Diabetes mellitus, commonly known as diabetes, is a metabolic disease characterized by high blood glucose levels. Type 1 diabetes results from the failure of the beta cells in pancreas to produce enough insulin [16]. Transplantation of pancreatic islets containing insulin producing β cells has recently been used to cure type 1 diabetes. However, allo-islet transplantation is limited owing to shortage of donors; instead, xenotransplantation using islets from non-human animals has emerged as an alternative source of donor tissue. Owing to their physiological similarities to humans, ease of mass breeding, and availability of breeding in pathogen-free facilities, pigs are considered an optimal animal model for xeno-islet transplantation [1]. Especially, the NPCCs have been used valuably as good as the APIs. Although the maturity of NPCCs is lower than that of APIs, NPCCs have some advantages over APIs including, having a relatively simple and inexpensive islet isolation procedure, ability to develop resistance to hypoxic environments and proliferation in vivo after transplantation [1–3]. For these reasons, we used 3-5-day-old neonatal pigs as the islet source in this study.
Unfortunately, once pig islets are implanted into human or nonhuman primate blood vessels, severe immune reactions such as IBMIR or hyperacute rejection often occur. IBMIR usually occurs due to several tissue factors (TF) expressed in pig islets that mediate coagulation in human blood vessels via activation of an extrinsic pathway. Alpha-galactose or non-gal antigens expressed on the surface of pig cells can also be targets of natural human antibodies, followed by a complementing cascade activation called hyperacute rejection. As a result, grafts are lost following hypoxia by clot formation from the coagulation pathway and cell death by complement activation in the host [3]. To solve these problems, encapsulation, a method of coating pancreatic islets with biocompatible materials to protect them against attack by antibody or complement reactions, has been tried. First, macro-encapsulation uses a device with a semipermeable membrane containing the islet and is implanted next to blood vessels where it releases insulin into the blood stream in response to blood glucose levels [4]. Second, micro-encapsulation, mainly using alginate, has selective permeability and can allow oxygen and nutrients to pass through its porous surface, while blocking multiple cytokines and immune cell infiltration. However, because they use the same size of capsules regardless of the size of islets, it is difficult to conformally coat the islets. Additionally, fibrosis may occur, enclosing the graft after transplantation [17]. Lastly, surface modification of islets (nano-encapsulation) mainly uses polyethylene glycol (PEG) that have “stealth effect” property that blocks the interaction of materials coated with “stealth” polymer (PEG) and components in the blood (immune cells) in vivo [11–18]. Our NPCCs nano-encapsulation strategy uses the modified PEG copolymers (PEG-b-PLA, Polymersome, PSome). Also, Psome has both hydrophilic and hydrophobic properties and can incorporate immunosuppressant or factors involved in cell differentiation or growth [8].
Nano-encapsulation of islets using PEG has been performed in basic HBSS buffer (pH 8.0 or above) to enhance the binding affinity between NHS on PEG and NH2 on ECM of islet [10–12]. However, since these conditions cannot provide suitable cell culture environment, we attempted nano-encapsulation in an environment mimicking the NPCC culture condition. To address the issues above, we tested plain F-10 media, NPCCs culture base medium, with physiological pH (without any supplements) used as the nano-encapsulation reaction buffer. Nano-encapsulation in F-10 with physiological pH showed a similar coating efficiency and maintained normal morphology of NPCCs when compared with the culture condition using basic HBSS buffer (Figure. 3). Therefore, we can propose a platform that minimizes NPCC damage during the nano-encapsulation in an NPCC culture mimicking environment.
Although the nano-encapsulation method for minimizing NPCC damage was established, the residual amount of NPCCs collected after nano-encapsulation decreased when the nano-encapsulation was performed in petri dishes. This means that you require more NPCCs for nano-encapsulation for transplantation. According to previous reports, islet yields were improved in some mouse strains by using BSA during isolation [19]. Also, BSA was used as a suspension culture by pre-coating the surface of cell culture dishes in rat hepatoma cell cultures [13]. Therefore, to increase the amount of NPCCs after nano-encapsulation, 0.25% BSA was added in F-10 nano-encapsulation reaction buffer, same to the concentration of BSA used for culturing NPCCs. As a result, the NPCC recovery rate increased significantly after nano-encapsulation (Figure. 4). This means that the correct number of islets (containing NPCCs) after the nano-encapsulation can be predicted and transplanted by minimizing islets (containing NPCCs) loss. In summary, this study using F-10 with 0.25% BSA for nano-encapsulation showed that i) the normal morphology of NPCCs was maintained, ii) the binding between NHS conjugated PSome and NH2 in ECM of NPCCs was not interfered and iii) the recovery rate of NPCCs after nano-encapsulation increased.
Finally, we attempted to enhance the stability of nano-encapsulation through i) conjugation between PSomes and ii) binding between the PSomes and ECM of NPCCs. First, NHS- and NH2-PEG-b-PLA polymers were mixed proportionally to form the bi-functional PSome (NHS-/NH2-PSome) that can bind to both PSome and ECM of NPCCs. We postulated that conjugation could be efficiently achieved by bi-functional groups within one PSome rather than conjugating two PSomes with mono-functional groups due to the potential interruption caused by binding between PSomes with the same functional group (NHS-NHS and NH2-NH2). As seen from the results, the conformal coating of NPCCs was achieved at a proportion of 9:1 of NHS-/NH2-PSome nano-encapsulated NPCCs, and viability and functionality were maintained (Figure. 5). However, further studies are needed to quantify the strength of the PSome bond, to prove that nano-encapsulation with bi-functional PSomes resulted in more stable encapsulation than that with the mono-functional PSome. Therefore, we suggest that our effective nano-encapsulation conditions mimicking the NPCC culture environment can be used in the nano-encapsulation strategy using islets containing NPCCs.