OSCA to Shed light on Concealed Actuality
Cut Solid human Lung Tissue into small
> 5mm pieces
Prepare enzyme mix of Multi Tissue Dissociation Kit 1 into a gentleMACS C Tube
Transfer the tissue pieces into the gentleMACS C Tube
Place the Tube onto the gentleMACS Dissociator and run the gentleMACS Program Multi_A
After the termination of the program, apply the cell suspension to a MACS SmartStrainer(70µm)
Resuspend lung tissue cell pellet in Matrigel® and aliquot into wells
40µl of the Matrigel® cell suspension mixture in the center of each well of a 24-well plate
Incubate the plate in the cell culture incubator for 15 mintues to allow Matrigel® to polymerize
Add 500µl of Lung Airway Organoid Expansion Medium per well (24-well plate)
The culture medium should be replaced with fresh medium every 2-3 days
Organoids should be passaged for continued culturing
Prepare 10% Matrigel® solution with appropriate volume of prechilled PBS
Precoat the insert of 24 well Corning Costar Transwell by adding 100µl of 10% Matrigel® solution into the apical chamber
Incubate the plate containg Transwell inserts with 10% Matrigel® solution for 1 hour in 37℃
Transfer the 24-well plate containing lung organoids to the cell culture hood
Aspirate the medium without disturbing the gel matrix containing organoids
Add 500 µl of TrypLE Select reagent per well and mechanically disturb the gel with pipette tip
Transfer TrypLE solution containing organoid suspension into a 15mL tube
Incubate the 15mL tube in a waterbath at 37 ℃ for 10 minutes
Halfway through the incubation, mechanically shear the organoid suspension using a p1000 tip with a p10 tip placed on top.
Add 10mL of AdDF+++ to the 15mL tube and centrifuge cell suspension at 400×g for 5 minutes at 4℃
Discard the supernantant
Resuspend the cell pellet with appropriate volume of Lung Airway Orgnaoid Medium (ensure cell concentration is
> 0.5M cells/mL)
Count the cell numbers and dilute the cell suspension to 0.5M cells/mL
Transfer the plate with precoated Transwell inserts to the cell culture hood.
Aspirate the 10% Matrigel solution from each Transwell insert
Add 250 µl cell suspension to each apical chamber (125,000 cells per insert)
Add 500 µl of Lung Airway Organoid Medium to each of the bottom chamber