Hybrid Microspheres For Cell Encapsulation

Cell microencapsulation is an active interdisciplinary research field. It requires the input from materials sciences, organic and physical chemistry, biology and medicine. Hydrogels are particularly suitable materials to form microspheres for cell entrapment. The present work describes the development of two novel types of hybrid microspheres, their physical properties, in vitro and in vivo biocompatibility as well as the application for the microencapsulation of three different cell types. The first part describes an approach to produce hybrid microspheres by combining fast ionotropic gelation of sodium alginate (Na-alg) with calcium ions and slow covalent crosslinking of poly(ethylene glycol) (PEG) derivatives. A one-step extrusion process yields under physiological conditions alginate-poly(ethylene glycol) hybrid microspheres (alg-PEG-M) consisting of a PEG chemical hydrogel network interpenetrating the physical calcium alginate hydrogel network. The physical properties of alg-PEG-M such as mechanical resistance, elasticity, permeability, and water uptake are adjustable by the macromolecular characteristics of the components, their concentration as well as the process conditions. In vitro cytotoxicity tests did not show cytotoxic effects for EC219 rat endothelial cells, ECp23 mouse endothelial cells and RAW264.7 murine macrophages upon incubation with alg-PEG-M. The immune response to alg-PEG-M intraperitoneally implanted into mice was similar as the response to injected medium used as control. The feasibility of cell microencapsulation within alg-PEG-M was confirmed by encapsulating two cell models, human islets of Langerhans and human mesenchymal stem cells (MSC). Encapsulated human islets continued insulin secretion upon stimulation. Insulin release revealed technology dependency. The viability of encapsulated MSC, their proliferation, and their differentiation into adipocytes was confirmed. In the second part, eleven heterobifunctional PEG derivatives having variable functionalities were synthesized as potential candidates for biomaterials modification. The synthesis involves selective monotosylation of symmetrical PEG. For both end groups, a degree of functionalization > 95 % was achieved in all cases. Heterobifunctional α-amine-ω-thiol PEG was selected and grafted onto alginate chain units as an example of pegylation technology. The grafted alginate molecules maintained their gelling capacity in presence of divalent cations, while a novel chemically cross-linked hydrogel was obtained via simultaneous spontaneous disulfide bond formation. The combination of these two gelling mechanisms yields Ca-alg-PEG hybrid microspheres in one step under physiological conditions. Microencapsulation of human hepatocellular carcinoma cells (Huh-7) in Ca-alg-PEG demonstrated cell viability, metabolic activity, and proliferation post encapsulation in vitro, while applying microgravity culture conditions during storage for two weeks as model for bioartificial liver development. The two novel types of hybrid microspheres avoid the incorporation of polycations for mechanical reinforcement and tuning of the permeability and can be produced by a one-step microencapsulation technology under physiological conditions. They thus fulfill important requirements for biocompatible cell encapsulation materials.

Alginate-Poly(ethylene glycol) Hybrid Microspheres for Primary Cell Microencapsulation

Redouan Mahou, Christine Wandrey

The progress of medical therapies, which rely on the transplantation of microencapsulated living cells, depends on the quality of the encapsulating material. Such material has to be biocompatible, and the microencapsulation process must be simple and not harm the cells. Alginate-poly(ethylene glycol) hybrid microspheres (alg-PEG-M) were produced by combining ionotropic gelation of sodium alginate (Na-alg) using calcium ions with covalent crosslinking of vinyl sulfone-terminated multi-arm poly(ethylene glycol) (PEG-VS). In a one-step microsphere formation process, fast ionotropic gelation yields spherical calcium alginate gel beads, which serve as a matrix for simultaneously but slowly occurring covalent cross-linking of the PEG-VS molecules. The feasibility of cell microencapsulation was studied using primary human foreskin fibroblasts (EDX cells) as a model. The use of cell culture media as polymer solvent, gelation bath, and storage medium did not negatively affect the alg-PEG-M properties. Microencapsulated EDX cells maintained their viability and proliferated. This study demonstrates the feasibility of primary cell microencapsulation within the novel microsphere type alg-PEG-M, serves as reference for future therapy development, and confirms the suitability of EDX cells as control model.

Mdpi Ag2014

Development of functionalized alginate-based hydrogels to reduce fibrosis in the transplantation of microencapsulated cells

François Rémi Pierre Noverraz

Nowadays, the development of cell therapy relies mainly on the advances made toward cell microencapsulation which allows to evade the need for immunosuppression during cell transplantation. Among the materials considered for cell encapsulation, hydrogels distinguish themselves by their exceptional properties. Due to its gelling properties in contact with divalent cations, the biopolymer sodium alginate (Na-alg), has been widely studied for the microencapsulation and transplantation of cells. However, several defects notably in permselectivity and durability in vivo is limiting the translation of alginate-based hydrogels in clinical applications. In addition, the transplantation of cells without immunosuppressive treatment have to face adverse host responses such as inflammation and fibrosis. At the moment, several approaches have been considered for the development of an ideal material for cell encapsulation; however many challenges still remain in this domain. Hence, this research project focused on the development of hydrogels in order to improve their properties toward cell encapsulation. The first part of this research focused on the development of new types of one-component hybrid alginate-based hydrogels. These hydrogels combine the gelling properties of Na-alg with covalent crosslinking within the same polymeric structure. In order to do so, a new synthetic approach was developed allowing the functionalization on the hydroxyl position of alginate with heterobifunctional poly(ethylene glycol) (PEG) linkers through carbamate bond. This allowed to maintain the carboxyl moieties available for the formation of electrostatic interactions. Two types of one-component hybrid hydrogel were developed with this method adding either thiols or lipoyl moieties on alginate leading to an improvement of mechanical properties while preserving a good biocompatibility. Yet, transplantation of encapsulated cells in a foreign host body have to face immune response, in particular inflammation and pericapsular fibrosis overgrowth (PFO) which ultimately leads to necrosis of the encapsulated cells and loss of graft functionality. Therefore, the second part of this research project focused on tuning the composition of the polymeric components of the hydrogels with anti-inflammatory agents which would reduce PFO in vivo. Different anti-inflammatory agents were considered for this purpose. The best candidate (ketoprofen) was then PEGylated via either ester or amide bond and grafted on the backbone of alginate to ensure a controlled release at the site of transplantation. By analyzing the fibrotic tissue around the microspheres, it was observed that the incorporation of ketopreofen on the structure of the hydrogel significantly reduced PFO 30 days after transplantation. In summary, Na-alg was functionalized with a new synthetic methodology allowing the incorporation of either functionalities reinforcing the mechanical resistance of the hydrogel or anti-inflammatory compounds improving the biocompatibility of the graft.

EPFL2018

Development of functionalized alginate-based hydrogels to reduce fibrosis in the transplantation of microencapsulated cells

François Rémi Pierre Noverraz

EPFL2018