ECM Micropatterning for Organoid Cultures

Organoid monolayers were confined in large circular ECM micropatterns (>900 μm). For this, PAA gels were functionalized with 2mg/mL of Sulpho-SANPAH (Cultek) irradiated for 7.5 minutes with UV light (365nm). Gels were then washed twice with HEPES 10mM (Gibco). For ECM micropatterning, Polydimethylsiloxane (PDMS) stencils with circular openings were used^{25 (link)}. PDMS stencils were incubated with Pluronic acid F127 2% in PBS (Sigma-Aldrich) for 1 hour. Then, they were washed twice with PBS and allowed to dry at RT for 20 minutes. Stencils were carefully placed on top of the functionalized PAA gels. A solution of 250 μg/mL rat tail type I Collagen (First Link UK) and 100 μg/mL Laminin I (Sigma-Aldrich) dissolved in PBS was added on top of the PDMS stencils and incubated overnight at 4°C. Finally, the ECM solution was aspirated, the gels were washed twice with PBS and the PDMS stencils were carefully removed.