. TiTannic gels were prepared (following the standard protocol described above) in 1 mL cut-off syringe tops for easy gel-moulding and removal. Samples were cut from the bulk gels using a biopsy punch and immersed in liquid nitrogen for 1 minute until completely frozen before freeze fracturing using a scalpel. A piece approximately 2×2×2 mm in size was taken for further processing. The sample was freeze substituted using a Leica EM AFS2 (Leica Microsystems, Germany) by immersion in pure acetone precooled to -90°C and the sample was ramped to 0°C at 5°C per hour. The sample was removed from the acetone and lyophilized, mounted on a SEM stub, and sputter coated with a 10 nm layer of gold. The sample was imaged using an Auriga Zeiss Crossbeam (Carl Zeiss AG, Germany) in SEM mode at 54° sample tilt (tilt corrected) using the in-lens secondary electron detector and 1.6 kV accelerating voltage. The images were processed using Fiji. Permeability assay. A porous well insert (membrane with 0.4 µm pores) was secured in a well of a 12-well cell culture plate filled with stirred 3.5 mL PBS solution. The well insert, containing either no gel (i.e., empty well control), or gel (either 0.2 mL of TiTannic gel or 0.2 mL of Pluronic F127 gel), was positioned so that the permeable bottom was fully immersed in the PBS solution. An in-house fabricated glucose sensor was used to monitor glucose concentration outside of the well insert (Figure ). After a few minutes of stabilization (as monitored by continuous read-out from the glucose sensor), 0.3 mL of either PBS (negative control) or 20 mM glucose solution (in PBS) was added on top of the well insert, i.e. on the opposite side of the gel in relation to the glucose sensor. Response of the glucose biosensor was continuously recorded over time with measurements acquired approximately once per second. Evaporation was prevented using a parafilm cover during the assay.