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The overall goal of this procedure is to generate lung tissue in culture that is morphologically and physiologically comparable to native lung tissue. This is accomplished by first harvesting the heart and lungs from an adult rat. The next step is to cannulate the pulmonary artery and the trachea and to connect the cannulated lungs to a bioreactor for decellularization.

After decellularization and rinsing, the lung is transferred to a sterile culture bioreactor and prepared for seating. The final step of the procedure is to seed the desired compartment of the lung with a prepared cell population. Ultimately, results can be obtained that show the decellularized extracellular matrix efficiently repopulated with cells that express regionally specific lung markers through immunofluorescence microscopy.

The main advantage of this technique over existing methods of culturing lung cells in vitro is that the cells maintain differentiated phenotypes for an extended period of time, so they can be studied for several weeks in a three dimensional biologically derived substrate. For decellularization, the lungs are connected to the bioreactor cap via the vascular cannula, and the cap is connected to the decellularization apparatus. The glass bottle above the lungs is held in place by a rings stand and attached clamp.

At this point, cellular material can be removed to produce an extracellular matrix scaffold, which can be repopulated with cellular material. The assembly of the bioreactor for seeding and culture of engineered lung tissue is shown in this diagram. The lure lock connector of the tracheal cannula connects to a breathing loop between the trachea reservoir and the main chamber.

The breathing loop includes two one-way valves. Positions such that medium follows a different path into and out of the lung. A small reservoir is temporarily incorporated into the perfusion line for seeding of endothelial cells.

A pulsatile pump is used to convey cells from the reservoir to the vascular compartment of the lung. Vascular perfusion is provided using a roller pump. Medium is perfused into the pulmonary artery via the attached cannula, flows through the lung vasculature and out the pulmonary vein directly into the main bioreactor where medium is drawn up for perfusion.

Pulmonary epithelium is seated by injection in the breathing loop. The main chamber containing the lung and the trachea reservoir are used for long-term lung culture. When the lungs have been extracted and the artery and trachea are cannulated correctly, the freshly extracted lungs should hold air without leaking.

This can be verified by inflating them with air while submerged in liquid. There should not be any bubbles indicating air leaks. To begin the protocol, the lungs are inflated with PBS containing sodium nitropress side until the lungs are full but not overinflated immediately cap the tracheal cannula so that the lungs remain inflated.

Next, perfuse the lungs with PBS and SNP for at least 15 minutes at 15 millimeters of mercury. After that, remove the stopper from the tracheal cannula to allow the lungs to deflate. Continue perfusion with PBS and SNP for 15 to 30 minutes if necessary.

Refill PBS and SNP to ensure profusion pressure is maintained at 10 to 15 millimeters of mercury. To begin the procedure for long decellularization, the bioreactor cap is connected to the decellularization apparatus. Then the lungs are connected to the cap.

Using the lure lock connections linked to the YS shaped cannula, the pulmonary artery cannula should connect to the perfusion line and the tracheal cannula should float free. To ensure full decellularization of the tissue, it is crucial to keep air out of the system. The one-way valves in the arterial and tracheal cannula help to clear air bubbles in the tubing by enabling reversal flow in the tubing.

Therefore, allowing air bubbles to be removed by syringe, Ensure all lines are clear of air. Begin perfusion with decellularization solution. Perfused with decellularization solution.

Until 500 milliliters of solution has perfused through the lungs, the optimal pressure is less than 15 millimeters of mercury. This will typically require 2.5 hours. Flow rates are typically very slow initially and rapidly increased during the second hour to approximately one milliliter per minute or greater.

During the perfusion periodically removed, used decellularization fluid from the bioreactor while ensuring enough fluid remains to support the lung and tracheal cannula. When perfusion with the decellularization fluid is complete, transfer the lungs and bioreactor to a tissue culture hood. To begin organ rinsing, remove the 500 milliliter jar that contains the decellularization fluid and replace it with a sterile jar containing up to one liter of sterile PBS.

Use vacuum suction or a syringe to ensure lines are clear of air perfuse PBS through the vasculature at 10 to 15 millimeters of mercury in the same manner. As for decellularization, using sterile techniques periodically remove waste PBS from the bioreactor and replace or refill the PBS jar. With fresh, sterile PBS continue rinsing until at least 2.5 liters of sterile PBS has perfused the lungs.

This will typically take two days from the beginning of the decellularization process when the rinsing is complete. Transfer the lungs to a new sterile bioreactor system containing fresh PBS plus 10%FBS plus 10%Pen strep ensure that both the entire perfusion loop and the entire airway lines are filled with fluid. From here on a pulsatile pump will be used to perfuse the lungs at five millimeters per minute.

Sterilize the lung extracellular matrix scaffold by overnight perfusion with PBS that has 10%FBS and 10%penicillin streptomycin. After completing this step, transfer the lungs to a 37 degree Celsius incubator for one hour or until the temperature is equilibrated. In preparation for treatment with Ben Nase, the next step is to treat the lungs with Ben Nase.

To remove remnant DNA for each lung, first fill one 10 milliliter syringe with Prewarm Ben Nase buffer only, and one 10 milliliter syringe with 90 units per milliliter. Ben Nase diluted in Ben Nase buffer. Stop perfusion of the lungs.

Then inflate the airway with Ben Nase buffer while avoiding injecting air into the lung. Allow the lung to deflate for one minute. Then inflate the lung with the Ben Nase solution.

Again, being careful not to introduce air into the lung after inflating with Ben Nase. Allow the lungs to sit without perfusion or ventilation at 37 degrees Celsius for one hour. At the end of the one hour resume perfusion with the PBS plus 10%FBS 10%penicillin streptomycin that is already in the bioreactor, and continue the perfusion overnight on the following day.

After rinsing and sterilization, the long extracellular matrix scaffold can be prepared for cellular repopulation. Replace the P-B-S-F-B-S benzo a solution with 250 milliliters of culture. Medium perfu for at least one hour before cells are introduced and replace with fresh culture medium directly before seeding cells.

Many cell sources can be utilized for organ receding. Here, seating with an epithelial cell population will be demonstrated. First, prepare a syringe containing 15 milliliters of a cell suspension of the desired epithelial cell population.

Next, fill the airway reservoir with 80 milliliters of culture medium and ensure all ventilation lines are clear of air. Then place the bioreactor in a 37 degrees Celsius tissue culture incubator and connect the syringe pump for ventilation. Seed the cells into the lungs using a single rapid bolus by injecting the 15 milliliter cell suspension into the tracheal cannula.

Ensure the air filters on the main bioreactor are capped off. Then immediately begin a single slow breath using the syringe pump to withdraw 60 milliliters of air from the main bioreactor at three milliliters per minute. This will last for approximately 20 minutes.

A allow the lungs to sit statically for approximately 18 hours after that. Begin slow vascular perfusion at approximately 0.5 milliliters per minute. During organ culture, perfusion is typically performed at one to three milliliters per minute.

Using a roller pump during epithelial culture, ventilation is typically provided at a continuous rate of one breath per minute and tidal volume of five to 10 milliliters. Using a syringe pump air is cyclically, withdrawn and injected into the bioreactor to affect breathing by alternating between negative pressure and atmospheric pressure inside the main chamber. Due to the need to maintain the bioreactor airtight during ventilation, ventilation should be paused daily and the air in the main chamber should be exchanged.

In addition, the medium should be changed approximately every three to four days. During culture, approximately half of the total volume should be exchanged each time by culturing pulmonary epithelium and vascular endothelium. Within the bioreactor mounted scaffold, we are able to generate lung tissue that is able to participate in gas exchange for short time intervals.

A standard hemat, toin and eoin stain of native rat lung is shown here for comparison with an H and e stain of a decellularized rat lung. The final decellularized extracellular matrix should be completely devoid of cellular materials and retain the gross, microscopic and ultra structural characteristics of native lung. The h and e stain will enable the visualization of any remnant DNA, sticking to the scaffold as a result of insufficient decellularization or rinsing optimal conditions for cell seeding and subsequent culture of the lung.

And the bioreactor should yield well distributed cells within all five lobes of the lung and should provide coverage of approximately 70%of the extracellular matrix scaffold. These images compare native lung to repopulated lung when stained by immunofluorescence. For key lung markers, the cultured cell population is positive for Clara cell secretory protein pro secretory protein C and Aquaporin five.

The markers of interest are shown in red and the DAPI stained nuclei are in blue in all panels. Once mastered this procedure from decellularization to receding, can be done in four to five days if performed properly.