Fibronectin fc010

Before cell loading, the PDMS molds were sterilized with 70% ethanol for 5 min by being submerged. Subsequently, the ethanol was aspirated and dried in an incubator. Immediately after sterilization, the surface to be seeded with cells was coated with 20 µg/cm² Fibronectin (FC010, Sigma Aldrich). On day 0, the cells were seeded in DMEM/F12 with the seeding medium replaced by differentiation medium#1, which was 10% FBS and 1% penicillin–streptomycin, and then the differentiation was performed. On day 1, differentiation medium#1 was prepared by adding 10 µM retinoic acid (RA) (RA; R2625, Sigma Aldrich) to DMEM/F12 with 10% FBS and 1% penicillin–streptomycin to induce neuronal differentiation. On day 3, differentiation medium#1 was replaced with differentiation medium#2, which contained 50 ng/mL brain-derived neurotrophic factor (BDNF) (B2795, Sigma Aldrich) plus DMEM/F12 with 10% FBS, 1% penicillin–streptomycin, and 10 µM RA to sustain the survival of the cell differentiation. Thereafter, the culture with GelMA was stopped on day 5 and prepared for fixation and further analysis, and, when we observed that the culture with PEGDA did not differentiate, it was discarded. The cultures for the cell seeding density experiment were stopped on day 6 and only analyzed by bright-field without fixation.

Immuno-surgery was performed on E4.5 embryos (Solter and Knowles, 1975 (link)). Embryos were recovered from mouse uteri and incubated for 15 min in advanced IVC medium supplemented with 20% anti-mouse serum (rabbit, M5774, Sigma Aldrich) for 20 min at 37°C. Following incubation, embryos were washed 3x in advanced IVC medium, placed in IVC medium supplemented with 20% complement (home-made rat serum, gift of Thorsten Boroviak) and incubated for 15 min at 37°C. Embryos were washed for 3x and thereby the trophectoderm lineage, which died through the antiserum and complement incubation, was removed through pipetting. Then, the embryos were placed in placed in hanging drops, 2-2.5 ul of advanced IVC medium supplemented with 30% of FBS and 1ug/ml of Fibronectin (FC010, Sigma Aldrich) for 48h. Hanging drop culture was carried out in order to prevent attachment to the dish and thereby spreading of the epiblast. Each embryo was cultured in a single drop to prevent merging of embryos. After 24h, the embryos were changed to fresh drops.