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

Summary

Whole-organ decellularization produces natural biological scaffolds that may be used for regenerative medicine. The description of a nonhuman primate model of lung regeneration in which whole lungs are decellularized and then seeded with adult stem cells and endothelial cells in a bioreactor that facilitates vascular circulation and liquid media ventilation is presented.

Abstract

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an attractive alternative to classic organ transplantation. This technology utilizes an organ's natural biological extracellular matrix (ECM) as a scaffold onto which autologous or stem/progenitor cells may be seeded and cultured in such a way that facilitates regeneration of the original tissue. The natural ECM is isolated by a process called decellularization. Decellularization is accomplished by treating tissues with a series of detergents, salts, and enzymes to achieve effective removal of cellular material while leaving the ECM intact. Studies conducted utilizing decellularization and subsequent recellularization of rodent lungs demonstrated marginal success in generating pulmonary-like tissue which is capable of gas exchange in vivo. While offering essential proof-of-concept, rodent models are not directly translatable to human use. Nonhuman primates (NHP) offer a more suitable model in which to investigate the use of bioartificial organ production for eventual clinical use.

The protocols for achieving complete decellularization of lungs acquired from the NHP rhesus macaque are presented. The resulting acellular lungs can be seeded with a variety of cells including mesenchymal stem cells and endothelial cells. The manuscript also describes the development of a bioreactor system in which cell-seeded macaque lungs can be cultured under conditions of mechanical stretch and strain provided by negative pressure ventilation as well as pulsatile perfusion through the vasculature; these forces are known to direct differentiation along pulmonary and endothelial lineages, respectively. Representative results of decellularization and cell seeding are provided.

Introduction

Bioengineering of tissues and organs is an attractive addition to the field of regenerative medicine. The creation of "lab-grown" organs that are suitable for transplant into patients to replace functionality of diseased organs is highly desirable in order to meet the current and future demand for transplantation needs. The principles of tissue engineering center around the seeding of desired cell types, or progenitors thereof, into a scaffold that supports the shape of the engineered tissue while supplying the appropriate growth factors and culture conditions necessary to mimic developmental or regenerative processes. While synthetic scaffolds have been used for tissue engineering, and the natural extracellular matrix (ECM) may be the best source of organ-specific scaffolds for this purpose. Whole-organ decellularization is a process which allows the removal of cells while leaving the chemical and structural aspects of the native ECM intact. The resulting acellular matrix scaffold can be used as a platform onto which regenerative cells can be seeded and cultured in vitro^1,2.

Several rodent models of lung decellularization and subsequent recellularization have been developed to study the feasibility of this technology^3-6. While offering essential proof-of-concept, rodent models are not directly translatable to human clinical needs. A recent study pointed out that genomic responses to traumatic injury (and related inflammation) do not correlate well between mice and humans; these findings raise questions of the validity of using mice as models for such complex biochemical processes in humans⁷. Nonhuman primate (NHP) models offer the advantage of closely resembling the biology of humans at the genomic, anatomic, and physiologic levels and allow more flexible manipulation for greater extrapolation to human use. The rhesus macaque has been used in a variety of preclinical applications and is an excellent model in which to study tissue engineering^8-11. We recently described the successful decellularization of rhesus macaque (Macacca mulatta) lungs utilizing a procedure that has minimum impact on the lung ECM¹². Lung decellularization is accomplished by treating the tissue consecutively with four decellularization solutions composed of detergents, salts, and enzymes with intermittent washing with deionized water (dH₂O) and PBS. We have optimized this procedure by modifying a protocol originally described by Price et al.⁴ A variety of histological and protein analytical techniques were used to characterize the components of resulting acellular matrices relative to native macaque lungs.

In this report, we demonstrate a detailed protocol for the decellularization of nonhuman primate lungs and the recellularization of the resulting acellular lung scaffolds in a large-organ bioreactor originally demonstrated by Calle et al.¹³ in JoVE. By modifying their original protocol to accommodate the size, ventilation, and perfusion requirements for large-animal lungs, the technology was successfully moved from the rodent model to the rhesus macaque model. All studies presented in this report were performed in accordance with the Institutional Biosafety Committee (IBC) policies in place at the Tulane National Primate Research Center. Demonstration of this technique is essential because identification of anatomical structures and physical manipulation of larger organs is sometimes difficult without visualizing the steps of the protocol. The studies made possible by these methods provide a basis for essential preclinical studies in decellularized rhesus macaque lungs in which recellularization with rhesus mesenchymal lineage stem cells and rhesus microvascular endothelial cells can be assessed. Our version of this bioreactor simulates the developmental environment and applies forces of mechanical stretch and strain in large-animal lungs and allows the investigation of lung recellularization under conditions known to facilitate pulmonary and endothelial development^13-15.

Protocol

1. Whole-organ Macaque Lung Decellularization

1. Preparation of Solutions
   1. Autoclave 10-15 L of deionized water (dH₂O) in 1-2 L bottles.
   2. Prepare "Triton solution" (0.1% Triton X-100 in dH₂O) by mixing 1 ml Triton X-100 in 999 ml of dH₂O while stirring on a magnetic stirrer. Filter the solution through a 0.22 µm filter apparatus. Store at room temperature.
   3. Prepare "SDC solution" (2% SDC in dH₂O) by slowly adding 20 g of sodium deoxycholate (SDC) to ~900 ml dH₂O while stirring. Once SDC is dissolved transfer the solution to a graduated cylinder and bring the volume to 1 ml with dH₂O. Filter the solution through a 0.22 µm filter apparatus. Store at room temperature.
   4. Prepare "NaCl solution" by adding 58 g of NaCl to ~900 ml of dH₂O and stir until completely dissolved. Add dH₂O to a final volume of 1 L, filter, and store at room temperature.
2. Organ preparation
   1. Utmost care must be taken to prevent infectious exposure incidents when working with macaque tissues. Put on the following personal protective equipment (PPE) before handling nonhuman primate tissues: surgical mask, face shield, surgical gloves, disposable gown, and a second layer of surgical gloves.
   2. Collect intact heart-lung blocs from animal necropsy in PBS containing 50 U/ml heparin to prevent coagulation of external blood.
   3. With the lungs lying flat in a dissecting tray, cannulate the pulmonary artery using a female Luer connector with an appropriately sized barb. Secure the cannula in place with an alcohol-sterilized zip tie.
   4. Slip a sterile zip tie around the trachea, insert a 0.250 in female Luer 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.
   5. Remove trapped air from the lungs by instilling PBS containing 30 U/ml heparin and 5 µg/ml sodium nitroprusside (SNP). Allow the solution to be expelled by natural recoil and repeat instillation twice more. After the third instillation, cap the tracheal Luer cannula with a Luer plug to hold the solution in the lungs.
   6. 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.
   7. Submerge the heart-lung bloc in dH₂O. Fill a 60 ml syringe with the PBS-heparin-SNP solution. Using forceps, submerge the arterial cannula so that all air bubbles escape from the vascular opening. Attach the syringe and carefully remove the plunger and allow the liquid to flow into the vasculature.
   8. Remove the plug from the tracheal cannula and allow the fluid in the airway to be expelled by natural recoil. Continue to add PBS-heparin-SNP solution to the syringe connected to the vascular cannula in order to maintain ~15-20 cm H₂O pressure above the artery. Continue perfusion until as much blood as possible is removed from the pulmonary vasculature.
3. Decellularization
   1. Day 1: Submerge the heart-lung bloc in dH₂O. Inflate and perfuse the lung with dH₂O using 60 ml syringes attached to the submerged tracheal and arterial cannulae, respectively. Repeat the airway and vascular washes with dH₂O 4x more for a total of five rinses.
   2. Remove the lungs from water and submerge the bloc in Triton solution. Inflate and perfuse the lungs with Triton solution as before. Repeat the instillation a second time, and incubate the submerged organs in Triton solution overnight at 4 °C.
   3. DAY 2: After overnight incubation, remove the lung bloc from Triton solution, wash externally with dH₂O, and then submerge the bloc in fresh dH₂O. Repeat dH₂O washes 5x as in step 1.3.1.
   4. Remove the lungs from dH₂O and submerge in SDC solution. Inflate and perfuse with SDC solution in the same manner as in step 1.3.2. Incubate submerged in SDC solution overnight at 4 °C.
   5. DAY 3: Wash the tissue again with dH₂O as in 1.3.1.
   6. Remove the tissue from dH₂O and submerge in NaCl solution. Inflate and perfuse with NaCl solution in the same manner as in step 1.3.2. Incubate submerged in NaCl solution for 1 hr at room temperature.
   7. Wash the lungs clean of NaCl solution with dH₂O as in step 1.3.1.
   8. Prepare fresh "DNase solution" (30 µg/ml DNase, 1.3 mM MgSO₄, 2.0 mM CaCl₂ in dH₂O) Bathe the lungs in this solution and instill into the airway and vasculature as in step 1.3.2. Incubate the lungs in DNase solution for 1 hr at room temperature.
   9. Prepare fresh "PBS solution" (1x PBS + 5x antibiotic/antimycotic). Remove the lungs from DNase solution and wash externally with PBS solution. Submerge the lungs in PBS solution and wash with this solution five times as in step 1.3.1.
   10. Store the lungs in PBS solution in a sealed container overnight at 4 °C. On the next day (DAY 4), wash the lungs in fresh, ice-cold PBS solution five times as in step 1.3.1 and store in PBS solution in a sterile, sealed container at 4 °C until use.

2. Large-organ Bioreactor

1. Assembly and Tissue Installation
   1. Assemble breathing loop and vascular loop structures as well as tracheal and vascular cannulae adapters ahead of time (Figure 1). With the exception of sterile-packaged air filters and syringe ports, autoclave all bioreactor components.
   2. Assemble the bioreactor components under a laminar flow hood according to the schematic in Figure 2. 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.
   3. Apply the tracheal and vascular cannulae adapters to the lung cannulae and install the organs in the main bioreactor chamber by bathing the lungs in the culture medium and attaching the cannulae 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 recellularization.
   4. Fill the tracheal reservoir approximately half-full with culture medium and affix the modified cap.
   5. Apply sterile 0.22 µm syringe filters, Luer plugs, and syringe ports to all chambers as indicated.
   6. Connect the trachea reservoir to the main chamber via the breathing loop apparatus. Attach the breathing loop tubing as indicated.
   7. Aspirate air from the tubing using the syringe ports and a 60 ml syringe fitted with an 18 G needle; move the directionality of the 3-way stopcocks to direct the flow of liquid into the syringe.
   8. 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 ~1 full "breath" every 2 min, and perfuse the vasculature via the peristaltic pump at approximately 10 ml/min for a total of ~30 min.
2. Airway seeding
   1. Prepare suspensions of cells to be seeded into the airway at the desired cell density (volume will depend on organ size).
   2. Inflate the lungs with the cell suspension by gently injecting through the syringe port attached to the three-way stopcock in the breathing loop.
   3. Hold the lungs statically at 37 °C, 5% CO₂ overnight without airway or vascular perfusion to allow cells to attach to the decellularized lung matrix.
   4. After overnight incubation, reinitiate the standard airway ventilation program, and culture the organs for 3-7 days (or other desired duration).
3. Vascular seeding
   1. Prepare suspensions of endothelial cell cultures as above for airway cell seeding.
   2. Transfer the cell suspension to the endothelial-seeding reservoir that contains a small magnetic stir bar.
   3. Change the directionality of the flow path from the main chamber to the endothelial seeding reservoir by rotating the valve on the stopcock positioned in the tubing connecting these two compartments, and seed endothelial cells gradually while gently stirring using the peristaltic pump.
   4. When seeding is complete (i.e. the volume of endothelial cell suspension is depleted in the endothelial seeding reservoir), stop perfusion and incubate statically for ~4-6 hr. Reinitiate vascular perfusion with main chamber medium at a rate of ~10 ml/min (or desired pressure if monitored).
   5. Culture for 3-7 days with continuous vascular perfusion.

3. Troubleshooting and Alternative Approaches

1. Tracheal cannulation: Sutures can be used in lieu of a zip tie to hold the Luer cannula in place if the trachea is too small. A ¼ in female Luer connection acts as a sufficient cannula for a wide range of trachea diameters.
2. Removing blood by pulmonary artery perfusion: Since tissues are collected from cadaveric animals, microthrombi are common and prevent some areas of the pulmonary capillary bed from being completely cleared of residual blood. During decellularization, however, these small vessels become permeable, and the decellularization reagents efficiently lyse and degrade these clots. Predecellularization perfusion should be performed to clear as much blood as possible, but it is not necessary to remove all traces of blood from the parenchyma.
3. Expulsion of fluids from the lungs: Macaque lungs retain strong natural recoil ex vivo, even when inflated with fluids. During decellularization, however, cells are lysed and release materials (including DNA) that increase the viscosity of the instilled fluids thereby making them more difficult to expel. Overnight incubation at 4 °C while submerged in the instilled fluid is usually sufficient to allow the lungs to completely expel the viscous fluid. Alternatively, allow as much fluid as possible to be expelled from the lungs and then proceed with instillation of the subsequent solutions; addition of detergents and deionized water washes usually facilitates the removal of the viscous fluids and allows decellularization to proceed efficiently.
4. Addition of antibiotics to detergent solutions: Antibiotics and antimycotics can be added to deionized water wash solutions as well as detergent solutions; however, penicillin/streptomycin precipitates in the presence of sodium deoxycholate, and these precipitates may interfere with the decellularization and washing processes. Moreover, the presence of antibiotics/antimycotics is not necessary during decellularization as the detergents used at these concentrations are able to lyse and kill microbes. After decellularization, rinsing and storage with PBS solution (containing 5x penicillin/streptomycin/amphotericin B) is sufficient to prevent most contamination.
5. Removal of air from bioreactor tubing: Air is efficiently aspirated from the bioreactor tubing using a syringe with a needle inserted into the needle ports in the three-way stopcocks. Alternatively, 1x PBS or culture media can be flushed through the tubing before installing the organs using a syringe attached in the same manner.
6. Ventilation and perfusion of lungs during and after recellularization: While the flow rates and times listed in this report were sufficient for cell attachment as well as airway liquid ventilation and vascular perfusion, these values should be determined empirically by the user. Culture times can also be adjusted based on the growth rates of the instilled cells.

Results

Results shown below represent separate experiments in which either airway or vascular compartments were seeded with rhesus macaque bone marrow-derived mesenchymal stem cells or rhesus lung-derived microvascular endothelial cells, respectively, that were isolated and characterized as previously described^16-18.

Throughout the decellularization process, macaque lungs displayed a progressive whitening culminating in a translucent appearance at the end of the process; however, the lungs ...

Discussion

Tissues can be efficiently and effectively decellularized by a number of methods employing physical, chemical, and enzymatic agents^12,20. The challenges of producing 3D biological matrices from large organs include the requirement for large volumes of decellularization solutions, expensive commercial equipment (i.e. bioreactors), and a dizzying amount of methodological perturbations required to achieve the final tissue-derived product. Our method provides a straightforward approach that minimizes phys...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Sodium nitroprusside	Sigma-Aldrich	71778-25G
Heparin sodium salt	Fisher	BP2425
Triton X-100	Fisher	BP151-100
Sodium deoxycholate	Fisher	BP349-100
Sodium chloride	Fisher	7647-14-5
Bovine pancreatic DNase	Sigma-Aldrich	DN25	Prepare stock, aliquot, and freeze
Magnesium sulfate	Fisher	10034-99-8
Calcium chloride	Fisher	C614-500
PBS (no Ca/Mg)	Gibco, Life Technologies	10010-031
Antibiotic-Antimycotic	Gibco Life Technologies	15240062
Cell Culture Media
Alpha MEM	Gibco Life Technologies	12561-072
Medium 199	Gibco Life Technologies	11150067
Premium Fetal Bovine Serum	Atlanta Biologicals	S11150
L-Glutamine 100x	Gibco Life Technologies	25030-081
Endothelial Cell Growth Supplement (ECGS)	ScienCell	1052
Antibiotic-Antimycotic	Gibco Life Technologies	15240062
Cell Seeding and Bioreactor Culture
Check valves	Cole-Parmer	EW-98553-20
Y-connectors	Cole-Parmer	ED-30614-08
3-Way stopcocks	Harvard Apparatus	721664
MasterFlex L/S 14 tubing	Cole-Parmer	96420-14
MasterFlex L/S 16 tubing	Cole-Parmer	96420-16
Male lock Luer 1/8 in	Cole-Parmer	EW-45505-04
Female Luer 1/8 in	Cole-Parmer	SI-45502-04
Male Luer lock plug	Cole-Parmer	EW-45505-56
Injection ports	Medi-Dose EPS	IV2004
Latex tubing	St. Louis Medical Suppy	HN10910
Hose clamp	Cole-Parmer	EW-06832-02
2 L Wide-mouth jar	Fisher	05-719-276
60 ml Syringes	Fisher	NC9035364