3D organoid cultures are proximal in distal lung epithelium developed using this protocol. Provide tractable model to study fundamental mechanisms of lung tissue maintenance or disease associated remodeling, and serves an effective platform for drug discovery and validation. This protocol is compatible with other tissue dissociation cellular fractionation approaches.
And since epithelial progenitor cells is isolated from different anatomic compartments maintain their positional identity in vitro, it can be used for modeling regional differences in lung responses to either exogenous or endogenous stimuli. This level technique can be used for the isolation of different subpopulation of epithelial cells including region specific progenitor cells that can be cultured to yield specialist differentiated progeny representative of the region of origin. Upon receive of the lung tissue, separate the proximal trachea and bronchi from the distal lung epithelium, which contains small airways of two millimeters in diameter or less, and the surrounding parenchymal tissue.
Place the samples in individual sterile 150 by 15 millimeter Petri dishes and dice the distal tissue sample into approximately one centimeter cubed pieces. Place the pieces into a clean 50 millimeter tube and wash the tissues three times with fresh chilled HBSS per wash. After the last wash, transfer the tissues to a new Petri dish and blot the pieces dry with sterile anti lint wipes.
Then use forceps and scissors to remove as much visceral pleura as possible before mincing the tissues into approximately two millimeter diameter fragments. For tissue fragment digestion, add 50 micrograms per milliliter of liberase and 25 micrograms per milliliter of DNAs into 25 milliliters of sterile HBSS in a 50 milliliter conical tube. And add approximately two to three grams of the minced tissue to the tube.
Then place the tissues at 37 degrees Celsius with continuous shaking at 900 revolutions per minute for 40 to 60 minutes. After 30 minutes, use a 10 milliliter syringe to triturate the digested tissue to prevent clump formation. At the end of the incubation, use a 10 milliliter syringe equipped with a 16 gauge needle to triturate the tissue five times.
Then use a wide bore pipette to pass the suspension through a series of cell strainers under vacuum pressure as indicated. After washing the last strainer with 20 milliliters of HBSS plus to collect any remaining cells, centrifuge the filtrates to collect the isolated cells. After discarding the supernatants, add one milliliter of red blood cell lysis buffer to the pellets with gentle pipetting before incubating the cells for one minute done ice.
At the end of the incubation, add 10 to 20 milliliters of HBSS plus buffer to neutralize the lysis and collect the remaining cells by centrifugation. Deplete CD31 positive endothelial cells and CD45 positive immune cells from the pool of total cells using the CD31 and CD45 microbeads conjugated to monoclonal anti-human CD31 and CD45 antibody and LS Columns in accordance to the manufacturers'protocol. For cell surface staining for FACS, add the appropriate primary antibodies of interest at the required concentration and incubate the cells for 30 minutes at four degrees Celsius in the dark.
At the end of the incubation, wash the cells with three milliliters of HBSS plus and labeled the cells with the appropriate concentration of fluorophore conjugated secondary antibody for a 30-minute incubation on ice as appropriate. At the end of the incubation, wash the cells with three milliliters of HBSS plus and resuspend the cells at a one times 10 to the seventh cells per milliliter of HBSS plus concentration before filtering the cells into five milliliter polystyrene tubes through a strainer cap to ensure the formation of a single cell suspension. Then add one microgram per milliliter of DAPI to stain the permeable cells.
For proximal tissue epithelial progenitor cell enrichment, dissect out the proximal airways from the lungs and use scissors to open the airways along their length to expose the lumen. Cover the tissues in 50 micrograms per milliliter of liberase for 20-minute incubation at 37 degrees Celsius with continuous shaking on a mixer. At the end of the incubation, transfer the tissue to a sterile 150 by 15 millimeter dish and use a scalpel to gently scrape the airway surface to completely strip the luminal epithelial cells from the tissue.
Next, wash the dish with five milliliters of sterile HBSS plus buffer to collect all of the dislodged luminal epithelial cells and transfer the cells to a 50 milliliter conical centrifuge tube. Successively triturate the suspension five times using a 10 milliliter pipette to obtain a single cell suspension. Then collect the suspension by centrifugation.
Then resuspend the pellet in fresh HBSS plus buffer on ice. Use scissors to cut the remaining tracheobronchial tissue along its rings to generate small strips of tissue. In a new dish, use a single sided razor blade to mince the strips into smaller pieces and transfer the tissue pieces into C tubes containing two milliliters of liberase per tube.
Then use human lung protocol two on a gentleMACS automated dissociator to mechanically dissociate the tissue further. At the end of the dissociation, transfer approximately two grams of minced proximal tissue to 50 milliliter conical tubes containing 50 micrograms per milliliter of liberase and 25 micrograms per milliliter of DNA solution. At the end of a 45-minute incubation at 37 degrees Celsius with shaking, filter the tissue slurry through a series of strainers as demonstrated for the distal lung tissue and add an equal volume of HBSS plus buffer to the filtrate to stop the digestion.
Add the isolated luminal proximal airway cells to the tube and collect the cells by centrifugation. The cells can then be enriched by microbead isolation and stained for cell surface markers of interest as demonstrated for the distal lung tissue cells. Proximal airways lined by a pseudo stratified epithelium include abundant basal progenitor cells that are immuno reactive for the membrane protein NGFR.
In contrast, epithelial cells lining the alveoli include a subset that shows apical membrane immunoreactivity with the HTII-280 monoclonal antibody suggestive of their alveolar type II cell identity. Magnetic bead depletion of contaminating red blood, immune, and endothelial cells results in a significant enrichment of epithelial cell populations in both distal and proximal tissue samples with corresponding increases in FACS efficiency. Further FACS depletion leads to highly enriched distal cell populations that can be additionally fractionated based on their NGFR or HTII-280 surface staining.
HTII-280 positive distal lung epithelial cell cultures yield rapidly expanding organoids with an average colony forming efficiency of 10%Immunofluorescence staining of day 30 cultures reveals that the lumen containing organoids are composed predominantly of HTII-280 and SPC positive distal lung epithelial cells. Proximal lung epithelial organoids cultured from NGFR positive cells results in the formation of lumen containing organoids at day 30 of culture with an average colony forming efficiency of 7.8%Empirical optimization of enzymatic tissue association is critical for preservation of surface epitopes allowing immunofluorescence staining and enrichment of viable epithelial cell subsets for subsequent generation of region specific organized cultures. This method can be adapted for the identification and enrichment of immune, vascular, stromal, and epithelial cell subsets and development of co-culture models using either 3D organoid chip platforms.
We have recently described the use of alveolar organoids generated using this technique as a platform to study SARS-CoV-2 infection of the human lung epithelium and for COVID-19 drug validation.
