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Here, we describe the preparation, structure, and properties of cryogel sponges, which represent a new type of macroporous biomaterial for tissue engineering. Cryogels were produced through freeze-thawing techniques, either from agarose alone or from agarose with grafted gelatin. The aim of this study was to evaluate agarose cryogel sponges as scaffolds for culturing both isolated pancreatic islets and insulinoma cells (INS-1E). In order to evaluate the effect of cell entrapment in artificial scaffolds, cell function reflected by insulin secretion and content was studied in cells cultivated for a 2-week period either in culture plastic plates or in cryogel sponge disks. Our results show that tumor-derived INS-1E cells grown either on plastic or on cryogels do not differ in their proliferation, morphology, insulin release, and intracellular insulin content. However, isolated pancreatic islets cultivated on cryogels sponge show 15-fold higher basal insulin secretion at 3.0 mM glucose than islets cultivated on plastic plates and fail to respond to stimulation with 16.7 mM glucose. In addition, these islets have about 2-fold lower insulin content compared to those grown in plastic plates. It is possible that the cell dysfunction noted in these in vitro experiments is due to the effect of the limited oxygen supply to the islets cultivated in cryogel sponge. Further in vivo studies are needed to clarify the nature of such an observation since according to previous reports, agarose and gelatin induce new vessel formation supporting enhanced oxygen supply.

Supermacroporous (spongy) agarose-based cryogels were prepared by a two-step freezing procedure (freezing at 2308C followed by incubation at a warmer subzero temperature) and subsequent thawing. The cryogels were formed as cylinders in plastic syringes and as platelike samples in flat metal molds. The characteristic feature of the gel matrices thus obtained was their heterogeneous spongelike morphology with a system of interconnected gross (50-250-lm and larger) pores. The influence of the cryogenic processing regimes on the properties and porous morphology of such agarose cryogels was explored by flow-through analysis, optical microscopy, thermometry, and high-sensitivity differential scanning calorimetry. These biocompatible, spongelike matrices were used as three-dimensional scaffolds for culturing insulin-producing rat insulinoma cells self-assembled in multicellular spherical aggregates (pseudoislets). The cell morphology and functional activity of such pseudoislets indicate that supermacroporous agarose-based cryogels can be useful as a tool for engineering biohybrid insulin-producing tissue.