Gibco™ IMDM, GlutaMAX™ Supplement

Protocol tips

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Defrost the Matrigel in advance on ice or in fridge

Upstream tips
Defrost the Matrigel in advance on ice or in fridge

Publication protocol

3D lung organoid formation, branching, and maturation. Thaw growth factor−reduced Matrigel on ice and place pipet tips in the freezer. Add 10 μM of Y‐27632 to each well from step 20 containing LPC induction medium. Aspirate LPC induction medium and wash 1× with PBS. Add Accutase (0.5 ml/12‐well) and incubate for 10 min at 37°C in a 5% CO2 incubator. Add 1 ml of quenching medium. Pipet up and down forcefully and make sure all cells are dislodged. Epithelial cells are very “sticky,” so make sure that all cells are dissociated from the well. Transfer individual wells into one 15‐ml conical tube and centrifuge for 5 min at 300 × g, room temperature. Resuspend in quenching medium plus 10 μM Y‐27632. Perform a cell count (see Current Protocols article: Phelan & May, 2017) and aliquot out the desired volume of quenching medium/Y‐27632 with cells into 1.5‐ml microcentrifuge tubes to obtain 4.0 × 104 cells per transwell (see steps below) for ESCs and 8.0 × 104 cells for iPSCs. Cell number must be optimized per cell line; the cells should not become overly confluent within 18 days of differentiation. Microcentrifuge the tubes for 5 min at 300 × g, room temperature. Aspirate off the medium and re‐suspend cells in cold Matrigel, adding 200 µl Matrigel for every 4.0 × 104 cells per transwell for ESCs and 8.0 × 104 cells for iPSCs. Keep Matrigel with cells on ice. Add 200 µl of Matrigel/cell mixture on to a 12‐well transwell insert suspended in a 12‐well plate. Place plate into the 5% CO2 humidified incubator at 37°C for 30‐60 min to allow gelling of Matrigel. Add 1 ml of 3D organoid induction medium with all additive plus 10 μM of Y‐27632 to the lower chamber of the well and change the medium every other day for 6 days. At the end of 3D organoid induction, there should be multiple clumps of cells beginning to show “sprouts.” On day 23, change the medium to 3D organoid branching medium. Change medium every other day for 6 days using 3D organoid branching medium. At day 6 of 3D branching differentiation, there should be multiple branching organoids (see Fig. Fig.22). On day 29, change the medium to 3D organoid maturation medium. Change medium every other day for 6 days using 3D organoid maturation medium. 24 hr after 3D maturation, the branching organoids should inflate into large, clear spheres. At the end of 3D maturation, the majority of organoids should be clear, large spheres (see Supporting Information, Movie #1).

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