TITLE: How to Culture Human Bronchial Epithelial Cells as Airway Organoids
AUTHORS: STEMCELL Technologies

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Human bronchial epithelial cells (HBECs) are typically cultured at the air-liquid interface (ALI) to functionally recapitulate the human airway. ALI cultures are formed by plating primary HBECs onto porous cell culture inserts and allowing the cells to achieve confluence prior to removing the culture medium from the apical surface of the cells. However, the requirement of porous culture inserts can limit the application of ALI culture to smaller scale experiments, thus largely precluding high-throughput drug screening of differentiated epithelial cultures. Alternatively, HBECs may be cultured and differentiated as spherical aggregates, providing a means for high-throughput study of the differentiated human airway. This protocol describes a method for the culture of differentiated HBECs as spherical structures known as broncospheres or airway organoids.</div></div>

[STEPS]
?. [Preparation of Reagents and Materials]
Prepare PneumaCult™-ALI Complete Base Medium by adding 50 mL PneumaCult™-ALI 10X Supplement to 450 mL PneumaCult™-ALI Basal Medium. This Complete Base Medium can be aliquoted and stored at -20°C for up to 6 months. Avoid additional freeze/thaw cycles.
Note: the PneumaCult™-ALI Medium kit contains:PneumaCult™-ALI Basal MediumPneumaCult™-ALI 10X SupplementPneumaCult™-ALI Maintenance Supplement
?. [Preparation of Reagents and Materials]
Prepare PneumaCult™-ALI Maintenance Medium by adding the following components per 1 mL PneumaCult™-ALI Complete Base Medium:10 μL PneumaCult™-ALI Maintenance Supplement2 μL Heparin Solution5 μL Hydrocortisone Stock Solution
Note: Prepare only enough PneumaCult™-ALI Maintenance Medium needed for immediate use.
?. [Preparation of Reagents and Materials]
Prepare a sufficient volume of a 40% Matrigel® solution by promptly mixing the following components (will require 500 μL/ well of a 24-well plate):300 μL cold PneumaCult™-ALI Maintenance Medium200 μL cold Matrigel®
?. [Preparation of Reagents and Materials]
Aliquot 500 μL 40% Matrigel® solution per well of a 24-well plate.
?. [Preparation of Reagents and Materials]
Incubate the plate at  in a humidified incubator at 5% CO2 for  to allow for the Matrigel® layer to solidify. Steps 6 through 12 can be completed during this 30 minute incubation.
37 °C
?. [Preparation of a Single Cell Suspension]
Prepare a sufficient volume of a 5% Matrigel® solution by mixing the following components (require 500 μL/well of a 24-well plate):475 μL cold PneumaCult™-ALI Maintenance Medium25 μL cold Matrigel®
?. [Preparation of a Single Cell Suspension]
Warm 5% Matrigel® solution, Ca2+/Mg2+-free PBS, 0.025% Trypsin-EDTA [1/2 dilution of 0.05% Trypsin-EDTA] and 1 mg/mL Soybean Trypsin Inhibitor to .
37 °C
?. [Preparation of a Single Cell Suspension]
Wash each T-25 flask of HBECs twice with 2 mL warm Ca2+/Mg2+-free PBS. Aspirate the Ca2+/Mg2+-free PBS and add 2 mL warm 0.025% Trypsin-EDTA to each flask.
?. [Preparation of a Single Cell Suspension]
Incubate flask at  for 3 - 5 minutes, until the cells can be dislodged with gentle tapping of the flask. Neutralize the Trypsin-EDTA by adding an equivalent volume of 1 mg/mL warm Soybean Trypsin Inhibitor to each T-25 flask.
37 °C
?. [Preparation of a Single Cell Suspension]
Collect the cell suspension into a 15 mL conical tube and centrifuge at
?. [Preparation of a Single Cell Suspension]
Remove the supernatant and resuspend the cell pellet in 1 - 2 mL warm 5% Matrigel® solution.
?. [Preparation of a Single Cell Suspension]
Perform a viable cell count using Trypan Blue and a hemocytometer.
?. [Airway Organoid Culture]
Prepare cells at a concentration of 180,000 cells/mL in 5% Matrigel® solution. Plate 500 μL cell solution per well of a 24-well plate (90,000 cells/well or approximately 50,000 cells/cm2).
Note: This seeding density has been optimized for use with P4 cells. If using lower passage number cells, the optimal seeding density may be lower.
?. [Airway Organoid Culture]
Incubate at , 5% CO2 in a humidified incubator. Medium changes should be performed three times per week (e.g. Monday/Wednesday/Friday) by gently aspirating the medium above the semi-solid Matrigel® layer and replacing with 500 μL fresh warm 5% Matrigel® solution.
37 °C
HBECs will grow into spheres within approximately one week, continually increasing in size throughout the entire four week culture period. Between week one and two, a lumen will become visible within some of the bronchospheres (Figure 1). By two weeks, the cells within the wall of each bronchosphere will start to differentiate and self-organize to create a pseudostratified epithelium with the apical surface of goblet and ciliated cells pointing into the lumen (Figure 2).   By three weeks, visualization under bright field microscopy will reveal beating cilia:Video 1. Visualization of Bronchospheres.Light microscope visualization showing a bronchosphere cultured in PneumaCult™-ALI. This video shows mucus in the lumen being swirled around by the beating of synchronized cilia lining the lumen of the bronchosphere.