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.


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