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Copyright © 2015 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.
Perfusion-Decellularization of Porcine Lung and Trachea for Respiratory Bioengineering *†¶Alexander Weymann, †¶Nikhil Prakash Patil, †Anton Sabashnikov, *Sevil Korkmaz, *Shiliang Li, ‡Pal Soos, §Roland Ishtok, *Nicole Chaimow, *Ines Pätzold, *Natalie Czerny, *Bastian Schmack, †Aron-Frederik Popov, †Andre Rüdiger Simon, *Matthias Karck, and *Gabor Szabo *Department of Cardiac Surgery, Heart and Marfan Center, University of Heidelberg, Heidelberg, Germany; †Department of Cardiothoracic Transplantation & Mechanical Circulatory Support, Royal Brompton and Harefield NHS Foundation Trust, Middlesex, UK; and ‡Heart and Vascular Center and §2nd Department of Pathology, Semmelweis University, Budapest, Hungary
Abstract: Decellularization of native organs may provide an acellular tissue platform for organ regeneration. However, decellularization involves a trade-off between removal of immunogenic cellular elements and preservation of biomechanical integrity. We sought to develop a bioartificial scaffold for respiratory tissue engineering by decellularization of porcine lungs and trachea while preserving organ architecture and vasculature. Lung–trachea preparations from 25 German Landrace pigs were perfused in a modified Langendorff circuit and decellularized by an SDC (sodium deoxycholate)-based perfusion protocol. Decellularization was evaluated by histology and fluorescence microscopy, and residual DNA quantified spectrophotometrically and compared with controls. Airway compliance was evaluated by endotracheal intubation and mechanical ventilation to simulate physiological breathing-induced stretch. Structural integrity was
evaluated by bronchoscopy and biomechanical stress/strain analysis by measuring passive tensile strength, all compared with controls. Decellularized lungs and trachea lacked intracellular components but retained specific collagen fibers and elastin. Quantitative DNA analysis demonstrated a significant reduction of DNA compared with controls (32.8 ± 12.4 μg DNA/mg tissue vs. 179.7 ± 35.8 μg DNA/mg tissue, P < 0.05). Lungs and trachea decellularized by our perfusion protocol demonstrated increased airway compliance but preserved biomechanical integrity as compared with native tissue. Whole porcine lungs–tracheae can be successfully decellularized to create an acellular scaffold that preserves extracellular matrix and retains structral integrity and three-dimensional architecture to provide a bioartifical platform for respiratory tissue engineering. Key Words: Decellularization—Tissue engineering—Whole organ concept—Lung—Trachea.
Lung transplantation is the definitive treatment for end-stage pulmonary disease, but remains limited by donor organ shortage and waiting-list mortality. Long-segment circumferential tracheal defects also pose a major problem for surgical reconstruction fol-
lowing malignancy, traumatic injury, or airway stenosis. While transplant recipients must live with the “necessary evil” of lifelong immunosuppression, often begetting hypertension, diabetes, renal failure, malignancy, and other sequelae of chronic immunosuppression (1,2), attempts at replacing the trachea with synthetic prosthetics and scaffolds entail complications like mucous build-up, inflammation, infection, stricture, and eventual stenosis (3). Bioartificial or “tissue engineered” lung/trachea offers the potential for long-term graft survival without the need for long-term immunosuppressive therapy (4). Decellularization of whole organs yields a biologic scaffold of three-dimensional extracellular matrix (ECM)
doi:10.1111/aor.12481 Received September 2014; revised December 2014. Address correspondence and reprint requests to Dr. Alexander Weymann, Department of Cardiac Surgery, Heart and Marfan Center—University of Heidelberg, Im Neuenheimer Feld 110, Heidelberg 69120, Germany. E-mail: weymann.alexander @googlemail.com ¶ These authors contributed equally to this work and are considered to be joint first authors. Artificial Organs 2015, 39(12):1024–1032
BIOARTIFICIAL LUNGS AND TRACHEAE that can be subsequently repopulated with donorspecific mature or stem cells for ex vivo regeneration of donor organs (5). Various approaches have been tried for lung decellularization, including physical agitation and exposure to chemical and enzymatic agents such as Triton-X, sodium deoxycholate (SDC), and vascular perfusion of CHAPS (3-[(3cholamidopropyl)dimethylammonio]-1-propanesulfonate) detergent (6). However, most studies are limited to mice or rats, and there are limited data on decellularization of lungs from larger mammals (7–10). Further, approaches utilized in rodent models may be insufficient for adequate decellularization of lungs obtained from larger animals and humans. We previously reported our perfusion decellularization protocol using sodium dodecyl sulfate (SDS) in pulmonic heart-valves (11), more recently applied to whole-hearts (12). In the present study, we attempted to decellularize porcine airways to create whole lung–trachea scaffolds with a perfusable vascular bed and preserved airway and alveolar geometry. MATERIALS AND METHODS Lungs and adjacent trachea were harvested under sterile conditions from 25 female German Landrace crossbred pigs (80–100 kg). All animals received humane care in accordance with the “Guide for the Care and Use of Laboratory Animals” prepared by the Institute of Laboratory Animal Resources (US NIH Publication No. 86-23, revised 1996). All procedures followed the European Agreement of Vertebrate Animal Protection for Experimental Use (86/609). This investigation was reviewed and approved by the concerned institutional review committees. Anesthetic and surgical procedures Intravascular access was secured via a superficial ear vein of the animal, as described previously (13). After intramuscular injection of 4 mg azaperone (Stresnil, Janssen, High Wycombe, UK) and 0.01 mg/kg fentanyl, 3–4 mg/kg Hypnomidate (Janssen-Cilag GmbH, Neuss, Germany) was administered intravenously, followed by intubation and ventilation with 40% FiO2. Muscle relaxation was achieved with pancuronium (0.3 mg/kg/h). After systemic heparinization and median sternotomy, the lungs and trachea were procured. Perfusion decellularization circuit The modified Langendorff decellularization model comprised a perfusion circuit and a pressure control
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module, as described previously (12). All components were sterilized by conventional autoclaving and connected with 3/8″ silicon tubes (Maquet, Rastatt, Germany). The pulmonary artery and trachea were connected with cannulas of adjustable diameter for controlled perfusion. The circuit was powered by a roller pump (Stöckert, München, Germany) controlled via a pressure transducer (Medex-Smith Medical, Kent, United Kingdom). A computer system (Engineo, Mainz, Germany) continuously recorded the perfusion pressure. We interposed an airtrap (Gambro Medical Line, Hechingen, Germany) to ensure air-free perfusate. Inlet and outlet ports permitted easy and quick detergent change. The returning (outflow) perfusate was collected into a reservoir (Maquet AR 28150), with total circuit volume of 2500 mL. Antibiotic-containing phosphate-buffered solution (PBS, Sigma, Seelze, Germany) enriched with 100 μg/mL penicillin-streptomycin (Biochrom, Berlin, Germany) was perfused through the lungs and tracheae at 1.5 L/min for 24 h. Subsequently, the lungs were perfused with 4% SDC (12 h) and 0.1% Triton X-100 in PBS (12 h) at 2.0 L/min for 5 days. The tracheae were subjected to 72 h immersion in 4% SDS solution at room temperature on a mechanical agitator, with a solution change at 24 h. After 72 h, a 0.1% peracetic acid and 4% ethanol wash was administered for 6 h on a mechanical agitator for further decellularization and disinfection. During the entire process, the lungs and trachea were washed with PBS once a day to remove residual substances. The final washing step included perfusion with PBS for another 24 h to remove remnant detergents and cell debris at 1.5 L/min.
Evaluation of lung biomechanical stability We performed pulmonary compliance testing on the decellularized lung scaffolds under quasi-clinical conditions by employing patient mechanical ventilators (Model Primus, Dräger Medical, Lübeck, Germany) for ventilating decellularized (n = 6) and native lungs (n = 6). The resultant pressure changes were monitored and compared. An endotracheal tube (Teleflex Medical, Kernen, Germany) was used for tracheal intubation and ventilation. The initial settings in the volume-controlled mode of the ventilator were kept to a frequency of 30 breaths/min at a tidal volume of 30 mL and a positive end expiratory pressure of 3 mbar. To determine the compliance of the lungs, the tidal volume was subsequently increased in 10 mL steps and the corresponding pressure values were measured. All values were Artif Organs, Vol. 39, No. 12, 2015
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captured by photo documentation to create a volume/pressure chart. Measurement of tracheal mechanical stability Mechanical stability of tracheal tissue was analyzed using a static material testing instrument (Zwick Roell, Ulm, Germany). Decellularized tracheal samples (n = 6) were stretched until complete tearing. Passive tensile strength was continuously recorded throughout the procedure. Samples from untreated native trachea (n = 6) were used as controls. Bronchoscopy Maintenance of major airways was assessed in decellularized porcine airway by bronchoscopy (Pentax flexible nasolaryngoscope [FNL-10RP3], Tokyo, Japan) and compared with controls. DNA quantification Lung sections were processed for spectrophotometric quantification to determine the concentrations of residual DNA in the decellularized group compared with the controls. Samples were normalized according to equivalent dry weight in mg. The total amount of DNA was silica-membrane-based purified (QIAamp DNA Mini Kit, Qiagen, Basel, Switzerland) following the manufacturer’s instructions and later quantified by spectrophotometry. Western blot analysis Tissue samples of 25–30 μg were homogenized with 150 mL PBS using a tissue lyser (Qiagen) and then digested in 150 μL 2× RIPA buffer (Melford Laboratories, Chelsworth, UK). Protein concentration was quantified via Bradford assay and then denatured with sample buffer (Life Technologies, Carlsbad, CA, USA) for 10 min at 70°C. The proteins were separated via SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membrane (Invitrogen). The membranes were blocked with 5% milk in Tris-buffered saline Tween 20 for 1 h at room temperature before incubation overnight at 4°C with primary antibodies specific to elastin (1:200, Abcam, Cambridge, UK) and Collagen1 (1:1000, Abcam). After washing the blots to remove excess antibody binding, membranes were incubated for 1 h with horseradish peroxidaseconjugated goat secondary antibody (1:5000, Santa Cruz, Dallas, TX, USA) followed by three washing steps. The immunoreactive protein bands were developed using enhanced chemiluminescence (PerkinElmer, Waltham, MA, USA) and were Artif Organs, Vol. 39, No. 12, 2015
detected with a Chemi-smart 5100TM imager (PEQLAB GmbH, Erlangen, Germany). Histology Samples of decellularized lung and trachea were analyzed with light microscopy and compared with native, untreated cross-sections. Tissue samples were fixed in 10% formalin, embedded in paraffin and sectioned following standard protocols. We cut tissue into 5 μm sections, stained them with 4′,6-diamidino2-phenylindole (DAPI, Sigma-Aldrich Inc., Munich, Germany) and hematoxylin and eosin (H&E) to determine if remnant nuclear structures could be observed after decellularization as compared with controls. The fluorescence method used has been described elsewhere (12,14). Moreover, Masson’s trichrome stain Verhoeff van Gieson stain, and Alcian Blue stain (Sigma-Aldrich, Hamburg, Germany) were used to visualize different ECM components such as collagen, elastin, and glycosaminoglycans in decellularized lung tissue and compared with controls. The sections were analyzed by routine bright field and fluorescence microscopy (Olympus Optical Co, BX 51 and CKX 41 microscopes, Tokyo, Japan). Images were acquired with the CellA Soft Imaging System (Olympus Soft Imaging Solutions). Statistical analysis All values in the figures and text are shown as mean ± SEM. A two-tailed Student’s t-test was performed to determine significant differences of the native lungs (control) versus the decellularized lungs, considering P values less than 0.05 as statistically significant. The commercially available SPSS statistical software package 13.0 for Windows (SPSS Inc., Chicago, IL, USA) was used for data analysis. RESULTS Decellularization treatment There was a progressive loss of pink pigmentation during decellularization, with fully decellularized whole lung–trachea scaffolds having a pale/whitish appearance and visible vascular conduits throughout the matrix (Fig. 1) produced by day 5 of the decellularization treatment. Bronchoscopy confirmed that the underlying macroscopic structure and branching of both airway and vessels remained intact after decellularization, with no evidence of gross structural damage, sloughed cell material, or cell debris (data not shown). Histological examination
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FIG. 1. Whole porcine lungs with adjacent trachea during decellularization after 1 (A), 3 (B), and 5 days (C). All gross structures including blood vessels and airway (D, E) were preserved with no evidence of major structural damage, sloughed cell material, or cell debris.
demonstrated that our decellularization treatment converted native porcine lungs and tracheae into an almost acellular neoscaffold as demonstrated by H&E staining and additionally DAPI staining for the lungs (Fig. 2). The lacunae in the cartilage rings were devoid of cells, nuclei, or nuclear material (Fig. 3). Thus, intracellular constituents containing potentially immunogenic DNA were effectively removed while preserving the integrity of the ECM. Further examination using Masson’s trichrome, Verhoeff van
Gieson, and Alcian Blue stain visualized different ECM components such as mesh-like collagen formations, elastin, and glycosaminoglycans (Fig. 4). Total DNA content of decellularized lungs was markedly decreased compared with untreated controls. After decellularization, the DNA content was reduced to 32.8 ± 12.4 μg DNA/mg tissue versus 179.7 ± 35.8 μg DNA/mg tissue in the native group (P < 0.05). Surgical experiments confirmed nativelike tissue properties with adequate structural
FIG. 2. H&E (A) and DAPI (C) staining of lung tissue before and after perfusion decellularization (B, D) showing no remnant nuclear structures with maintained ECM. Scale bars, 200 μm.
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FIG. 3. Images of decellularized porcine trachea after 72 h immersion in 4% SDS solution at room temperature on a mechanical agitator (A, B) with retained gross morphology and stability. After decellularization, native tracheal matrix (C, H&E stain) turned into an acellular scaffold (D, H&E stain) with preservation of ECM integrity. Scale bars, 200 μm.
integrity for surgical purposes with suture application for lungs and tracheae. Biomechanical tests The stress–strain curve of decellularized porcine trachea tissue in comparison with controls is shown in Fig. 5. Maximal stress (equivalent to maximal tensile strength) was defined by the peak of the curve, while tissue resistance to stretch was assessed by the slope of the curve. It is evident that porcine tracheae
treated using the decellularization treatment described here have stress resistance similar to that of native trachea. Volume/pressure measurements showed that decellularized lungs did not exhibit statistically relevant changes in peak pressure, dynamic compliance, or static compliance when compared with native lungs (Fig. 6). In decellularized lungs, higher ventilatory volumes were achieved with lesser airway pressures compared with native lungs, indicating
FIG. 4. Masson’s trichrome (A, B), Verhoeff van Gieson (C, D), and Alcian Blue staining (E, F) of lung tissue before (A, C, E) and after perfusion decellularization (B, D, F), demonstrating retained ECM components such as mesh-like collagen formations, elastin, and glycosaminoglycans. Scale bars as indicated.
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FIG. 5. Stress–strain curves of native and decellularized trachea demonstrating similar stress resistance. The peak of the curve defines maximal tensile strength, with tissue resistance to stretch represented by the slope of the curve.
increased compliance in decellularized lungs. Thus, no substantial stiffening or weakening of the ECM occurs in the decellularized lungs as compared with native lungs. Western blot analysis Western blot analysis results are depicted in Fig. 7. As shown, protein bands for collagen 1 and elastin have the same intensity in decellularized and native samples. Thus, there is no obvious difference in the content of elastin or collagen 1 between decellularized and native lungs. DISCUSSION Several approaches for decellularization have been tried, including continuous whole lung perfusion in a bioreactor chamber (15), manual instillation of fluids (8), and physically agitating excised sheets of lung tissue during exposure to decellularizing solvents (16). However, there are no consensus criteria for defining adequate/optimal decellularization achieved by different protocols. Nichols et al. (15) used a combination of freezing and SDS washes to achieve decellularization, with organs stored at −80°C for at least 1 month prior to decellularization. However, it is not known whether an additional freeze-thaw
cycle confers any advantage as compared with less complicated methods for decellularization. O’Neill and colleagues compared different methods for decellularizing lung sections (16). The authors did not work with whole-lung preparations but evaluated human and porcine lung slices in a two-dimensional system and found that acellular scaffold slices allowed cell attachment and survival. Importantly, though, human cells thrived equally on porcine and human matrix sections—a welcome finding considering the abundant supply of porcine lungs as a test bed for organ regeneration and also for potential “offthe-shelf” organ scaffolds (16). Porcine lungs are of a size comparable with human lungs, with similar physiology and anatomical features. A relatively short generation-interval of pigs means that a considerable number of bred porcine offspring can be procured in a reasonable time period. Pigs have long been used in xenotransplantation studies, with genetically modified pigs shown to be significantly less susceptible to human complement-dependent cytotoxicity (17). Also, studies with cardiac ECM have demonstrated remarkable homology between porcine and human hearts (18). In addition, porcine organs may be advantageous with regard to product development, ethical considerations, and regulatory approval. All Artif Organs, Vol. 39, No. 12, 2015
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FIG. 6. Volume/pressure measurements of decellularized lungs compared with native lungs. Decellularized lungs did not exhibit statistically relevant changes in peak pressure, dynamic compliance, or static compliance when compared with native lungs.
FIG. 7. Western blot analysis for collagen-1 and elastin. The protein bands for collagen-1 and elastin have the same intensity in decellularized and native lung samples with no obvious difference in the content of collagen-1 or elastin after decellularization. The first five bands demonstrate one native lung (one sample per lobe) representative for native lung tissue. The next 12 bands represent six decellularized lungs (each two samples, one from left lung and one from right lung). The last three bands are controls.
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BIOARTIFICIAL LUNGS AND TRACHEAE these factors highlight the importance of porcine airways in the attempt to develop a human-sized bioartificial lung–trachea scaffold. Whereas Wagner et al. (19) reported their findings with different perfusion protocols for decellularization of porcine and human lungs, they did not use whole organs—rather, they utilized small segments (1–3 cm3) and lobes for their experiments. Unlike our previous work with hearts (12), however, we found that there is an important difference to keep in mind when trying to decellularize lungs and trachea— there is no normal egress from the airway, unlike the arteriovenous egress of vasculature. Hence, maintaining optimal pressures is critical to avoid barotrauma-induced disruption of intercellular junctions when decellularizing the airway, because there is no egress for the SDC (or any solution) used for perfusion-decellularization. This is an important caveat, which underscores the importance of accurate and efficient pressure modulation control in decellularization protocols for lung– trachea preparations. Preserving the biomechanical integrity is critical if a decellularized matrix is to serve as an effective recellularization scaffold. In this study, decellularized tracheae had stress resistance similar to that of native trachea, and there was no substantial stiffening or weakening of the ECM in decellularized lungs as compared to native lungs. In fact, in decellularized lungs, higher ventilatory volumes could be achieved with lesser airway pressures compared with native lungs, indicating increased compliance in decellularized lungs. These findings are in contrast to another contemporary protocol that resulted in lungs having lower specific compliance after decellularization (20). Our results demonstrate that porcine lung–trachea can be decellularized to generate whole-organ acellular scaffolds for organ regeneration. Our protocol employs a simple and inexpensive constantpressure perfusion system to generate an acellular lung–trachea matrix that retains biomechanical strength and three-dimensional architecture with the advantage of enhanced airway compliance. Further strides in decellularization protocols and conjunctory recellularization techniques will be critical in making bioengineered transplantable organs a clinical reality. Study limitations This experimental work deals with lungs and tracheae sourced from pigs. Even though there is significant anatomical and physiological similarity between human and porcine respiratory systems,
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results obtained in animal experiments with large mammals like pigs may not be accurately reflective of or extrapolatable to human organ-milieu. CONCLUSIONS Whole porcine lungs-tracheae can be successfully decellularized to create an acellular scaffold that preserves extracellular matrix and retains structral integrity and three-dimensional architecture to provide a bioartifical platform for respiratory tissue engineering. Acknowledgments: The expert technical assistance of Patricia Kraft, Lutz Hoffmann, and Karin Sonnenberg is gratefully acknowledged. Source of funding: This study was supported by the Land Baden-Württemberg, Germany, the Medical Faculty of the University of Heidelberg, Germany (to S.K.). Disclosures: None. REFERENCES 1. Kobashigawa JA, Patel JK. Immunosuppression for heart transplantation: where are we now? Nat Clin Pract Cardiovasc Med 2006;3:203–12. 2. Tonsho M, Michel S, Ahmed Z, Alessandrini A, Madsen JC. Heart transplantation: challenges facing the field. Cold Spring Harb Perspect Med 2014;4:pii:a015636. 3. Weymann A, Dohmen PM, Grubitzsch H, Dushe S, Holinski S, Konertz W. Clinical experience with expanded use of the Ross procedure: a paradigm shift? J Heart Valve Dis 2010;19: 279–85. 4. Soto-Gutierrez A, Wertheim JA, Ott HC, Gilbert TW. Perspectives on whole-organ assembly: moving toward transplantation on demand. J Clin Invest 2012;122:3817–23. 5. Song JJ, Ott HC. Organ engineering based on decellularized matrix scaffolds. Trends Mol Med 2011;17:424–32. 6. Wallis JM, Borg ZD, Daly AB, et al. Comparative assessment of detergent-based protocols for mouse lung de-cellularization and re-cellularization. Tissue Eng Part C Methods 2012;18: 420–32. 7. Song JJ, Kim SS, Liu Z, et al. Enhanced in vivo function of bioartificial lungs in rats. Ann Thorac Surg 2011;92:998–1005, discussion 1005–1006. 8. Petersen TH, Calle EA, Zhao L, et al. Tissue-engineered lungs for in vivo implantation. Science 2010;329:538–41. 9. Tapias LF, Ott HC. Decellularized scaffolds as a platform for bioengineered organs. Curr Opin Organ Transplant 2014;19: 145–52. 10. Price AP, England KA, Matson AM, Blazar BR, Panoskaltsis-Mortari A. Development of a decellularized lung bioreactor system for bioengineering the lung: the matrix reloaded. Tissue Eng Part A 2010;16:2581–91. 11. Weymann A, Radovits T, Schmack B, et al. In vitro generation of atrioventricular heart valve neoscaffolds. Artif Organs 2014;38:E118–28. 12. Weymann A, Loganathan S, Takahashi H, et al. Development and evaluation of a perfusion decellularization porcine heart model—generation of 3-dimensional myocardial neoscaffolds. Circ J 2011;75:852–60. Artif Organs, Vol. 39, No. 12, 2015
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13. Weymann A, Sabashnikov A, Patil NP, Konertz W, Modersohn D, Dohmen PM. Eprosartan improves cardiac function in swine working heart model of ischemiareperfusion injury. Med Sci Monit Basic Res 2014;20:55–62. 14. Cebotari S, Mertsching H, Kallenbach K, et al. Construction of autologous human heart valves based on an acellular allograft matrix. Circulation 2002;106(12 Suppl. 1):I63–i68. 15. Nichols JE, Niles J, Riddle M, et al. Production and assessment of decellularized pig and human lung scaffolds. Tissue Eng Part A 2013;19:2045–62. 16. O’Neill JD, Anfang R, Anandappa A, et al. Decellularization of human and porcine lung tissues for pulmonary tissue engineering. Ann Thorac Surg 2013;96:1046–55, discussion 1055–46.
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17. Hara H, Long C, Lin YJ, et al. In vitro investigation of pig cells for resistance to human antibody-mediated rejection. Transpl Int 2008;21:1163–74. 18. Barallobre-Barreiro J, Didangelos A, Schoendube FA, et al. Proteomics analysis of cardiac extracellular matrix remodeling in a porcine model of ischemia/reperfusion injury. Circulation 2012;125:789–802. 19. Wagner DE, Bonenfant NR, Sokocevic D, et al. Threedimensional scaffolds of acellular human and porcine lungs for high throughput studies of lung disease and regeneration. Biomaterials 2014;35:2664–79. 20. Price AP, Godin LM, Domek A, et al. Automated decellularization of intact, human-sized lungs for tissue engineering. Tissue Eng Part C Methods 2015;21:94–103.
Copyright © 2015 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.
Perfusion-Decellularization of Porcine Lung and Trachea for Respiratory Bioengineering *†¶Alexander Weymann, †¶Nikhil Prakash Patil, †Anton Sabashnikov, *Sevil Korkmaz, *Shiliang Li, ‡Pal Soos, §Roland Ishtok, *Nicole Chaimow, *Ines Pätzold, *Natalie Czerny, *Bastian Schmack, †Aron-Frederik Popov, †Andre Rüdiger Simon, *Matthias Karck, and *Gabor Szabo *Department of Cardiac Surgery, Heart and Marfan Center, University of Heidelberg, Heidelberg, Germany; †Department of Cardiothoracic Transplantation & Mechanical Circulatory Support, Royal Brompton and Harefield NHS Foundation Trust, Middlesex, UK; and ‡Heart and Vascular Center and §2nd Department of Pathology, Semmelweis University, Budapest, Hungary
Abstract: Decellularization of native organs may provide an acellular tissue platform for organ regeneration. However, decellularization involves a trade-off between removal of immunogenic cellular elements and preservation of biomechanical integrity. We sought to develop a bioartificial scaffold for respiratory tissue engineering by decellularization of porcine lungs and trachea while preserving organ architecture and vasculature. Lung–trachea preparations from 25 German Landrace pigs were perfused in a modified Langendorff circuit and decellularized by an SDC (sodium deoxycholate)-based perfusion protocol. Decellularization was evaluated by histology and fluorescence microscopy, and residual DNA quantified spectrophotometrically and compared with controls. Airway compliance was evaluated by endotracheal intubation and mechanical ventilation to simulate physiological breathing-induced stretch. Structural integrity was
evaluated by bronchoscopy and biomechanical stress/strain analysis by measuring passive tensile strength, all compared with controls. Decellularized lungs and trachea lacked intracellular components but retained specific collagen fibers and elastin. Quantitative DNA analysis demonstrated a significant reduction of DNA compared with controls (32.8 ± 12.4 μg DNA/mg tissue vs. 179.7 ± 35.8 μg DNA/mg tissue, P < 0.05). Lungs and trachea decellularized by our perfusion protocol demonstrated increased airway compliance but preserved biomechanical integrity as compared with native tissue. Whole porcine lungs–tracheae can be successfully decellularized to create an acellular scaffold that preserves extracellular matrix and retains structral integrity and three-dimensional architecture to provide a bioartifical platform for respiratory tissue engineering. Key Words: Decellularization—Tissue engineering—Whole organ concept—Lung—Trachea.
Lung transplantation is the definitive treatment for end-stage pulmonary disease, but remains limited by donor organ shortage and waiting-list mortality. Long-segment circumferential tracheal defects also pose a major problem for surgical reconstruction fol-
lowing malignancy, traumatic injury, or airway stenosis. While transplant recipients must live with the “necessary evil” of lifelong immunosuppression, often begetting hypertension, diabetes, renal failure, malignancy, and other sequelae of chronic immunosuppression (1,2), attempts at replacing the trachea with synthetic prosthetics and scaffolds entail complications like mucous build-up, inflammation, infection, stricture, and eventual stenosis (3). Bioartificial or “tissue engineered” lung/trachea offers the potential for long-term graft survival without the need for long-term immunosuppressive therapy (4). Decellularization of whole organs yields a biologic scaffold of three-dimensional extracellular matrix (ECM)
doi:10.1111/aor.12481 Received September 2014; revised December 2014. Address correspondence and reprint requests to Dr. Alexander Weymann, Department of Cardiac Surgery, Heart and Marfan Center—University of Heidelberg, Im Neuenheimer Feld 110, Heidelberg 69120, Germany. E-mail: weymann.alexander @googlemail.com ¶ These authors contributed equally to this work and are considered to be joint first authors. Artificial Organs 2015, 39(12):1024–1032
BIOARTIFICIAL LUNGS AND TRACHEAE that can be subsequently repopulated with donorspecific mature or stem cells for ex vivo regeneration of donor organs (5). Various approaches have been tried for lung decellularization, including physical agitation and exposure to chemical and enzymatic agents such as Triton-X, sodium deoxycholate (SDC), and vascular perfusion of CHAPS (3-[(3cholamidopropyl)dimethylammonio]-1-propanesulfonate) detergent (6). However, most studies are limited to mice or rats, and there are limited data on decellularization of lungs from larger mammals (7–10). Further, approaches utilized in rodent models may be insufficient for adequate decellularization of lungs obtained from larger animals and humans. We previously reported our perfusion decellularization protocol using sodium dodecyl sulfate (SDS) in pulmonic heart-valves (11), more recently applied to whole-hearts (12). In the present study, we attempted to decellularize porcine airways to create whole lung–trachea scaffolds with a perfusable vascular bed and preserved airway and alveolar geometry. MATERIALS AND METHODS Lungs and adjacent trachea were harvested under sterile conditions from 25 female German Landrace crossbred pigs (80–100 kg). All animals received humane care in accordance with the “Guide for the Care and Use of Laboratory Animals” prepared by the Institute of Laboratory Animal Resources (US NIH Publication No. 86-23, revised 1996). All procedures followed the European Agreement of Vertebrate Animal Protection for Experimental Use (86/609). This investigation was reviewed and approved by the concerned institutional review committees. Anesthetic and surgical procedures Intravascular access was secured via a superficial ear vein of the animal, as described previously (13). After intramuscular injection of 4 mg azaperone (Stresnil, Janssen, High Wycombe, UK) and 0.01 mg/kg fentanyl, 3–4 mg/kg Hypnomidate (Janssen-Cilag GmbH, Neuss, Germany) was administered intravenously, followed by intubation and ventilation with 40% FiO2. Muscle relaxation was achieved with pancuronium (0.3 mg/kg/h). After systemic heparinization and median sternotomy, the lungs and trachea were procured. Perfusion decellularization circuit The modified Langendorff decellularization model comprised a perfusion circuit and a pressure control
1025
module, as described previously (12). All components were sterilized by conventional autoclaving and connected with 3/8″ silicon tubes (Maquet, Rastatt, Germany). The pulmonary artery and trachea were connected with cannulas of adjustable diameter for controlled perfusion. The circuit was powered by a roller pump (Stöckert, München, Germany) controlled via a pressure transducer (Medex-Smith Medical, Kent, United Kingdom). A computer system (Engineo, Mainz, Germany) continuously recorded the perfusion pressure. We interposed an airtrap (Gambro Medical Line, Hechingen, Germany) to ensure air-free perfusate. Inlet and outlet ports permitted easy and quick detergent change. The returning (outflow) perfusate was collected into a reservoir (Maquet AR 28150), with total circuit volume of 2500 mL. Antibiotic-containing phosphate-buffered solution (PBS, Sigma, Seelze, Germany) enriched with 100 μg/mL penicillin-streptomycin (Biochrom, Berlin, Germany) was perfused through the lungs and tracheae at 1.5 L/min for 24 h. Subsequently, the lungs were perfused with 4% SDC (12 h) and 0.1% Triton X-100 in PBS (12 h) at 2.0 L/min for 5 days. The tracheae were subjected to 72 h immersion in 4% SDS solution at room temperature on a mechanical agitator, with a solution change at 24 h. After 72 h, a 0.1% peracetic acid and 4% ethanol wash was administered for 6 h on a mechanical agitator for further decellularization and disinfection. During the entire process, the lungs and trachea were washed with PBS once a day to remove residual substances. The final washing step included perfusion with PBS for another 24 h to remove remnant detergents and cell debris at 1.5 L/min.
Evaluation of lung biomechanical stability We performed pulmonary compliance testing on the decellularized lung scaffolds under quasi-clinical conditions by employing patient mechanical ventilators (Model Primus, Dräger Medical, Lübeck, Germany) for ventilating decellularized (n = 6) and native lungs (n = 6). The resultant pressure changes were monitored and compared. An endotracheal tube (Teleflex Medical, Kernen, Germany) was used for tracheal intubation and ventilation. The initial settings in the volume-controlled mode of the ventilator were kept to a frequency of 30 breaths/min at a tidal volume of 30 mL and a positive end expiratory pressure of 3 mbar. To determine the compliance of the lungs, the tidal volume was subsequently increased in 10 mL steps and the corresponding pressure values were measured. All values were Artif Organs, Vol. 39, No. 12, 2015
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captured by photo documentation to create a volume/pressure chart. Measurement of tracheal mechanical stability Mechanical stability of tracheal tissue was analyzed using a static material testing instrument (Zwick Roell, Ulm, Germany). Decellularized tracheal samples (n = 6) were stretched until complete tearing. Passive tensile strength was continuously recorded throughout the procedure. Samples from untreated native trachea (n = 6) were used as controls. Bronchoscopy Maintenance of major airways was assessed in decellularized porcine airway by bronchoscopy (Pentax flexible nasolaryngoscope [FNL-10RP3], Tokyo, Japan) and compared with controls. DNA quantification Lung sections were processed for spectrophotometric quantification to determine the concentrations of residual DNA in the decellularized group compared with the controls. Samples were normalized according to equivalent dry weight in mg. The total amount of DNA was silica-membrane-based purified (QIAamp DNA Mini Kit, Qiagen, Basel, Switzerland) following the manufacturer’s instructions and later quantified by spectrophotometry. Western blot analysis Tissue samples of 25–30 μg were homogenized with 150 mL PBS using a tissue lyser (Qiagen) and then digested in 150 μL 2× RIPA buffer (Melford Laboratories, Chelsworth, UK). Protein concentration was quantified via Bradford assay and then denatured with sample buffer (Life Technologies, Carlsbad, CA, USA) for 10 min at 70°C. The proteins were separated via SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membrane (Invitrogen). The membranes were blocked with 5% milk in Tris-buffered saline Tween 20 for 1 h at room temperature before incubation overnight at 4°C with primary antibodies specific to elastin (1:200, Abcam, Cambridge, UK) and Collagen1 (1:1000, Abcam). After washing the blots to remove excess antibody binding, membranes were incubated for 1 h with horseradish peroxidaseconjugated goat secondary antibody (1:5000, Santa Cruz, Dallas, TX, USA) followed by three washing steps. The immunoreactive protein bands were developed using enhanced chemiluminescence (PerkinElmer, Waltham, MA, USA) and were Artif Organs, Vol. 39, No. 12, 2015
detected with a Chemi-smart 5100TM imager (PEQLAB GmbH, Erlangen, Germany). Histology Samples of decellularized lung and trachea were analyzed with light microscopy and compared with native, untreated cross-sections. Tissue samples were fixed in 10% formalin, embedded in paraffin and sectioned following standard protocols. We cut tissue into 5 μm sections, stained them with 4′,6-diamidino2-phenylindole (DAPI, Sigma-Aldrich Inc., Munich, Germany) and hematoxylin and eosin (H&E) to determine if remnant nuclear structures could be observed after decellularization as compared with controls. The fluorescence method used has been described elsewhere (12,14). Moreover, Masson’s trichrome stain Verhoeff van Gieson stain, and Alcian Blue stain (Sigma-Aldrich, Hamburg, Germany) were used to visualize different ECM components such as collagen, elastin, and glycosaminoglycans in decellularized lung tissue and compared with controls. The sections were analyzed by routine bright field and fluorescence microscopy (Olympus Optical Co, BX 51 and CKX 41 microscopes, Tokyo, Japan). Images were acquired with the CellA Soft Imaging System (Olympus Soft Imaging Solutions). Statistical analysis All values in the figures and text are shown as mean ± SEM. A two-tailed Student’s t-test was performed to determine significant differences of the native lungs (control) versus the decellularized lungs, considering P values less than 0.05 as statistically significant. The commercially available SPSS statistical software package 13.0 for Windows (SPSS Inc., Chicago, IL, USA) was used for data analysis. RESULTS Decellularization treatment There was a progressive loss of pink pigmentation during decellularization, with fully decellularized whole lung–trachea scaffolds having a pale/whitish appearance and visible vascular conduits throughout the matrix (Fig. 1) produced by day 5 of the decellularization treatment. Bronchoscopy confirmed that the underlying macroscopic structure and branching of both airway and vessels remained intact after decellularization, with no evidence of gross structural damage, sloughed cell material, or cell debris (data not shown). Histological examination
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FIG. 1. Whole porcine lungs with adjacent trachea during decellularization after 1 (A), 3 (B), and 5 days (C). All gross structures including blood vessels and airway (D, E) were preserved with no evidence of major structural damage, sloughed cell material, or cell debris.
demonstrated that our decellularization treatment converted native porcine lungs and tracheae into an almost acellular neoscaffold as demonstrated by H&E staining and additionally DAPI staining for the lungs (Fig. 2). The lacunae in the cartilage rings were devoid of cells, nuclei, or nuclear material (Fig. 3). Thus, intracellular constituents containing potentially immunogenic DNA were effectively removed while preserving the integrity of the ECM. Further examination using Masson’s trichrome, Verhoeff van
Gieson, and Alcian Blue stain visualized different ECM components such as mesh-like collagen formations, elastin, and glycosaminoglycans (Fig. 4). Total DNA content of decellularized lungs was markedly decreased compared with untreated controls. After decellularization, the DNA content was reduced to 32.8 ± 12.4 μg DNA/mg tissue versus 179.7 ± 35.8 μg DNA/mg tissue in the native group (P < 0.05). Surgical experiments confirmed nativelike tissue properties with adequate structural
FIG. 2. H&E (A) and DAPI (C) staining of lung tissue before and after perfusion decellularization (B, D) showing no remnant nuclear structures with maintained ECM. Scale bars, 200 μm.
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FIG. 3. Images of decellularized porcine trachea after 72 h immersion in 4% SDS solution at room temperature on a mechanical agitator (A, B) with retained gross morphology and stability. After decellularization, native tracheal matrix (C, H&E stain) turned into an acellular scaffold (D, H&E stain) with preservation of ECM integrity. Scale bars, 200 μm.
integrity for surgical purposes with suture application for lungs and tracheae. Biomechanical tests The stress–strain curve of decellularized porcine trachea tissue in comparison with controls is shown in Fig. 5. Maximal stress (equivalent to maximal tensile strength) was defined by the peak of the curve, while tissue resistance to stretch was assessed by the slope of the curve. It is evident that porcine tracheae
treated using the decellularization treatment described here have stress resistance similar to that of native trachea. Volume/pressure measurements showed that decellularized lungs did not exhibit statistically relevant changes in peak pressure, dynamic compliance, or static compliance when compared with native lungs (Fig. 6). In decellularized lungs, higher ventilatory volumes were achieved with lesser airway pressures compared with native lungs, indicating
FIG. 4. Masson’s trichrome (A, B), Verhoeff van Gieson (C, D), and Alcian Blue staining (E, F) of lung tissue before (A, C, E) and after perfusion decellularization (B, D, F), demonstrating retained ECM components such as mesh-like collagen formations, elastin, and glycosaminoglycans. Scale bars as indicated.
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FIG. 5. Stress–strain curves of native and decellularized trachea demonstrating similar stress resistance. The peak of the curve defines maximal tensile strength, with tissue resistance to stretch represented by the slope of the curve.
increased compliance in decellularized lungs. Thus, no substantial stiffening or weakening of the ECM occurs in the decellularized lungs as compared with native lungs. Western blot analysis Western blot analysis results are depicted in Fig. 7. As shown, protein bands for collagen 1 and elastin have the same intensity in decellularized and native samples. Thus, there is no obvious difference in the content of elastin or collagen 1 between decellularized and native lungs. DISCUSSION Several approaches for decellularization have been tried, including continuous whole lung perfusion in a bioreactor chamber (15), manual instillation of fluids (8), and physically agitating excised sheets of lung tissue during exposure to decellularizing solvents (16). However, there are no consensus criteria for defining adequate/optimal decellularization achieved by different protocols. Nichols et al. (15) used a combination of freezing and SDS washes to achieve decellularization, with organs stored at −80°C for at least 1 month prior to decellularization. However, it is not known whether an additional freeze-thaw
cycle confers any advantage as compared with less complicated methods for decellularization. O’Neill and colleagues compared different methods for decellularizing lung sections (16). The authors did not work with whole-lung preparations but evaluated human and porcine lung slices in a two-dimensional system and found that acellular scaffold slices allowed cell attachment and survival. Importantly, though, human cells thrived equally on porcine and human matrix sections—a welcome finding considering the abundant supply of porcine lungs as a test bed for organ regeneration and also for potential “offthe-shelf” organ scaffolds (16). Porcine lungs are of a size comparable with human lungs, with similar physiology and anatomical features. A relatively short generation-interval of pigs means that a considerable number of bred porcine offspring can be procured in a reasonable time period. Pigs have long been used in xenotransplantation studies, with genetically modified pigs shown to be significantly less susceptible to human complement-dependent cytotoxicity (17). Also, studies with cardiac ECM have demonstrated remarkable homology between porcine and human hearts (18). In addition, porcine organs may be advantageous with regard to product development, ethical considerations, and regulatory approval. All Artif Organs, Vol. 39, No. 12, 2015
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FIG. 6. Volume/pressure measurements of decellularized lungs compared with native lungs. Decellularized lungs did not exhibit statistically relevant changes in peak pressure, dynamic compliance, or static compliance when compared with native lungs.
FIG. 7. Western blot analysis for collagen-1 and elastin. The protein bands for collagen-1 and elastin have the same intensity in decellularized and native lung samples with no obvious difference in the content of collagen-1 or elastin after decellularization. The first five bands demonstrate one native lung (one sample per lobe) representative for native lung tissue. The next 12 bands represent six decellularized lungs (each two samples, one from left lung and one from right lung). The last three bands are controls.
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BIOARTIFICIAL LUNGS AND TRACHEAE these factors highlight the importance of porcine airways in the attempt to develop a human-sized bioartificial lung–trachea scaffold. Whereas Wagner et al. (19) reported their findings with different perfusion protocols for decellularization of porcine and human lungs, they did not use whole organs—rather, they utilized small segments (1–3 cm3) and lobes for their experiments. Unlike our previous work with hearts (12), however, we found that there is an important difference to keep in mind when trying to decellularize lungs and trachea— there is no normal egress from the airway, unlike the arteriovenous egress of vasculature. Hence, maintaining optimal pressures is critical to avoid barotrauma-induced disruption of intercellular junctions when decellularizing the airway, because there is no egress for the SDC (or any solution) used for perfusion-decellularization. This is an important caveat, which underscores the importance of accurate and efficient pressure modulation control in decellularization protocols for lung– trachea preparations. Preserving the biomechanical integrity is critical if a decellularized matrix is to serve as an effective recellularization scaffold. In this study, decellularized tracheae had stress resistance similar to that of native trachea, and there was no substantial stiffening or weakening of the ECM in decellularized lungs as compared to native lungs. In fact, in decellularized lungs, higher ventilatory volumes could be achieved with lesser airway pressures compared with native lungs, indicating increased compliance in decellularized lungs. These findings are in contrast to another contemporary protocol that resulted in lungs having lower specific compliance after decellularization (20). Our results demonstrate that porcine lung–trachea can be decellularized to generate whole-organ acellular scaffolds for organ regeneration. Our protocol employs a simple and inexpensive constantpressure perfusion system to generate an acellular lung–trachea matrix that retains biomechanical strength and three-dimensional architecture with the advantage of enhanced airway compliance. Further strides in decellularization protocols and conjunctory recellularization techniques will be critical in making bioengineered transplantable organs a clinical reality. Study limitations This experimental work deals with lungs and tracheae sourced from pigs. Even though there is significant anatomical and physiological similarity between human and porcine respiratory systems,
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results obtained in animal experiments with large mammals like pigs may not be accurately reflective of or extrapolatable to human organ-milieu. CONCLUSIONS Whole porcine lungs-tracheae can be successfully decellularized to create an acellular scaffold that preserves extracellular matrix and retains structral integrity and three-dimensional architecture to provide a bioartifical platform for respiratory tissue engineering. Acknowledgments: The expert technical assistance of Patricia Kraft, Lutz Hoffmann, and Karin Sonnenberg is gratefully acknowledged. Source of funding: This study was supported by the Land Baden-Württemberg, Germany, the Medical Faculty of the University of Heidelberg, Germany (to S.K.). Disclosures: None. REFERENCES 1. Kobashigawa JA, Patel JK. Immunosuppression for heart transplantation: where are we now? Nat Clin Pract Cardiovasc Med 2006;3:203–12. 2. Tonsho M, Michel S, Ahmed Z, Alessandrini A, Madsen JC. Heart transplantation: challenges facing the field. Cold Spring Harb Perspect Med 2014;4:pii:a015636. 3. Weymann A, Dohmen PM, Grubitzsch H, Dushe S, Holinski S, Konertz W. Clinical experience with expanded use of the Ross procedure: a paradigm shift? J Heart Valve Dis 2010;19: 279–85. 4. Soto-Gutierrez A, Wertheim JA, Ott HC, Gilbert TW. Perspectives on whole-organ assembly: moving toward transplantation on demand. J Clin Invest 2012;122:3817–23. 5. Song JJ, Ott HC. Organ engineering based on decellularized matrix scaffolds. Trends Mol Med 2011;17:424–32. 6. Wallis JM, Borg ZD, Daly AB, et al. Comparative assessment of detergent-based protocols for mouse lung de-cellularization and re-cellularization. Tissue Eng Part C Methods 2012;18: 420–32. 7. Song JJ, Kim SS, Liu Z, et al. Enhanced in vivo function of bioartificial lungs in rats. Ann Thorac Surg 2011;92:998–1005, discussion 1005–1006. 8. Petersen TH, Calle EA, Zhao L, et al. Tissue-engineered lungs for in vivo implantation. Science 2010;329:538–41. 9. Tapias LF, Ott HC. Decellularized scaffolds as a platform for bioengineered organs. Curr Opin Organ Transplant 2014;19: 145–52. 10. Price AP, England KA, Matson AM, Blazar BR, Panoskaltsis-Mortari A. Development of a decellularized lung bioreactor system for bioengineering the lung: the matrix reloaded. Tissue Eng Part A 2010;16:2581–91. 11. Weymann A, Radovits T, Schmack B, et al. In vitro generation of atrioventricular heart valve neoscaffolds. Artif Organs 2014;38:E118–28. 12. Weymann A, Loganathan S, Takahashi H, et al. Development and evaluation of a perfusion decellularization porcine heart model—generation of 3-dimensional myocardial neoscaffolds. Circ J 2011;75:852–60. Artif Organs, Vol. 39, No. 12, 2015
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17. Hara H, Long C, Lin YJ, et al. In vitro investigation of pig cells for resistance to human antibody-mediated rejection. Transpl Int 2008;21:1163–74. 18. Barallobre-Barreiro J, Didangelos A, Schoendube FA, et al. Proteomics analysis of cardiac extracellular matrix remodeling in a porcine model of ischemia/reperfusion injury. Circulation 2012;125:789–802. 19. Wagner DE, Bonenfant NR, Sokocevic D, et al. Threedimensional scaffolds of acellular human and porcine lungs for high throughput studies of lung disease and regeneration. Biomaterials 2014;35:2664–79. 20. Price AP, Godin LM, Domek A, et al. Automated decellularization of intact, human-sized lungs for tissue engineering. Tissue Eng Part C Methods 2015;21:94–103.
