Methacrylation of glass surface

The protocol for methacrylation of glass surfaces was described in [16]. The goal of glass methacrylation is to covalently bind methacrylate functional groups to the glass bottom of the culture vessel. This allows the glass surface to participate in the photocrosslinking reaction and thus to covalently bond the PEGDA gel for longitudinal culture. Without methacrylation, the gel may detach from the glass surface within hours or days. Here, gels were directly fabricated on the surfaces of glass-bottomed dishes (Cellvis, California, USA) or 18-well μ-Slides (Ibidi GmbH, Gräfelfing, Germany). These vessels are pre-cleaned and sterilized for tissue culture by the supplier, and no further cleaning was needed before methacrylation. To methacrylate glass surfaces, a methacrylated silane coupling agent (3-(Trimethoxysilyl) propyl-methacrylate; TPM) was mixed with ethanol (1: 20) and dilute acetic acid (1:30). The dilute acetic acid was prepared by mixing acetic acid with MilliQ H₂O (1: 10).

To each 35 mm glass bottom dish, 2 mL silane solution was added to cover the bottom; to each well of the 18 well μ-Slide, 150 μL silane solution was added. The filled dishes or slides were covered to reduce the evaporation rate of the silane solution and incubated for 12–24 hours in a chemical hood at room temperature. After incubation, the remaining silane solution was removed. The dishes or slides were then rinsed with ethanol two times and sonicated in a 70% ethanol solution for 20 minutes. After air drying with N₂, the dishes or slides were baked at 60°C for 5 hours. The methacrylated glass-bottomed dishes or slides were either used immediately or stored at 4°C and protected from light (Fig 1A(i)).

(a) Schematic illustration of the fabrication process (i) Glass surface methacrylation; (ii) PEGDA pre-hydrogel solution was added onto the glass surface, and then exposed to UV light (wavelength = 405 μm) with the virtual photomask embedded; (iii) Micropatterned PEGDA gel was fabricated on the glass surface. Uncrosslinked pre-hydrogel solution was removed by multiple PBS washes. (b) Schematic illustration of hESC seeding and culture (i) Laminin coating was applied on the micropatterned surface. Unattached laminin was washed away after 2.5-hr-incubation at 37°C; (ii) hESC suspension was added at desired density on the laminin coated surface. Unattached cells were washed away after 45-min-incubation at 37°C. Following seeding, media with desired reagents was added to the dish for further culture or induction.