Functionalization of polyacrylic hydrogel scaffold for 3D cell culture

Traditionally, cell cultures have been performed on 2-dimensional surfaces, but recent evidences have demonstrated that 2D-cultured cells are morphologically different from natural cells, which may induce alterations in cellular biochemical processes and trafficking. On the contrary, in a 3D culture, cells retain their shape and the relevant microenvironment, more similar to the in vivo one, favors greater culture stability and longer lifespans [1]. We have recently patented a new hydrogel scaffold based on Carbopol® 980, obtained by compression of a mixture of the polyacrylic polymer with the porogen agents NaHCO3 and NaCl and subsequent thermal treatment, which leads to the formation of a continuous matrix; finally, a leaching process removes the water-soluble components and turns the matrix into a transparent, porous hydrogel scaffold, which can be autoclaved. The 3D scaffold has been successfully tested for culturing various cell types [2]. Our current research has focused on the functionalization of the polymer network by exploiting its acidic groups for covalent tethering of molecules that might further improve cell adhesion and proliferation. On this purpose, the carboxylic functions have been activated by EDC/NHS coupling, followed by reaction with ethylenediamine (EDA), to generate free amino groups suitable for further binding with acidic molecules, and with glycine (GLI), as a proof of concept to verify the feasibility of functionalization with peptides or proteins (Figure 1). The functionalized scaffolds, after lyophilization, have been characterized by IR spectroscopy, which confirmed the formation of amide bonds. The degree of functionalization has been quantified by hydrolysis in acidic medium, followed by titration of released EDA/GLI by ninhydrin reaction [3]. In the case of EDA, the amount of amine groups present of the scaffold surface has also been measured by reaction with 4- nitrobenzaldehyde [4]. Finally, the functionalized scaffolds have been analysed for their mechanical and swelling properties in aqueous medium, in comparison with neat ones. Further research is ongoing to functionalize the neat scaffold with RGD peptides, and the EDA scaffold with chondroitinsulfate, both known as effective biomolecules for scaffold surface modification.