Transplantation of Bioengineered Lung Using Decellularized Mouse Lungs and Primary Human Endothelial Cells

Bioengineering human-sized organs requires a large amount of resources, which often exceeds the capacity of academic labs. Reducing the cost of iterative protocol development will accelerate the research progress in this field. The aim of this study is to provide the standardized protocol for lung bioengineering using mouse heart-lung blocks. It is still unknown what cells are appropriate for organ bioengineering, and how those cells should be integrated into the organ bioreactor culture. Multiple types of cells and conditions need to be compared in order to establish scalable and reproducible protocols.

Mice are small and easy-to-handle experimental animals. However, although they are small, the basic architecture of the lung is similar across mammals. By optimizing the protocol on this small platform, we can scale up the laser animal models by simply multiplying the optimized results according to the size of the animals. This mouse-size lung bioengineering platform allows researchers to test their stem cells efficiently. These genetically modified cells can be precious or expensive to use for the whole lung engineering. Additionally, patient-derived cells can also be used to create disease models in vitro.

Current efforts involve testing multiple types of engineered stem cells and progenitor cells, to figure out the required proliferation potential, and differential capacity for lung bioengineering. Once suitable cells are identified, this research will be extended to a larger animal model such as swine.

[Narrator] To begin, place the euthanized mouse in a supine position on a surgical table, and fix the limbs. Spray 70% ethanol evenly over the chest and abdominal surface to sterilize it. Next, open the abdominal cavity along the median line extending to the neck, using stainless scissors, and split the sternum. Resect the diaphragm from the thoracic wall. Cut the ventral chest wall to expose the thoracic cavities and remove the thymus. Using a 4/0 silk suture, legate the inferior vena cava and the right superior vena cava. Cut the abdominal aorta with stainless scissors for drainage. Using a five-milliliter sterile syringe equipped with a 27 gauge needle, inject three milliliters of sterile PBS into the right ventricle by puncturing the ventricle wall. Use Dumont forceps to loop a 4/0 silk suture around the main pulmonary artery. Next, cut a two-millimeter window beneath the pulmonary artery valves, using spring scissors, to access the right ventricle wall. Insert the pulmonary artery catheter through the window, and secure it with the pre-looped 4/0 silk suture. Slowly inject two milliliters of PBS through the pulmonary artery catheter, using a five-milliliter syringe. Cannulate the trachea with a tracheal catheter, and tie it in place with a 4/0 silk suture. Now, slowly inject two milliliters of air through the tracheal catheter using an empty five-milliliter syringe. Hold the air for 10 seconds to ensure no air leakage from the lungs. Remove the heart and lungs end block. Next, transfer the resected heart-lung block to a 10-centimeter diameter plastic Petri dish containing deionized water, for the decellularization of the lungs. Incubate the block for one hour at four degrees Celsius. After incubation, using a five-milliliter syringe, inject two milliliters of sterile deionized water through the tracheal catheter three times. Pause after each injection to allow the liquid to exit as the lungs recoil. Similarly, inject two milliliters of water through the pulmonary artery catheter. Inject two milliliters of 0.1% Triton X-100 solution into both tracheal and pulmonary artery catheters. Transfer the heart-lung block to a Petri dish, and incubate statically in Triton X-100 solution overnight at four degrees Celsius. The next day, to remove the Triton X-100 solution from the lungs, inject two milliliters of deionized water into the tracheal catheter and pulmonary artery catheter, and repeat these with two milliliters of 2% deoxycholate solution. After removing the deoxycholate solution from the lungs, inject two milliliters of one molar sodium chloride solution into both tracheal and pulmonary artery catheters. Incubate the heart-lung block for one hour. After removing the sodium chloride solution with deionized water, inject two milliliters of DNase I working solution into the tracheal and pulmonary artery catheters. Incubate the heart-lung block for one hour. Inject sterile PBS into the tracheal and pulmonary artery catheters to remove the DNase I solution. After decellularization, confirm that the lungs are white and transparent at the edges. Add 70 milliliters of Endothelial Cell Growth Medium 2, or EGM-2 culture media, to the glass canister, and place the silicon stopper on top of the canister to seal it. Assemble a silicon stopper, a glass canister, GL45 screw cap with tubing, a 250-milliliter autoclaveable glass bottle, and L/S 14 tubing with lure fittings using three-way stopcocks. Insert a 20 gauge needle into the silicon septum of the GL45 screw cap. Fill tubing A, B, and C with culture media using a 10-milliliter syringe connected to stopcocks one and three. Attach the pulmonary artery catheter of the decellularized heart-lung block to tubing C via a lure fitting. Ensure no air bubbles are present in the catheter or tubing. For the gravity-driven injection of endothelial cells, place a cell reservoir containing human umbilical vein endothelial cells, or HUVECs, on a magnetic stirrer, ensuring the reservoir is 30 centimeters above the organ chamber. Turn on the magnetic stirrer at a speed of 120 RPM. Open stopcocks one and two to allow the cell suspension to flow into the decellularized scaffold, via tubing A, tubing C, and the pulmonary artery catheter. For the perfusion of organ culture, place the organ chamber in a carbon dioxide incubator. Attach tubing B to a pump head connected to a pulsatile pump. Close the incubator glass door. Ensure that the tubing is properly positioned between the door and the rubber seal. Incubate the decellularized scaffold at 37 degrees Celsius for three hours to allow the endothelial cells to settle within the scaffold. Start the pump at a rate of six RPM, resulting in two milliliters per minute media perfusion using L/S 14 tubing. Observe the decellularized lung slightly expanding due to media perfusion. Then, close the incubator door. To perform media change, stop the pump drive. Attach a 50-milliliter sterile syringe to stopcock three, and withdraw 50 milliliters of media from the chamber. Fill another 50-milliliter sterile syringe with 50 milliliters of pre-warmed media. Inject the solution into a glass canister, and transfer the media into the chamber via stopcock three, ensuring the stopcock is switched appropriately. Restart the pulsatile pump. Harvest the recullularized heart-lung block after two days of perfusion organ culture. Mouse lungs appeared white and translucent following decellularization, indicating the removal of cellular components, while preserving the overall lung structure. Histological analysis of decellularized mouse lungs revealed intact alveolar structures, with no remaining cellular components. Recellularized mice lungs with three times 10 to the power of seven HUVECs after two days of perfusion-based bioreactor culture, exhibited homogeneous distribution of HUVECs. HUVECs migrated into peripheral alveolar areas, forming a capillary like network, as observed in recullularized lungs.