Advanced DMEM

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Defrost the Matrigel in advance on ice or in fridge	Seed 2 × 104 cells/well of the flat bottom 24-well plates Grow 3D organoids for 10–14 days	Collect organoids for histological analysis

Upstream tips
Defrost the Matrigel in advance on ice or in fridge
Protocol tips
Seed 2 × 104 cells/well of the flat bottom 24-well plates Grow 3D organoids for 10–14 days
Downstream tips
Collect organoids for histological analysis

Publication protocol

To generate 3D organoids, each piece of the biopsies was rinsed in saline (0.9% NaCl) (S7653, Sigma-Aldrich, St. Louis, MO), and minced immediately with scissors and incubated for 45 minutes at 37°C in a 1-mL cocktail comprising 6.25-mg/mL dispase I (354235, BD Biosciences, San Jose, CA), 5-mg/mL collagenase (17018029, Thermo Fisher Scientific), 10-mM ROCK inhibitor Y27632 (1254, R&D Systems, Minneapolis, MN), and 0.5-mg/mL Fungizone (15290018, Thermo Fisher Scientific) with a periodical mixing (∼once every 10 minutes) via Vortex-Genie 2 (Scientific Industries, Bohemia, NY). The solution was replaced with - mL 0.25% trypsin/EDTA (R001100, Thermo Fisher Scientific) and incubated for 10 minutes at 37°C. The trypsin activity was then neutralized with 6-mL 250 mg/L soybean trypsin inhibitor (T9128, Sigma-Aldrich) in Dulbecco's phosphate-buffered saline (DPBS) (10010023, Thermo Fisher Scientific). The enzymatically dissociated tissue was forced through a 70-μm cell strainer (22-363-548, Thermo Fisher Scientific) and cells were pelleted via centrifugation at 2000 rpm for 5 minutes at room temperature. Cells were then resuspended in 1-mL advanced Dulbecco’s modified Eagle medium (DMEM)/F12 (12491015, Thermo Fisher Scientific) containing 100-U/mL penicillin and 100-μg/mL streptomycin (15140122, Thermo Fisher Scientific), 1×GlutaMAX (35050061, Thermo Fisher Scientific), and 1×HEPES (15630080, Thermo Fisher Scientific) (aDMEM/F12+). The Countess II FL Automated Cell Counter (Thermo Fisher Scientific) was used to count cells where Trypan blue exclusion test was done to determine cell viability. The cell suspension (4 × 105/mL) was mixed further with 10× volume of ice-cold Matrigel (354234, Corning, Corning, NY) and 2 × 104 cells in 50 μL of Matrigel were seeded into each well of the flat bottom 24-well plates. After solidification, 500 μL of aDMEM/F12+ supplemented with 1×N2 (17502048, Thermo Fisher Scientific), 1× B27 supplements (17504044, Thermo Fisher Scientific), 0.1-mM N-acetyl-L-cysteine (A7250, Sigma-Aldrich), 50-ng/mL recombinant human EGF (236-EG, R&D Systems), 2% Noggin/R-Spondin–conditioned media, 100-ng/mL recombinant human Wnt3A (5036-WN, R&D Systems), 500-nM A83-01 (2939, R&D Systems), 10-nM SB202190 (1264, R&D Systems), 10-nM gastrin (3006, R&D Systems), 10-nM nicotinamide (N0636, Sigma-Aldrich), and 10-μM Y27632, was added, a composition described for murine esophageal 3D organoids22 and replenished every other day. 3D organoids were grown for 10–14 days at 37°C in a humidified atmosphere of 5% CO2. When ESCC cell lines were used to generate 3D organoids, monolayer cultures were trypsinized to prepare cell suspensions. Under an inverted phase-contrast microscope (Apotome, Carl Zeiss, Jena, Germany), growing organoids were observed and photomicrographed to determine their number and size. Organoid formation rate was defined as the average number of ≥50-μm spherical structures at day 14 that was divided by the total number of cells seeded in each well at day 0. When indicated, 3D organoids were grown in the presence or absence of 5FU (F6627, Sigma-Aldrich) or chloroquine (CQ) (C6628, Sigma-Aldrich) at indicated concentrations after organoid structure was established at day 8. Dimethyl sulfoxide (BP231-1, Thermo Fisher Scientific) was used as vehicle for 5FU. CQ was reconstituted in water as described.13 3D organoids were recovered by digesting Matrigel with Dispase I (354235, BD Biosciences, 1U/mL), embedded in iPGell (GSPG20-1, GenoStaff, Tokyo, Japan) and fixed overnight in 4.0% paraformaldehyde for histological analyses. Alternatively, isolated organoids were dissociated by incubation with 0.25% Trypsin-EDTA, Y27632 and DNase I (1010415901, Sigma-Aldrich) into single cell suspensions and passaged.

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