세포주 정보

DMEM/F-12, 52.5%; FBS, 40%; DMSO, 7.5%

40%W/V basal culture medium, 50%W/V L-WRN conditioned medium, 1 x B27,50ng/mL human EGF, 10ng/mL human FGF-10, 10 mM nicotinamide, 1.25mM N-acetylcysteine, 500nM A83-01, 3 µM SB202190, 10nM prostaglandin E2, N2 supplement ,5ug/ml insulin

40%W/V basal culture medium, 50%W/V L-WRN conditioned medium, 1 x B27,50ng/mL human EGF, 10ng/mL human FGF-10, 10 mM nicotinamide, 1.25mM N-acetylcysteine, 500nM A83-01, 3 µM SB202190, 10nM prostaglandin E2, N2 supplement ,5ug/ml insulin

The organoid-containing dome (Corning, Matrigel; Trevigen, Cultrex BME; Merck, ECM; Thermofisher, Geltrex) was mechanically pipetted using animal origin-free, recombinant enzyme (Thermofisher사 TrypLE Express solution 등) and organoids were collected in a 15 mL conical tube. The organoid-containing dome was mechanically dissociated with intense pipetting, and the tube containing the organoids and organoid-containing dome mixture was incubated at 37°C for approximately 5 - 10 min. The organoids were centrifuged at 1,000 rpm for 3 min, and the supernatant was aspirated. Once organoid-containing dome was removed, the cell pellet was resuspended with fresh organoid-containing dome, seeded in a T-25 flask and the flask was incubated at 37℃ for 10 min. Once organoid-containing dome was solidified, 5 mL of the culture medium was added to the flask to overlay the organoid-containing dome and cells were incubated in a 37℃ and 5% CO2 culture incubator.

The medium is aspirated. Care was taken not to destroy the BME dome. 5 mL of medium was added, and cells were incubated in a 37℃ and 5% CO2 culture incubator.