Supermacroporous (spongy) agarose-based cryogels were prepared by a two-step freezing procedure (freezing at -30 degrees C 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-mu m 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. (C) 2008 Wiley Periodicals, Inc.