End-stage lung disease is the third leading cause of death worldwide, accounting for 400,000 deaths per year in the United States alone. To reduce the morbidity and mortality associated with lung disease, new therapeutic strategies aimed at promoting lung repair and increasing the number of donor lungs available for transplantation are being explored. Because of the extreme complexity of this organ, previous attempts at bioengineering functional lungs from fully decellularized or synthetic scaffolds lacking functional vasculature have been largely unsuccessful. An intact vascular network is critical not only for maintaining the blood-gas barrier and allowing for proper graft function but also for supporting the regenerative cells. We therefore developed an airway-specific approach to removing the pulmonary epithelium, while maintaining the viability and function of the vascular endothelium, using a rat model. The resulting vascularized lung grafts supported the attachment and growth of human adult pulmonary cells and stem cell–derived lung-specified epithelial cells. We propose that de-epithelialization of the lung with preservation of intact vasculature could facilitate cell therapy of pulmonary epithelium and enable bioengineering of functional lungs for transplantation.

( A ) EVLP circuit and circuit elements. ( B ) Double lung cannulation method enabling ventilation of one lung (native) and airway de-epithelialization of contralateral lung (de-epith). R, right; L, left. ( C and D ) Schematic of experimental approach demonstrating (C) native airway, interstitium, and adjacent pulmonary vasculature and (D) denuded airway basement membrane following de-epithelialization with intact interstitium and preservation of vascular viability and function. ( E ) Components of vascular perfusate (see Materials and Methods for the complete list of components) and airway de-epithelialization solution.

Given the essential need for intact and functional pulmonary vasculature ( 13 ), we developed an airway-specific approach to removing the pulmonary epithelium while preserving the lung vasculature, ECM, and other supporting cell types (for example, fibroblasts, myocytes, chondrocytes, and pericytes). We demonstrated the outcomes of our method by using a rodent lung model. Following lung cannulation, lungs were ventilated and perfused on an ex vivo lung perfusion (EVLP) circuit ( Fig. 1A ). A mild detergent solution was delivered intratracheally to an isolated single lung ( Fig. 1B ) to remove epithelial cells and obtain a functional vascularized lung scaffold ( Fig. 1 , C and D). We investigated the utility of this model in (i) removing pulmonary epithelial cells, (ii) maintaining and preserving pulmonary vascular integrity and function, and (iii) creating a vascularized lung scaffold supporting the delivery and engraftment of human airway epithelium and stem cell-derived lung-specified epithelial cells.

Gas exchange, the major function of the lung, requires close interaction between functional epithelium, intact alveolar-capillary membrane, and viable endothelium. Lung epithelium is highly susceptible to pathological insults and is specifically implicated in a number of congenital and acquired diseases. After injury, lung epithelium may either activate physiologic repair mechanisms or undergo aberrant remodeling. When repair mechanisms fail, epithelial-vascular-mesenchymal interplay can lead to dysfunctional wound healing dominated by fibrosis ( 3 ). The period following injury that determines the fate of lung epithelium may serve as an optimal window of time for therapeutic intervention to either promote healthy physiologic repair or allow for cell replacement. Tissue engineering strategies are currently under development to regenerate or replace injured lungs ( 5 – 8 ). Because of the extreme complexity of the lung, with its hierarchical three-dimensional architecture, diverse cellular composition, highly specialized extracellular matrix (ECM), and region-specific structure and function ( 4 , 9 , 10 ), bioengineering a functional lung is still an elusive goal ( 11 , 12 ). The lungs bioengineered by full decellularization and recellularization have shown a limited and temporary function, largely due to blood clotting and pulmonary edema, which have led to lung failure within a few hours following transplantation. To date, whole-organ engineering methods using lung grafts with denuded vascular networks have failed to produce functional grafts.

Lung injury, whether acute or chronic, can lead to end-stage lung disease, a condition that affects millions of patients in the United States and accounts for approximately 400,000 deaths per year ( 1 ). Lung transplantation—the only definitive treatment for patients with end-stage lung disease—remains limited by a severe shortage of donor organs such that only 20% of patients waiting for a donor lung undergo transplantation ( 2 ). Strategies aimed at increasing the number of transplantable lungs would have an immediate and profound impact. In addition, early intervention with gene or cell therapy may offer even greater benefits by promoting lung repair and regeneration, thus slowing the progression of disease and ultimately avoiding the need for transplantation ( 3 , 4 ).

As a translational alternative to SAECs, vascularized lung scaffolds were recellularized with mRNA hiPSC–derived lung-specified epithelial cells fluorescently labeled at days 40 to 45. The cells contained 71.25 ± 3.34% FOXA2 + NX2.1 + cells [over 4′,6-diamidino-2-phenylindole–positive (DAPI + ) population] of which approximately half expressed SPB ( 22 , 23 ). Twenty-four and forty-eight hours after intratracheal delivery, cells were uniformly distributed and attached to the alveolar basement membrane ( Fig. 6 , O to T). Notably, hiPSC-derived lung-specified epithelial cells attached to the native lung matrix ( Fig. 6 , O and P, and fig. S7, G and H) and expressed apical membrane markers specific to alveolar type I (HT1-56) ( Fig. 6 , Q and R) ( 24 , 25 ) and type II (HT2-280) cells ( Fig. 6 , S and T, and fig. S7, I to L) ( 25 – 27 ).

Survival and proliferation of delivered CFSE-labeled SAECs were assessed by immunostaining for Ki67 (proliferation marker) ( Fig. 6M and fig. S7, E and F) and activated caspase-3 (apoptosis marker) ( Fig. 6N ). SAECs in recellularized lung maintain their proliferation state: 75 of 856 and 31 of 1030 Ki67-positive cells among CFSE-labeled SAECs and native cells, respectively (n = 3; P < 0.0001, Student’s t test). The fractions of apoptotic cells in recellularized and intact lungs were comparable: 9 of 543 or 5 of 538 caspase-3–positive cells among CFSE-labeled SAECs, respectively (n = 3; P = 0.41, Student’s t test).

( A ) Bioreactor schematic. R, reservoir of perfusate, cell culture medium; green circles, human SAECs or hiPSC-derived lung-specified epithelial cells delivered into de-epithelialized lung. ( B and C ) Delivery and attachment of SAECs in de-epithelialized lungs. Global distribution demonstrated with transpleural imaging of NIR-labeled cells (B) (dotted line indicates pleura). CFSE-labeled cells are distributed in the respiratory zone (C). ( D to G ) Lung section and histologic analysis of CFSE-labeled cells. H&E staining of native lung demonstrating intact pseudostratified respiratory epithelium (arrowhead) and vasculature (asterisk) (D) and of repopulated lung with intact native vasculature (asterisk) (E). CFSE-labeled SAECs attachment in the alveoli (F and G). ( H to J ) Immunostaining of native (H) and recellularized lungs with CFSE-labeled SAECs (I) with the epithelial marker Pankeratin. Inset: Higher-magnification images. Quantification of human and rat epithelial cells in recellularized lungs (n = 3; percentage of normalized to total Pankeratin-positive epithelial cells) (J). ( K ) Metabolic activity of native and recellularized lungs by resazurin assay (n = 3; P = 0.87, Student’s t test; error bars represent means ± SD of experimental values). OD, optical density. ( L ) Dynamic lung compliance measured by pressure-volume loops of native, de-epithelialized, and recellularized lungs (n = 3; native, 0.066 ± 0.0022 ml/cmH 2 O; de-epithelialized, 0.0169 ± 0.0021 ml/cmH 2 O; recellularized, 0.0257 ± 0.0015 ml/cmH 2 O; compliance decreased by ~74.20 ± 3.872% after de-epithelialization and increased by ~53.38 ± 20.83% after recellularization). Values are expressed as means ± SD. ( M and N ) Ki67 and caspase-3 (Cas 3) staining (indicated by filled arrowheads) demonstrating viable CFSE-labeled SAECs following 24 hours of culture in the bioreactor. Endogenous cells are indicated by the empty arrowhead. ( O to T ) Day 40 to 45 hiPSC-derived lung-specified epithelial cells, which were differentiated as described in Materials and Methods, were delivered into and attached in the de-epithelialized lung (O and P), expressing the human alveolar type I cell marker HT1-56 (Q and R) and the alveolar type II cell marker HT2-280 (S and T). (R) and (T) show higher magnification of boxed areas.

To assess whether de-epithelialized lungs could serve as a vascularized lung scaffold supporting therapeutic human cells, we developed a lung bioreactor system to investigate the engraftment and survival of human small airway epithelial cells (SAECs) and human-induced pluripotent stem cell (hiPSC)–derived lung-specified epithelial cells ( 22 , 23 ) within de-epithelialized lung scaffolds ( Fig. 6A ). SAECs were labeled with near-infrared (NIR) dye before intratracheal delivery to enable real-time transpleural imaging as a means of visualizing and monitoring cell distribution immediately after delivery ( Fig. 6B ). Twenty-four hours after delivery, carboxyfluorescein diacetate succinimidyl ester (CFSE)–labeled SAECs were detected throughout the tracheobronchial tree and in subpleural regions ( Fig. 6C ). Histological analysis of recellularized lungs confirmed the preservation of intact blood vessels ( Fig. 6 , D and E, fig. S7, A and B) and the presence of cells with squamous morphology attached to the basement membrane reconstituting the alveolar epithelium ( Fig. 6 , F and G, and fig. S7, C and D). Vascularized lung grafts were effectively repopulated with CFSE-labeled SAECs. A total of 51.98 ± 2.78% epithelial cells in the lung graft were human (n = 3) ( Fig. 6 , H to J). Notably, vascularized lung scaffolds recellularized with SAECs displayed a level of metabolism similar to native lungs (P = 0.87) ( Fig. 6K ). DNA content of the recellularized lung was similar to that of the native lung (n = 3; native, 596.18 ± 386.12 ng per tissue biopsy; recellularized, 884.58 ± 596.37 ng per tissue biopsy; P = 0.40, Student’s t test). Furthermore, 24 hours after recellularization, vascularized lung scaffolds demonstrated improved compliance (native, 0.066 ± 0.002 ml/cmH 2 O; de-epithelialized, 0.017 ± 0.002 ml/cmH 2 O; recellularized, 0.026 ± 0.0015 ml/cmH 2 O). Compliance decreased by 74.2 ± 3.9% following de-epithelialization but increased by 53.4 ± 21% following recellularization by SAECs ( Fig. 6L ).

To assess alveolar-capillary barrier function in de-epithelialized lungs, 500-kDa FITC-dextran was administered via the PA and collected from the pulmonary veins (PVs) and airways ( 21 ). Native lungs and fully decellularized lungs retained 96 ± 6% and 21 ± 4%, respectively, of the total dextran administered to the vascular compartment. De-epithelialized lungs retained 62 ± 9.5% of the total dextran, indicating significant preservation of the alveolar-capillary barrier ( Fig. 5E ). Histologic assessment documented the differences between native, de-epithelialized, and fully decellularized lungs ( Fig. 5F ). To evaluate the integrity of the pulmonary microvasculature, 0.2-μm-diameter fluorescent microspheres were administered to de-epithelialized lungs through the PA and were seen in capillaries and larger vessels but not in either the alveoli or bronchoalveolar lavage (BAL) samples, indicating the integrity of the vascular tree ( Fig. 5 , G to I, and movies S3 and S4).

To assess the viability and function of vascular smooth muscle cells following de-epithelialization, a vasoconstrictor and a vasodilator were subsequently administered, while pressure was measured in the pulmonary artery (PA). De-epithelialized lungs demonstrated a marked vasoconstrictive response to endothelin-1, with an 89% pulmonary arterial pressure increase from baseline within 10 min. Response to the vasodilator treprostinil reversed most of the effects of endothelin-1 and demonstrated a notable decrease in arterial pressure after 20 min, resulting in an arterial pressure of 31.9% of baseline ( Fig. 5D and fig. S6, F and G).

( A ) Immunofluorescence staining demonstrating the preservation of endothelial cells (vWF and CD31), vascular smooth muscle (SMA), pericytes (NG2), and tight and gap junction proteins (ZO-1 and Cx43) of the vascular bed following de-epithelialization. ( B ) Viability of endothelial cells by capture of acetylated LDL (aLDL) and loss of alveolar type I cells (Aq5) after de-epithelialization. ( C ) Nonapoptotic endothelial cells following de-epithelialization shown by TUNEL and vWF costaining; apoptotic cells (negative for vWF) in de-epithelialized lungs are indicated by arrowheads. ( D ) Intravascular administration of endothelin-1 and treprostinil demonstrating the preservation of vasoresponsiveness in native and de-epithelialized lungs (n = 3; Student’s t test; error bars represent means ± SD of experimental values). ( E ) Integrity of the pulmonary vascular bed shown by quantification of fluorescein isothiocyanate (FITC)–conjugated dextran recovered from the pulmonary venous drainage in native lung, following 4 and 8 mM CHAPS treatment (n = 3 for each group; *P = 0.01 and **P < 0.01, Student’s t test; error bars represent means ± SD of experimental values). ( F ) H&E histologic comparison of native lung and de-epithelialization by 4 and 8 mM CHAPS treatment. ( G to I ) Transpleural imaging to visualize pulmonary microvasculature. (G) Imaging setup with water-dipping lens. Images are captured during vascular perfusion of 0.2-μm fluorescent microspheres in (H) native and (I) de-epithelialized lungs (see movies S3 and S4). Blood vessels are indicated by asterisks.

Integrity of the pulmonary vascular bed and the alveolar-capillary membrane is critical for lung function ( 13 , 20 ). Immunostaining of de-epithelialized lung revealed preservation of markers for endothelial cells (vWF and CD31), smooth muscle cells (SMA), pericytes (NG2), tight junctions [zonula occludens–1 (ZO-1)], and gap junctions [connexin 43 (Cx43)] throughout the pulmonary vasculature ( Fig. 5A and fig. S6A). The function of endothelial cells in the de-epithelialized lung was confirmed by the uptake of acetylated low-density lipoprotein (LDL) ( Fig. 5B and fig. S6, B and C). The fractions of endothelial cells that were apoptotic in the TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling) assay were comparable for the native and de-epithelialized lungs (n = 3, 82 of 521 and 33 of 385, respectively; P = 0.16) ( Fig. 5C and fig. S6, D and E).

To assess the viability of airway smooth muscle cells and their responsiveness to physiological signals, we subjected de-epithelialized lungs to methacholine challenge similar to that used diagnostically for asthmatic patients ( 18 , 19 ). Following administration of methacholine, dynamic compliance significantly decreased in both native lungs [from 0.190 ± 0.046 ml/cmH 2 O to 0.048 ± 0.007 ml/cmH 2 O (reduction of 74.7 ± 8.7%)] and de-epithelialized lungs [from 0.065 ± 0.036 ml/cmH 2 O to 0.031 ± 0.004 ml/cmH 2 O (reduction of 45.3 ± 19%)]. These data demonstrated a clear bronchoconstrictive response to stimulation of muscarinic receptors in smooth muscle cells of the de-epithelialized lung airway ( Fig. 4 , G and H, and fig. S5, D to G). Following de-epithelialization, lungs showed reduced lung compliance before methacholine challenge (fig. S5, F and G).

Preservation of bronchial structure of native and de-epithelialized lungs ( A to F ). Pentachrome stain (A and B). SMA immunofluorescence staining (C and D). Airways are indicated by arrows, and vessels are indicated by stars. Scanning electron microscopy of airway casts (E and F). ( G and H ) Airway responsiveness during intravascular administration of methacholine measured by pressure-volume loops. Lung compliance in native (G) (0.190 ± 0.046 ml/cmH 2 O; after methacholine, 0.048 ± 0.007 ml/cmH 2 O; reduction of ~73.48 ± 8.704%) and de-epithelialized (H) (0.065 ± 0.036ml/cmH 2 O; after methacholine, 0.031 ± 0.004 ml/cmH 2 O; reduction of 45.28 ± 18.71%) lungs. Values are expressed as means ± SD.

Bronchial architecture, smooth muscle, and ECM components play pivotal roles in regulating gas exchange in the respiratory zone of the lung. Pentachrome staining of large bronchi showed de-epithelialized airways with histologic appearance otherwise similar to that of native airways. Collagen abundance in submucosal interstitium and airway cartilage is shown in yellow, with elastic fibers highlighted in black ( Fig. 4 , A and B). Smooth muscle actin (SMA) was observed in native and de-epithelialized lungs surrounding large airways and arteries ( Fig. 4 , C and D). Similarities in underlying airway smooth muscle between native and de-epithelialized lungs were observed regardless of the presence or absence of airway epithelium ( Fig. 4 , C and D, arrowheads). Airway casts imaged by scanning electron microscopy showed no microscopic differences in airway structure between native and de-epithelialized lungs. Thin parallel striations visible along the length of bronchi within airway casts suggested that the natural wrinkles present in native lung airway lining (which render airways capable of accommodating increases in airway diameter up to 30% during inspiration) were also preserved following de-epithelialization ( Fig. 4 , E and F). Therefore, although the epithelial cells were effectively removed, it did not compromise the structure of the basement membrane lining of airways. Clusters of alveolar sacs were visible in airway casts of both native and de-epithelialized lungs ( Fig. 4 , E and F).

( A ) Immunofluorescence staining demonstrating preservation of collagen IV, elastin, fibronectin, and laminin in de-epithelialized lung. ( B to I ) H&E staining (B and C) and special staining of native and de-epithelialized lung: trichrome (D and E), Alcian blue (F and G), and van Gieson (H and I). ( J ) Quantification of sGAGs (n = 3; P = 0.15), HOP (n = 3; P = 0.71), elastin (n = 3; P = 0.26), and DNA (native, n = 5; de-epithelialized, n = 5; decellularized, n = 4; *P < 0.05 and **P < 0.0001). ( K ) Scanning electron microscopy of native and de-epithelialized lungs. ( L ) Morphometry and stereology of native and de-epithelialized lungs: airspace volume (n = 3; P = 0.27), septal volume fraction (n = 3; P = 0.31), surface density (n = 3; P = 0.27), and septal thickness (n = 3; P = 0.98). Data shown were analyzed by Student’s t test. Error bars represent means ± SD of experimental values.

Immunostaining, scanning electron microscopy, and quantitative analyses suggested that the de-epithelialized lung retains a native-like structure and ECM ( Fig. 3 ). Distributions of ECM components retained in the de-epithelialized lung were similar to those in the native lung ( Fig. 3A and fig. S4A), as well as the vessel structure ( Fig. 3 , B to E), histomorphology of airway submucosa and cartilage ( Fig. 3 , D to G), preservation of elastic fibers ( Fig. 3 , H and I), and architecture of the alveoli (fig. S4A). Further quantification of ECM components demonstrated no significant differences between native and de-epithelialized lungs with respect to hydroxyproline (HOP) (P = 0.72) and elastin (P = 0.26), indicating that de-epithelialization does not significantly reduce the concentrations of critical lung matrix components ( Fig. 3J ). Sulfated glycosaminoglycan (sGAG) content was decreased in de-epithelialized lungs compared to native lungs, although not significantly (P = 0.15). Quantification of DNA showed a 23.4% decrease in DNA content of de-epithelialized lungs compared to native lungs (P = 0.04) ( Fig. 3J ). In comparison, full decellularization resulted in the removal of >80% of DNA content compared to native lungs and 60% compared to de-epithelialized lungs (P < 0.0001, when compared to both native and de-epithelialized lungs). Scanning electronic microscopy confirmed the preservation of bronchial, alveolar, and vascular structures ( Fig. 3K and fig. S4, B to G). Morphometric and stereologic analyses did not show any significant differences between native and de-epithelialized lungs with respect to airspace volume fraction (native, 0.76 ± 0.05; de-epithelialized, 0.70 ± 0.08; P = 0.27; values are expressed as means ± SD throughout), septal volume fraction (native, 0.24 ± 0.05; de-epithelialized, 0.29 ± 0.08; P = 0.31), septal thickness (native, 7.1 ± 0.7 μm; de-epithelialized, 7.1 ± 0.5 μm; P = 0.98), and surface density (native, 0.067 ± 0.009; de-epithelialized, 0.081 ± 0.017; P = 0.15) ( Fig. 3L ). Because of the loss of alveolar surfactant and epithelial cells, dynamic lung compliance decreased after de-epithelialization (fig. S5, A to C).

( A to D ) Hematoxylin and eosin (H&E) staining of conducting zone in native and de-epithelialized lungs with intact pseudostratified epithelium (arrowheads) in the native lung (A and C) and denuded basement membrane (arrowheads) following the removal of respiratory epithelium in the de-epithelialized lung (B and D). Vessel is indicated by asterisk. ( E and F ) Immunofluorescence staining confirming the removal of bronchial epithelial cells by decreased EpCAM in de-epithelialized lung, with preservation of vessels indicated by vWF. ( G and H ) H&E staining of respiratory zone in native and de-epithelialized lungs. ( I to L ) Immunofluorescence staining confirmed the removal of alveolar type I cells by decreased Aq5, alveolar type II cells by decreased SPC, with preservation of vessels indicated by vWF in de-epithelialized lungs (J and L). ( M and N ) Western blot of CD31 and EpCAM from native and de-epithelialized lungs (asterisk indicates nonspecific band) (M). Quantification data indicate that de-epithelialized lungs contained five times less EpCAM than native (n = 3, values normalized to levels in native lung; error bars represent means ± SD of experimental values) (N). ( O and P ) TEM of native (O) and de-epithelialized (P) lungs showing intact microvessels and endothelial cell nuclei (asterisks) but absent alveolar cells (arrowheads) in de-epithelialized lungs.

Microscopic and Western blot analyses confirmed the removal of pulmonary epithelium ( Fig. 2 ). In the conductive zone of the lung, histologic analysis indicated the removal of pseudostratified epithelium from the airway ( Fig. 2 , A to D). Immunostaining revealed the preservation of blood vessels retaining the endothelial cell marker Von Willebrand factor (vWF) and the interstitial fibroblast marker vimentin (fig. S3, A to D), adjacent to large airways stripped of the epithelial cell marker epithelial cell adhesion molecule (EpCAM) ( Fig. 2 , E and F). Similarly, in the respiratory zone, lung epithelium was removed from the bronchioles and alveoli ( Fig. 2 , G and H), with the preservation of small blood vessels retaining vWF and a significant reduction in the epithelial cell marker EpCAM (fig. S3, E and F), the alveolar type I cell marker aquaporin 5 (Aq5) ( Fig. 2 , I and J), and the type II cell marker surfactant protein C (SPC) ( Fig. 2 , K and L). Immunoblotting of epithelial (EpCAM) and endothelial (CD31) cell markers in de-epithelialized lungs verified preservation of intact endothelial cells, along with a significant loss (70%) of epithelial cells ( Fig. 2 , M and N, and fig. S3I). Furthermore, de-epithelialized lungs showed significant reductions in lung epithelial cell–specific markers, including Aq5 (bronchial epithelial and alveolar type I cell marker), SPC (alveolar type II cell marker), acetylated tubulin (ciliated cell marker) ( 16 , 17 ), and CC-10 (club cell marker) (fig. S3, G and H). Transmission electron microscopy (TEM) of de-epithelialized lungs showed denuded basement membrane due to the loss of alveolar epithelium and retention of both the alveolar-capillary basement membrane and microvascular endothelial cells in pulmonary capillaries ( Fig. 2 , O and P, and fig. S3, J to M).

On the basis of previous studies in our laboratory ( 14 ), a 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) detergent–based solution was used for airway de-epithelialization. Concentrations of CHAPS and sodium chloride were optimized to enable efficient removal of the pulmonary epithelium while minimizing effects on the pulmonary interstitium and vasculature ( Fig. 1E and fig. S2, A and C). To enhance distribution of the solution throughout the airway and to improve epithelial cell removal by physical agitation, we ventilated lungs at low tidal volumes (TV) [0.03 ml/100 g; normal value, 0.3 ml/100 g body weight ( 15 )] and a high frequency [150 to 180 breaths per minute (bpm)], mimicking the high-frequency oscillatory ventilation (HFOV) used clinically in acute respiratory distress syndrome (ARDS) patients (movies S1 and S2).

Rat lung epithelium was removed by infusing a detergent solution only through the airway compartment. Following cannulation (fig. S1A), the animal was placed on an EVLP circuit (fig. S1B) and primed with venous blood. Ventilation on 21% oxygen resulted in normal gas exchange (ΔpO 2 = 121 ± 10 mmHg and ΔpCO 2 = −184 ± 6 mmHg), normal pH (pre-EVLP, 6.74 ± 0.3; EVLP, 7.4 ± 0.4), and a change to normal blood color (fig. S1C). The airway in one lung was isolated by cannulation of the main bronchus and ventilated to serve as control, whereas the other lung was subjected to de-epithelialization ( Fig. 1B ). Single lung ventilation maintained physiologic pH, pO 2 , and pCO 2 (fig. S1C). Stable EVLP was achieved using a physiologic vascular perfusate composed of electrolytes, an energy substrate, and an oncotic regulator ( Fig. 1E ) that supported the lung during de-epithelialization (fig. S1D).

DISCUSSION

End-stage lung disease has a profound impact on the quality and duration of countless lives and represents a growing burden to health care systems worldwide (28). Despite meritorious efforts to better understand the pathogenesis of lung disease and identify targets and strategies to reverse disease trajectory, many patients suffer significant impairment and ultimately require lung transplantation (2). Although increasing the pool of donor lungs by implementing tissue engineering strategies has tremendous promise, several key obstacles have slowed the progress and even questioned the validity of this approach. The absence of functional vascular networks has been a major limitation in creating functional, nonthrombogenic lung constructs (5, 11, 12). Given the complexity of the lung, the needs for nutrient and oxygen delivery require vascular supply (13). Global lung function depends on the intricate and extensive capillary network, which could not be recapitulated or preserved using available bioengineering methods.

We demonstrate a transformative approach to obtaining functional vascularized lung grafts. By intratracheal targeting of the airway, we removed lung epithelium in a manner that preserves the surrounding cells, matrix, basement membrane, and vasculature. De-epithelialized lungs with intact, functional vasculature could serve as physiologic scaffold by (i) enabling the delivery of nutrients, oxygen, growth factors, and signaling molecules; (ii) providing key biophysical and mechanical signals via perfusion (flow and shear) and ventilation (strain); and (iii) maintaining the ECM (biochemical moieties, adhesion molecules, and matricryptic peptides) and the interstitial and support cells (fibroblasts, pericytes, endothelial, mesothelial, and lymphatic cells).

Previously established methods for decellularization of the entire organ were designed to remove both the epithelium and endothelium and could only be applied ex vivo (5, 11, 12, 14). This study developed the first procedure for the removal of epithelium from the lung airway with the full preservation of vascular epithelium, which could be applied in vivo to treat diseases of lung epithelium (for example, ARDS and idiopathic pulmonary fibrosis). Whole lung scaffolds with an intact vascular network may also allow for recellularization using patient-specific cells and bioengineering of chimeric lungs for transplantation capable of gas exchange. In addition to the clinical potential, lung scaffolds lacking an intact epithelial layer but with functional vascular and interstitial compartments may also serve as a valuable physiological model for investigating (i) lung development, (ii) the etiology and pathogenesis of lung diseases involving pulmonary epithelium, (iii) acute lung injury and repair, and (iv) stem cell therapies.

We demonstrated the removal of airway epithelium by general histology (Fig. 2, A to D and G and H); immunostaining of EpCAM, Aq5, and SPC (Fig. 2, E and F and I to L); immunoblotting of EpCAM and other lung epithelial cell–specific markers (Fig. 2M and fig. S3G); and a 24% decrease in the DNA content (Fig. 3J). Given the cellular composition in the parenchyma of rat lung (43% endothelial cells, 32% interstitial cells, and 22% epithelial cells) (29), these results demonstrated that the treatment effects are well contained to the epithelium. Typically, full organ decellularization results in 75 to 98% DNA reduction, consistent with the depletion of all cell types (14, 30–32).

Tissue ECM is known to provide the structural and mechanical support for resident cells, along with biochemical cues: growth factors, chemokines, and cytokines. The structure, composition, and mechanics of the ECM play major biological roles, making tissue-specific ECM an ideal scaffold for cell attachment, differentiation, and functional organization (11, 33–37). In this respect, the proposed method results in retention of the lung architecture and matrix proteins, as evidenced by immunostaining (collagen IV, collagen I, elastin, fibronectin, and laminin), quantification of HOP and elastin (Fig. 3, A and J, and fig. S4A), scanning electron microscopy (Fig. 3K), and morphometric and stereologic analyses (Fig. 3L). Although not statistically significant, the measured decrease in sGAG content of de-epithelialized lungs (Fig. 3J) may be due to the loss of epithelial cell–associated sGAGs rather than matrix-bound sGAGs because our approach uses CHAPS at pH 8, known to be more protective of matrix-bound sGAGs compared to treatments with solutions at higher pH (31). The preserved matrix features in de-epithelialized lungs support diverse lung functions: elastic recoil during ventilation, cell adhesion, and barrier function.

The endothelial cells and functional vasculature were preserved in de-epithelialized lungs, as demonstrated by histology (Fig. 2, A and B), immunostaining for vWF (Fig. 2, E and F and I and J), and immunoblotting for CD31 (Fig. 2, M and N). The alveolar-capillary membrane remained intact, as shown by TEM and immunostaining for gap (Cx43) and tight (ZO-1) junctions (Figs. 2, O and P, and 5A and figs. S3, J to M, and S6A). Viability and function of endothelial cells in the macro- and microvasculature were shown by the capture of acetylated LDL (Fig. 5B and fig. S6, B and C). Blood vessels maintained responsiveness to vasoconstrictors/vasodilators, indicating that de-epithelialized lung scaffolds are capable of regulating blood flow (Fig. 5D). Integrity of the pulmonary vascular tree was well preserved, as demonstrated by the infusion of FITC-dextran (Fig. 5E) and microspheres (Fig. 5, H and I, and movies S3 and S4). The leakage of dextran in de-epithelialized lung scaffolds (~30%) was higher than that in an intact lung but much lower than that in a fully decellularized lung. This leakage, indicative of weakened endothelial tight junctions, may be transient and recoverable, given the presence of preserved endothelial cells and pericytes, an expectation that will need to be confirmed in long-term studies.

Ultimately, a challenge for using de-epithelialized lung as a scaffold for lung bioengineering is lung regeneration following infusion of fresh epithelial cells. Here, de-epithelialized lung scaffolds with preserved functional vasculature enabled the delivery and topographic distribution of several types of human cells, including human SAECs and hiPSC-derived lung-specified epithelial cells (Fig. 6). Cells attached throughout the distal lung, assuming a flattened morphology that normally precedes reconstitution of airway epithelium (Fig. 6, F and G). Fifty-two percent of all epithelial cells in our recellularized and vascularized lung scaffolds were human (Fig. 6, H to J). Following 24 hours of incubation in a bioreactor, cells were viable (Fig. 6, M and N) and metabolically active (Fig. 6K). Recellularized lungs with SAECs showed higher numbers of Ki67-positive cells compared to native lungs. This result is likely a reflection of the relatively low regeneration rate in native lungs (4) when compared to a cell line like SAECs.

Lung compliance showed a trend of improvement (Fig. 6L) that we attribute to recellularization and reconstitution of the alveolar membrane, rather than surfactant production, because we would not expect SAECs to produce surfactants over this time course. In previous studies of full decellularization and recellularization with rat lung fetal or neonatal epithelial cells (11, 12), lung constructs showed production of SPC and SPA only at 4 to 5 days of bioreactor culture. Finally, hiPSC-derived lung-specified epithelial cells expressed markers specific to alveolar type I and type II cells (Fig. 6, Q to T), indicating that the vascularized lung scaffold supported their survival and phenotypic gene expression.

There are several limitations to the present study, which open new avenues for future research. To preserve the architecture and viability of the vascular compartment, we carefully titrated our de-epithelialization solution to only remove, as much as possible, epithelial cells. This titrated balance, which enabled vasculature preservation, also resulted in the retention of some endogenous epithelial cells. Nevertheless, we believe that these few remaining cells may also provide important instructive cues to cells newly introduced into the airway. Although our de-epithelialization approach targets airway epithelial removal, we cannot exclude some detergent leak in the vascular compartment. Although vascular viability and the attachment of exogenous cells confirmed a functional scaffold, long-term studies are needed to investigate the presence of residual detergent after de-epithelialization and its effects on scaffold functionality and cell viability. Immunological changes during de-epithelialization and recellularization were not investigated, given that human cells were studied in nude rats. It would be of interest to investigate the role of the innate immune system in chimeric lung constructs and define the host response to de-epithelialization in situ. In terms of recellularization, our hypothesis is that human pluripotent stem cell progenitor cells seeded into de-epithelialized regions of the lung, with the support of functional lung vasculature, will enable regeneration and remodeling of the donor lung. This could lead to a gradual increase in the fraction of the recipient’s cells and contribute to global lung functionality. Unless patient-specific hiPSC cells can be used in the future, immunosuppression would be necessary during this transient period (extent and duration remain to be determined). Although this study used healthy lungs, the method is intended for application to injured lungs. We anticipate that diseased epithelium may have enhanced response to de-epithelialization and that the treatment may thus need to be shortened to achieve equivalent efficacy. Cell delivery and engraftment potential were demonstrated over a period of 24 to 48 hours. Vascularized lung scaffolds demonstrated a high level of recellularization, a parameter that can be experimentally manipulated by varying the number of human cells delivered. Additional studies are needed to determine optimal cell dosage and the degree to which exogenous cells expand and repopulate the recipient lung. Future studies are needed to evaluate the remodeling and function of recellularized lung scaffolds over prolonged periods and investigate their function in subsequent transplantation.

Lung decellularization has resulted in substantial advances in lung bioengineering and the ability to create acellular scaffolds for tissue engineering applications (11, 12, 21). We believe that our methodology can address some of the challenges that have slowed the progress in lung bioengineering by (i) preserving the vascular endothelium throughout the lung (from large vessels to capillaries) and (ii) targeting the removal of airway epithelium while maintaining structural and cellular components essential for lung repair. Further developments may involve the therapeutic use of de-epithelialization by targeting specific types of damaged/defective cells. Cell replacement therapy may have potential for repairing acutely injured epithelium (for example, following ARDS and acute injury), defective epithelium (for example, Hermansky-Pudlak syndrome and SFTPC disease), and conditions wherein the damaged epithelium activates aberrant remodeling (for example, chronic interstitial lung disease and Birt-Hogg-Dubé syndrome) (3). The use of patient-specific lung progenitor cells and the transplantation of chimeric organs may help mitigate rejection and offer a more personalized approach to lung transplantation (fig. S8) (4, 38). Additionally, de-epithelialization could be applied to other organs with dual flow, such as the liver or kidney. In summary, the creation of de-epithelialized whole lungs with functional vasculature may open new frontiers in lung bioengineering and regenerative medicine.