Rapid Porcine Lung Decellularization Using a Novel Organ Regenerative Control Acquisition Bioreactor

Abstract

To regenerate discarded lungs that would not normally be used for transplant, ex vivo reseeding after decellularization may produce organs suitable for clinical transplantation and therefore close the donor gap. Organ regenerative control acquisition (Harvard Biosciences, Holliston, MA), a novel bioreactor system that simulates physiological conditions, was used to evaluate a method of rapid decellularization. Although most current decellularization methods are 24–72 hours, we hypothesized that perfusing porcine lungs with detergents at higher pressures for less time would yield comparable bioscaffolds suitable for future experimentation. Methods involved perfusion of 1% Triton X-100 (Triton) and 0.1% sodium dodecyl sulfate at varied physiological flow rates. Architecture of native and decellularized lungs was analyzed with hematoxylin and eosin (H&E) staining, transmission electron microscopy (TEM), and scanning electron microscopy (SEM). Dry gas and liquid ventilation techniques were introduced. Our 7 hour decellularization procedure removes nuclear material while maintaining architecture. Bioscaffolds have the microarchitecture for reseeding of stem cells. Hematoxylin and eosin staining suggested removal of nuclear material, whereas SEM and TEM imaging demonstrated total removal of cells with structural architecture preserved. This process can lead to clinical implementation, thereby increasing the availability of human lungs for transplantation.

Although the number of people in need of a lung transplant continues to rise, less than 20% of donor lungs are considered suitable for transplantation.^1-3 Regenerating unused donor lungs will increase the number of transplantable organs to help meet increasing demand.⁴ Recently, decellularization of whole organs through ex vivo lung perfusion has been explored as a feasible route to tissue regeneration.^5-8 Decellularized organs provide a scaffold for future reseeding with various populations of cells, such as stem cells and tissue-specific cells.^7,9-12 However, significant challenges remain for developing a vascularized lung suitable for clinical transplantation.

Here, we describe an effective and rapid decellularization method for engineering acellular lungs, particularly the porcine lung. To monitor organ decellularization, a novel organ regenerative control acquisition (ORCA) bioreactor system was applied to simulate physiological conditions ex vivo. The circuit consists of a temperature-regulated organ chamber with two temperature probes, pressure monitoring and peristaltic pumps controlled by operation software. Decellularization methods using Triton and sodium dodecyl sulfate (SDS) with porcine lungs have been previously reported^12-14 with resulting organs appearing grossly white or translucent, indicating the loss of cellular components.¹⁵ These reported organ decellularizations call for protocols ranging from 24 to 72 hours.^13,16 Acknowledging previously established literature on bioengineering,¹⁷ we hypothesized that subjecting the lungs to the detergents for a shorter period of time with higher flow rates would yield a homogenously decellularized porcine lung.

To simulate physiological conditions, two types of ventilation were used in this study: dry gas and liquid ventilation.¹⁸ The dry ventilation entails using a clinical grade ventilator to perform consistent inspiration and expiration. Liquid ventilation involved completely filling and ventilating (aqueous) all lung fields with fluid using a pulsatile pump through a secured endotracheal tube.

Materials and Methods

Harvest of Porcine Lungs and Surgical Preparation

Adult porcine lungs were obtained from healthy porcine at the Food Product and Safety Laboratory of the University of Arizona. At the time of collection, each whole animal weighed between 127.01 and 136.08 kg.

Lungs were surgically dissected from adjacent attachments to the esophagus and thoracic vasculature using size 11 and 15 scalpels, within one surgical field. The pulmonary artery (PA), along with the ascending and descending aortic portions, was separated. The PA was dissected at the proximal pulmonary trunk and the left atrium was excised, leaving the pulmonary veins intact. Cannulation of the PA was completed via straight barbed coupler and anchored with clamps. A 60 ml syringe with a suction catheter was inserted into the right and left bronchus for aspiration.

ORCA Bioreactor Apparatus

Organ regenerative control acquisition consists of a heat reservoir and temperature control system, the organ chamber, and peristaltic pumps controlled by operation software. The most comprehensive function of the system includes the advanced sensor options on the bioreactor, which allows monitoring of the decellularization process. By using this system, multiple parameters such as temperature, flow rate, and pressure could be monitored, regulated, and recorded. We also evaluated respiratory mechanics, including total lung volumes and lung pressures as well as compliance.

Lung Decellularization

The prepared lung was attached to the ORCA system and the left lung clamped to block perfusion for use as a control. The lung was flushed with 5 L of reagent-grade deionized (DI) water for 30 minutes at a rate of 1 L/minute. The DI water was replaced with 1× phosphate-buffered saline (PBS, pH 7.2) at 1 L/minute for 30 minutes, followed by another DI water wash for 30 minutes at the same flow rate. Triton (1%) was then applied via perfusion at 1.2 L/minute for 1 hour at room temperature. After Triton, DI water was perfused through the lung for 30 minutes followed by 1× PBS for 30 minutes to aid in cell lysis and removal of cellular debris and chemical residues. SDS (0.1%) was used next at 1.3 L/minute for 2 hours at room temperature. The final flush was completed using DI water, followed by 1× PBS both for 30 minutes at 1.3 L/minute. Fluid pressures were monitored at the PA and trachea during the entire decellularization process. Temperature was monitored via the ORCA system and kept room temperature ±2°C throughout the experiment. The decellularized lung was removed from the ORCA organ chamber and was allowed to passively drain at 4°C before postdecellularization mechanical properties testing.

Lung Mechanical Testing

Functional properties of native and decellularized lungs were assessed using conventional clinical ventilation. Excised porcine lungs were intubated with a 10 mm endotracheal tube and attached to the ventilator. Pressure controlled ventilation (GALILEO, Hamilton Medical, Inc., Bonaduz, Switzerland) was delivered to all lungs. Ventilation began at 5 breaths per minute, a driving pressure of 20 cm H₂O, a positive end-expiratory pressure (PEEP) of 5 cm H₂O, and a 1:1 inspiratory to expiratory ventilation ratio. Hamilton Medical data logger facilitated biomechanical feedback recording properties every 32 ms. Lung volumes, pressures, and compliance per group (native versus acellular) were analyzed to examine physiological variation. To this aim, each ventilator parameter inflating the lungs was collected for 20 complete breaths and their usage was analytically determined by means of an automated procedure. Afterward, the trending data were collected and plotted with 5 breaths per group and statistically compared.

Histology Assessment

Decellularized and native lung samples were excised for morphological analysis. The specimens were fixed overnight with 10% formalin, then switched into 70% ethanol, and embedded with paraffin. Thick sections (10 μm) were cut and stained with hematoxylin and eosin (H&E) and were used to evaluate the presence of nuclear material by standard light microscope. The slides were captured on a Leica microsystem.

Transmission Electron Microscopy

From both decellularized and native lungs, we cut 5 mm³ specimens for processing and analysis. Samples were fixed in 2.5% glutaraldehyde in piperzine-N,N′-bis(2-ethanesulfonic acid) (PIPES) buffer (pH 7.4) overnight. Thereafter, they were washed three times for 10 minutes with PIPES and fixed in 1% osmium tetroxide along with two washes in DI water. Samples were block stained in 2% uranyl acetate and dehydrated through graduated ethanol (50%, 70%, 90%, 100%, 3×). After infiltration with Spurr′s Resin (50/50, 100%, 3×), blocks were polymerized at 60°C overnight. Sections (70 nm) were cut on a Leica EMUC6 ultra microtome onto 150 mesh copper grids. Sections were stained with 2% lead citrate and viewed in an FEI Tecnai Spirit electron microscope operated at 100 kV. Tiff images (8 bit) were collected via an AMT 4M pixel camera.

Scanning Electron Microscopy

From both decellularized and native lungs, we cut 5 mm³ specimens for processing and analysis, using a standardized protocol. The specimens were first fixed with 2.5% glutaraldehyde in PBS (pH 7.4) at 4°C overnight and fixed in 1% osmium tetroxide for 1 hour; then, they were dehydrated in graded ethanol solutions (50%, 70%, 90%, and 100%, 3×) v/v in DI water at 10 minute intervals for each concentration. After two 10 minute incubations in hexamethyldisiloxzane, specimens were air-dried. To dry the specimens, we used the critical point drying apparatus (Polaron model 3100, Energy Beam Sciences, East Granby, CT); then, to mount them on aluminum stubs, we used a carbon double-sided tape. To provide surficial conduction, we sputter-coated the dried specimens with a 5 nm thin layer of gold (Pelco SC4, Ted Pella, Inc., Redding, CA).

DNA Quantification

Approximately 100 mg of native and decellularized porcine lungs was incubated with 400 μl cell lysis buffer and 8 μl of 1 mg Proteinase K (Viagen, 20 mg/ml) for overnight in a water bath at 55°C. Samples were then placed in 90°C for 10 minutes to ensure inactivation of Proteinase K. The digest was vortexed for 1 minute, then centrifuged at 13,000 rpm for 10 minutes. The supernatant was transferred to a new tube and the DNA precipitated with isopropanol. Centrifugation was performed to create a DNA pellet, which was then rinsed with 70% ethanol, dried, and resuspended in nuclease-free water. DNA was quantified using spectrophotometry. DNA samples were evaluated in triplicate by 1% agarose gel electrophoresis, visualized with ethidium bromide and ultraviolet transillumination.

Results

Decellularization of Porcine Lung by Using ORCA Bioreactor

One of the main goals of organ decellularization is the removal of all cellular material without adversely affecting the composition, biologic activity, or mechanical integrity of the remaining 3D matrix.^2,9,14,15,19 In this study, porcine lung decellularization process was performed under ORCA bioreactor control (Figure 1). The effect of decellularization on whole porcine lung architecture naturally varies depending on which detergents are applied but was also influenced by multiple quantitative parameters including perfusion flow rate and pressure. Flow rates were recorded at 1.2 L/minute when we decellularized lung using 1% Triton while maintaining pressure under 20 mm Hg. After Triton administration, the lung had a grossly pink appearance, suggesting that decellularization was occurring. The next 3 hours of decellularization, which entailed increasing the flow rate to 1.3 L/minute, resulted in the lung becoming more translucent during treatment with 0.1% SDS. The results showed that the gross color of the lung lightened and became translucent at the completion of the described 7 hour protocol (Figure 2), indicating the loss of cellular material comparable to similar protocols of 14 and 24 hours.

Figure 1.

Organ regenerative control acquisition (ORCA) bioreactor. ORCA bioreactor consists of a heater reservoir and temperature control system (A), the organ chamber (B), and peristaltic pumps (C) controlled by the operation software.

Figure 2.

Porcine lung gross images. Gross image of intact decellularized porcine lungs after 24 hours (A), 14 hours (B), and 7 hours (C) of decellularization. Images demonstrate the half of the lung that has not been decellularized juxtaposed with the decellularized half, where the degree of translucency indicates the loss of cellular material following decellularization.

Lung Mechanics

Pulmonary inflation volumes (Figure 3A), pressures (Figure 3B), and compliance (Figure 3C) values were determined with driving pressures of 20 cm H₂O and PEEP of 5 cm H₂O. Decellularized porcine lungs displayed both significantly higher mean pulmonary volumes 709 ± 9.2 ml and mean lung compliance values 27.9 ± 0.44 ml/cm H₂O than that of native lungs (p < 0.0001). The mean compliance values were 61.3 ± 1.25 ml/cm H₂O and 89.2 ± 1.69 ml/cm H₂O for native lung and decellularized lung, respectively. The mean pressure values were 26.1 ± 0.1 cm H₂O and 26.3 ± 0.1 cm H₂O for native lung and decellularized lung, respectively (p = 0.081). There were no significant differences in pressure values between groups, but observed inflation patterns of decellularized lungs were shaped differently compared with the group of native lungs (Figure 3B).

Figure 3.

Lung mechanics. Assessment and comparison of inflation volumes (A), pressure (B), compliance (C) of functional lung tissue responses and behavioral trends of bioscaffold.

Histology Assessment

Hematoxylin and eosin and toluidine blue staining revealed no visible basophilic staining representative of cellular nuclear material after 7 hours under the detergent-mediated decellularization protocol. In contrast, cell nuclei were visible in native control lung (Figure 4), as evidenced by the presence of basophilic staining. The tissue morphology was not perturbed by the decellularization. Lung architecture and collagen staining were representative of native, un-decellularized lungs. The decellularization process did not disrupt lung architecture despite the removal of cellular material.

Figure 4.

Assessment of whole porcine lung, 7 hour decellularization. A: Native lung H&E stain (400× magnification). B: Native lung toluidine blue stain (400× magnification). C: Native lung TEM (2,650× magnification). D: Decellularized lung H&E stain (400× magnification). E: Decellularized lung toluidine blue stain (400× magnification). F: Decellularized lung TEM (2,550× magnification). H&E, hematoxylin and eosin; TEM, transmission electron microscopy.

Transmission Electron Microscopy and Scanning Electron Microscopy

Transmission electron microscopy (TEM) imaging indicated that there are no nuclei at the completion of the 7 hour decellularization process. However, tissue basement membrane is intact and architectural integrity is maintained while cellular material has been predominantly removed (Figure 4).

Scanning electron microscopy (SEM) analysis (Figure 5) indicated the maintenance of architecture after intact porcine lung 7 hour decellularization. The pleural surface of the porcine lung in normal control (Figure 5A) showed intact tissues. The parenchymal surface of lung in normal control (Figure 5C) showed visible bronchioles with alveoli (arrow). The pleural surface of the decellularized lung tissues (Figure 5B) indicated a topographic variance and intact extracellular matrix (ECM) fibers. The parenchymal surface of decellularized lung showed that the matrix is still intact, whereas no cells are present (Figure 5D). The ECM components, particularly collagen, are preserved.

Figure 5.

Maintenance of architecture after intact porcine lung 7 hour decellularization. Scanning electron micrographs show intact tissues in normal lungs (A and C, 10,000× magnification) and topographic variance in the decellularized lung (B and D, 10,000× magnification). Panels (B) and (D) indicate no visible cells, but the matrix and collagen fibrils are maintained in the decellularized lung.

DNA Quantification

DNA quantification analysis from native and decellularized lungs showed a significant decrease in the amount of DNA present in decellularized lung tissue compared with native lung tissue. Using a spectrophotometer, it was determined that 97% of DNA was removed after the 7 hour decellularization procedure. Equal amounts of DNA were loaded into 1% agarose gel containing ethidium bromide and were observed under ultraviolet transillumination. These data indicated that there was no obvious DNA in the decellularized lung tissue, whereas the native control lung tissue contained DNA (Figure 6).

Figure 6.

DNA Assessment. Quantitative analysis of DNA content (A) shows a significant decrease in DNA content of the decellularized lung as compared to the native lung. In addition, no residual DNA was observed in the decellularized lung as compared to native lung on a 1% agarose gel (B) stained with ethidium bromide. A DNA ladder was loaded as a control.

Discussion

As the gap between the number of people on the transplant list and the number of donors widens,^1,3,4 it is imperative that a feasible option be implemented to address this crucial need. Because of the intricate complexity associated with the recreation of the bronchi alveolar network, alveolus–capillary interface, functional vasculature, and airway systems, lung tissue engineering has had limited successful applications.²⁰ Decellularization provides a conduit for exploration because decellularized lung bioscaffolds may be able to be reseeded with the recipient′s autologous stem cells to create a recipient-compatible organ²¹ and therefore eliminate immunorejection and the recipient′s need for chronic immunosuppression. Hence, this time-efficient decellularization process that results in an intact bioscaffold is a significant step toward organ regeneration.

There are currently no effective standardized and rapid methods for clinical lung decellularization for lung regeneration. The current progress of lung tissue engineering has been limited because of the difficulty of biological cooperation between both microanatomy and lung function. One aim of this study was to explore a rapid protocol to decellularize large animal organs, specifically the porcine lung. To monitor the decellularization process, the current study applied the ORCA bioreactor system. Organ regenerative control acquisition is designed to address regenerative medical research in both the decellularization and recellularization processes of various organs, which is central to organ regeneration. In this system, in vivo physiological conditions can be mimicked through control algorithms that regulate flow rate, profiles, and pressure. Critical readings can be taken from both inside the organ as well as in the support environment through the use of sensors. The concurrent software allows assessment of the entire decellularization process.

Before this study, our protocol optimization included evaluating the effectiveness of 1% Triton and 0.1% SDS in the decellularization of porcine lung. Concentrations of, or above, 1% SDS will result in ECM component degradation, and the ultrastructure and composition of the cell basement membrane will be negatively impacted.^22,23 A decrease in elastic recoil and mechanical function is observed when SDS is administered at 1% concentration. Our protocol requires a significantly lower concentration as 0.1% SDS is used. The results yielded significant nuclear material removal, which was confirmed by quantitation of residual DNA content. Transmission electron microscopy showed that the architecture is still preserved while all nuclear material has been removed; SEM indicated that the matrix is still intact, whereas no cells are present, only the matrix and collagen are preserved. Data supported our hypothesis that increased perfusion rates for shorter amounts of time in the ORCA system can produce a decellularized bioscaffold with minimal damage to the native architecture.

In the absence of the thorax, the lung parenchyma encounters no opposing forces of the native chest wall. With the greater ease of expansion, lower pressures are required for ex vivo ventilation than in vivo. Decellularizing ex vivo lung parenchyma entails removing immeasurable quantities of intracellular content from the entire organ. The postdecellularization process results in an increase of intrapulmonary volume during both inspiration and expiration from the native state. This observation could potentially be caused by the loss of cellular mass and warrants the investigation of mass to volume relationships to better optimize future lung decellularization methods.

Whether lung function is evaluated ex vivo or in vivo, lung volumes and lung compliance values are key contributors in driving proper lung function.^24-26 One of the objectives of this protocol was to minimize the disruption of bioscaffold integrity to preserve its future potential for recellularization. This objective was achieved by limiting tracheal pressures to levels that did not exceed 20 mm Hg (approximately 25–27 cm H₂O). Our value is below published pressures of 30 cm H₂O that are known to damage lung tissue if exceeded while being mechanically ventilated.²⁷ To avoid barotrauma, it is important to maintain consistent airway portal pressures and alveolar recruitment while transitioning from gas inflation to liquid detergent application. A pressure value ensured alveolar recruitment during liquid pulsations of detergents, which facilitated a homogenous decellularization through the controlled distribution of liquids. We discovered lung compliance retains its direct relationship with lung volume in bioscaffolds and generates progressively different inflation trends (Figure 3, A and C) comparative to native trends (Figure 3, A and C). These findings may give rise to information regarding the intrinsic pulmonary behaviors, as ventilation is kept fixed for these bioscaffolds with respect to time.²⁸

In this study, we achieved the production of acellular lung bioscaffolds using a time-efficient and clinically minded protocol. Use of the ORCA system leads to an adequate, homogenous decellularization, by applying high perfusion pressures of detergents. The removal of cellular components combined with the maintenance of lung bioscaffold architecture implies that the DNA components have been eradicated while ECM structure remains intact. Presumably, the decellularization process removes cellular antigens responsible for immune rejection; however, this has not been tested clinically. Clinical translatability is the major limitation of the study, given that these bioscaffolds have not yet been reseeded. However, in the future, these bioscaffolds can potentially be reseeded and subsequently used in autologous transplantation. Successful reseeding of decellularized rodent lungs has recently been reported in the literature,¹² and similar reseeding methods will be applied in future experiments. If larger-scale reseeding experiments are successful, lung engineering, starting with the creation of decellularized bioscaffolds, may be applied to a clinical setting and implemented as an intervention for those awaiting a lung transplant.

Conclusion

A 7 hour protocol is adequate for lung decellularization, removing all nuclear materials and preserving architecture. The use of a clinical ventilator before and after the decellularization process may create a more comprehensive picture of the lung′s complex alveolar systems and the ex vivo bioscaffold physiology necessary for reseeding of stem cells. The optimization of this process will ultimately increase the availability of human lungs available for transplantation.