This protocol enables the repeatable use of intact lung extracellular matrix as a three-dimensional tissue culture substrate and moderate throughput. The main advantage of the engineered lung tissue system is the flexibility of the platform. ELTs can be adapted to use various tissue scaffolds, cells, or culture media of interest.
Once the lungs are extracted, fill a 10-milliliter syringe with agarose and HBSS without phenol red. Inflate the lungs with approximately 10 milliliters of air via the trachea cannula, then immediately inject the prepared agarose via the trachea cannula until the most distal tips of the lung lobes are inflated. Cap the trachea by attaching the white cap from a four-way stopcock to the female luer lock of the tracheal cannula.
Place the lung in a 150-millimeter Petri dish on ice to allow the agarose to solidify. Prepare the lung slices according to the protocol in the manuscript, and then fill a 100-millimeter Petri dish with 1/3 volume of PBS. Transfer cassettes and tabs to the dish using forceps.
If the slices are frozen, thaw one dish at a time by pouring on room temperature PBS. Then, transfer a thawed slice to a 150-millimeter Petri dish. Gently unfold the slice using fine forceps or a hemostat and carefully aspirate excess PBS from around the tissue.
Then, cut a three-millimeter-wide, at least nine-millimeter-long strip from the slice by pressing the full length of a razor blade firmly against the dish and slightly rocking it side to side. To clip the tissue strip into the cassette, float it above the cassette, centering it carefully to extend past the holes in the clips at either end. Place a tab partly into the hole at one end with fine forceps, gently straighten the tissue, and set the second tab.
Finally, use forceps to press each tab in completely to secure the tissue. After clipping all the sides, transfer the 100-millimeter dish containing the cassettes to a laminar flow hood. Transfer cassettes to six-well plates containing the first decellularization solution using a curved hemostat to grasp the notched sides.
Then, place the six-well plate on an orbital shaker at 30 RPM for 10 minutes. Aspirate the fluid from each well, then replace it with three milliliters of the second decellularization solution per well. Place the plate on an orbital shaker at 30 RPM and incubate for five minutes.
Repeat this process with each solution as outlined in the decellularization protocol in the manuscript. After the final rinse with PBS, transfer tissues to sterile six-well plates containing fresh PBS with antibiotics and antimycotics and incubate at 37 degrees Celsius for 48 hours. After sterilization with antibiotics and antimycotics, lung tissue scaffolds can be seeded immediately or stored at four degree Celsius for up to 30 days.
Prior to utilizing for culture, incubate scaffolds stored at four degree Celsius with fresh PBS and antibiotics and antimycotics overnight at 37 degrees Celsius as an additional sterilization step. Then, rinse scaffolds with sterile PBS, five milliliters per well, three times, for five minutes each. Examine scaffolds under a phase contrast microscope at 5X magnification to select tissues for seeding.
Prepare the endothelial cell suspension in endothelial medium at 5 million cells per milliliter with sufficient cells to seed 500, 000 endothelial cells per slice. Place autoclaved seeding baths in 100-millimeter Petri dishes and carefully transfer the rinse scaffolds upside down into the seeding baths. Gently swirl the prepared cell suspension to mix.
Then, carefully pipette 100 microliters of cells directly on top of each tissue at the base of the wells, taking care not to damage the tissue with the pipette tip. Transfer the seeded tissues to the cell culture incubator. 24 hours after endothelial cell seeding, add 900 microliters of pre-warmed culture medium to each well using a manual pipette, then return the plate to the incubator.
After 24 hours of cell seeding, remove the medium and replace the one milliliter of fresh endothelial medium per well. 72 hours after seeding the endothelial cells, count AEC2s, or alveolar epithelial type II cells, and fibroblasts using a hemocytometer and prepare a 1:1 cell suspension in AEC2 growth medium at 5 million cells per milliliter. Pipette the medium from each seeding bath well and gently swirl the prepared cell suspension.
Pipette 100 microliters of cells directly on top of each tissue at the base of the well. Transfer the seeded tissues to the cell culture incubator. After two hours, add 900 microliters of pre-warmed AEC2 growth medium to each well, then return the plate to the incubator.
After 24 hours of culture with AEC2s and fibroblasts, prepare a 12-well plate with one milliliter of pre-warmed AEC2 growth medium per well, per cassette. Pipette 800 microliters of medium from each well of the seeding bath, then remove the cassettes from the seeding bath and transfer them right side up to the prepared 12-well plate, one cassette per well. Change culture medium carefully using a glass Pasteur pipette every other day for the desired culture duration.
Monitor the degree of tissue repopulation via phase contrast microscopy at 5X magnification throughout the culture duration. H and E staining of tissue scaffolds showed preserved alveolar architecture after decellularization with no visible cell nuclei. Alveolar tissue was observed when decellularized extracellular matrix scaffolds were viewed at 5X magnification by phase contrast microscopy.
Large branching airways and vessels were also visible in some slices. On day seven of culture, phase contrast microscopy showed that the pattern of recellularization in engineered lung tissues emulated the tissues alveolar structure in cases of successful repopulation. Poor recellularization was also visible.
H and E staining on day seven or eight showed repopulation of the alveolar septa. Immunofluorescence standing revealed pro-collagen type I alpha 1-positive fibroblasts, ABCA3-positive AEC2s, CD31-positive endothelial cells, ans abundant SPB-positive AEC2s. Moreover, the thymidine assay showed many proliferating AEC2s.
This technique has facilitated the development of strategies for lung tissue engineering and has enabled investigations into queues supporting type II cell proliferation and differentiation within the alveolus.
