Control of cellular adhesiveness in an alginate-based hydrogel by varying peroxidase and H2O2 concentrations during gelation

An aqueous solution of alginate possessing phenolic hydroxyl (Alg-Ph) groups is gellable via a horseradish peroxidase (HRP)-catalyzed oxidative crosslinking reaction between Ph groups, consuming H2O2 as an electron acceptor. This study evaluates the effect of H2O2 and HRP concentrations on cellular adhesiveness and proliferation on the resultant enzymatically crosslinked Alg-Ph gels. After 4 h of seeding, 81.1% of L929 fibroblast cells adhere to an Alg-Ph hydrogel prepared with 1 U ml−1 HRP and 1 mM H2O2. Increasing the concentration of H2O2 to 15 mM decreases the percentage of adhering cells to 28.4%. The cellular adhesion at this H2O2 concentration is increased to 82.6% by increasing the HRP concentration to 10 U ml−1. The cells adhering to the Alg-Ph hydrogels with higher cellular adhesiveness establish a confluent monolayer during 168 h of culture. A cell sheet can then be harvested within 5 min of immersion in a medium containing alginate lyase at 1.0 mg ml−1. The harvested cell sheet re-adhere, and the cells contained in the sheet proliferate after being transferred to another cell culture dish.