 43.1, islets require a protective semipermeable coating to immunoisolate them and preserve their viability and functionality upon transplantation. This approach opens up possibilities for allo- and xenotransplants, and thus has the potential to overcome the shortage of islets while addressing the issue of transplant rejection. To summarize briefly, the BAP construct is not only a viable option to address the human islet shortage but also offers tremendous benefits to the
Regenerative Medicine Applications in Organ Transplantation. © 2014 Elsevier Inc. All rights reserved.
43.3.4 Oxygen Requirements of Islets and the Effect of Transplantation Site on BAP Function 43.4 Potential Role of ECM-Based Technology in the Development of the BAP 43.5 Conclusions Acknowledgment References
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transplant recipient such as relief from long-term use of immunosuppressant drugs. In this review, we will focus on two technologies which may enhance the development of a viable BAP. The first is the use of alginate to microencapsulate islets. Alginate is a widely used naturally occurring biopolymer for islet microencapsulation [3]. The second technology, which is currently in its infancy, is the use of extracellular matrix (ECM) to influence the viability and function of islets in BAP constructs. ECM is a three-dimensional meshwork of proteins and polysaccharides that impart structure and mechanical stability to tissues [3], which can be obtained after decellularization of tissue and organs. Alginate has been extensively used because it has unique properties and advantages for islet microencapsulation such as its mechanical strength, hydrophilic nature, and ability to cross-link at physiological conditions, ECM is a relatively new and upcoming technology where the tissue’s own natural scaffold can be used to promote the interactions between the islets and a construct matrix.
43.2 ISLET ISOLATION In fabricating a BAP, a critical starting point is the procurement of pancreatic islets from an abundant source using reliable techniques to preserve β-cell function. Thus, isolation of whole islets without inflicting any
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FIGURE 43.1 Concept of a BAP. A protective semipermeable coating to immunoisolate islets (iso-, allo-, or xenosource) and preserve their viability and functionality upon transplantation [1].
significant damage to the cells is a key component of developing a viable BAP. A critical balance of composition, process, and duration of collagenase digestion is required for isolating islets with integrity, viability, and high purity with a significant yield. This overall process has tremendous impact on the clinical outcome of islet transplants [4]. The pancreas is digested with combined collagenase and protease action, which disintegrates the intercellular matrix of collagen, releasing islets. These islets are isolated, purified, tested for viability, and sometimes cultured before being transplanted in the patient. Collagenase digestion disrupts islet exocrine tissue adhesive contacts [5]. Thus, shorter duration or lower concentration of collagenase would lead to incomplete digestion of islets from exocrine tissue, leading to reduced yield on purification. On the other hand, extended duration of incubation or higher concentration of collagenase would adversely affect the islet cell cell adhesion, leading to loss of islet integrity and viability. Intra-islet cell cell adhesion is protease sensitive, while extra-islet cell matrix adhesion is collagenase sensitive. In the pig pancreas, very little periinsular capsule is present, and the structural integration of the porcine islet in the exocrine pancreas is almost exclusively cell cell adhesion. In canine, the islets are almost exclusively encapsulated with very little exocrine endocrine cell cell contact. In rodent and human, the situation is intermediate with a tendency toward predominance of cell matrix adhesion. The presence of protease in the collagenase preparations has been reported to reduce the yield and quality of isolated islets from rats [6], however, it is more efficient for the isolation of pig islets [7].
43.3 ALGINATE-BASED MICROENCAPSULATION OF ISLETS It is crucial that the biomaterial used to encapsulate islets must be biocompatible and permeable (for hormonal, nutrient, and oxygen exchange). Hydrogels are such
PART | VI
Endocrine Pancreas and Islets of Langerhans
semisolid materials, which not only are soft but remain stable under mechanical stress. Hydrogels are very attractive for making microcapsules. Hydrogels also provide higher permeability for low-molecular weight nutrients and metabolites. Furthermore, the soft and pliable features of the gel reduce the mechanical or frictional irritations to surrounding tissue [8]. Alginate is one such hydrogel which has been widely used owing to its many excellent properties conducive to islet transplants. Alginate molecules are linear block copolymers of β-D-mannuronic (M) and α-L-guluronic acids (G). It forms a gel in the presence of divalent ions like Ca21 and Ba21. Recent studies have shown that divalent ions cross-link not only G blocks but also blocks of alternating M and G (M G blocks) [9]. Mainly calcium is used for gelling, as barium is known to be toxic and concerns have been raised about patients’ safety if it is used as the crosslinking agent. Alginate is one of the few biopolymers that allow cell encapsulation at physiological conditions. The encapsulation can be done at room or body temperature, at physiological pH, and in isotonic solutions. Alginatebased capsules have been shown to be stable for years in both animals and humans [10 12]. Also, since alginate materials are negatively charged, the attachment of immune cells to the microcapsule is limited due to the negative charge on the cell surface thus making alginate very highly biocompatible [13]. In most tissues it has been shown that maximum diffusion distance for effective oxygen and nutrient diffusion from blood capillary to cells is about 200 μm. Absence of this convection inside a capsule induces a nutrient gradient from the capsule surface to center of cells. Present insights suggest microcapsules as a preferable system over macrocapsules due to their high surface to volume ratio for fast exchange of hormones and nutrients. Microencapsulation uses the interfacial precipitation predominantly, where a polyanionic polymer (alginate) gels with a divalent cation (Ca21, Ba21). Cells are suspended in an alginate solution and its droplets are generated by air jet spray method [14], electrostatic generators [15,16], submerged oscillating coaxial extrusion nozzles [17], conformal coatings [18], and spinning disk atomization [19]. Of these methods, the air jet spray method, which uses a two-channel air droplet microencapsulator, is most commonly used. Two-channel air droplet microencapsulators operate by allowing the alginate cell suspension to drip through an inner channel of the device while the outer channel uses an air jacket to shear off the alginate droplet. Using this method, the diameters of the inner and outer channels, the flow rate of the alginate, and air pressure of the outer channel can be adjusted to vary the microcapsule size [14]. In order to prevent hypoxic damage to cells, microencapsulation must be done relatively quickly even at lower temperature such as 4 C.
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Microencapsulation Technology
A reduction in capsule size would benefit the cells and also exponentially decrease the total transplant volume. Therefore, much work has been done with various new technologies to make beads as small as 185 μm (diameter) which is about four times smaller than conventional beads (800 μm). The smaller the diameter of the capsules the better the diffusion of nutrients and oxygen to the cells, and it has been shown that microcapsules with a diameter of 600 6 100 μm had improved stability in vivo compared to larger capsules with diameters of 1000 6 100 μm [20]. Uncoated non-permselective alginate microbeads have been reported to have a high permeability (.600 kD). Uptake studies with IgG (150 kD) and thyroglobulin (669 kD) suggested that they permeated uncoated alginate microbeads. Similarly, uncoated alginate microbeads implanted in peritoneum were positive for both IgG and C3 components after only 1 week [21]. Therefore, various immunoreactive molecules from macrophages and T-cells to smaller cytokine molecules such as IL-1β, TNF-α, and IFN- can easily penetrate into the microcapsules and can damage or destroy the encapsulated islets [22]. The role of permselective coating of alginate microcapsules cannot be overemphasized, and this issue can be illustrated with studies of encapsulated islet xenografts in the spontaneously diabetic nonobese diabetic (NOD) mouse. While studies performed with pig islet xenografts encapsulated with permselective alginate microcapsules showed prolonged reversal of hyperglycemia in immunocompetent diabetic NOD mice [23,24], another study using uncoated alginate microcapsules to encapsulate fish islets showed rapid destruction of the xenografts in NOD mice [25], thus highlighting the need to provide immunoisolation for islets within alginate microcapsules designated for transplant studies. Encapsulated islets may incite a host inflammatory response. There are two general targets of the hostderived responses: 1. Inflammatory reaction against the capsule material: With the present technology these reactions can be successfully prevented by applying purification steps to the materials to be used [8]. 2. Host response against the allogenic or xenogenic cellderived bioactive factors or antigens that leak out of the capsules. It results in overgrowth by macrophages and lymphocytes on a small portion (B10%) of the capsules and in a humoral immune response against the encapsulated tissue. It has long been known that islets secrete cytokines upon stress [26]. Encapsulated islets have been shown to produce the cytokines MCP-1, MIP, nitric oxide (NO), and IL-6 under stress (stress induced by adding IL-1β and TNF-α), and these cytokines are well known to contribute to the recruitment
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and activation of inflammatory cells [27]. Also, it has been demonstrated that activated macrophages on the 2 10% microcapsules with overgrowth do secrete the cytokines IL-1β and TNF-α when cultured with encapsulated islets but not with empty capsules [28]. This activation of inflammatory cells results in the production of cytokines, which are deleterious not only to the islet cells in the overgrown capsules but also the islets in the vast majority of transplanted, clean, and nonovergrown capsules.
43.3.1 Semipermeable Membrane Coating Techniques for Alginate To provide immunoisolation for the microcapsules, it is essential to apply a permeability barrier between the encapsulated cells and the host immune system. Applying a polyamino acid layer, followed by an additional outer coating of alginate, creates an adequate barrier from the host system. The positively charged polyamino molecules will readily bind to the negatively charged alginate molecules forming a complex membrane [29,30], which significantly reduces the pore size of the microcapsule and prevents immune cells from entering into it [31 33]. In order to prevent interactions of nonbound polyamines to host tissue, a thin second layer of alginate is added. This polyamino acid barrier also acts as a shell, providing mechanical stability to the microcapsule, allowing for the liquefaction of the inner alginate core [34]. The thickness and pore size of this barrier can be varied through adjustments in incubation time and concentration of the polymer used [35]. The most researched permselective biomaterial is poly-L-lysine (PLL) which was the first material used to generate this barrier [36]; however, more recent research has shown that poly-L-ornithine (PLO) has markedly reduced immune response and provides more mechanical support to the microcapsules. Like PLL, PLO is a positively charged polyamine which, when applied to alginate microcapsules, forms a semipermeable membrane which significantly reduces the porosity of the microcapsules, allowing for immunoisolation without impairing oxygen and nutrient diffusion. PLO has been shown to evoke less of an immune response as well as to have improved mechanical properties in comparison to PLL [37 41]. When compared to alginate PLL microcapsules, alginate PLO microcapsules have been shown to better resist swelling and bursting under osmotic stress [37]. Bead swelling is an important factor to take into consideration because it can cause increases in pore size and permeability, as well as in shear stress, leading to decreased islet viability [42,43]. It has been hypothesized that the improved mechanical properties of alginate PLO
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microcapsules over alginate PLL microcapsules are due to the improved bonding of PLO to alginate owing to the shorter monomer structure of PLO [37,44]. Also, while PLL seems to bind to M G sequences, PLO has been shown to prefer M M sequences [45]. Long-term studies, in which empty alginate PLO microcapsules were injected intraperitoneally in rodents, dogs, or pigs have always resulted in retrieval of intact and overgrowth-free microcapsules up to 1-year postimplant [46]. Usually after the coating with a cationic poly(amino acid), e.g., PLL or PLO is followed by a surface coating of low viscosity alginate, resulting in a microcapsule morphology that presents encapsulated cells in a sol layer of alginate, followed by PLL/PLO coating and gel layer of alginate on exterior, thus creating an alginate PLL/PLO alginate construct known as APA microcapsules.
43.3.2 Studies of Microencapsulated Islet Grafts in Large Animals and Humans The technique of microencapsulation of islets prior to transplantation has shown promise in both large animal trials and pilot clinical trials. Multiple canine and primate studies have been conducted and have demonstrated the ability of encapsulated islets to maintain insulin independence [47 49]. A study conducted by Sun et al. demonstrated the ability of encapsulated islet xenografts to reverse diabetes for periods of time greater than 800 days [11]. A more recent study by Dufrane et al. demonstrated the ability of encapsulated islets to survive and produce insulin in the kidney capsule of Cynomolgus macacus for up to 6 months [51]. Several pilot clinical studies [10,12,45,50] have been conducted in humans. While these trials have failed to establish long-term insulin independence in any of the subjects, they have shown that the implantation of viable encapsulated islets can stabilize the blood glucose levels and reduce the required amount of exogenous insulin required. In a study by Soon-Shiong et al., a long-term type 1 diabetic patient was implanted with 15,000 encapsulated islet equivalents per kilogram body weight and evaluated for up to 9 months posttransplantation. In this study, average blood glucose levels were maintained at 135 mg/dL, and daily insulin requirements decreased from 0.69 6 0.01 U/kg to 0 U/kg, and hyperglycemic episodes (.200 mg/dL) decreased from 11.7% to 6.14% at 9 months. Furthermore, the patient’s quality of life was evaluated and shown to have greatly improved over the duration of the study [11]. The study by Calafiore et al. evaluated two individuals 60 days after receiving the encapsulated allografts. Although insulin independence was not attained, there was a significant reduction in the daily insulin requirements as well as a significant
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Endocrine Pancreas and Islets of Langerhans
reduction in the number of hypoglycemic events [45]. A third human study by Elliot et al. evaluated the effectiveness of porcine xenograft encapsulated islets up to 9.5 years after implantation. In this study, immediately after implantation, the daily insulin dosage was reduced by 30%, and C-peptide was present in urine samples up to 14 months posttransplantation. Retrieval of the capsules 9.5 years later revealed that the islets were still capable of producing insulin, however, the levels of insulin were significantly reduced and C-peptide could not be measured [12]. Tuch et al. studied the safety and viability of human islets microencapsulated in non-permselective alginate microbeads and found that allografts of these encapsulated islets were safe but had no efficacy in diabetic patients [50]. In addition to these small pilot trials, larger clinical trials are underway in New Zealand and Russia by Living Cell Technologies Limited (LCT). LCT is currently performing phase I and II clinical trials with DIABECELLs which are encapsulated neonatal porcine islets that are injected into the peritoneal cavity via laparoscopy at doses of 10,000 20,000 islet equivalents/kg. Currently, the shortterm and long-term safety and effectiveness as well as proper dosage are being evaluated [51].
43.3.3 Microencapsulation Devices One major limitation to the development of the microencapsulated islet technology is the scarcity of highthroughput devices. Current available microencapsulation devices are incapable of efficiently encapsulating large numbers of islets in a reasonable amount of time. This may result in hypoxic stress and loss of islet viability or islet function [3]. A newly proposed alternative procedure for islet microencapsulation utilizes multichannel air jacket microfluidic devices. These devices have the advantage of rapidly encapsulating large numbers of islets into microcapsules at speeds in excess of eight times of those of conventional methods without affecting the functionality of the islets. Additionally, this microfluidic approach can be used to produce microcapsules in the size range of 300 500 μ in diameter and are easily scaled up to increase production rates and can be cost effectively produced using rapid prototyping technology [52]. Figure 43.2 is a picture of PLO-coated APA microcapsules made with a prototype microfluidic device in our laboratory. An important consideration in the evaluation of the devices is the ability to produce microcapsules with uniformity in shape and size as the morphology of the microcapsules used to encapsulate islets plays a critical role in the performance of the BAP construct. Spherical microcapsules are necessary for long-term functionality; irregularities or imperfections in the microcapsules can cause an immune response and result in loss of islet function [53].
Chapter | 43
Microencapsulation Technology
FIGURE 43.2 Alginate PLO alginate (APA) microcapsules made with a prototype microfluidic device. Islet stained with dithizone.
43.3.4 Oxygen Requirements of Islets and the Effect of Transplantation Site on BAP Function Although islets constitute approximately 1% of the pancreas, they receive about 6 10% of its blood flow [54], indicating a disproportionate level of perfusion in which islets receive and consume lots of oxygen. The usual high oxygen requirement of islets is interrupted during the process of islet isolation and processing when islets are used for transplantation, and studies have shown that hypoxia has significant deleterious effects on the survival and function of islets [55]. In the immediate posttransplant period, isolated islets are forced to depend upon diffusion of oxygen and nutrients through peripheral perfusion from the surrounding tissue within the site of transplantation [56], until revascularization by angiogenesis, a process that requires 7 10 days [57]. The peritoneal cavity is a commonly used site for implantation of microencapsulated islets. An advantage of the peritoneal site is the ease of transplantation, but the site has a number of disadvantages, including a low vascular density. Observations suggest that engraftment is slow and inefficient within the peritoneal cavity, probably because of the low vascular density and an extended period of hypoxia before engraftment occurs. Therefore, the death of most of the encapsulated islet grafts owing to severe hypoxia results in the need for large quantities of microencapsulated islets to achieve normoglycemia in studies performed in large animals and humans [3]. Considering the issue of adequate nutrient supply as discussed above, it is necessary to find a site where encapsulated islets are in close contact with the blood stream. Unfortunately, it is difficult to find such a site since it should combine the capacity to bear a large
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graft volume in the immediate vicinity of blood vessels. Transplantation of encapsulated islets is most commonly done intraperitoneally, as it offers the advantages of laparoscopic implantation or through injection and allows ample room to implant numerous microcapsules [58]. In addition to the problem of avascular supply discussed earlier, another major disadvantage is that microcapsules that are implanted intraperitoneally are vulnerable to an immune response from intraperitoneal T-cells and macrophages [59 61] and have less access to the vasculature. This results in an increased likelihood of fibrotic growth over encapsulated islets, a loss of graft functionality, and a delay in insulin uptake into the blood circulation [62]. Consequently, alternative transplantation sites have been investigated, including transplanting into liver [63], kidney capsule, subcutaneously, and into an omentum pouch [3,51,64 66]. In the study conducted by Toso et al., microcapsules were injected into the portal veins of rats; however, the results of the study showed that immunosuppressants were necessary to prevent fibrotic overgrowth, and the risk of hepatic thrombosis makes this approach impractical. The studies by Dufrane et al. that investigated implant sites such as subcutaneous and the kidney capsule showed that encapsulated islets implanted in these two sites had less cellular overgrowth compared to encapsulated islets implanted intraperitoneally. The studies by Dufrane et al. demonstrated the functionality of encapsulated islets implanted within the kidney capsule of primates [51]; however, clinical application would be difficult given the limited space within this site [8]. The attraction for the omentum pouch is that like the kidney capsule, it offers a well-vascularized site for transplantation but has more space for microcapsules and is easier to access [67]. In addition, microencapsulated islets transplanted in the omentum pouch are easily retrievable for posttransplant evaluation [3]. Taking the oxygen requirements of islets into consideration, a recent study has described a promising approach that involves enclosure of microencapsulated islets in a macrochamber specifically engineered for islet transplantation. The subcutaneous implantable device allows for controlled and adequate oxygen supply and provides immunological protection of donor islets against the host immune system. This minimally invasive implantable BAP was shown to normalize blood sugar in streptozotocin-induced diabetic rodents for up to 3 months after subcutaneous transplantation [68]. In another study, investigators showed that encapsulation of solid calcium peroxide within hydrophobic polydimethylsiloxane resulted in sustained oxygen generation that lasted for more than 6 weeks and was enough to prevent hypoxiainduced cell dysfunction and death in insulin-producing cells [69].
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43.4 POTENTIAL ROLE OF ECM-BASED TECHNOLOGY IN THE DEVELOPMENT OF THE BAP In organ bioengineering, seeding of cells on supporting scaffolding material offers an exciting opportunity to enhance the clinical application of bioartificial organs [70 73]. In the last decade, more than 50 patients have received an organ manufactured from autologous cells which were seeded on supporting scaffolding material, with no use of immunosuppression at any time after the implantation. With this groundbreaking, history-making achievement tissue engineering has shown the potential to dramatically impact solid organ transplantation by successfully addressing the two major barriers to solid organ transplantation, namely the need for a new, ideally inexhaustible source of organs and for immunosuppressionfree status posttransplantation. Scaffolds may be synthetic or natural. Natural scaffolds consist of the innate ECM of animal or human organs and can now consistently be produced by perfusion of detergent solutions through the organ’s vasculature, a process called decellularization [74]. Innate ECM represents a biochemically, geometrically, and spatially ideal platform for bioengineering investigations, because it is biocompatible [75], it has both basic components (proteins and polysaccharides) and matrix-bound growth factors and cytokines preserved [76]. It retains an intact and patent vasculature which—when implanted in vivo— sustains the physiologic blood pressure [75], and it is able to drive differentiation of progenitor cells into an organspecific phenotype [77]. Importantly, when cells are seeded on ECM samples, they attach and expand well; when cells are seeded within whole, intact ECM scaffolds and allowed to mature into bioreactors, cells proliferate and show signs of active metabolism and effective function [78 81]. Pancreatic islet transplantation seems to be an ideal ground for the implementation of ECM-based bioengineering technology, because the role of matrix integrin interactions on beta-cell survival and function is well known. This was demonstrated more than a decade ago by Wang and Rosenberg. In their classic study [82], the ability of canine islets in culture to attach to a collagen matrix was shown to decline progressively over 6 days. This decline was accompanied by a decrease in integrin expression and beta-cell function and an increase in apoptosis; yet it could be prevented or delayed by exposure of islets to matrix proteins. This observation provided evidence that the disruption of the cell matrix relationship following pancreatic islets isolation can be prevented by restoration of a culture microenvironment that includes matrix proteins. Later, several investigations were focused on either increasing the survival of islets in vitro through
PART | VI
Endocrine Pancreas and Islets of Langerhans
support in a solid matrix or on restoring the ECM environment and determining the effect of cell matrix and cell cell interaction on survival. In one study, Daoud et al. tried to identify the factors responsible for postisolation islet survival and promotion of function in vitro [83]. By investigating the effects of collagen I and IV, fibronectin, and laminin on human islet adhesion, survival, and functionality, the authors observed that collagen I/IV and fibronectin are essential for cell adhesion, while fibronectin is the only ECM protein capable of maintaining islet structural integrity and insulin content distribution. Some groups are currently using pancreas ECM to support and enhance the islet viability in vivo in small animal models. Rat pancreata were minced and decellularized to obtain acellular ECM. When seeded on ECM patches, islets adhered well to the pancreatic matrix, maintained their long-term viability and function, and showed a constant glucose-induced insulin release during long-term in vitro incubation. In contrast, islets cultured on plastic or on non-pancreatic matrix showed a progressive reduction [84]. Moreover, when acellular matrix/islet cultures were inserted into poly(vinyl alcohol)/poly(ethylene glycol) tubes to obtain implantable devices, an in vitro constant insulin release could be detected. When the devices were implanted into diabetic rats, a reduced insulin requirement was noted suggesting insulin secretory activity of islets contained in the device. Later, immunofluorescence confirmed the presence of insulinand glucagon-producing cells in the explanted devices. From the foregoing illustration of the important role of ECM on islet function, we envisage a scenario where coencapsulation of micro/nanoparticles of ECM with islets in alginate microcapsules would result in significant enhancement of the function and longevity of encapsulated islet grafts. Indeed, it has recently been shown that the coencapsulation of islets with ECM proteins and mesenchymal stromal cells in a silk hydrogel resulted in enhanced function of the islets in in vitro studies [85]. Overall, ECM-based bioengineering technology holds a great promise for pancreatic islet transplantation research because of the essential role played by ECM islet interactions for islet integrity. Nevertheless, knowledge of pancreas ECM biology and of the interactions with pancreatic islets remains inadequate and represents the biggest hurdle to overcome before it can be used in the development of a BAP for the treatment of diabetes. Acellular pancreas ECM can be produced effectively and consistently; however we still do not know how much damage is caused, and whether, in doing so, we destroy molecular domains that are essential for cells/islet to attach and grow. Also, in other scenarios, innate ECM has been used to drive differentiation of progenitor cells toward an organ-specific phenotype. Therefore, in the
Chapter | 43
Microencapsulation Technology
future, pancreas ECM may be used also to obtain insulinproducing cells from different strains of progenitor cells, a situation which would enhance the availability of islets for BAP constructs.
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[8]
[9]
43.5 CONCLUSIONS Alginate-based and ECM-based materials offer complementary technologies, which working together have the potential to advance the development of a viable BAP and provide metabolic function and a cure for type 1 diabetes. At present, we know more about the encapsulation biomaterials and semipermeable membrane materials. We can transplant more volume (smaller microcapsules size) as well as purified alginates/polymers which are biocompatible. With recent developments in technologies for the BAP as discussed in this chapter, there is tremendous hope that routine use of BAP constructs will become a clinical reality in the not so distant future.
[10]
[11]
[12]
[13] [14]
ACKNOWLEDGMENT The authors would like to acknowledge financial support from the National Institutes of Health (RO1 DK080897) and the Vila Rosenfeld Estate, Greenville, NC for the work in Dr. Opara’s laboratory at the Wake Forest Institute for Regenerative Medicine.
[15]
[16]
LIST OF ABBREVIATIONS BAP bioartificial pancreas ECM extracellular matrix
[17]
[18]
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