Shrna lentiviral particles

MISSION shRNA Lentiviral transduction particles were purchased from Sigma-Aldrich (St. Louis, MO). 2 × 10⁴ LTC cells were plated in a 96-well plate and incubated overnight (ON). Fresh experimental media were added containing 110 μl 0.8 μg Hexadimethrine bromide (Sigma, St. Louis, MO) and 10 μl of lentiviral shRNA particles to each well and incubated ON. Following the transduction, experimental media were replaced with normal media ON, and puromycin (10 μg/ml) was added to remove any non-transduced cells.

Mature huTGOs were collected in ice-cold DPBS, centrifuged at 400× g for 5 min, and the pellet was resuspended in Accutase. The organoids were incubated at 37 °C for 10–12 min, and gently syringed (26G needle) 10 times. Prewarmed complete growth medium was added to neutralize the Accutase before centrifuging at 400× g for 5 min; then the pellet was resuspended in 1 mL 3D growth medium. The empty vector (EV) or the shRNA lentiviral particles (Sigma-Aldrich) were added to the cells using the appropriate MOI unit along with 8 mg/mL hexadimethrine bromide, and incubated overnight at 37 °C. An equal volume of fresh medium was added to the cells and they were centrifuged at 400× g for 5 min. Cells were resuspended in Matrigel and plated in 12-well tissue culture plates. A 1mL volume of 3D gastric growth medium was added to each well after 13 min of incubation at 37 °C. Three days later the organoids were selected by adding puromycin (Thermo Fisher Scientific) into the culture. The control (EV) and knockdown (KD) cells were grown for at least 7–10 days. The efficiency of the knocking down of the organoids was tested either using an immunofluorescence and/or Western blot technique.