**1. Introduction**

Type 1 diabetes (T1D) is a chronic autoimmune disease representing a major health care problem worldwide (Tierney et al., 2002). T1D is caused by islet-reactive immune T cells that destroy insulin-producing pancreatic β-cells. Transplantation of insulin-producing pancreatic islets by their injection in vascularized organs has been recently recognized as a promising path to curing diabetes (Meloche, 2007; Robertson, 2000). However, despite the significant promise, the clinical application of the procedure remains limited due to (a) limited supply of islets suitable for transplantation, (b) a hypoxia because of a low tension of oxygen at the implantation sites and (c) an acute rejection during transplantation. One of the challenges is associated with isolation and culturing islets *in vitro* before injection. In the pancreas, endocrine cells of the islet clusters are separated from exocrine cells by a discontinuous mantle of collagen fibers defining their respective basement membrane. During collagenase isolation of islets from the pancreas, further disruption of the islet mantle results in preparations exhibiting various morphological changes (islet fragmentation, fusion) under routine tissue culture conditions, particularly in human islets (Lacy & Kostianovsky, 1967). Attenuation of islet viability and functionality accompanies these morphological changes. The second issue is associated with islet transplantation which requires immunosuppression to protect the donor islets from the host immune response and prevent implant rejection and post-surgery inflammations (Ricordi & Strom, 2004). Despite the fact that a range of immunosuppressive drugs have demonstrated pharmacologically inhibitory effects on pro-inflammatory cytokines (Riachy et al., 2002; Contreras et al., 2002; Lv et al., 2008; Stosic-Grujicic et al., 2001), the use of immunosuppressive molecules is very specific since they can induce non-specific suppression of the immune system resulting in serious side effects and increased risk of infection which can work against the benefits of a transplant (Narang & Mahato, 2006). These issues have inspired the development of a number of strategies to prevent immunogenic reactions and stabilize islet morphology and functionality, both *in vitro* and following transplantation *in vivo* (Chandy et al., 1999; Abalovich et al., 2001). Two major approaches have been introduced to prevent immunogenic reactions on the islet surfaces: macro and microencapsulation of the islet cells and islet cell surface modification (Fig. 1) (De Vos et al., 2003; Panza et al., 2000; Scott & Murad, 1998; Opara et al., 2010).

Encapsulation and Surface Engineering of Pancreatic Islets: Advances and Challenges 5

factors generated by cells damaged during these processes, providing sufficient oxygen and nutrients to allow islet cells to recover. After isolation of islets from donors, it is crucial to maintain islet viability and functionality until transplantation to give sufficient time to perform microbiological tests as well as donor matching and recipient pre-conditioning.

Modifying the islet preparations for reducing immunogenicity by altering temperature (Kim et al., 2005; Stein et al., 1994), or media composition is one of the advantages for islet preculture (Ricordi et al., 1987; Murdoch et al., 2004). For example, supplementation of culture media with lactogen hormones has been shown to minimize β-cell loss during pretransplant culture leading to a higher β-cell survival rather than proliferation (Yamamoto et al., 2010; Nielsen, 1982). When islets were cultured in media supplied with recombinant human prolactin (rhPRL) for 48 h, production of interferon-gamma (IFN-γ), tissue necrosis factor-alpha (TNF-α), interleukins cytokines, IL-6, IL-8 and microphage inflammatory protein-1-β was comparable with the control group of islets with no increase in proinflammatory mediators in the presence of rhPRL suggesting no elevated immunogenicity. Furthermore, the PRL treatment of islet preparations resulted in decreased apoptosis in βcell subsets, suggesting β-cell specific anti-apoptotic effects of rhPRL (Yamamoto et al., 2010). Another possible issue with the pre-cultured islets is the possibility of islet fusion during incubation, which may lead to hypoxia and starving of the cells. Those result in central necrosis of fused islet aggregates causing a significant loss of islet potency and

Apoptosis of human islets after isolation from supporting extracellular matrix is a very common cell pathway *in vitro*. During the first steps towards apoptosis integrin expression is diminished and, consequently, phenotype characteristics are lost and islets stop secreting insulin (Ris et al., 2002). Exploring the parameters important for preventing pre-apoptotic events should help in preserving islet viability and function for long periods of time. The effects of two types of collagen, type I and type IV, and fibronectin, proteins that are generally present in the cell-supporting matrix have been explored (Daoud et al., 2010). Islets have a tendency to spread and form a monolayer on surfaces *in vitro*. The islet monolayer can still be viable without preserving the phenotype characteristics, however, the normal insulin secretion of islets will be lost. Daoud et al. showed that integrity and insulin production of islets can be preserved by presence of fibronectin in the medium (Daoud et al., 2010). Both types of collagen increased the viability of islets from 24 to 48 hours *in vitro*. Several studies revealed an increasing survival of islets *in vitro* when embedded in a solid matrix. Culture in collagen I gels obtained from rat tail and fibrin gels have shown promising for prolonging islet survival

**3. Approaches to prevent immunogenic reactions on the islet surfaces** 

The immune reactions against encapsulated islets can be divided into non-specific immune activation initiated by surgery; a host response against the encapsulating materials and implanted islets provokes the immune response by releasing the bioactive molecules.

The instant blood-mediated inflammatory reaction (IBMIR) is an inflammatory reaction that occurs when isolated islets come in contact with human blood. This process is responsible for islets destruction together with overall failing of transplantation. One of the major

viability (Ichii et al., 2007).

(Wang & Rosenberg, 1999; Beattie et al., 2002).

Islet macro/microencapsulation strategy is based on embedding islets in solid matrices, allowing for the creation of a semi-permeable environment around islets capable of immune-protection and for mass and oxygen transfer (Beck et al., 2007; Weber et al., 2007). For that, the isolated islets are usually entrapped individually or as islet clusters in thick gels, for example, high-viscous alginate droplets stabilized with divalent ions of barium or calcium (Zimmermann et al., 2001). Islet surface modification strategy involves covalent conjugation of molecules to islet cell surfaces. However, this technology is limited to the introduction of specified functional small molecules to cells and might interfere with cell physiology (Rabuka et al., 2008; Paulick et al., 2007). Layer-by-layer (LbL) technique has been recently applied as a new approach to modify islet surfaces (Krol et al., 2006; Wilson et al., 2008). The technique is based on alternating LbL deposition of water soluble polymers on surfaces from aqueous solutions which results in nano-thin coatings of controllable thickness and composition (Decher & Schlenoff, 2002; Kharlampieva & Sukhishvili, 2006; Tang et al., 2006).

Unlike bulk encapsulating materials, the ultrathin conformal coating affords a faster response to stimulation and the possibility to bind factors or protective molecules to the protective ultrathin shell with the later slow triggered release of these molecules (Chluba et al., 2001). By selecting specific pairs of polyelectrolytes, a defined cutoff of the coating (Kozlovskaya & Sukhishvili, 2006) is possible, as is inhibitor binding to prevent graft rejection, microphage attacks, or antibody recognition (Kim & Park, 2006). Here, we review methods and devices designed for protecting isolated islets from host immune responses while allowing transport of essential nutrients. We also discuss challenges of various approaches developed for encapsulation of individual islets in thin coatings that conform to the islet surfaces, fabricated using a number of physical and chemical processes.
