**3. Biomaterials used for islet encapsulation**

One of the important steps to bring islet encapsulation into widespread clinical use is to develop a standard for the type of biomaterial used and the dose of islets to be infused. The type of biomaterial has also been shown to affect graft survival. A test of several encapsulation methods using alginate with or without poly L-lysine (PLL) as well as with high guluronic (G) or mannuronic (M) acid in mouse recipients showed that significant results were achieved with PLL-free high M microcapsules, showing sustained normoglycemia for 8 weeks [24]. Likewise, improved capsule integrity and graft function could be achieved by altering the concentration of alginate in their xenotransplants into diabetic Lewis rats [25].

Currently, the most common employed method for islet encapsulation involves alginate microcapsules [24, 26, 27]. The original device was developed over three decades ago as capillary fibers in a culture-coated medium [28], shaped as arterial-venous shunts into diabetic canines. These devices showed promising results with several canines achieving reduced insulin requirements [29, 30]. Vascular shunts are limited by the volume and number of islets that can be contained within the fibers. Elongation of the fibers resulted in increased fibrosis, leading to abandonment of this device as the higher dose of islets needed for human recipients would require such large fibers that resulted in a large amount of fibrosis [31]. Other macroscale devices have seen less use due to their increased immunogenicity as well as the larger diffusion parameters required for oxygen and nutrients to reach the cell.

Nanoencapsulation has an advantage compared to other techniques because of its more efficient diffusion capability. With a better surface area to volume ratio, this means that nanoencapsulation can improve insulin response time to blood glucose levels, offering the protection of encapsulation without compromising tissue function due to the physical barrier. PEG has been used for nanoencapsulation devices and can be cross-linked through exposure to UV or visible light. This characteristic also allows for a reduction in the amount of damage done to the capsule's inner cells normally achieved by other cross-linking methods. On the other hand, PEG biocompatibility still leaves much to be desired compared to other hydrogels, and complete protection from cytokines is still not achieved [32]. Despite these concerns, some success has been attained with these gels [33].

By far the most common encapsulation device is a microscale vehicle. These capsules have mechanical stability, optimal surface area to volume ratio, and have enhanced immunologic profiles [26, 33]. Microscale device is also easily made using standard droplet-based encapsulators that produces consistent size and shape of the resulting capsules [34, 35], as shown in **Figure 1**.

Microcapsules can also be easily made using materials other than alginate. The most common synthetic chemicals used for microcapsule production are poly ethylene oxide, poly acrylic acid, poly vinyl alcohol, polyphosphazene, and polypeptides and their derivatives. Natural occurring hydrogels include gelatin, fibrin, agarose, hyaluronate, chitosan, and alginate [36, 37]. Poly glycolic and lactic acid polymers continue to be the most commonly used synthetic materials used in medical devices.

Regardless of the materials used, capsule materials still face the fundamental flaw of being foreign materials. Thus there will always be the possibility they will elicit a greater immune response, eventually leading to fibrosis and loss of the encased cells. As such, it is important to ensure that the materials are nontoxic and purified prior to microcapsule production.

The type of biomaterial has also been shown to affect graft survival. A test of several encapsulation methods using alginate with or without poly L-lysine (PLL) as well as with high guluronic (G) or mannuronic (M) acid in mouse recipients showed that significant results were achieved with PLL-free high M microcapsules, showing sustained normoglycemia for 8 weeks [24]. Likewise, improved capsule integrity and graft function could be achieved by altering the concentration of alginate in their xenotransplants into diabetic Lewis rats [25].

Currently, the most common employed method for islet encapsulation involves alginate microcapsules [24, 26, 27]. The original device was developed over three decades ago as capillary fibers in a culture-coated medium [28], shaped as arterial-venous shunts into diabetic canines. These devices showed promising results with several canines achieving reduced insulin requirements [29, 30]. Vascular shunts are limited by the volume and number of islets that can be contained within the fibers. Elongation of the fibers resulted in increased fibrosis, leading to abandonment of this device as the higher dose of islets needed for human recipients would require such large fibers that resulted in a large amount of fibrosis [31]. Other macroscale devices have seen less use due to their increased immunogenicity as well as the

Nanoencapsulation has an advantage compared to other techniques because of its more efficient diffusion capability. With a better surface area to volume ratio, this means that nanoencapsulation can improve insulin response time to blood glucose levels, offering the protection of encapsulation without compromising tissue function due to the physical barrier. PEG has been used for nanoencapsulation devices and can be cross-linked through exposure to UV or visible light. This characteristic also allows for a reduction in the amount of damage done to the capsule's inner cells normally achieved by other cross-linking methods. On the other hand, PEG biocompatibility still leaves much to be desired compared to other hydrogels, and complete protection from cytokines is still not achieved [32]. Despite these concerns, some

By far the most common encapsulation device is a microscale vehicle. These capsules have mechanical stability, optimal surface area to volume ratio, and have enhanced immunologic profiles [26, 33]. Microscale device is also easily made using standard droplet-based encapsulators that produces consistent size and shape of the resulting capsules [34, 35], as shown

Microcapsules can also be easily made using materials other than alginate. The most common synthetic chemicals used for microcapsule production are poly ethylene oxide, poly acrylic acid, poly vinyl alcohol, polyphosphazene, and polypeptides and their derivatives. Natural occurring hydrogels include gelatin, fibrin, agarose, hyaluronate, chitosan, and alginate [36, 37]. Poly glycolic and lactic acid polymers continue to be the most commonly used synthetic

Regardless of the materials used, capsule materials still face the fundamental flaw of being foreign materials. Thus there will always be the possibility they will elicit a greater immune response, eventually leading to fibrosis and loss of the encased cells. As such, it is important to ensure that the materials are nontoxic and purified prior to microcapsule production.

larger diffusion parameters required for oxygen and nutrients to reach the cell.

success has been attained with these gels [33].

materials used in medical devices.

in **Figure 1**.

222 Challenges in Pancreatic Pathology

**Figure 1.** Porcine islet encapsulated in alginate. Isolated juvenile porcine islets (from 22 to 24 days old pigs, matured for 7 days) were encapsulated in 2.5% low viscosity mannuronate (Pro-Nova UPLVM) alginate (Novamatrix) using an electrostatic gas-driven encapsulator (Nisco Engineering AG).

Because of their tolerability, biologically derived materials have been of interest for islet encapsulation. One possible material is collagen, a naturally derived polymer that is the most widely used in medical devices today. However, collagen gels exhibit poor strength, which are expensive and have high variability of purity, making standardization of the process a problem [37]. Comparatively, alginate has excellent biocompatibility, hydrophilic properties, easy gelation process, stable architecture, and relatively low cost. Alginate is polysaccharide derived from seaweed, which can be highly purified to prevent foreign body response [38]. Impure alginate has been implicated in islet cell necrosis and recruitment of inflammatory mediators [39].

Alginate is a polymer of 1-4 linked β-D-mannuronic acid (M) and 1-4 linked α-L-guluronic acid (G). This polysaccharide can contain varying concentrations of M and G carbohydrates, which provides a variety of molecular weight, stability, permeability, and immunogenicity. High G alginates form gels, which are smaller and stronger than high M alginates [38]. High-M alginate was often avoided when immunosuppression was the desired outcome, because mannuronic acid tends to provoke both innate and antibody-mediated immune response, independent of the type of cation used for cross-linking (Ca2+ or Ba2+). High-M also triggers macrophages to secrete pro-inflammatory cytokines including IL-1, IL-6, and TNF-α through interactions with the monocyte CD14 receptor [39]. However, recent studies seem to contradict these earlier findings, reporting a higher amount of cellular adhesion to high-G alginate capsules when compared to high-M alginate [40, 41]. It is likely that the observed difference in the immune response depends not on the identity of the alginate material, but instead on the quality of the alginate purification method [42, 43].

Using surface modification, poly-methyl co-guanidine-cellulose sulfate/poly l-lysine-sodium alginate (PMCG)-CS/PLL was used for syngeneic transplant into T1DM canine recipients [44]. Their study reported normoglycemia in the canines for approximately 160 days, with one canine achieving euglycemia for 214 days [45].

Alginate converts into a gel form by ionic cross-linking with bivalent cations such as calcium, magnesium, and more commonly barium [46]. Cross-linking establish a mesh of porous material that allows bidirectional flow of materials, including oxygen, nutrients, and hormones (especially insulin). However, hydrogel polymerization does not result in uniform pore size, while internal permeability tends to vary between batches [38].

An increase in the degree of cross-linking results in gels that have superior mechanical strength but inversely reduces the size of the pores available for diffusion. It is possible to artificially organize the islets in alginate gels into clusters mimicking natural islets [47].

Surface modification using polycations and anions can change the permeability and mechanical strength of alginate, but the polarity tends to increase the immune response. Common molecules used for this purpose include: poly-d-lysine (PDL), polyethylene glycol (PEG), poly-L-ornithine (PLO), and poly-L-lysine (PLL). This effect can be counteracted by adding another layer of alginate to prevent direct contact with a polar surface [38], or by modifying the alginate [48].

Capsule fibrosis was the most significant problem encountered when utilizing alginate capsules [23, 49–51]. Theoretically, immune isolation is achieved by encapsulation of the cells, but some levels of immune rejection and foreign body response still occur. Also, while oxygen and nutrients are able to freely diffuse across a matrix, studies have shown that at the time of explant, histology showed a necrotic core in the encapsulated islets without evidence of fibrosis. This suggests inadequate oxygen diffusion into the center of the encapsulated islets [52].

The results demonstrated by these prior studies suggest that there are key points to be considered during engineering of the encapsulation vehicle. The raw and the purified capsule material must be nontoxic, while the purification method needs to be reproducible across batches. The polymerization of the capsule material needs to be noncytotoxic to the islets. If there is any degradation of the material, it must follow physiological tissue growth and its products must not adversely affect the coated cells or human body. For clinical application, it would be important for the capsule engineering to be easily scalable, while maintaining good manufacturing practices (GMP) adherence to satisfy regulatory standards.
