Ln 111

The thin-film PEOT-PBT and poly(ester-urethane) microwell array devices were shortly exposed to oxygen plasma at an oxygen pressure of 1.0 bar, at 40 mA, and 200 mTorr for 10 and 20 s, respectively. The oxygen plasma treatment was used to modify the surface chemistry of the microwell scaffold and generates, for example, aldehyde groups that can react with molecules containing free amine groups present in the ECM proteins. Subsequently, microwell scaffolds and tissue culture polystyrene plates were coated with 10 mg/mL of ECM proteins. The ECM proteins include FN, Col4, LN111 (Merck), LN332 (Biolamina, Sweden), and a mixture of LN111, 211, 121, 311, 411, and 511 (Merck). With a total protein concentration of 10 mg/ mL, the pairwise compositions of varying ratios of FN, Col4, and LN (5:5, 2:8, and 8:2) were coated at room temperature for 2 h. After three times rinsing in phosphatebuffered saline (PBS) to remove any unbound proteins, the scaffolds were placed in cell culture inserts and transferred to ultra-low-attachment cell culture plates (Corning).

Peptide-coated wells/inserts were incubated with 1% (w/v) heat-inactivated bovine serum albumin (BSA, Sigma) to block non-specific adhesion, and used for assessment of ES-NSPC adhesion, viability, migration, and differentiation (details are provided in Supporting Information). Wells incubated in parallel with 1% (w/v) heatinactivated BSA were used as negative control, while wells incubated with 100 µg/mL of PDL, 20 µg/mL of laminin 111 from Engelbreth-Holm-Swarm murine sarcoma (LN 111, Sigma), or 20 µg/mL of human recombinant laminin 511 (LN 511, Biolamina), were used as positive controls. Glass coverslips previously coated with 10 µg/mL of PDL and then with 5 µg/mL of LN 111 (PDL-LN 111) were also prepared and used as an additional positive control in 24h experiments or longer.