OSCA to Shed light on Concealed Actuality

Lung Organoid Establishment Protocol

  • 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

Lung Organoid 3D Culture to ALI Culture Protocol

  • 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