Cryogel and Hydrogel Formation with ELRs

The ELRs were^{27 (link)} dissolved in 10% (v/v) Ethanol (1015, Solveco) in PBS at a concentration of 64.28 mg/ml for the HE5-C and 35.71 mg/ml for the HRGD6-N3 and left at 4 °C overnight. MilliQ water (Millipore Reference, Merck) was sprayed using a spray bottle on top of liquid nitrogen and sieved through strainers with mesh sizes 500, 200, and 100 µm (Pluristrainer, 43-50500-03, 43-50200-03, 43-50100-51, pluriSelect Life Science) to collect ice crystals of desired size. Ice crystals were added to a mold (using a syringe (1 ml syringe, 329654, BD Bioscience)). Ice crystal and dissolved ELR were left in a cryostat (HM500M, Microm) at −5 °C to equilibrate. The HE5-C and HRGD6-N3 ELRs were mixed at a volume ratio of 1:1 by pipetting using cold tips, then this mixture was added to an equal volume of ice crystals and incubated at −5 °C for 15 min to fully form the cross-linkage, and then moved to −80 °C overnight. The samples were extracted from the mold and the edges were removed while being kept cold, and then samples were freeze dried (Freezone 4.5 Plus, Labconco) overnight. Hydrogels were made by dissolving each ELR in PBS at the same concentrations as for cryogels and mixed at a 1:1 volume ratio using cold tips. The mixture was pipetted into the desired molds and incubated at 4 °C for 15 min followed by 10 min at 37 °C to complete hydrogel formation.