Gibco™ DMEM, high glucose

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Defrost the growth factor reduced Matrigel in advance, on ice or in fridge	Seed esophageal organoid units in ∼2 mL per well of growth factor reduced Matrigel, spreading over the bottom of a six-well plate Culture for 10 days with medium changed every other day	Collect organoids for microscopic analysis

Upstream tips
Defrost the growth factor reduced Matrigel in advance, on ice or in fridge
Protocol tips
Seed esophageal organoid units in ∼2 mL per well of growth factor reduced Matrigel, spreading over the bottom of a six-well plate Culture for 10 days with medium changed every other day
Downstream tips
Collect organoids for microscopic analysis

Publication protocol

Esophageal organoid unit isolation:Human surgical samples were obtained as waste tissue after esophageal resection, collected at the time of surgery. All esophageal tissue was washed several times with 4°C Hank's balanced salt solution (Invitrogen, Carlsbad, CA) on ice, sedimenting between washes, to remove debris and mucous. Each lavaged esophagus was minced into <1 mm3 pieces with sterile scissors. Tissue fragments were enzymatically digested with 0.12 mg/mL dispase (Invitrogen) and 800 U/mL collagenase type 1 (Sigma-Aldrich) at 37°C on an orbital shaker for 20 min. Tissue fragments were further mechanically digested with a 10 mL pipet for trituration. Digestion was stopped with 4°C 4% sorbitol (Sigma-Aldrich), 10% fetal bovine serum (FBS) (Invitrogen) in high glucose Dulbecco's modified Eagle's medium (DMEM) (Invitrogen). After centrifugation at 39 g for 10 min, the supernatant was poured off, the pellet resuspended in 4°C 10% FBS in DMEM, and then centrifuged at 100 g for 5 min. The resulting pellet contained the isolated OU, heterogeneous multicellular clusters of epithelium and mesenchyme. Isolated EOU were suspended in an equal volume of growth factor reduced Matrigel (BD Biosciences, Franklin Lakes, NJ) and evenly spread over the bottom of a six-well plate at a total of ∼2 mL per well. The suspension was solidified by incubation for 20 min at 37°C at which point medium consisting of DMEM with 10% FBS, 1× MEM nonessential amino acids (Life Technologies, Grand Island, NY) and 1× antibiotic-antimycotic (Life Technologies) was added. The EOU cultures were incubated at 37°C with 5% CO2 for 10 days with culture medium changed every other day. After 10 days the EOU were freed from the wells with a cell scraper and centrifuged at 39 g for 5 min. The pellet was then mounted in a liquid mold of low melting agarose (Gold Biotechnology, St. Louis, MO) for microscopic analyses. Alternatively, the cultured EOU were seeded onto a polymer scaffold and implanted, as above, to generate TEE.

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