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and J. Agresti for experimental assistance; and E. Rubin, L. Cai, and J. Elf for helpful discussions. This work was supported by the Gates Foundation (X.S.X.), the NIH Pioneer Director's Award (X.S.X.), and the Canadian

Institutes of Health Research (MOP-77639) (A.E.). Y.T. acknowledges additional support from the Japan Society for the Promotion of Science, the Uehara Memorial Foundation, and the Marubun Research Promotion Foundation, and P.J.C. from the John and Fannie Hertz Foundation.

Supporting Online Material www.sciencemag.org/cgi/content/full/329/5991/533/DC1 Materials and Methods SOM Text Figs. S1 to S23 Tables S1 to S6 References

16 February 2010; accepted 4 June 2010 10.1126/science.1188308

Tissue-Engineered Lungs for in Vivo Implantation Thomas H. Petersen,1,2 Elizabeth A. Calle,1 Liping Zhao,3 Eun Jung Lee,3 Liqiong Gui,3

MichaSam B. Raredon,1 Kseniya Gavrilov,4 Tai Yi,5 Zhen W. Zhuang,6

Christopher Breuer,5 Erica Herzog,6 Laura E. Niklason1,3*

Because adult lung tissue has limited regeneration capacity, lung transplantation is the primary therapy for severely damaged lungs. To explore whether lung tissue can be regenerated in vitro, we treated lungs from adult rats using a procedure that removes cellular components but leaves behind a scaffold of extracellular matrix that retains the hierarchical branching structures of airways and vasculature. We then used a bioreactor to culture pulmonary epithelium and vascular endothelium on the acellular lung matrix. The seeded epithelium displayed remarkable hierarchical organization within the matrix, and the seeded endothelial cells efficiently repopulated the vascular compartment. In vitro, the mechanical characteristics of the engineered lungs were similar to those of native lung tissue, and when implanted into rats in vivo for short time intervals (45 to 120 minutes) the engineered lungs participated in gas exchange. Although representing only an initial step toward the ultimate goal of generating fully functional lungs in vitro, these results suggest that repopulation of lung matrix is a viable strategy for lung regeneration.

L ung diseases account for some 400,000 deaths annually in the United States (1). Human lungs do not generally repair or

regenerate beyond the microscopic, cellular level. Currently, the only way to replace lung tissue is to perform lung transplantation, which is an ex- pensive procedure that achieves only 10 to 20% survival at 10 years and one that is hampered by a severe shortage of donor organs (2). Recently, techniques have been developed to quantitatively decellularize complex organs such as heart, liver, and kidney (3–5). Acellular matrices can provide attractive scaffolds for repopulation with lung- specific cells for lung engineering because the extracellular matrix template should contain ap- propriate three-dimensional (3D) architecture and

regional-specific cues for cellular adhesion. To be functional in vivo, an engineered lung should (i) contain lung-specific cells, (ii) display the

branching geometry of the airways and contain a perfusing microvasculature, (iii) provide bar- rier function to separate blood from air, and (iv) have mechanical properties that allow ventila- tion at physiological pressures.

Here, we describe our progress toward the construction of a functional tissue-engineered lung, using rat as a model system. Our approach is summarized in Fig. 1. We first decellularized native lung tissue in order to remove all immu- nogenic cellular constituents (Fig. 1, A and B). We found that after careful decellularization, the tissue retained its alveolar micro-architecture, its ability to function as a barrier to particulates, and its tissue mechanics. Repopulation of the acellular lung matrix with mixed populations of neonatal lung epithelial cells resulted in regional-specific epithelial seeding in correct anatomic locations. To enhance the survival and differentiation of lung epithelium, we cultured the matrix in a bioreactor designed to mimic certain features of the fetal lung environment, including vascular perfusion and liquid ventilation (Fig. 1, C and D) (6). Lastly, we tested the functionality of the engineered lung tissue by implanting it for short time periods in a syngeneic rat model (Fig. 1E).

1Department of Biomedical Engineering, Yale University, New Haven, CT 06520, USA. 2Department of Biomedical Engineer- ing, Duke University, Durham, NC27708, USA. 3Department of Anesthesia, Yale University, New Haven, CT 06520, USA. 4De- partment of Cellular and Molecular Physiology, Yale University, New Haven, CT 06520, USA. 5Department of Surgery, Yale Uni- versity, New Haven, CT 06520, USA. 6Department of Internal Medicine, Yale University, New Haven, CT 06520, USA.

*To whom correspondence should be addressed. E-mail: [email protected]

Fig. 1. Schema for lung tissue engineering. (A) Native adult rat lung is cannulated in the pulmonary artery and trachea for infusion of decellularization solutions. (B) Acellular lung matrix is devoid of cells after 2 to 3 hours of treatment. (C) Acellular matrix is mounted inside a biomimetic bioreactor that allows seeding of vascular endothelium into the pulmonary artery and pulmonary epithelium into the trachea. (D) After 4 to 8 days of culture, the engineered lung is removed from the bioreactor and is suitable for implantation into (E) the syngeneic rat recipient.

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Preparation of a decellularized lung scaffold. We harvested lung tissue from adult Fischer 344 rats and treated the lungs in the vascular and airway compartments with detergent solutions (Fig. 1 and movie S1). The decellularization solution contains 3-[(3-cholamidopropyl)dimethylammonio]-1- propanesulfonate(CHAPS),azwitterionicdetergent, in a phosphate buffer at 1.0 M salt concentration (7). Vascular perfusion pressure was maintained below 20 mmHg, with total time of tissue exposure typically in the range of 2 to 3 hours. Analysis of the decellularized lung matrices by means of high- resolution micro-computed tomography (micro- CT) revealed intact lung architecture and an intact arterial tree and microvasculature (Fig. 2, A to C). The cells and nuclear material were removed with the decellularization process, but the alveolar sep- tal architecture remained undisturbed (Fig. 2, E and F). Fluorometric DNA assay confirmed that

approximately 99% of DNAwas removed by the decellularization process (Fig. 2G), and immuno- blotting for major histocompatibility complex I and II (MHC-I and MHC-II) as well as b-actin confirmed that decellularized lung matrices were depleted of these cellular markers (Fig. 2D). Scanning electron microscopy (SEM) confirmed that alveolar cells, red blood cells, and cellular proteins present in native lung were absent in the decellularized scaffold (Fig. 2, H and I, and fig. S3). Analysis of the matrix by means of trans- mission electron microscopy (TEM) revealed that the ultrastructure of the alveolar septae and the delicate microvessels surrounding the alveoli re- mained intact (Fig. 2J and fig. S3). Immuno- fluorescence and histochemical staining indicated that collagen, elastin, and laminin were preserved in decellularized matrices (figs. S4 and S5). Ad- ditionally, quantitative assays showed that extra-

cellular matrix collagen was preserved, whereas elastin was partially depleted by the decellulari- zation process (40% of elastin remains in acel- lular matrices), and sulfated glycosaminoglycans (GAGs) were >90% removed (Fig. 2G and fig. S4). Hence, the lung decellularization protocol produces an acellular matrix scaffold that retains the gross, microstructural, and ultrastructural properties of native lung, yet removal of antigenic cellular components is essentially complete.

Properties of the lung bioreactor. The lung bioreactor contains a main reservoir in which the decellularized lung is mounted, using cannulae that are inserted into the trachea and pulmonary artery (fig. S1A). Culture medium is perfused into the pulmonary artery at physiological pressures (fig. S1B) (7). To provide negative-pressure ven- tilation of the engineered lung, a syringe pump withdraws a defined volume of air from the main bioreactor to create a negative pressure (Fig. 1C and figs. S1C and S2). Lung inflation under neg- ative pressure is accompanied by inhalation of liquid medium from a trachea reservoir via a “breathing loop.” For exhalation, the syringe pump returns air to the main reservoir, causing lung exhalation of liquid medium into the trachea reservoir.

To repopulate the decellularized matrix, we injected cells into the vascular or airway compart- ments, or both, and maintained the matrix in cul- ture for up to 8 days (Fig. 1C). This produced an engineered lung tissue that could then be removed from the bioreactor for further analysis or that could be orthotopically transplanted into a rat recipient (Fig. 1, D and E).

Repopulation of acellular matrix to produce an engineered lung. Seeding of the decellularized matrix with mixed populations of neonatal rat lung epithelial cells (fig. S7) into the airway com- partment generally resulted in good adherence of the cells to alveolar structures, as well as to small- and medium-sized airways (Fig. 3A). Micro- vascular lung endothelial cells, when injected into the pulmonary artery of acellular scaffolds, adhered throughout the scaffold vasculature (Fig. 3B). Seeded pulmonary epithelial cells replicated rapidly and rarely displayed markers of apoptotic cell death (Fig. 3, C and D), despite the difficulty in culturing such cells on standard tissue culture plastic in vitro (8). This observation suggests that substrate cues on the acellular lung matrix are important for pulmonary epithelial cell attach- ment and replication.

In the biomimetic bioreactor, vascular perfu- sion greatly enhanced endothelial adhesion and survival on the lung matrix (fig. S9, A and B). Negative pressure ventilation had multiple ben- eficial effects on cultured lung epithelium, in- cluding enhanced survival of epithelium in distal alveoli and clearance of epithelial secretions from the airway tree (fig. S9, C to F). Clearance of airway secretions through ventilation indicates that the developing epithelium is in communica- tion with the airway tree and is not growing ran- domly within the matrix. In addition, ventilation

Fig. 2. Characterization of acellular lung matrix. (A) 3D micro-CT of the acellular matrix airway compartment. Large airways are in green. (B) Micro-CT angiography of vascular compartment, thresholded to visualize only large vessels. In (A) and (B), voxel size is 58 mm; scale bar, 4 mm. (C) Micro-CT angiography of smaller vessels in acellular lung. Voxel size, 6.5 mm; scale bar, 500 mm. (D) Immunoblot for MHC-1, MHC- II, and b-actin in native (Nat) and decellularized (Dec) lungs, showing removal of cellular proteins. (E) Hematoxylin and eosin (H&E) stain of native rat lung. (F) H&E stain of acellular lung matrix. Scale bar, 50 mm in (E) and (F). (G) Collagen (Coll), elastin (Elas), glycosaminoglycan (GAG), and DNA contents of native lung (black bars) and acellular matrices (hatched bars). Values are mean T SD per lung (n ≥ 4 lungs for all measures), scaled to 1 for native, with asterisk indicating P < 0.05 for difference between native and acellular matrices. (H) SEM of native rat lung. (I) SEM of acellular matrix. Scale bar, 10 mm in (H) and (I). (J) TEM of acellular lung matrix. Asterisk indicates capillaries in alveolar septa. Scale bar, 5 mm.

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with air—as opposed to with culture medium— increased the numbers of type I alveolar epithelial cells, as well as the numbers of ciliated columnar epithelial cells (fig. S9, G to J). Engineered lungs also produce pro-surfactant proteins B and C

(pro-SPB, pro-SPC), although we could not de- tect mature SPB (Fig. 3E). Surfactant proteins are critical for reducing alveolar surface tension and enabling lung inflation at physiologically normal pressures.

We performed compliance testing on the engineered lungs under quasi-static conditions by injecting air into the lungs and monitoring result- ant pressure changes. Typical compliance curves for native lung, acellular matrix, and repopulated engineered lungs are shown in Fig. 3, F to H. The compliance values were, respectively, 0.35 T 0.08 (n =10measures),0.09T 0.02(n=4measures),and 0.14 T 0.06 (n = 5 measures) mL/mmHg (mean T SD, P < 0.001 for difference between native and both decellularized and engineered compliances). Compliance values were measured at initial filling of the lungs (arrowheads in Fig. 3, F to H), and thus lower compliances for decellularized matrix and engineered lung mean that these two tissues have higher “opening pressures,” and less functional surfactant, than does native lung. Despite this dif- ference, the overall stress-strain relationships and the ultimate tensile stresses were similar between the three groups (Fig. 3I and fig. S6). Thus, no substantial stiffening or weakening of the extra- cellular matrix occurs in the repopulated, engineered lungs as compared with native adult lungs.

To evaluate the distribution and phenotype of cells in the engineered lungs, we performed fluo- rescent immunohistochemical staining (fig. S8). Endothelial cells seeded into the vasculature were extensively distributed and expressed CD-31, as did comparable cells in native rat lung (fig. S8A). TEM analysis revealed the presence of tight junctionswithintheengineeredendothelium,which is consistent with the development of some bar- rier function (fig. S10). With respect to the seeded lung epithelium, Clara cell secretory protein (CCSP)–positive cells, which in native lung are found in small airways, were found primarily in very small airway structures after 4 days and in larger structures after 8 days in engineered lungs (fig. S8B). Pro-SPC, a marker of type II alveolar epithelium, is normally present at the vertices of alveoli in native lung. In the engineered lungs, pro-SPC expression was diffuse in alveoli and in small airways after 4 days of culture, but at 8 days it showed a more native expression pattern at the vertices of alveoli (fig. S8C). In contrast, aquaporin- 5—a specific marker for type I epithelium—was found diffusely throughout native alveoli and in engineered lungs after 4 days but was largely absent after 8 days (fig. S8D). This observation is consistent with previous work on neonatal rat development, which showed that type I cells do not fully differentiate until the post-natal period, when air breathing commences (9). Indeed, engineered lungs that were ventilated with air, as opposed to liquid culture medium, displayed more aquaporin-5 expression in alveoli after 8 days of culture (fig. S9, G and H) and also contained sparse ciliated epithelial cells (fig. S9, I and J). Additional cell types noted included mes- enchymal cells and the airway epithelial progen- itor basal cells (fig. S8, E and F). Hence, the engineered lungs contained many of the impor- tant cell types of native lung tissues. In addition, the spatial distribution of the various cell types was regional-specific, and with extended culture

Fig. 3. Repopulation of the matrix with lung epithelial and endothelial cells and mechanical assessment of the engineered lungs. (A) H&E stain of mixed neonatal pulmonary epithelium seeded into acellular matrix and cultured for 8 days. (B) H&E stain of lung microvascular endothelium seeded into vascular compartment and cultured for 8 days. Scale bars, 100 mm in (A) and (B). (C) Proliferating cell nuclear antigen stain of epithelial culture after 8 days; brown nuclei are dividing. (D) Terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling stain of epithelium after 4 days of culture detects no apoptotic cells (positive cells stain brown; green indicates nuclear counterstain). Scale bars, 50 mm in (C) and (D). (E) Immunoblots for pro-SPC indicates similar expression for native (Nat) and engineered (Eng) lung. Engineered lung expresses SPB precursor proteins at 43 and 90 kD, but no mature SPB. b-actin confirms similar protein loading. (F to H) Quasi-static compliance curves for (F) typical native, (G) acellular, and (H) engineered lungs; arrows indicate inflation arm of loop. (I) Mean ultimate tensile strengths (UTS) of native (n = 4), acellular (n = 10), and engineered (n = 5) lungs. Error bars are SD; there were no significant differences between any groups.

Fig. 4. Implantation of engineered lungs into rats. (A) Tissue-engineered left lung was implanted into adult Fischer 344 rat recipient and photographed ~30 min later. (B) X-ray image of rat showing the implanted engineered left lung (white arrow) and the right native lung. (C) H&E stain of explanted lung. Red blood cells perfusing septa are evident, and some red blood cells are present in airspaces. Scale bar, 50 mm.

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periods in the bioreactor the overall pattern of cellular distribution and differentiation became more similar to that in native lung tissue.

To determine whether the decellularization and repopulation methodologies used in our studies of rat lungs were applicable to human tissues, we obtained human lung segments from a tissue bank and treated them with decellularization solutions for up to 6 hours (7). Histological stain- ing showed that complete cellular removal was achieved, with preservation of alveolar architecture (fig. S11, A and B). We seeded the acellular matrices with A549 human epithelial carcinoma cells and endothelial cells derived from human cord-blood endothelial progenitor cells (7). The A549 cells adhered well to alveolar surfaces, and the endothelial cells adhered to the vasculature (fig. S11, C and D), supporting the notion that these approaches may also be applicable to hu- man lung tissues.

Implantation of engineered lungs into rats. To determine whether engineered rodent lungs were implantable and functional for gas exchange, we performed orthotopic left lung transplantation in four animals (7). Acellular matrices were seeded with neonatal rat lung epithelium and lung micro- vascular endothelium and cultured for approximate- ly 1 week in the bioreactor. For lung implantation, the native lungs were exposed via left thoracoto- my, and the left lung was excised. After anticoagula- tion with heparin, the left mainstem bronchus, left pulmonary vein, and left pulmonary artery of the engineered lung were anastomosed to the recip- ient, the lung was ventilated with 100% oxygen, and blood flow was reestablished.

In all cases, the engineered lungs were easily suturable to the recipient and were ventilated with no visible air leak from the parenchyma (Fig. 4A and movie S2). All engineered lungs became perfused with blood over a period of seconds to minutes, with blood visibly turning from dark to bright red as the hemoglobin became oxygenated. Implantation times for engineered lungs ranged from 45 min to 2 hours. After perfusion and ven- tilation, blood gas samples were drawn from the pulmonary artery, left and right pulmonary veins (veins were clamped and samples drawn from each lung individually), and the unclamped pul- monary vein so as to document the extent of gas exchange occurring in the native and engineered lungs (Table 1).

Chest x-ray confirmed that the engineered lung was inflated with air, but the level of inflation was

less than that of the native right lung (Fig. 4B). Histological evaluation of explanted engineered lungs revealed red blood cells in large blood vessels and septal microvessels, and some bleed- ing into airways, although this was modest (Fig. 4C). Blood gas analysis revealed that the tissue- engineered lungs were effective in exchanging oxygen and carbon dioxide (Table 1). Partial pres- sures of oxygen (PO2) increased from 27 T 7 mmHg in the pulmonary artery to 283 T 48 mmHg in the left pulmonary vein, indicating complete hemo- globin saturation and oxygenation. Hemoglobin saturation was 100% for both engineered left lung and native right lung venous samples. In ad- dition, carbon dioxide removal was efficient, with CO2 falling from 41 T 13 mmHg in the pulmonary artery to 11 T 5 mmHg in the left, engineered pulmonary vein. Although the PO2 in the right pulmonary vein was higher than in the left (634 T 69 versus 283 T 48 mmHg), this difference may not be of substantial physiological consequence because hemoglobin saturation is complete above oxygen pressures of 100 mmHg (10).

Discussion. To date, cell therapy and tissue engineering have been applied less extensively to lung than to other tissues and organs (11–13). Many efforts in lung regeneration have involved synthetic scaffolds or simple in vitro culture sys- tems. Such systems can regenerate certain micro- scopic features of alveolar architecture but have not yet produced tissue that can participate in gas exchange (14, 15). Use of the decellularization para- digm for respiratory tissue was described in 2008, when Macchiarini and colleagues implanted a re- seeded tracheal matrix into a patient with severe bronchomalacia (16).

In the current work, we have demonstrated the feasibility of producing an engineered lung that displays much of the microarchitecture of native lung and that can effect gas exchange for short periods of time when implanted into rats. Al- though these results are encouraging, multiple issues remain to be addressed before long-term engineered lung function can be realized. For ex- ample, alveolar barrier function must be improved so as to prevent any leakage of blood components into the airways. This can be accomplished through iterative improvement in the decellulari- zation procedure in order to minimize alveolar septal damage (17). Production of surfactant should be increased (18), and differentiated co- lumnar ciliated epithelium should be enhanced by more prolonged air breathing in culture (19). In

addition, the efficiency of vascular endothelial cov- erage must be very high throughout the engineered lung vasculature. This is to prevent exposure of collagen-containing basement membrane to the circulation, with consequent thrombosis, because some clotting was noted at explant. Lastly, a de- cellularization strategy for lung regeneration will only become clinically useful when a suitable, autologous source of pulmonary epithelium can be identified, such as a resident lung stem cell or induced pluripotent stem cell (20–24).

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M. Liu, Am. J. Physiol. Lung Cell. Mol. Physiol. 292, L510 (2007).

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21. J. Yu et al., Science 318, 1917 (2007). 22. C. F. Kim et al., Cell 121, 823 (2005). 23. H. J. Rippon et al., Proc. Am. Thorac. Soc. 5, 717

(2008). 24. B. Roszell et al., Tissue Eng. Part A 15, 3351 (2009). 25. We thank M. Colehour for help with bioreactor

development and Western blotting, R. Homer for histologic assessment, and B. Stripp for the CCSP antibody. This work was funded by Yale University Department of Anesthesia and by NIH grant HL 098220 (to L.E.N.). T.H.P. is supported by NIH T32 GM007171. L.E.N. holds stock in Humacyte, a regenerative medicine company. The authors (L.E.N., T.H.P., and E.A.C.) and Yale University have filed a patent application related to tissue engineering of lungs.

Supporting Online Material www.sciencemag.org/cgi/content/full/science.1189345/DC1 Materials and Methods SOM Text Figs. S1 to S11 Table S1 References

9 March 2010; accepted 14 June 2010 Published online 24 June 2010; 10.1126/science.1189345 Include this information when citing this paper.

Table 1. Gas exchange in engineered lungs implanted into rats. Shown are blood gas values and oxygen saturations for samples taken from rats implanted with an engineered lung. Values are mean T SD; n = 3 samples for each sample type except for right pulmonary vein, in which n = 2 samples. Sat, saturation.

Sample location pH PO2 (mmHg) O2 Sat (%) PCO2 (mmHg)

Pulmonary artery 7.30 T 0.06 27 T 7 44 T 20 41 T 13 Right pulmonary vein 7.53 T 0.08 634 T 69 100 T 0 20 T 1 Left (implant) pulmonary vein 7.68 T 0.28 283 T 48 100 T 0 11 T 5 Mixed pulmonary veins 7.58 T 0.08 495 T 174 100 T 0 18 T 3

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