A new hydrogel sponge scaffold suitable for 3D cell culture
Abstract: The three-dimensional (3D) culturing techniques decrease the gap between cell culture system and the cellular physiology, helping to understand better various cell functions such as adhesion, viability, morphology, microenvironment, and response to drugs or hazardous substances. We developed a transparent hydrogel sponge scaffold (HS)  , based on a polyacrylic acid, able to resist to freeze-drying and sterilization by moist heat process in autoclave. SEM analysis of HS evidenced a highly interconnected porous structure suitable for cell migration and diffusion nutrients and metabolic wastes. HS exhibited suitable mechanical strength and flexibility to support the attachment of the cells, also withstanding the stress suffered during manipulation. The molecular diffusion through the HS has been evaluated using Franz diffusion cells. The diffusion coefficient of a small hydrophilic template molecule was calculated, mimicking the fast diffusion of the cultured medium components, metabolic products, or vital dyes. Moreover, several human cell lines, as HaCaT, HECV and HeLa, were grown into the HS without any coating proof of healthy and proliferating cells, up to 20 days of culture: MTS viability index  showed no toxic effects of HS, and Alamar Blue assay , evidenced an increase of metabolic state, . during the experimental cellular growth. The scaffold transparency allowed to confirm the wellness of the cultures: no evident alterations of cell morphology or necrotic figures were observed at contrast phase microscopy throughout the 20 days of culture. Our HS exhibits several positive features: good optical transparency, high porosity and suitability to let cells grow for at least 20 days. Moreover, the surface of the HS might be covalently linked to components of the extracellular matrix to enhance its biocompatibility, due to the presence of free carboxylic groups, or mineralized for bone tissue regeneration.