Generating 3D Spheres and 2D Air-Liquid Interface Cultures of Human Induced Pluripotent Stem Cell-Derived Type 2 Alveolar Epithelial Cells

This protocol outlines a method for culturing and passaging an important cell population in the lung and establishes a physiologically-relevant platform for environmental exposure. The main advantage of this technique is that it can be used to investigate lung biology in the context of homeostasis and disease. This method can provide insights, especially into pulmonary diseases affecting lung alveoli, including those caused by genetic variants, environmental exposures, infectious agents, or a combination of these factors.

Demonstrating the procedure will be myself and Rhiannon Werder, assisted by Kristy Abo, an MD, PhD student, and Olivia Hix, a lab manager from our laboratories. First, aspirate all the CK DCI medium from the 3D matrix droplets containing alveolospheres derived from directed differentiation in the 12-well plate. Add one milliliter of dispase per droplet.

Gently pipette the droplet into the dispase using a P-1000 pipette. After incubation transfer the dissociated organoids from one matrix droplet in the dispase to a 15-milliliter conical tube. Add 10 milliliters of Iscove's Modified Dulbecco's Medium and centrifuge at 300 x g for five minutes at room temperature.

Aspirate the supernatant as much as possible. Resuspend the cells in one milliliter of 0.05%trypsin per droplet and transfer it back to the 12-well plate. Incubate this at 37 degrees Celsius for 12 to 15 minutes before observing the dissociation under the microscope and avoid over pipetting the cells.

At the end of the incubation a single cell suspension is obtained. Stop the action of trypsin with an equal volume of fetal bovine serum containing medium, followed by centrifuge at 300 x g for five minutes. Wash the cells with 10 milliliters of IMDM before next centrifugation.

Resuspend the cells in an appropriate volume for counting. And then count the cells using a hemocytometer. Generate alveolospheres by plating single cell suspension of iAT2 cells in the 3D matrix or on cell culture inserts for air-liquid interface culture.

After counting the cells, determine the number of desired cells to replay in the 3D matrix and centrifuge the cells at 300 x g for five minutes at room temperature. Remove as much supernatant as possible using a pipette and quickly resuspend the cells in the 3D matrix. Dispense one 3D matrix droplet into a well of prewarmed 12-well plate using a P-200 pipette without creating bubbles in the matrix droplet, and without allowing the cell suspension to settle while dispensing multiple droplets.

Place the plate in a 37 degree Celsius incubator for 20 to 30 minutes to allow the matrix droplets to polymerize, then add one milliliter of CK DCI 10 micromolar of Y-27632 medium per well to cover the matrix droplet. Change the medium to CK DCI without 10 micromolar of Y-27632 after 72 hours and replace it with fresh CK DCI every 48 to 72 hours. Prepare freshly coated 6.5 millimeter cell culture inserts one hour before the use by diluting the 2D matrix in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 to the working solution.

Then add 100 microliters of the diluted matrix per 6.5 millimeter cell culture insert. Allow the matrix to polymerize in a 37 degree Celsius incubator for 30 minutes or at room temperature from one hour. Aspirate the excess matrix from the cell culture inserts using a pipette.

Rinse it with DMEM F-12, and immediately aspirate this wash before adding the cells. Centrifuge the cells at 300 x g for five minutes and remove as much supernatant as possible. Resuspend the cells in the appropriate volume to seed with 100 microliters of CK DCI 10 micromolar Y-27632 per cell culture insert and add 100 microliters to the apical compartment of the cell culture insert.

Gently agitate the plate in a cross pattern to ensure the even distribution of cells, and check it under a microscope at 10X objective. Add 500 microliters of CK DCI 10 micromolar of Y-27632 to the basolateral compartment of each cell culture insert, and 48 hours after seeding, aspirate the apical medium using a pipette to initiate the air-liquid interface. After 72 hours change the basolateral medium to CK DCI without 10 micromolar Y-27632, followed by replacing the basolateral medium with fresh CK DCI every 48 to 72 hours for up to 28 days post plating.

The representative flow cytometry results demonstrate the ease of visualization for the SPC2 line having a tdTomato reporter targeted to the endogenous surfactant protein C locus and tracking of type 2 alveolar epithelial cells program over time in culture. The IPSC-derived alveolar type 2 cell surfactant protein C tdTomato expression was maintained when replated in the 3D matrix as spheres and when plated on cell culture inserts. Transepithelial electrical resistance can be measured to determine the integrity of the air-liquid interface culture.

When attempting this procedure avoid excessive dissociation by either mechanical pipetting or enzymatic digestion as this can lead to low cell viability and poor survival. Additional methods performed after this procedure are measurements of transepithelial electrical resistance to assay barrier integrity, addition of viruses or compounds to stimulate the cells, immunofluorescence, and RNA, protein, or supernatant collection. This technique paved the way for researchers to explore new lung biology and disease questions, such as how viruses like SARS COVID-2 or compounds like electronic cigarette vapors affect the alveolar epithelium.