Abstract: The present disclosure describes two click reactions to sequentially polymerize and then functionalize hydrogel microparticles to provide therapeutic agent-functionalized crosslinked hydrogel microparticles. The two click reactions have one common reactive group, and are non-interfering with respect to one another. The therapeutic agents can be released from the hydrogel microparticles over time as the hydrogel degrades, or the therapeutic agents can be released from the hydrogel microparticles by enzymatic degradation or hydrolysis. In some embodiments, the biological function of the therapeutic agents can be preserved or enhanced when covalently bound to the hydrogel microparticles. The present disclosure also provides methods for the production of hydrogel cell culture substrates. The methods overcome the hurdle of protein functionalization through the implementation of an advanced but simple chemical strategy that involves modifying a protein of interest with chemical groups (e.g.