For many cell types, substrates that exhibit intermediate elastic moduli are better able to support in vivo-like cell function than standard tissue culture plastic surfaces. To create such surfaces, polysaccharide hydrogels are a promising alternative to polyethylene glycol (PEG) or polyacrylamide (PAAM) and do not require ultra violet light exposure to initiate gelation. We are exploring the utility of two polysaccharides, agarose and alginate, as well-defined surfaces for presenting protein ligands to target cells and are characterizing the elastic modulus of these substrates at the submicron scale using atomic force microscopy nanoindenation measurements.

Agarose is a thermoreversible hydrogel whose elastic modulus can be controlled by adjusting the total weight% of the gel solution. Using different sources of agarose, linear increases in elastic modulus values from 0.5 to 50 kPa can be achieved by adjusting the total gel concentration from 0.5 to 2.0wt/vol%. The point to point variability for any individual sample was less than 5%. To make the agarose more amenable to ligand modification, the hydroxyl moieties were modified to form a carboxymethyl ether group via Williams ether synthesis. The carboxy groups are then amenable to EDC-NHS mediated attachment of amine containing ligands. Carboxyl modification results in an approximately 60% decrease in elastic modulus at each of the concentrations tested. Titrating unmodified gel into modified gel results in linearly increasing elastic moduli from 0% unmodified to 100% unmodified gels. The variables of total gel concentration and modified to unmodified ratios allow the creation of hydrogels with varied elastic modulus and constant carboxyl densities. The elastic modulus of samples stored at 37degC in serum containing media did not decrease over incubation periods of up to 1 week. Surfaces modified with fibronectin induced fibroblast cell spreading whereas no spreading was seen on carboxyl modified substrates.

Alginates gel with the addition of Ca2+. The challenge of using alginate gels is the creation of uniformly gelled substrates with well defined elastic moduli. Bulk mixing of Ca2+ and alginate can result in single sample variability of as much as 80%. To create more uniform gels, we are using a diffusion method to slowly release Ca2+ into the bulk alginate solution. This method results in single sample variability of less than 20%. Because alginate gels already contain a carboxyl moiety, they are easily modified with amine containing ligands. We are modifying these gels with cadherins and using them to study the adhesion and differentiation of the P19 cell line into neurites.

Supported by the NSF CHE-0402420 and US Dept of E DEFG02-91-ER45439