Substrate Preparation for Cell Culture

The glass cover slips were treated with 3-aminopropyltrimethoxysilane and 0.5% glutaraldehyde. Then, 8% acrylamide (sigma, USA) was mixed with varying concentrations of bisacrylamide (0.1% and 0.7%) (Sigma, USA). Polymerization was initiated with N,N,N′,N′-tetramethylethylenediamine (TEMED) and ammonium persulfate (sigma, USA). Then, 0.2 mg/mL N-sulfosuccinyimidyl-6-(4′-azido-2′-nitrophenylamino) hexanoate (sulfo-SANPAH) (Thermo, USA) dissolved in 10 mM HEPES (pH 8.5) was applied to cover polyacrylamide (PAAM) gel and exposed to 365 nm ultraviolet light for 70 min for photo activation in 24-well plates. The PAAm sheet was washed for three times with PBS to remove excess reagent and incubated with fibronectin solution (1 μg/cm²; Sigma, USA) overnight at 4° C. Before the cells were plated, the PAAm matrices were soaked in PBS and then in DMEM at 4° C. The Young’s modulus of the PAAm hydrogels was quantified using a biomechanical testing machine under contact load at a strain rate of 0.5 mm/s [45 (link)].