Establishing Human Lung Organoids and Proximal Differentiation to Generate Mature Airway Organoids

We are introducing a method to establish the organoid culture of human airway epithelium in culture plates. We derive human lung organoid directly from primary lung tissues with high efficiency. The derived human lung organoid are stably expanded for over one year, and a proximal differentiation protocol allows the generation of mature airway organoids that can faithfully simulate the human airway epithelium to a near physiological level.

Therefore, the culture system allows scientists to reconstruct and expand the human airway epithelium in culture plates. Demonstrating the procedure will be Man Chun Chiu and Yifei Yu, PhD students in our lab To begin the cell isolation from human lung tissues for 3D organoid culture, mince the lung tissues into one-millimeter small pieces with a sterile scalpel and wash the tissue pieces with 10 milliliters of cold basal medium. Centrifuge the tissues at 400 times G for five minutes at four degrees Celsius and discard the supernatant before resuspending the pellet in eight milliliters of basal medium supplemented with collagenase at a final concentration of two milligrams per milliliter.

Then, digest the tissue pieces by shaking the tube at 120 RPM for 30 to 40 minutes at 37 degrees Celsius. After digestion, pipette up and down 20 times using a 10-milliliter serological pipette to shear the digested tissue pieces. Then, filter the suspension with a 100-micron strainer into a 50-milliliter tube.

Recover and transfer the tissue pieces from the strainer to a 15-milliliter centrifuge tube with the basal medium. To terminate the digestion, add fetal bovine serum to the flow-through to a final concentration of 2%Then, centrifuge the tube and resuspend the cell pellet in 10 milliliters of the basal medium as described before. After centrifugation, resuspend the pellet in 80 to 160 microliters of cold basement matrix medium and place the tubes on ice.

Next, dispense 40 microliters of the suspension to each well of a pre-warmed 24-well suspension culture plate. Incubate the plate at 37 degrees Celsius for 10 to 15 minutes. Allow the basement matrix to solidify and form a droplet.

After incubation, add 500 microliters of human lung organoids expansion medium supplemented with five-nanomolar of heregulin-beta 1 to each well and incubate the plate in a cell culture incubator. After three days, remove the old medium while keeping the droplet intact and carefully add fresh medium. After 10 to 14 days of passaging, observe the organoids under a microscope and ensure that the organoids are not embedded with a very high cell density.

To passage the lung organoids with shearing, break the droplets by pipetting up and down with a one-milliliter tip. Then, transfer the organoids along with the medium to a 15-milliliter centrifuge tube and adjust the volume to 10 milliliters with a cold basal medium. Centrifuge the tube at 300 times G for five minutes at four degrees Celsius, discard the supernatant, and wash the organoids with 10 milliliters of cold basal medium.

Then, resuspend the organoids in two milliliters of cold basal medium and shear into small pieces with a Pasteur pipette. Next, add basal medium to a total volume of 10 milliliters and centrifuge before resuspending the organoid fragments with a cold basement matrix sufficient to enable a 1:3 to 1:5 expansion and place the tubes on ice. Dispense 40 microliters of organoid suspension in each well of a pre-warmed 24-well plate to incubate at 37 degrees Celsius to allow the basement matrix to solidify for 10 to 15 minutes.

Later, add 500 microliters of lung organoid expansion medium to each well and incubate in a cell culture incubator. Refresh the medium every three days and passage the organoids every two weeks. To passage lung organoids with trypsinization, resuspend harvested lung organoids in one milliliter of dissociation enzyme.

Incubate the organoid in a 37-degree-Celsius water bath for three to five minutes. Then, add one milliliter of basal medium to the tube and shear the organoids mechanically by pipetting up and down. Check the size of organoid pieces under a microscope at 100X magnification before terminating the digestion with 40 microliters of fetal bovine serum.

Make up the volume to 10 milliliters with basal medium and centrifuge before resuspending the organoid pieces in a cold basement matrix with a volume sufficient to passage with a ratio of 1:5 to 1:10. Later, dispense 40 microliters of organoid suspension in each well of a 24-well plate and culture the plate as demonstrated before. To generate 3D airway organoids, incubate the lung organoids in the expansion medium for seven to 10 days after passaging via mechanical shearing.

Then, replace the expansion medium with proximal differentiation medium and incubate the organoids in a cell culture incubator. After 14 days, discard the medium in each well and add cell lysate buffer to harvest the differentiated airway organoid for RNA extraction and detection of cellular gene expression by RT-qPCR assay. Pre-incubate the inserts in the cell culture incubator with 250 and 500 microliters of the basal medium in the top and bottom chamber of a 24-well plate, respectively.

Then, add one milliliter of basal medium to the tube and pipette up and down to shear the organoids into single cells. Observe the cells under a microscope before terminating the digestion with 40 microliters of fetal bovine serum. Filter the cells through a 40-micron strainer into a 50-milliliter tube and transfer the filtered cell suspension to a 15-milliliter tube.

Top up the suspension with basal medium to a total volume of 10 milliliters. After centrifugation, resuspend the pellet collected from 24 droplets in 1 to 2.5 milliliters of lung organoid expansion medium, depending on the cell density. Count the number of cells with the cell counter under a microscope, then adjust the cell concentration to 1.3 times 10 to the six per milliliter for 24-well inserts.

Remove the basal medium from the top and bottom chambers before adding 500 microliters of expansion medium in the bottom chamber. Seed 100 microliters of cell suspension prepared earlier onto the apical chamber of the 24-well insert and incubate. After two days, replace the expansion medium with proximal differentiation medium in both the apical and bottom chamber of the plate.

Incubate the organoids for 14 days and refresh the medium every three days. Measure the trans-epithelial electrical resistance every day using an electrical resistance measurement system. In the representative microphotograph, the freshly isolated lung cells can be seen embedded in reduced growth factor basement membrane matrix type two.

The cystic organoids were appeared and grown over time. The lung organoids after the fourth passage are demonstrated here. Within two hours of mechanical shearing, the organoid fragments embedded in the basement membrane extract formed the cystic domains.

The microphotograph of the same field on day five confirmed organoid growth over time. The expanding organoids harbored all the four major airway epithelial cell types, including ACCTUB-positive or FOXJ1-positive ciliated cell, P63-positive basal cell, CC10-positive club cell, and MUC5AC-positive goblet cell in a premature state. The organoids incubated in the expansion and proximal differentiation medium developed distinct morphology over time.

After two weeks of differentiation in the proximal differentiation medium, motile cilia were discernible in every organoid. It was observed that the synchronously beating celia drove the cell debris inside the organoid lumen and swirled them unidirectionally to remove the inhaled particles. Further, the flow cytometry analysis demonstrated that the differentiated organoids accommodated four airway epithelial cell types in proximal differentiation and expansion medium.

After two weeks of differentiation, 2D airway organoids developed an intact epithelial barrier. A dextran blockage assay was performed to assess the integrity of the epithelial barrier formed in 2D airway organoids. The 2D organoids contained abundant ciliated cells.

The long-term expandable lung organoid and the differentiated airway organoid provide a physiologically active and universal model system for studying respiratory biology and pathology, including COVID-19.