Microfluidic Hydrogel Encapsulation

Flow-focusing microfluidic geometry was utilized to form polymer droplets. Both shielding and crosslinker phases consisted of light mineral oil (Sigma) with 2% SPAN80 (Sigma). The crosslinker phase also contained an emulsion, at a ratio of 1:15, of 20 mg/mL dithiothreitol (DTT) (Sigma) in PBS. A co-flowing shielding phase protected the macromer solution – a 5% PEG-4MAL (10 kDa or 20 kDa, Laysan Bio) solution containing molecules or cells to be encapsulated – from the crosslinker phase until droplets of the macromer solution were formed. DTT rapidly diffused into macromer droplets, forming crosslinked microgels. To functionalize hydrogel with GRGDSPC (‘RGD’, AAPPTec), macromer was reacted for 20 minutes before encapsulation with 2.0 mM RGD in buffer solution containing 4 mM triethanolamine (Sigma). After formation, microgels were washed 5 times by centrifugation to remove mineral oil and excess DTT.