**4. Improvement on islet encapsulation engineering**

#### **4.1. Co-encapsulation**

Co-encapsulation is the process of adding additional molecules to the capsule to enhance the performance of the encapsulated islets. Encapsulation of islets along with dexamethasone, a corticosteroid serving as local immune suppression, can improve islet survival in mice recipients compared to those islets alone [53]. In another study, co-encapsulation of mouse monocyte macrophage cells and hamster kidney cells with ibuprofen improved the encapsulated cell survival both *in vitro* and *in vivo* [54].

While encapsulation protects the cells inside from large molecules such as antibodies as well as direct cellular contact, smaller molecules such as pro-inflammatory cytokines can still diffuse across most hydrogel gradients due to their smaller molecular weight. To achieve this, an attempt at islet encapsulation with a silicon nanopore membrane found observed cytokine protection and islet viability for over 6 hours, with the islets remaining responsive to glucose levels [55]. Thus, protection from these cytokines may promote capsule survival. In an *in vitro* study performed by Leung, capsules with anti-TNF alpha were able to remove active TNF-α, a pro-inflammatory cytokine from a culture medium, which resulted in better encapsulated cell survival [56].

#### **4.2. Protection against hypoxia**

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,

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

Co-encapsulation is the process of adding additional molecules to the capsule to enhance the performance of the encapsulated islets. Encapsulation of islets along with dexamethasone, a corticosteroid serving as local immune suppression, can improve islet survival in mice recipients compared to those islets alone [53]. In another study, co-encapsulation of mouse monocyte macrophage cells and hamster kidney cells with ibuprofen improved the encapsulated

While encapsulation protects the cells inside from large molecules such as antibodies as well as direct cellular contact, smaller molecules such as pro-inflammatory cytokines can still diffuse across most hydrogel gradients due to their smaller molecular weight. To achieve this, an attempt

manufacturing practices (GMP) adherence to satisfy regulatory standards.

**4. Improvement on islet encapsulation engineering**

**4.1. Co-encapsulation**

224 Challenges in Pancreatic Pathology

cell survival both *in vitro* and *in vivo* [54].

while internal permeability tends to vary between batches [38].

In order to improve oxygen supply to the cell, access to a rich vascular bed is essential. Addition of the angiogenic factor, fibroblast growth factor 1 (FGF-1), into capsule was able to affect a continuous FGF-1 release for a 1-month period *in vitro* [57]. In another study, encapsulation of solid peroxide within polydimethylsiloxane resulted in sustained oxygen release from the matrix for approximately 6 weeks [58].

Extracellular matrix components, derived from laminin, have been shown to improve islet human islet function for encapsulated islet transplants. These extracellular matrix components are also found in native islets located in the pancreas prior to islet isolation. In this case, a variety of laminin-derived peptides or collagen were co-encapsulated with human islets and islet function was measured *in vitro*. Islet viability and insulin response to glucose were improved by the addition of laminin-derived peptide or collagen [59].

#### **4.3. Prevascularization**

Prevascularization of islet implant consists of establishing a well-vascularized matrix or scaffold, by implanting the scaffold, then encouraging angiogenesis that leads to scaffold penetration by microcapillaries. Angiogenesis is promoted by addition of fibrin at the time of scaffold implant. Islets were then added to the scaffold after a certain duration that has been shown to allow significant vascularization, as shown in **Figure 2**. This method was shown to improve subcutaneous islet efficacy in restoring normoglycemia when compared to subcutaneous transplants of islets alone [60].

**Figure 2.** Prevascularized scaffold for islet transplant. Device is implanted subcutaneously 28 days before the introduction of the β-cell clusters during which the foreign body response and neovascularization are completed (A). The device contains polyethylene rods with high hydrophobicity to avoid cell adhesion. Upon removal of the rods, the islets can be infused (B).

#### **4.4. Toward GMP standard**

One of the key issues facing the engineering of encapsulating material for islet transplantation would be to define standards for the materials. The standards required contain the choice of raw material, the purification method and quality control of the purification, the shape of the device used for encasing the islets, and the quality of the encased islets. The lack of such standards is likely to account for the current variability in the results reported in the literature on the encapsulated islet transplant.

As an example of the standard necessary for clinical translation of the encapsulation technology, commercially available alginates used to create islet capsules have been found to contain pathogen-associated molecular patterns (PAMPS). PAMP such as peptidoglycan, lipoteichoic acid, and flagellin among other proteins, endotoxins, and polyphenols [61] can trigger recognition by the innate immune system. PAMPS are recognized by toll-like receptors (TLRs) and pattern-recognition receptors (PRRs) [61, 62], leading to pericapsular fibrotic overgrowth (PFO) [63] as the immune system attempts to isolate the graft. PFO severely hinders graft survival by preventing diffusion of nutrients and waste.

In addition to cellular adhesion and PFO, death of encapsulated islets may also be caused by chemokines and cytokines that are small enough to pass through the permeable capsules [64]. TLRs, upon recognition and binding of PAMPS to the receptor surface, initiate an intracellular signaling cascade ultimately resulting in the secretion of a host of inflammatory cytokines attributed to translocation of the NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) into the nucleus [65].

Before alginate can be used for clinical transplantation, it will need further development in the GMP manufacturing and purification of the raw materials, to ensure a low amount of PAMP detectable by the recipient's immune system. In addition, the production of the encapsulated islets, including the islet isolation and the encapsulation process, needs to achieve a threshold of standard of quality to ensure a consistent and reliable result, to make it possible to compare the effect of the variety of encapsulation techniques and improvements.
