Nonhuman Primate Lung Decellularization and Recellularization Using a Specialized Large-organ Bioreactor

The overall goal of this procedure is to isolate whole organ biological matrices from the lungs of Reus Maccas and to use these matrices as scaffolds for investigating lung tissue engineering applications. This is accomplished by first decellularizing whole Reuss Maca lungs with detergents, salts, and enzymes. The second step is to install the decellularized lung scaffolds into a bioreactor capable of both ventilating the airway and perfusing the pulmonary vasculature with liquid cell culture media.

Next, the lung scaffolds are seated with stem cells via the airway and endothelial cells via the pulmonary vasculature. The final step is to culture the cells within the lung scaffold under the ventilation and perfusion conditions provided by the bioreactor for the desired amount of time, followed by analysis of the resulting cellular and tissue morphology. Ultimately re ization of acellular Maca lung scaffolds with stem cells and endothelial cells results in engineered tissue that mimics the anatomical and histological features of native lung tissues.

This method can help answer key questions in the lung tissue Engineering fields such as whether stem or progenitor cells can be used to regenerate pulmonary tissue either alone or in concert with mature pulmonary epithelial and endothelial cells. The implications of this technique extend toward therapy for end stage lung diseases because ization of acellular lung matrices with patient derived stem or progenitor cells has the potential to increase the number of donor lungs available for lung transplantation. Moreover, the resulting lungs engineered using patient's own stem cells may be resistant to immune rejection.

We first had the idea for this method when we viewed a previously published Joe video by members of Dr.Laura Nicholson's laboratory at Yale University. Here we present modifications of their procedure for rodents to fit the needs of our large animal Resus Meac model. Utmost care must be taken to prevent infectious exposure incidents when working with maca tissues.

Before handling non-human primate tissues, put on personal protective equipment including surgical mask, face shield, one layer of surgical gloves, disposable gown, and a second layer of surgical gloves with the lungs lying flat in a dissecting tray, cannulate the pulmonary artery using a female lure connector with an appropriately sized barb. Secure the cannula into place with an alcohol sterilized zip tie. Slip a plastic zip tie around the trachea.

Insert a female lure connector into the tracheal opening and tighten the zip tie around the barb of the connector to hold the trachea in place around the barb. Remove trapped air from the lungs by instilling phosphate buffered saline or PBS containing 30 units per milliliter of heparin and five micrograms per milliliter of sodium nitropress side. Abbreviated SNP allow the solution to be expelled by natural recoil before repeating.

Installation twice more after the third airway installation with the PBS Heparin SNP solution. Cap the tracheal lure cannula with a lure plug. Cut off the apex of the heart and irrigate the internal ventricles with PBS Heparin SNP to remove residual blood lacerate both atria to facilitate drainage of the pulmonary circuitry upon perfusion.

Attach a 60 cc syringe filled with the PBS Heparin SNP solution to the pulmonary artery cannula and carefully remove the plunger to allow the liquid to flow into the vasculature. Remove the plug from the tracheal cannula and allow the fluid in the airway to be expelled by natural recoil. Continue perfusion until as much blood as possible is removed from the pulmonary vasculature.

The most difficult aspect of the procedure is effectively rinsing and washing the lung airway With liquids, liquid flow is inhibited relative to gases as airways are not meant to conduct liquids, time and patients are necessary to accomplish these steps, we advise continuing with the protocol, despite residual liquid remaining in the airway as decellularization progresses and cellular debris is flushed out, fluid viscosity will remain low and the lungs will be able to efficiently expel liquids On day one. Submerge the heart lung block in deionized water, inflate and perfuse the lung with deionized water using 60 cc syringes attached to the submerged tracheal and arterial cannula respectively. Effectively repeat the airway and vascular washes with deionized water.

Four more times for a total of five rinses of approximately 0.5 to one liter each after five washes, remove the lungs from water and submerge the block in Triton solution. Inflate and perfuse the lungs with Triton solution. As before, repeat the installation a second time and incubate the submerged organs in Triton solution overnight at four degrees Celsius.

Following an overnight incubation, remove the lung block from Triton solution and wash externally with deionized water. Then submerged the block in fresh deionized water. Instill the deionized water into the trachea and pulmonary artery five times to wash out the Triton solution and cell debris.

Remove the lungs from deionized water and submerge in 2%Sodium deoxy coate or SDC solution. Inflate and perfuse with SDC solution in the same manner. Submerge the lungs in SDC solution overnight at four degrees Celsius on day three.

Wash the tissue again with deionized water externally and through the airway and vasculature five times. As before, remove the tissue from deionized water and submerge in the one molar sodium chloride solution. Inflate and perfuse with sodium chloride solution as before, and submerge the tissue in sodium chloride solution for one hour at room temperature after washing the lungs clean of sodium chloride solution with five rinses of deionized water.

As before, bathe them in fresh DNA solution and instill this solution into the airway and vasculature. Incubate the lungs in DNA solution for one hour at room temperature. Then remove the lungs from the DNA solution and wash externally with PBS solution.

Submerge the lungs in PBS solution and wash this solution five times through the airway and vasculature. As before, store the lungs in PBS solution in a sealed container overnight at four degrees Celsius on the fourth day. Wash the lungs in fresh ice cold PBS solution five times through the airway and vasculature.

Then store them in PBS solution in a sterile sealed container at four degrees Celsius until use. Assemble the bioreactor components under a laminar flow hood according to the schematic. In the text protocol, fill the main chamber with culture medium that has been equilibrated to the 5%CO2 atmosphere of a cell culture incubator immediately prior to use in the bioreactor, apply the tracheal and vascular cannula adapters to the lung cannula.

Install the organs in the main bioreactor chamber by bathing the lungs in the culture medium and attaching the cannula adapters to the appropriate ports in the modified lid. Once connected, affix the lid securely and tightly. The chamber will not be opened again for the duration of reation aspirate air from the tubing using the syringe ports and a 60 CCC syringe fitted with an 18 gauge needle moving the directionality of the three-way stop cocks to direct the flow of liquid into the syringe.

Move the sealed contiguously connected bioreactor chambers with installed organs to a tissue culture incubator to equilibrate temperature and gas. Ventilate the lungs using a syringe pump attached to the main chamber at approximately one full breath every two minutes, and perfuse the vasculature via the peristaltic pump at approximately 10 milliliters per minute For a total of 30 minutes for airway seating. Inflate the lungs with the cell suspension by gently injecting through the syringe port attached to the three-way stop cock in the breathing loop.

Hold the lungs statically at 37 degrees Celsius, 5%CO2 overnight without airway or vascular perfusion to allow cells to attach to the decellularized lung matrix. After overnight incubation, reinitiate the standard airway ventilation program and culture, the organs for three to seven days for vascular seating. The directionality of the flow path can be changed from the main chamber to an endothelial seating reservoir by rotating the valve on the stop cock positioned in the tubing connecting these two compartments seed endothelial cells gradually using the peristaltic pump while gently stirring using the magnetic stir when seeding is complete.

Stomp perfusion and incubates statically for approximately four to six hours. Reinitiate vascular perfusion with main chamber medium at a rate of approximately 10 milliliters per minute. Culture for three to seven days with continuous vascular perfusion concomitantly with the airway ventilation culture period.

Throughout the decellularization process, MCCA lungs displayed progressive whitening culminating in a translucent appearance at the end of the process. However, the lungs maintained their gross anatomical features and remained largely elastic and able to produce natural recoil after inflation with liquid. At the microscopic level.

The histologic ultra structure remained intact after decellularization. That is bronchial respiratory bronchials alveolar sacs. Blood vessels and capillaries were still distinguishable quite clearly by low power microscopy.

Histological microanatomy, however, demonstrated that while the gross anatomy and ultra structure of the lung were mildly disturbed by decellularization, the tissue completely lacked intact cells as seen here. Only trace amounts of DNA remained in decellularized tissues. Moreover, the trace DNA, which was concentrated by alcohol precipitation and visualized in a 0.8%AROS gel was composed of mostly low molecular weight degraded fragments.

The efficiency of cellular protein removal was assessed by western blot analyses of both native and decellularized lung protein. Lysates using an antibody to beta actin, beta actin was easily detected in native lung lysates, but not in decellularized lung lysates suggesting that decellularization dramatically depleted cells and removed cell associated protein material 14 days after airway seeding with maca bone marrow derived mesenchymal stem cells or BMCs and bioreactor culture with approximately one inspiration expiration cycle every two minutes. The parenchyma of Decellularized Maca lungs was effectively ized.

BMCs lined the alveolar septi while maintaining a clear and open alveolar lumen. The denuded matrix of large airways was also ized by BMCs using the bioreactor, the luminal surface of a main stem bronchus was lined with a monolayer of squamous like BMCs after 14 days of culture. In the bioreactor shown here is a decellularized reus lung lobe five days after seeding the vasculature with microvascular endothelial cells and providing constant vascular perfusion with endothelial culture medium at five milliliters per minute.

Histological analysis revealed cells lining the small vasculature in the lung parenchyma. In some instances, cells appeared to be attached to the matrix via several cellular projections across the lumen, while other cross sections of vessels showed cells lining the endothelial surface with clear lumen. Following this procedure.

Other methods like immunohistochemistry, western blot, and quantitative real-time PCR can be performed in order to answer additional questions like whether the seeded stem cells differentiate into pulmonary lineage cells once cultured on the acellular lung matrix or with mature epithelial and endothelial cells within the bioreactor. After watching this video, we should have a good understanding of how to decellularize non-human primate lungs and subsequently use the resulting acellular lungs as scaffolds for culturing stem cells, epithelial cells, and endothelial cells in a bioreactor system. Don't forget that working with non-human primate tissues can be extremely hazardous and precautions such as wearing full personal protective equipment, including a face shield and a dual layer of latex or nitrile Gloves should always be taken while performing this procedure.