Biocompatible Coating of Encapsulated Cells Using Ionotropic Gelation

et al. (2013) Biocompatible Coating of Encapsulated Cells Using Ionotropic Gelation. PLoS ONE 8(9): e73498. doi:10.1371/journal.pone.0073498 Biocompatible Coating of Encapsulated Cells Using Ionotropic Gelation Friederike Ehrhart 0 Esther Mettler 0 Thomas Bse 0 Matthias Max Weber 0 Julio Alberto Vsquez 0 Heiko 0 Mrio A. Barbosa, Instituto de Engenharia Biomdica, University of Porto, Portugal 0 1 Biophysik und Kryotechnologie , Fraunhofer IBMT, Sankt Ingbert, Germany, 2 Schwerpunkt Endokrinologie und Stoffwechselerkrankungen, Universitatsmedizin Mainz, Mainz, Germany , 3 Departmento Biologia Marina, Universidad Catolica del Norte, Coquimbo, Chile, 4 Lehrstuhl fur Molekulare und Zellulare Biotechnologie/Nanotechnologie, Universitat des Saarlandes , Saarbrucken , Germany The technique of immunoisolated transplantation has seen in the last twenty years improvements in biocompatibility, long term stability and methods for avoidance of fibrosis in alginate capsules. However, two major problems are not yet solved: living cellular material that is not centered in the capsule is not properly protected from the hosts' immune system and the total transplant volume needs to be reduced. To solve these problems, we present a method for applying fully biocompatible alginate multilayers to a barium-alginate core without the use of polycations. We report on the factors that influence layer formation and stability and can therefore provide data for full adjustability of the additional layer. Although known for yeast and plant cells, this technique has not previously been demonstrated with mammalian cells or ultra-high viscous alginates. Viability of murine insulinoma cells was investigated by live-dead staining and live cell imaging, for murine Langerhans' islets viability and insulin secretion have been measured. No hampering effects of the second alginate layer were found. This multi-layer technique therefore has great potential for clinical and in vitro use and is likely to be central in alginate matrix based immunoisolated cell therapy. Current address; Institut fr Pathologie; Johannes Gutenberg Universitt; Mainz; Germany - Funding: The authors thank Bundesministerium fr Bildung und Forschung (BMBF) for granting (Grant No. 031581B (given to HZ)). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interest exist. Encapsulation of cells has a long history in biotechnology especially for the immunoisolated transplantation of endocrine cells or tissues. The encapsulation and implantation of Langerhans islets with alginate for diabetes therapy is possibly the most famous example for this technique [1-5]. Alginate is the preferred material for this application because it is biocompatible, polymerizes under gentle conditions and is non toxic in both the polymerized state and in solution. It is an unbranched polymer containing manuronic- (M) and guluronic (G) acid. The ionotropic gel is liquid only if monovalent cations are available to saturate the hydroxyl groups of M or G. Alginate solution is viscous but can be used to suspend cells. Capsule formation occurs when droplets of liquid alginate fall into a polymerization bath containing divalent cations, whereas different methods and techniques for droplet formation are yet established [3,6]. Ca2+ or Ba2+ cross link the alginate chains and form a stable hydrogel. Other di- or multivalent cations form rather weak gels which cannot be used for immobilization purposes [7-8]. Hydrogels made of UHV (ultra high viscous) alginates, characterized by extraordinary high molecular weight, have special properties, which makes them ideal for biomedical applications especially in the field of long term immunoisolation of transplanted cells [4,9,10]. The high molecular weight (high viscosity) alginates are intrinsically more biocompatible than those of low molecular weight (low viscosity) [11]. UHV alginate sheets can be very stable and under special conditions withstand pressures of 2 bar [12]. A broad variety of cell and tissue types have been successfully used for encapsulation including genetically engineered cells, stem cells and stem cell derived surrogate tissue [5,13]. Nevertheless, there are some problems, which hamper the immediate start of clinical trials. Long term stability and fibrosis of the capsule is probably the most commonly addressed problem which is dealt in many publications and shall not be a point of discussion in this work. Here, we consider two other issues: firstly, embedded living material in decentralized position, which is not properly protected against the hosts immune system because of too thin alginate layer and secondly, the total transplant volume. The first issue is a question of transplant safety and long term stability. Donor cells at the capsules surface are in danger from the hosts immune system and an overacting or long-term immune reaction can cause other problems like local inflammation and graft failure. Reduced life time of the transplant, the need for repeated transplantations and other medical problems could outweigh the benefits of immunoisolated transplantation. The second issue, the transplant volume, depends mainly on the amount of tissue which is necessary to restore functionality. The ratio of cell/ matrix material is usually quite low to avoid cells at the capsules surface resulting in a quite high transplantation volume. E.g. to achieve normal blood glucose regulation in a human adult metabolism, about 1 x 106 Langerhans islets are necessary. If every islet is singly encapsulated in a 500 m capsule the total transplantation volume would be about 100 ml. Putting more than one islet in a capsule would increase the risk of an islet, to be placed at the capsules surface with the above described risks. Coated capsules offer a solution to these problems. If a defined layer protects all embedded cells and tissues, higher cell loads are possible, reducing the total transplantation volume. By exploiting the polyanionic character of alginate, polycations are used in alternation with alginate (or another polyanions) to build up a poly-electrolyte shell which increases the stability and protection properties of the capsule [14-16]. Poly-L-Lysine (PLL) is the most popular polycation for this purpose [17-18] but other substances like polyacrylamide (PAA), pectin and chitosan can be used as well for coating a cell loaded alginate core [19-20]. However, PLL is highly immunogenic and has been shown to induce fibrotic overgrowth after implantation in animal models [21]. A final layer of alginate is necessary to mask the PLL layer (so called alginate-PLL-alginate (APA) capsules). Such APA capsules are still more immunogenic than massive alginate capsules, leading to rapid graft failure as the fibrotic overgrowth cuts off the nutrient supply [21-25]. Using sensitive m (...truncated)