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Efficient and functional endothelial repopulation of whole lung organ scaffolds. Andrew Viet Le, Go Hatachi, Arkadi Beloiartsev, Mahboobe Ghaedi, Alexander J Engler, Pavlina Baevova, Laura E. Niklason, and Elizabeth A Calle ACS Biomater. Sci. Eng., Just Accepted Manuscript • Publication Date (Web): 16 May 2017 Downloaded from http://pubs.acs.org on May 20, 2017

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ACS Biomaterials Science & Engineering

Efficient and functional endothelial repopulation of whole lung organ scaffolds.

Andrew V. Le a, Go Hatachi b, Arkadi Beloiartsev a, Mahboobe Ghaedi a, Alexander J. Engler c, Pavlina Baevova a, Laura E. Niklason a, c, Elizabeth A. Calle c

a

b

Department of Anesthesiology, Yale University, New Haven, CT 06519 Division of Surgical Oncology, Department of Surgery, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan c Department of Biomedical Engineering, Yale University, New Haven, CT 06519

Corresponding Author: Elizabeth A. Calle [email protected] 10 Amistad St., Room 314K New Haven, CT 06519

ACS Paragon Plus Environment


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Abstract To date, efforts to generate engineered lung tissue capable of long-term function have been limited by incomplete barrier formation between air and blood and by thrombosis of the microvasculature upon exposure of blood to the collagens within the decellularized scaffold. Improved barrier function and resistance to thrombosis both depend upon the recapitulation of a confluent monolayer of functional endothelium throughout the pulmonary vasculature. This manuscript describes novel strategies to increase cell coverage of the vascular surface area, compared to previous reports in our lab and others, and reports robust production of multiple anticoagulant substances that will be key to long-term function in vivo once additional strides are made in improving barrier function.. Rat lung microvascular endothelial cells were seeded into decellularized rat lungs by both the pulmonary artery and veins, with the use of low-concentration cell suspensions, pulsatile, gravity-driven flow, and supraphysiological vascular pressures. Together, these strategies yielded 72.44% +/- 10.52% endothelial cell nuclear coverage of the acellular matrix after 3-4 d of biomimetic bioreactor culture, compared to native rat lung. Immunofluorescence, Western blot, and PCR analysis of these lungs indicated robust expression of phenotypic markers such as CD31 and VE-Cadherin after time in culture. Endothelial seeded lungs had CD31 gene expression of 0.074±0.015 vs. 0.021±0.0023 for native lungs, p=0.025; and VE-Cadherin gene expression of 0.93±0.22, as compared to native lung 0.13±0.02, p=0.023. Precursors to antithrombotic substances such as tissue plasminogen activator, prostacyclin synthase, and endothelial nitric oxide synthase were expressed at levels equal to or greater than native lung. Engineered lungs reseeded with endothelial cells were implanted orthotopically and contained patent microvascular networks that had greater gas exchange function during mechanical ventilation on 100% O2 than that of decelluarized lungs. Taken together, these data suggest that these engineered constructs could be compatible with long-term function in vivo when utilized in future studies in tandem with improved barrier function.

Keywords: decellularization; tissue engineering; lung engineering; organ engineering; endothelialization; microvascular endothelial cells


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1. Introduction The single greatest challenge to whole organ engineering is the provision of a stable, perfusable microvascular network to the tissue of interest. Patent microvasculature is critical for nutrient and waste transport and for the exchange of oxygen and carbon dioxide. Maintenance of this network requires a capacity to resist thrombosis and to create a selectively permeable barrier between the vascular compartment and the interstitium and organ parenchyma.

Endothelial cells (ECs) play a critical role in both barrier function and resistance to thrombosis. ECs prevent coagulation and thrombus formation by shielding collagen from interaction with blood, which would lead to rapid platelet aggregation and clot formation.1 In addition, endothelial cells actively inhibit platelet activation, aggregation, and adhesion – key steps in thrombus formation – by the production of nitric oxide (NO), derived from endothelial nitric oxide synthase (eNOS), and by the secretion of prostacyclin (PGI2) generated by prostacyclin synthase (PGI2S). Endothelial cells also form adherens junctions, mediated by VE-cadherin and other adhesion proteins, and tight junctions, both of which prohibit translocation of particles and molecules.2

Recently, there have been a few reports of efforts to improve endothelial cell population of decellularized lung extracellular matrix scaffolds. Work by Ren et al. showed perhaps the greatest improvements in endothelial seeding of decellularized lung scaffolds by using a twophase culture protocol and by the addition of perivascular cells.3 This work demonstrated the importance of paracrine signaling in supporting proliferation of endothelium within the vascular compartment of the scaffold and in maturation of endothelial-mesenchymal barrier. Stabler et al.



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investigated the effect of lung posture on cell distribution within the lobes of rodent lungs and evaluated the types of integrins expressed by seeded endothelium and demonstrated their importance in promoting adhesion to the acellular scaffolds. Despite adding mechanistic knowledge to the field, none of the conditions investigated in this work yielded consistent and even distribution of endothelial cells throughout the alveolar capillary beds.4 Scarritt and colleagues evaluated the effects of gravity-driven vs. perfusion-driven endothelial cell delivery to the vascular compartment. They also assessed the effect of using endothelial cells isolated from different portions of the pulmonary vasculature; this yielded the observation of relatively homogenous and fairly dense distribution of cells throughout the lung when using microvascular endothelial cells, but not when endothelial cells of arterial or venous origin.5 Both of these groups (Stabler and Scarritt) also evaluated barrier function after seeding; both noted some persistent leak from the vascular compartment.

Prior work in our laboratory resulted in successful adhesion of rat lung microvascular endothelial cells within an acellular lung scaffold, but demonstrated aggregation of endothelial cells (EC) in medium- and small-caliber vessels and an overall deficiency in the amount of extracellular matrix surface area covered with endothelium. Upon implantation, clots formed in both small and large vessels, which rendered the engineered lung grafts nonfunctional after several hours in vivo;6 this result was likely related to the insufficient lining of vascular endothelium at that time.

Herein, we describe several key strategies for improved delivery of endothelial cells to the microvascular network of decellularized lung extracellular matrices. These strategies, while applied to lung in this work, are inherently general, and may be applied to other decellularized


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organ scaffolds as well. We demonstrate robust nuclear coverage of endothelial cell-seeded lung: (72.44% +/- 10.52%) when compared to native. We evaluate the endothelial cell-seeded lungs for production of the antithrombotic compounds NO and PGI2 by measuring eNOS and PGI2S. We show that expression of key endothelial markers (CD31, VE-cadherin, vWF, VEGFR1, VEGR2) in a repopulated lung scaffold is comparable to those expressed in native rodent lung. Furthermore, endothelial-repopulated lung matrices resist thrombosis after in vivo implantation, in contrast to previous engineered lung implants which suffered from rapid intra-vascular coagulation.6 Taken together, the techniques described here represent a significant advancement in the strategies available to promote endothelial cell seeding of decellularized organ scaffolds, and achieve near-native endothelial repopulation in terms of cell number, cell distribution, and markers that define endothelial phenotype and support the possibility of effective endothelial cell function.



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2. Materials and Methods 2.1 Lung Harvest All animal work was performed in accordance with AAALAC guidelines and was approved by the Yale Institutional Animal Care and Use Committee (IACUC). Lungs were harvested from adult (~300 gram) Fischer 344 (F344) or Sprague Dawley male rats as described previously.6 Briefly, a transverse incision was made below the costal margin to enter the abdominal cavity. The chest was entered through the diaphragm, and the heart was perfused with phosphate buffered saline (PBS) containing heparin (100 U/mL) and sodium nitroprusside (SNP, 10 µg/mL) via the right ventricle. The heart, lungs, and trachea were removed en bloc. The lungs were first inflated with air, then with PBS containing heparin and SNP. Lungs were perfused with the same solution via the pulmonary artery (PA), then mounted in a bioreactor for decellularization or saved for analysis as native samples.

2.2 Decellularization of Whole Rat Lungs Lungs were perfused with antibiotics/antimycotics, and blood was cleared by additional perfusion with PBS containing heparin and SNP. Following a subsequent rinse with PBS containing Ca2+ and Mg2+ (hereafter referred to as “PBS with ions”), lungs were decellularized using sodium deoxycholate and Triton X-100 detergents according to the protocol recently adopted by our lab7 and first used by Madri et al. for human lung,8 and by Price et al. for rodent lung.9 After decellularization, the cannulated heart-lung bloc was mounted in a sterile bioreactor and perfused with antibiotics/antimycotics for 48 h at 37°C as a final sterilization step. Lungs were stored at 4ºC until reseeding, or were processed for histology and analysis by western blot.


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2.3 Rat Lung Microvascular Endothelial Cell Culture Primary rat lung microvascular endothelial cells (RLMVECs), isolated from Sprague Dawley male rats 6-8 weeks in age (VEC Technologies), were cultured and expanded on fibronectincoated tissue culture flasks (1 µg/cm2) in MCDB-131 Complete medium prior to seeding on decellularized lung matrices. Medium was changed every 3-4 days. Cells were used at passage 36.

2.4 Labeling of Endothelial Cells with diI Membrane Dye Serum-containing medium was removed from adherent RLMVECs in culture and cells were washed with 1X PBS. DiI lipophilic membrane dye was initially reconstituted in dimethyl sulfoxide (DMSO). Cells were incubated with diI membrane-incorporating dye, and diluted in serum-free medium at a concentration of 1:1000. After 1 hr at 37ºC, cells were washed, trypsinized with 0.25% trypsin/ ethylenediaminetetraacetic acid (EDTA), and re-suspended in culture medium for seeding onto decellularized lung extracellular matrices.

2.5 Perfusion of Acellular Scaffolds with Washed Red Blood Cells Washed red blood cells (RBCs) were prepared from whole blood as previously described.10 Briefly, whole rat blood was centrifuged at 500 g at 4°C for 10 min and the plasma, buffy coat, and uppermost erythrocytes were removed by aspiration and discarded. The remaining erythrocytes were washed three times in wash buffer containing (in mM) 21.0 tris(hydroxymethyl)aminomethane, 4.7 KCl, 2.0 CaCl2, 140.5 NaCl, 1.2 MgSO4, and 5.5 glucose and 0.5% bovine albumin fraction V, final pH 7.4. Suspensions of washed red blood cells, which should pass easily through the capillaries of the native lung as they do in vivo, were introduced



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into the scaffold at a concentration similar to that of rat whole blood (about 8 million RBC/µL),11 and at a 100-fold reduction in cell density (80,000 RBC/µL). Cells were introduced to the pulmonary artery by a column of fluid designed to apply a pressure of ≤ 30 cm H2O.

2.6 Bi-directional Endothelial Cell Seeding To visualize the location of seeded EC seeded in the PA vs. pulmonary vein (PV), cells introduced via the PA were left unlabeled while cells introduced via the PV were labeled with red diI membrane-incorporating dye. A total of 160 million RLMVECs were seeded into the lung at a concentration of 1 million cells/mL; 80 million cells were seeded into the PA, followed directly by the seeding of 80 million cells into the PV. Cells were delivered with a constant pressure head of ≤ 30 cm H2O under gravity-driven flow. Cells distribution was evaluated after 30 min of static culture and after 6 hours of culture with pulsatile perfusion at 2 mL/min (first three hours) and 4 mL/min (hours 4-6).

2.7 Endothelial Cell Seeding Utilizing Sequential, Bi-directional, High Pressure, Low Cell Concentration, Pulsatile Technique: Whole decellularized lungs were mounted in a sterile bioreactor containing 50% MCDB-131 medium and 50% DMEM with 10% FBS. The perfusion loop of the bioreactor (Fig. 1A) was connected to a reservoir (Fig. 1B) containing a solution of single cells (Fig. 1C), which contained a stir bar and was mounted on a stir plate (Fig. 1D). The perfusion loop tubing passed through a solenoid valve (Fig. 1E) en route from the reservoir to the bioreactor to generate pulsatile flow at 60 beats per minute (bpm). The height of the cell reservoir was fixed at 60 cm above the bioreactor (Fig. 1F, ~44 mm Hg). RLMVECs were suspended in endothelial cell culture medium


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at a concentration of 250,000 cells/mL. Lungs were seeded with half of the total EC via the PA (50 million) by gravity-driven flow. Following PA seeding, the bioreactor-mounted lungs were transferred to an incubator and perfused with media via the PA at 37ºC for 1 h at 1 mL/min using a pulsatile pump. EC were then seeded via the PV with the remaining cells (50 million), in the same manner as described for PA seeding. After seeding, the lung was again perfused, this time via the PV at 37ºC for 1 h at 1 mL/minute. Thereafter, the lungs remained in the incubator statically for 60 minutes. The PA cannula was then reconnected to the bioreactor cap and the PV and trachea cannulae were freely suspended in the bioreactor chamber. Arterial perfusion was initiated with a pulsatile pump at a flow rate of 1 mL/min. The perfusion rate was increased 1 mL/min every hour until perfusion reached 4.5 mL/min.

During the ensuing 4 days of culture, half of the medium was exchanged every day with media that was comprised of 50% MCDB-131 and 50% DMEM and that contained no serum; this was repeated until the culture serum concentration reached 2.5% total. Lungs were harvested on day 4 and processed for analysis by histology, western blot, PCR or implanted in a rat recipient in a left lung orthotropic position.

2.8 Quantification of Endothelial Cell Nuclear Coverage on Lung Matrix

A series of overlapping 5x images were taken on a microscope to capture all geometry within an entire lobe. The images were stitched together using Image Composite Editor and exported as a TIFF file. The TIFF file was imported into MATLAB, where the image was transformed into a Hue-Saturation-Value matrix. Images were displayed according to intensity value, and three thresholds were selected to distinguish the following components: 1) dark background vs. purple



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nuclei (a threshold intensity value of 0.1); 2) purple nuclei vs. pink extracellular matrix (0.65); 3) pink extracellular matrix vs. bright background (0.75). In addition, the saturation image was displayed, and a background threshold was obtained (an intensity value of ~0.1). Purple pixels were defined as pixels between the two intensity thresholds for purple pixels and above the saturation threshold, and pink pixels were defined as pixels between the two intensity thresholds for pink pixels and above the saturation threshold. The percent nuclear coverage was calculated using the formula:

% Nuclear Coverage = 1 −

1 # purple pixels 1+ # pink pixels

Percent nuclear coverage values were averaged for three separate native lungs and three separate endothelial seeded lungs. Native and endothelial seeded percent nuclear coverage values were averaged and endothelial seed lungs were normalized to native by dividing the endothelial seeded lung average by the native lung average.

2.9 Left Orthotopic Lung Implantation of Endothelial Cell-Seeded and Decelluarized Rat Lungs

Left orthotopic lung transplantation was performed using decellularized (n=3) and endothelial seeded (n=6) rat donor lungs. Decellularized and engineered lungs were removed from the bioreactor and placed in a petri dish on ice. The lungs were then connected to rodent ventilator and mechanically ventilated with a tidal volume of 5 mL/kg for 10 minutes with room air. Positive end expiratory pressure (PEEP) was set to 6 cm H2O. After administration of 50 mg/kg R (surfactant) via the tracheal tube, the lungs were mechanically ventilated with 8 of Survanta○


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mL/kg tidal volume to get complete expansion, and PEEP was decreased to 3 cm H2O gradually. The left hilar vessels and main bronchus in the lungs were then isolated and transected to mount cuffs. A clip was put on the left main bronchus to maintain left lung inflation. The lung remained on ice until implantation.

Recipient Sprague-Dawley male rats weighing 270-350 g were anesthetized with 75 mg/kg of ketamine and maintained with 2% of isoflurane. In addition, 0.6 mg/kg of rocoronium as muscle relaxant and 0.04 mg/kg of buprenorphine as analgesia were administrated intraperitoneally. A continuous infusion of heparin was administered throughout the case (15 U/h). Left lung transplantation was performed following standard rodent surgical techniques.6, 12 Four mL/h of lactated Ringer’s solution was infused via a venous catheter. Left thoracotomy was performed and left hilum was dissected. Recipient hilar vessels and bronchus were clamped using micro clamps. Donor vessels and bronchus were inserted into the recipient vessels and bronchus and secured by 7-0 silk suture. After anastomosis and confirmation that the engineered or decelluarized donor lung could be ventilated, the pulmonary vein and pulmonary artery were declamped in turn.

Arterial blood gas (ABG) analysis was performed before removal of the left native lung (baseline) using a i-STAT handheld blood analyzer (Abbot) and CG4+ cartridge. ABG was then performed at 15 minutes, 1 hour, 2 hours, and 3 hours after implantation of the left engineered lung. Blood samples were collected from the carotid artery in vivo in order to access oxygenation (SaO2) and gas exchange (PaO2 and PaCO2) in the systemic circulation of anesthetized and mechanically ventilated rats who survived to the 3 h post-implant time-point.



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2.10 Immunofluorescence and Immunohistochemistry Tissue was fixed in 10% neutral buffered formalin (NBF) for 2-4 h and then transferred to PBS. Paraffin-embedded sections of native, decellularized, and engineered lungs were baked at 65°C for 30 minutes, deparaffinized in xylenes, and rehydrated with a decreasing ethanol gradient. After performing antigen retrieval with citrate buffer, sections were permeabilized in PBS with 0.2% Triton X-100 for 15 minutes and blocked in PBS with 0.75% glycine and 5% bovine serum albumin (BSA) for 1 h at room temperature. Blocked sections were incubated overnight at 4ºC with primary antibodies diluted in blocking buffer (Supplemental Table 1). After rinsing with PBS, secondary antibodies were applied at 1:500 dilution for 1 h at room temperature. Sections were stained with 4',6-diamidino-2-phenylindole (DAPI), mounted with polyvinyl alcohol with DABCO (PVA-DABCO), and dried overnight. Stained sections were imaged with a Zeiss Axiovert zoom inverted microscope, a Hamamatsu camera, and Volocity software.

For PCNA staining, 5 µm sections were rehydrated with a decreasing ethanol gradient, taken through antigen retrieval with citrate buffer (10 mM citric acid, 0.05% Tween 20, pH 6.0), permeabilized in PBS with 0.2% Triton X-100 for 15 minutes, and blocked in PBS with 0.75% glycine and 5% BSA for 60 minutes at room temperature. Blocked sections were incubated overnight at 4ºC with a primary PCNA antibody diluted in blocking buffer (Abcam, 1:1000 dilution), and a secondary antibody was applied at a 1:500 dilution. Sections were stained with 4',6-diamidino-2-phenylindole (DAPI, 1:1000 in Millipore ddH2O), mounted with Fluoromount (Sigma), and imaged.


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For TUNEL staining, samples were permeabilized following rehydration using a solution of 0.1% Triton X-100 and 0.1% sodium citrate, and incubated at room temperature for 8 minutes. 100 µL of an enzyme solution was added to 900 µL of a label solution from a TUNEL kit (Thermo Fischer Scientific) and kept on ice. Fifty µL of the TUNEL reaction mixture were added to each slide, after which sections were covered with a coverslip and incubated for 60 minutes at 37ºC in a humidified atmosphere in the dark. Samples were then rinsed with PBS, costained with DAPI, mounted, and imaged.

For histological quantification, 40x images were taken of each slide, n=3 per biological sample x 3 biological replicates, and the percentage of positive cells per high-powered field was calculated. Unpaired two-tailed parametric t-tests were performed between each combination of groups, with equal variance assumed in all cases. Statistical significance was characterized by p < 0.05. All statistics were performed using the GraphPad Prism statistical analysis software.

2.11 Western Blot The lower right lobes of decellularized, native, and endothelial cell-seeded lungs were homogenized in equal volumes of RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% [v/v] Triton X-100, 0.5% [w/v] SDC, and 0.1% [w/v] sodium dodecyl sulfate (SDS)) containing 1% protease inhibitor. Loading equal volumes of lung lysate allows for the comparison of endothelial cell-seeded lungs against native lung as the same proportions of endothelial proteins are present regardless of the cellular composition. Equal volumes of protein were loaded on 412% gradient polyacrylamide gels, run for 90 min, then transferred to a nitrocellulose membrane and blocked for 1 h at room temperature in 5% non-fat dry milk (NFDM). Primary antibodies were applied overnight at 4ºC in 2.5% NFDM in TBST buffer (0.05% Tween-20 in Tris-buffered



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saline (TBS)) at concentrations listed in Supplemental Table 1. After rinsing with TBST, secondary antibodies were applied at a dilution of 1:3000 for 1 h at room temperature. Protein was detected using enhanced chemiluminescence (Thermo Fischer Scientific).

2.12 Real time Quantitative Reverse transcriptase (RT)-qPCR The extraction of total RNA was performed using the RNeasy Mini RNA extraction Kit from QIAGEN Kit and the generation of cDNA was performed using Superscript First-Strand Synthesis System from Invitrogen following the manufacturer’s instructions. The PCR reactions were performed with 12.5 µM of iQ SYBR Green Supermix (Bio-Rad), 10.5 µM of dH2O and 0.5 µM of forward and reverse primers in a total volume of 25 µl including 1 µl of cDNA. PCR conditions started in an initial denaturation step of 4 min at 95ºC followed by 40 PCR-cycles consisting of 15s of denaturation at 95ºC, 30s of annealing at 60ºC, and 30s extension at 72ºC. Rat β-actin was used as a housekeeping gene control. Native rat lung and rat lung microvascular cells (RLMEC) were used as a positive control. Average Ct values from the triplicate PCR reactions for each gene of interest were normalized against average β-actin Ct values from the same cDNA sample. ∆Ct = Ct (gene of interest) – Ct (β-actin) and individual points for expression of genes of interest relative to β-actin are shown for each biological replicate as 2

-∆Ct (sample)

. RT-PCR results were expressed as abundance relative to β-actin gene expression,

and were not normalized. After removing outliers as described, n=5 biological replicates for native lung for CD31, VEGFR1, PTGIS, eNOS, tPA, PLAU, and vWF; n=6 biological replicates for native lung for VEGFR2, VE-CAD. N=5 biological replicates for EC-seeded engineered lung for all genes, and n=6 independent cultures for all RLMVEC cell samples; p 10 microns, red blood cells have a diameter of only 5 µm and should easily pass through the decellularized lung microvasculature. Infusion of washed red blood cells into the PA with a hematocrit similar to that of whole rat blood (8 x 106 RBCs per µL) showed many areas of packed RBCs at the levels of both the capillaries and larger vessels (Fig. 2D). However, dilution of the RBCs by a factor of 100x resulted in distribution of RBCs throughout the vasculature and into the alveolar microvessels, predominantly as single cells (Fig. 2E), though there were still some areas of dense packing (Fig. 2F). These results suggest that, in the setting of leaky vascular conduits, such as decellularized vessels, cell size may not be the predominant cause of aggregated clusters of cells.

3.3 Endothelial Cell Seeding for Whole Lung Engineering


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Guided by the results of the pilot studies described above, RLMVEC were seeded sequentially through the PA and PV at a relatively dilute concentration of 250,000 cells/mL (normal hematocrit for rats = 8 x 106 cells/µL), a perfusion pressure of 60 cm H2O, and pulsatile flow. Previous reports have used cell concentrations as high as 3 million cells/mL.13 Histological evaluation of lungs seeded with these parameters resulted in endothelial cells adhering to the extracellular matrix scaffold throughout the lung (Fig. 3A, B), with an absence of cell clustering and occlusive aggregation. Quantification of cell nuclear coverage of the matrix revealed that the endothelial cell seeding methods resulted an average of 72.44% +/- 10.52% nuclear coverage for endothelial only seeded lungs when normalized to native lungs (Fig. 3C). Approximately 50% of adherent endothelial cells were positive for proliferating cell nuclear antigen by immunofluorescence staining (PCNA; Fig. 3D, E), indicating high proliferation after 4 days in culture. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) indicated few to no positive cells (Fig. S1).

3.4 Endothelial Cell Protein Expression in Endothelialized Lungs After 4 days in culture, immunofluorescent staining showed that CD31-positive endothelial cells were abundantly distributed in engineered lung, including lining the luminal surface of large and medium-caliber vessels (Fig. 4A). Immnofluorescent staining for the adherens junction protein VE-cadherin reveals the presence of VE-cadherin protein expression in lungs recellularized with endothelium (Fig. 4B). However, the protein detected is diffusely distributed throughout the cytoplasm, as opposed to localized to adherens junctions located at cell-cell junctions, as would be expected in native lung (Fig. S2). Western blot reveals lower quantities (12.66% ± 6.84%) of



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VE-cadherin protein are present when compared to equal volumes of native and endothelial cellseeded lung scaffolds (Fig. 4G, H).

The endothelial cells seeded in our decellularized constructs also express eNOS and PGI2S, two proteins that are important for generating proteins that inhibit platelet activation, aggregation, and adhesion. Immunofluorescent staining for eNOS indicates an abundance of protein uniformly expressed by the endothelial cells cultured in the extracellular matrix scaffold (Fig. 4C). Analysis by western blot indicates quantities of eNOS protein 152.20% ± 37.72% greater than that in matching volumes of native lung (Fig. 4E, H). Finally, immunofluorescent staining indicates abundant expression of PGI2S by seeded cells (Fig. 4D). Immunoblot indicates 49.04% ± 9.64% of protein expression, compared to native rat lung (Fig. 4F, H). These data suggest that these cells possess some ability to prevent platelet aggregation and adhesion that could lead to local thrombus formation.

3.5 Gene Expression of Endothelial Cell Markers in Native Lung, in Cultured RLMVEC, and in Endothelialized Lung Evaluation of gene expression by RT-qPCR indicated expression of CD31 that was higher than that in native lung 0.074 ± 0.015 vs. 0.021 ± 0.0023, p = .025 (Fig. 5); while expression of von Willebrand factor 0.038 ± 0.0062 vs. 0.027 ± 0.0025, p=0.15; VEGFR1 0.027 ± 0.007 vs. 0.02 ± 0.01, p = 0.6; and VEGFR2 0.033 ± 0.01 vs. 0.07 ± 0.042, p=0.44, were not significantly different than native lung. Expression levels of VEGFR2, PTGIS, VE-Cad, and vWF in ECseeded lung, expressed as abundance relative to β-actin gene expression, increased compared to cultured RLMVEC grown in tissue culture flasks, while tPA decreased, and CD31, VEGFR1,


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eNOS, and PLAU were not significantly different. CD31 and VEGFR2 in particular are fairly specific vascular endothelial cell markers14. Assessment of VE-cadherin gene expression by RTqPCR indicates higher expression of this gene in endothelial cell seeded lungs (0.93 ± 0.22), as compared to native lung (0.13 ± 0.02); p=0.023.

Finally, examination of 4 genes important to endogenous anticoagulation indicated that all were expressed at levels similar to, or higher than, those observed for native rat lung. Tissue plasminogen activator (tPA) 0.0022 ± 0.0003 vs. 0.09 ± 0.052, p=0.16; urokinase-type plasminogen activator gene (PLAU) 0.017 ± 0.0021 vs. 0.082 ± 0.049, p=0.25; and PTGIS 0.054 ± 0.017 vs. 0.026 ± 0.024, p=0.37, were not significantly different from native lung, while eNOS expression 0.031 ± 0.01 vs. 0.002 ± 0.0004, p=0.041, was significantly greater in engineered than in native lung. The abundant expression of the enzymes producing key anticoagulants suggests that the re-seeded lung scaffolds will suitably resist thrombosis after in vivo implantation.

3.6 Implantation of Endothelialized Rat Lungs To evaluate whether endothelial cell seeded lungs had functional vascular perfusion and gas exchange compared to an acellular lung scaffold, we implanted either acellular scaffolds or endothelial cell-seeded left lungs into syngeneic rat recipients in orthotopic fashion. In total, the results of the implanted lungs included 3 decellularized lung implants and 6 recellularized lung implants. All 3 rats who received decellularized lobes survived for the full 3 h post-implant while mechanically ventilated. Of the rats that received endothelial-cell seeded lungs, there were various lengths of time to survival. The first two rat recipients of recellularized left lungs died



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shortly after extubation at 15 minutes. Thereafter, the remaining 4 rats remained intubated in order to evaluate the function of the graft for some limited time. Of these, 2 additional rats died shortly after implantation, and 2 were sustained to the 3 h post-implant time-point.

Photographs taken 15 minutes after implantation in recipients that survived to the 3 h postimplant time grossly capture areas of deficient perfusion in implanted acellular scaffolds (Fig. 6A, indicated by areas that remain white; arrows), despite intra-operative heparin. This suggests that coagulation may occur almost immediately after blood contacts the collagen-containing scaffold and must initiate such a potent coagulation response that heparin at the dose given is insufficient to prevent thrombus formation. In the setting of widespread clot formation any perfusion of areas of lung distal to the thrombus would be thwarted. This effect appears to be diminished in endothelial cell seeded lungs that are implanted; areas of un-perfused lung are much less apparent (Fig. 6B).

Though gas exchange was similar between acellular scaffolds and endothelial cell seeded lungs after 180 min of perfusion in vivo, there was a trend toward increased carotid PaO2 and decreased carotid PaCO2 in rats that received endothelial cell-seeded lungs, as compared to acellular scaffolds (Fig. 6C-F). In acellular lungs, PaCO2 gradually increased over 3 hours of implantation, while PaO2 tended to fall. The lower PaO2 for non-seeded scaffolds implies both an increase in V/Q mismatch due to vascular thrombosis and an increase in shunt fraction due to the translocation of blood and blood components from the acellular vasculature into the airspaces.


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Indeed, histological evaluation of the post-implant lungs shows large intravascular clots in many of the large vessels of the post-implant decellularized lung extracellular matrix (Fig. 7A), and occasional areas of distal lung that are poorly perfused (Fig. 7B). Images of post-transplant histology from implanted recellularized lung, on the other hand, include large vessels that contain abundant red blood cells, but fewer formed clots or massively occlusive clot within the vascular spaces (Fig. 7C). The microvasculature is highly perfused (Fig. 7D), but the airspaces also contain an abundance of blood. These data are consistent with the outcome overall for rodents that received recellularized left lung implants. In addition to impairment of gas exchange in the implanted left lung, the rats that received recellularized implants also likely had impaired right lungs. In addition to the possibility of “overflow” of airspace blood from the left into the right lung that could impair gas exchange in the remaining “good” native lung of these recipients, the exposure of blood to large areas of collagen surface, with platelet and leukocyte activation may also lead to additional physiological disturbance that would diminish gas exchange in the native right lung, compounding blood gas disturbances observed in these animals. The appearance of the explanted right lungs from decellularized lung recipients and recellularized lung recipients is noticeably different by gross observation (Supplemental Figure 3). That is, the right lung of decellularized lung recipients retained the overall appearance of intact native rat lung (Supplemental Fig. 3A), while that of recellularized lung recipients appears to have a greater amount of retained blood (Supplemental Fig. 3B).

Overall, the outcomes of these implantation studies suggest that, while none of the implanted lungs were able to sustain the rodent recipients long-term, the results do suggest that endothelial cells seeded into and cultured within acellular lung scaffolds do have an effect on the interaction



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of the engineered construct with the body – specifically, with the blood to which it is exposed. Indeed, the recellularized lungs do, in fact, seem to resist thrombus formation, compared to decellularized lungs – an important component of any functional lung tissue eventually created for long term implantation.

4. Discussion Recellularization of the vascular compartment of the decellularized lung extracellular matrix is principally a delivery challenge. Effective delivery of endothelial cells to all branches of the vascular tree, especially the delicate capillaries, depends critically on managing the effects of an extensive surface area of alveolar capillary network that must be adequately covered by a confluent monolayer of vascular endothelium, a permeable capillary basement membrane that causes vascular flow to diminish as it traverses this network, and relatively high vascular resistance conferred by the both this decline in flow and by the inherent state of the architecture of these small vascular conduits.

To enhance overall coverage of the ECM surface area in the vascular compartment, endothelial cells were introduced into both the arterial and venous networks of the vasculature, consistent with other reports in the literature.3,

5

To mitigate the effect of leaky vascular conduits, we

focused on diluting our cell suspension to avoid a filtration effect – essentially, preferential loss of fluid over particles (cells) that, if severe enough, can cause concentration, packing, and stasis of the cells initially suspended in solution.15 In addition, we applied arterial perfusion pressures of nearly three times normal physiological pressure in order to counteract the rapid loss of fluid upon entry into the capillaries. Increasing the arterial seeding pressure also likely increases


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capillary diameter, which lowers individual capillary resistance, thereby further facilitating the entry of cultured endothelial cells into the conduits. The use of pulsatile flow rather than steady flow at the time of seeding also assists in capillary recruitment – i.e. the pulsatile delivery of fluid may “open up” collapsed capillaries during pulsatile perfusion and thereby permit endothelial cell seeding of these otherwise inaccessible conduits.16 Increased recruitment should also decrease net capillary resistance during seeding due to increased cross sectional area available for flow. The combination of these techniques resulted in a lung matrix that had 72.44% +/- 10.52% nuclear coverage. The coverage we achieved may actually be too high; if we consider the total surface area of all alveolar capillaries in aggregate to approximately equal that of the alveolar surface area, then total endothelial cell surface area should comprise ~50% of the total area of extracellular matrix in the alveolar region. However, this projected goal of ~50% coverage also depends on the assumption that both microvascular endothelial cells and alveolar epithelial cells have nuclear to cytoplasm ratios of 1:1, which may not be the case in a fully recellularized lung. Nevertheless, future seeding studies may rely on a lower starting population of cells while allowing more time for endothelial cells to mature and form complete barriers.

With regard to cell phenotype, we observed that seeded endothelial cells express key markers of vascular endothelium such as CD31, vWF, and VEGFR-1 and -2 over 4 days in culture. The endothelial cells within the engineered lungs also show gene and protein expression of VEcadherin, a marker for both vascular endothelial cell phenotype and the potential for barrier function. The lack of organization of the VE-cadherin molecules and apparent permeability of the barrier may be the result of several factors, encompassed by the effects of physical stimuli (shear stress, cell density, and cell-cell contacts) and biochemical factors (growth factors,



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paracrine signaling thereof). These aspects of culture may also play important roles in the regulation of other genes and proteins evaluated here. Investigation into whether physical stimuli may play a role in the recellularized scaffolds or refinement with regard to the timing17 and magnitude of shear stress as the construct develops may be worthy of future consideration.

Increased cell density, achieved by potential migration and proliferation of the seeded cells such that a confluent monolayer of endothelium is established would also assist in localization of VEcadherin molecules at the interfaces between cells. Indeed, compared to sparsely distributed cells, confluent monolayers of endothelium have lower total levels of VE-cadherin, but a greater abundance of stable intercellular bonds.18 Large quantities of VE-cadherin protein in the cytosol is also likely related to the degree of cell proliferation; we observed a high amount of cell proliferation (50%) when compared to native lungs which are largely quiescent (5%). Many endothelial cell types express both VE-cadherin and CD31 cytosolically and constitutively during migration and proliferation.

Proteins that aid in the ability of endothelial cells to prevent thrombosis of the microvascular channels were also examined. These include those that prevent the aggregation of platelets and subsequent formation of thrombosis. In addition to promoting vasodilation, NO produced by eNOS and prostacyclin,generated by prostacyclin synthase work synergistically to inhibit or even reverse platelet aggregation.19 In addition to the prostacyclin produced by microvascular endothelial cell in native lung, both alveolar type II epithelial cells20 and macrophages21-22 express abundant prostacyclin synthase as well. Endothelial cells also produce enzymes that can lyse small clots once they form (tPA and urokinase plasminogen activator). The production of


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molecules that prevent the aggregation of platelets in the microvasculature of engineered lung likely contributed to the ability of the endothelialized constructs to maintain patent vascular conduits for several hours when implanted orthotopically, ventilated, and perfused.

The work presented here demonstrates effective delivery of endothelial cells to the majority of the microvascular beds present in decellularized lung extracellular matrix scaffolds. In doing so, this work supports similar results demonstrated by Ren et al. and bolsters the feasibility of the approach that has dominated the field of whole lung engineering thus far – that of repopulating existing microvascular conduits rather than generating vascular tubes de novo.

Additionally, this study is the first, to our knowledge, to perform in vitro analysis of molecular markers of functional capacity, including the production of antithrombotic and thrombolytic substances. In addition, we present the results of both decellularized and recellularized lung implants and report noticeable differences in the mode of failure for these two types of constructs that are consistent with our molecular data. That is, in the setting of pre-implant data that suggests that the recellularized lungs may be capable of resisting clot formation, we do, in fact, observe less frequent and less occlusive thrombus in the recellularized lungs, compared to the decellularized lungs.

Clearly, additional work needs to be done to elucidate the molecular status of the endothelium in greater detail and to evaluate the functional criteria outlined here - including barrier function and resistance to clot formation - quantitatively. These evaluations may make use of quantitative assessments of barrier function such as those described by Calle et al. and others,3-4, 23 and will



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also likely require the development of new or adaptation of existing assays used by other fields. However, these data add important pieces to the overall question of how recellularized constructs behave when implanted in vivo and what, if any, effect these recellularized constructs have on the interaction between the construct and the physiology of the organism into which they are implanted. Ultimately, by simultaneously pursuing greater understanding at the molecular and organ levels, we may be able to correlate molecular profiles in vitro with global function in vivo and thereby improve our ability to employ more targeted strategies to improve function.

6. Conclusions We have described several empiric strategies that to improve endothelialization of decellularized rodent lung extracellular matrix scaffolds. Endothelial cells introduced to the scaffold using these approaches were widely distributed throughout the matrix, were retained in the vascular compartment, and did not aggregate in small vessels or capillaries. These cells were able to proliferate in the lung scaffold, with minimal signs of cell death, and they maintained an endothelial cell phenotype throughout culture. Finally, seeded cell constructs expressed molecules that are important to the essential functions of endothelial cells in vivo - barrier function and anti-coagulation – at levels similar to or greater than levels found in native lung. These constructs were implanted for up to 3 hours and were highly perfusable. These strategies and techniques advance the field of lung tissue engineering and may enable advancement of endothelialization of many other acellular organs as well, including liver and kidney constructs.

Author Contributions A.L. - Performed experiments, data analysis, figure preparation, manuscript writing G.H. - Performed surgical procedures, data analysis


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A.B. - Performed surgical procedures, data analysis, figure preparation M.G. - Performed experiments, data analysis A.E. - Performed data analysis, figure preparation, manuscript writing P.B. - Performed experiments L.N. - Idea and Conceptualization, data analysis, overseeing entire project, funding source E.C. - Idea and Conceptualization, performed experiments, data analysis, figure preparation, manuscript writing, corresponding author

Acknowledgements We appreciate the assistance of Ashley L. Gard with early endothelial cell-seeding work not shown here, are grateful for critical reading of the manuscript and graphical table of contents by Katherine L. Leiby, and thank Adam Pissaris for assistance with immunostaining.

This work was funded by 1U01HL111016-01, and by T32GM086287 (Niklason).

Supporting Information -

1 supplemental table

-

3 supplemental figures with captions: •

S1-Sn: TUNEL staining for endothelial cell seeded lungs

•

S2-Sn: Native lung immunofluorescence staining

•

S3-Sn: Post-transplant histology for decellularized and recellularized constructs

Conflicts Of Interest:



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LEN has a founder and shareholder in Humacyte, Inc, which is a regenerative medicine company. Humacyte produces engineered blood vessels from allogeneic smooth muscle cells for vascular surgery. LEN’s spouse has equity in Humacyte, and LEN serves on Humacyte’s Board of Directors. LEN is an inventor on patents that are licensed to Humacyte and that produce royalties for LEN. LEN has received an unrestricted research gift to support research in her laboratory at Yale. Humacyte did not fund these studies, and Humacyte did not influence the conduct, description or interpretation of the findings in this report.


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References 1. Vlodavsky, I.; Eldor, A.; HyAm, E.; Atzom, R.; Fuks, Z., Platelet interaction with the extracellular matrix produced by cultured endothelial cells: a model to study the thrombogenicity of isolated subendothelial basal lamina. In Thrombosis Research, 1982; Vol. 28, pp 179-191. 2. Pober, J. S.; Sessa, W. C., Evolving functions of endothelial cells in inflammation. Nat Rev Immunol 2007, 7 (10), 803-15. 3. Ren, X.; Moser, P. T.; Gilpin, S. E.; Okamoto, T.; Wu, T.; Tapias, L. F.; Mercier, F. E.; Xiong, L.; Ghawi, R.; Scadden, D. T.; Mathisen, D. J.; Ott, H. C., Engineering pulmonary vasculature in decellularized rat and human lungs. Nat Biotechnol 2015, 33 (10), 1097-102. 4. Stabler, C. T.; Caires, L. C., Jr.; Mondrinos, M. J.; Marcinkiewicz, C.; Lazarovici, P.; Wolfson, M. R.; Lelkes, P. I., Enhanced Re-Endothelialization of Decellularized Rat Lungs. Tissue Eng Part C Methods 2016, 22 (5), 439-50. 5. Scarritt, M. E.; Pashos, N. C.; Motherwell, J. M.; Eagle, Z. R.; Burkett, B. J.; Gregory, A. N.; Mostany, R.; Weiss, D. J.; Alvarez, D. F.; Bunnell, B. A., Re-endothelialization of rat lung scaffolds through passive, gravity-driven seeding of segment-specific pulmonary endothelial cells. J Tissue Eng Regen Med 2016. 6. Petersen, T. H.; Calle, E. A.; Zhao, L.; Lee, E. J.; Gui, L.; Raredon, M. B.; Gavrilov, K.; Yi, T.; Zhuang, Z. W.; Breuer, C.; Herzog, E.; Niklason, L. E., Tissue-Engineered Lungs for in Vivo Implantation. In Science, 2010. 7. Balestrini, J. L.; Gard, A. L.; Liu, A.; Leiby, K. L.; Schwan, J.; Kunkemoeller, B.; Calle, E. A.; Sivarapatna, A.; Lin, T.; Dimitrievska, S.; Cambpell, S. G.; Niklason, L. E., Production of decellularized porcine lung scaffolds for use in tissue engineering. Integr Biol (Camb) 2015, 7 (12), 1598-610. 8. Lwebuga-Mukasa, J. S.; Ingbar, D. H.; Madri, J. A., Repopulation of a human alveolar matrix by adult rat type II pneumocytes in vitro. A novel system for type II pneumocyte culture. Exp Cell Res 1986, 162 (2), 423-35. 9. Price, A. P.; England, K. A.; Matson, A. M.; Blazar, B. R.; Panoskaltsis-Mortari, A., Development of a decellularized lung bioreactor system for bioengineering the lung: the matrix reloaded. Tissue Eng Part A 2010, 16 (8), 2581-91. 10. Hanson, M. S.; Stephenson, A. H.; Bowles, E. A.; Sridharan, M.; Adderley, S.; Sprague, R. S., Phosphodiesterase 3 is present in rabbit and human erythrocytes and its inhibition potentiates iloprost-induced increases in cAMP. Am J Physiol Heart Circ Physiol 2008, 295 (2), H786-93. 11. Sharp, P. E.; La Regina, M. C., Sharp: Veterinary care - Google Scholar. In The Laboratory Rat, 1998. 12. Kiser, A. C.; Ciriaco, P.; Hoffmann, S. C.; Egan, T. M., Lung retrieval from non-heart beating cadavers with the use of a rat lung transplant model. J Thorac Cardiovasc Surg 2001, 122 (1), 18-23. 13. Ott, H. C.; Clippinger, B.; Conrad, C.; Schuetz, C.; Pomerantseva, I.; Ikonomou, L.; Kotton, D.; Vacanti, J. P., Regeneration and orthotopic transplantation of a bioartificial lung. In Nat Med, Nature Publishing Group: 2010; Vol. 16, pp 927-933. 14. Holmes, K.; Roberts, O. L.; Thomas, A. M.; Cross, M. J., Vascular endothelial growth factor receptor-2: structure, function, intracellular signalling and therapeutic inhibition. Cell Signal 2007, 19 (10), 2003-12.



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15. Sun, C.; Jain, R. K.; Munn, L. L., Non-uniform plasma leakage affects local hematocrit and blood flow: implications for inflammation and tumor perfusion. Ann Biomed Eng 2007, 35 (12), 2121-9. 16. Presson, R. G., Jr.; Baumgartner, W. A., Jr.; Peterson, A. J.; Glenny, R. W.; Wagner, W. W., Jr., Pulmonary capillaries are recruited during pulsatile flow. J Appl Physiol (1985) 2002, 92 (3), 1183-90. 17. Inoguchi, H.; Tanaka, T.; Maehara, Y.; Matsuda, T., The effect of gradually graded shear stress on the morphological integrity of a huvec-seeded compliant small-diameter vascular graft. Biomaterials 2007, 28 (3), 486-95. 18. Lampugnani, M. G.; Corada, M.; Andriopoulou, P.; Esser, S.; Risau, W.; Dejana, E., Cell confluence regulates tyrosine phosphorylation of adherens junction components in endothelial cells. J Cell Sci 1997, 110 ( Pt 17), 2065-77. 19. Wu, K. K.; Thiagarajan, P., Role of endothelium in thrombosis and hemostasis. Annu Rev Med 1996, 47, 315-31. 20. Panos, R. J.; Voelkel, N. F.; Cott, G. R.; Mason, R. J.; Westcott, J. Y., Alterations in eicosanoid production by rat alveolar type II cells isolated after silica-induced lung injury. Am J Respir Cell Mol Biol 1992, 6 (4), 430-8. 21. Chandler, D. B.; Fulmer, J. D., Prostaglandin synthesis and release by subpopulations of rat alveolar macrophages. J Immunol 1987, 139 (3), 893-8. 22. Hsueh, W., Prostaglandin biosynthesis in pulmonary macrophages. Am J Pathol 1979, 97 (1), 137-48. 23. Calle, E. A., Alveolar barrier formation in engineered lung tissue. p xxiii, 266 leaves.


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Figure Captions Figure 1. Bioreactor components and methods for endothelial cell seeding. For endothelial cell seeding of a decellularized scaffold, the perfusion loop of a decellularized lung scaffold mounted in a bioreactor (B) is connected to a reservoir (B) of endothelial cells suspended in a single-cell solution (C). The reservoir contains a stir bar and is positioned on a magnetic stir plate (D) to maintain cells in suspension over the course of seeding. A solenoid valve (E) provides pulsatile perfusion while the arterial pressure is maintained at (F) a constant pressure of 60 cm H2O. Figure 2. Effect of endothelial cell seeding strategies on distribution and density. (A) RLMVECs seeded into the lung via the PA and the PV in series. Unlabeled cells counterstained with DAPI (blue) seeded via the PA; cells labeled with diI membrane dye (red) and counterstained with DAPI (blue) seeded via the pulmonary vein, shown at 30 min post-seeding. (B) Same lung after 6 h of pulsatile perfusion culture. (C) Decellularized lung scaffold seeded with vascular endothelium at a concentration of 1 x 106 cells/mL. Cells were seeded at 30 cm H2O of pressure. (D) Decellularized lung scaffold perfused with washed red blood cells at a concentration of 8 x 106 RBCs/µL or (E), a dilute solution of washed red blood cells, 8 x 104 RBCs/µL. (F) Inset of lung shown in (E); highlights rare persistence of aggregated cells. Scale bars = 100 µm (A); 400 µm (B); 200 µm (C, D, E, F). Figure 3. Decellularized rat lung scaffold seeded with endothelial cells using optimized techniques. (A) Hematoxylin and eosin image of a lobe of decellularized rat lung seeded with RLMVECs at low power and (B) high power. (C) Quantification of nuclear coverage on recellularized lungs. (D) Endothelial cell-seeded lung stained for proliferating cell nuclear antigen (PCNA; red), counterstained with DAPI (blue). (E) Quantification of PCNA-positive nuclei. Scale bars = 1 mm (G); 200 µm (B, D). Figure 4. Phenotype characterization of endothelial cell-seeded decellularized rat lung scaffolds, seeded with optimized endothelial cell seeing methods. Lungs cultured for 4 days. (A) Immunofluorescence for CD31 (red), (B) VE-cadherin (VE-cad; red).. (C) enodothelial nitric oxide synthase (eNOS; red), and (D) prostacyclin synthase (PGI2S; red). All nuclei counterstained with DAPI (blue; A-D). (E) Western blot for eNOS in native, decellularized, and endothelial cell seeded (engineered) lung. (F) Western blot for PGIS in native, decellularized (signal is from native spill over), and endothelial cell seeded (engineered) lung. (G) Western blot for VE-cad in native, decellularized and endothelial cell seeded (engineered) lung. (H) Densitometry for eNOS, PGI2S, and VE-cadherin westerns. Scale bars = 50 µm (A – D). Figure 5. Gene expression of endothelial cells seeded into decellularized lung scaffolds. Gene expression by quantitative real-time reverse transcription polymerase chain reaction (qRTPCR) for CD31 (p = 0.03); von-Willibrand Factor (vWF; p = 0.15); vascular endothelial growth factor receptor-1 (VEGFR1; p = 0.60); - 2 (VEGFR2; p = 0.44); VE-cadherin (VE-CAD; p = 0.02); tPA (p = 0.16); urokinasetype plasminogen activator (PLAU; p = 0.25); endothelial nitric oxide synthase (eNOS; p = 0.04); prostacyclin synthase (PTGIS; p = 0.37). Gene expression is the arithmetic ratio between the gene of interest and β-actin and is therefore represented as relative to β-actin expression in the same sample. Relative expression is on the y-axis. Sample is



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indicated below the x-axis. N=6 independent samples for native lung and RLMVEC. N=5 independent samples for engineered (endothelial cell seeded) lung. “NS” indicates not statistically significant, as determined by Student’s t-test. Significant differences are indicated with a single asterisk, along with the p value. p < 0.05 is considered significant (*). Figure 6. Implantation of acellular and recellularized rat lung scaffolds. (A) Decellularized left lung implanted orthotopically in an adult rat. Arrows indicate areas of the lung that are not perfused with blood. (B) Endothelial cell-seeded left lung implanted orthotopically. (C) Partial pressure of carbon dioxide (PaCO2) in the carotid artery before and after implantation of a decellularized lung scaffold. (D) Partial pressure of oxygen (PaO2) in the carotid artery of a recipient of a recellularized lung scaffold. (E) PaCO2 in the carotid artery with a decellularized lung scaffold. (F) PaO2 in the carotid artery of a recipient of a recellularized lung scaffold. For CF, time after incision is indicated on the x-axis. Figure 7. Hematoxylin and eosin histological images of left rat lung implants postimplantation. (A) Large vessels of explanted decellularized left lung after implant contain large intravascular clots, along with extravascular blood. (B) Areas of microvasculature in explanted decellularized left lung after implant contain relatively few red blood cells. (C) Large vessels in explanted endothelial cell-seeded left lungs after implant contain red blood cells; red blood cells are also located extravascularly. (D) Microvasculature of explanted endothelial cell-seeded left lung after implantation contains abundant red blood cells. Scale bars = 200 µm.


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Supplementary Table 1: Antibody Concentrations Used for Immunofluorescence Staining Antigen of Interest

Antibody Source

Primary Antibody Concentration for Immunofluorescence

Primary Antibody Concentration for Western Blot

CD31 (PECAM-1)

Santa Cruz

1:200

1:500

Endothelial nitric oxide synthase (eNOS)

Abcam

1:100

1:1000

Prostacylcin synthase (PGI2S)

Santa Cruz

1:50

1:1000

VE-cadherin (VE-cad)

Santa Cruz

1:50

1:1000

PCNA

Abcam

1:1000

N/A

GAPDH

Abcam

N/A

1:3000



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Efficient and functional endothelial repopulation of whole lung organ scaffolds.

Fig.%1.%Cartoon%that%highlights%key%improvements%in%endothelial%cell%seeding%of% decellularized% lung%extracellular%matrix%(ECM)%scaffolds.% B

Single cells

D

C

A

Solenoid valve E 60 cm H2O

Perfusion loop

! F Strategy Figure 5.1: Bioreactor components and Rationale methods for endothelial cell seeding. (A) Lungs are decellularized and stored in a bioreactor with a perfusion loop. (B) For cell seeding, the perfusion loop is Seedtovia PA andofPV in series Increase access vascular compartment and connected a reservoir endothelial cells in suspension. (C) Thetoreservoir contains a stir bar and is increase ECs, (D) A positioned on a magnetic stir plate to maintain cells in surface suspensionarea over covered the course by of seeding. solenoid valve provides pulsatile perfusion while the arterial pressure isvia maintained compared to seeding PA onlyat (E) a constant pressure.

Reduce cell concentration

Alleviate “filtration effect” that leads to occlusion of the vasculature after cell seeding.

pressure head culture flasks Improve capillary recruitment; andCells CellsIncrease were removed from tissue with 0.25% trypsin/EDTA and distend centrifuged. increase driving pressure in small vessels. were re-suspended in culture medium and filtered through a 40 µm cell strainer to remove cell

Increase time in culture

Resolve initial clustering of cells in arterioles,

as cells spread and aggregates and debris. The prepared cell suspension was placed in migrate the cell seeding reservoir, with Use pulsatile perfusion for

Recruit a greater number of capillaries,

a stirseeding bar to prevent settling or aggregationcompared of the cells. to Cell suspensions steady flow were stirred at 120 RPM for the duration of seeding, and remained viable for at least 90 min, as assessed by Trypan blue. Cells were seeded into the decellularized lung extracellular matrix scaffold over the course of several hours, under conditions designed to combat the complicating factors that accompany a decellularized scaffold. Cell seeding variables are included in Table 5.1.

203

Andrew V. Le a, Go Hatachi b, Arkadi Beloiartsev a, Mahboobe Ghaedi a, Alexander J. Engler c, Pavlina Baevova a, Laura E. Niklason a, c, Elizabeth A. Calle c

a

b

Department of Anesthesiology, Yale University, New Haven, CT 06519 Division of Surgical Oncology, Department of Surgery, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan c Department of Biomedical Engineering, Yale University, New Haven, CT 06519


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Figure 1. Bioreactor components and methods for endothelial cell seeding. For endothelial cell seeding of a decellularized scaffold, the perfusion loop of a decellularized lung scaffold mounted in a bioreactor (B) is connected to a reservoir (B) of endothelial cells suspended in a single-cell solution (C). The reservoir contains a stir bar and is positioned on a magnetic stir plate (D) to maintain cells in suspension over the course of seeding. A solenoid valve (E) provides pulsatile perfusion while the arterial pressure is maintained at (F) a constant pressure of 60 cm H2O. 153x168mm (300 x 300 DPI)



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Figure 2. Effect of endothelial cell seeding strategies on distribution and density. (A) RLMVECs seeded into the lung via the PA and the PV in series. Unlabeled cells counterstained with DAPI (blue) seeded via the PA; cells labeled with diI membrane dye (red) and counterstained with DAPI (blue) seeded via the pulmonary vein, shown at 30 min post-seeding. (B) Same lung after 6 h of pulsatile perfusion culture. (C) Decellularized lung scaffold seeded with vascular endothelium at a concentration of 1 x 106 cells/mL. Cells were seeded at 30 cm H2O of pressure. (D) Decellularized lung scaffold perfused with washed red blood cells at a concentration of 8 x 106 RBCs/µL or (E), a dilute solution of washed red blood cells, 8 x 104 RBCs/µL. (F) Inset of lung shown in (E); highlights rare persistence of aggregated cells. Scale bars = 100 µm (A); 400 µm (B); 200 µm (C, D, E, F). 558x82mm (300 x 300 DPI)


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Figure 3. Decellularized rat lung scaffold seeded with endothelial cells using optimized techniques. (A) Hematoxylin and eosin image of a lobe of decellularized rat lung seeded with RLMVECs at low power and (B) high power. (C) Quantification of nuclear coverage on recellularized lungs. (D) Endothelial cell-seeded lung stained for proliferating cell nuclear antigen (PCNA; red), counterstained with DAPI (blue). (E) Quantification of PCNA-positive nuclei. Scale bars = 1 mm (G); 200 µm (B, D). 431x165mm (300 x 300 DPI)



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Figure 4. Phenotype characterization of endothelial cell-seeded decellularized rat lung scaffolds, seeded with optimized endothelial cell seeing methods. Lungs cultured for 4 days. (A) Immunofluorescence for CD31 (red), (B) VE-cadherin (VE-cad; red).. (C) enodothelial nitric oxide synthase (eNOS; red), and (D) prostacyclin synthase (PGI2S; red). All nuclei counterstained with DAPI (blue; A-D). (E) Western blot for eNOS in native, decellularized, and endothelial cell seeded (engineered) lung. (F) Western blot for PGIS in native, decellularized (signal is from native spill over), and endothelial cell seeded (engineered) lung. (G) Western blot for VE-cad in native, decellularized and endothelial cell seeded (engineered) lung. (H) Densitometry for eNOS, PGI2S, and VE-cadherin westerns. Scale bars = 50 µm (A – D). 185x273mm (300 x 300 DPI)


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Figure 5. Gene expression of endothelial cells seeded into decellularized lung scaffolds. Gene expression by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) for CD31 (p = 0.03); vonWillibrand Factor (vWF; p = 0.15); vascular endothelial growth factor receptor-1 (VEGFR1; p = 0.60); - 2 (VEGFR2; p = 0.44); VE-cadherin (VE-CAD; p = 0.02); tPA (p = 0.16); urokinasetype plasminogen activator (PLAU; p = 0.25); endothelial nitric oxide synthase (eNOS; p = 0.04); prostacyclin synthase (PTGIS; p = 0.37). Gene expression is the arithmetic ratio between the gene of interest and β-actin and is therefore represented as relative to β-actin expression in the same sample. Relative expression is on the y-axis. Sample is indicated below the x-axis. N=6 independent samples for native lung and RLMVEC. N=5 independent samples for engineered (endothelial cell seeded) lung. “NS” indicates not statistically significant, as determined by Student’s t-test. Significant differences are indicated with a single asterisk, along with the p value. p < 0.05 is considered significant (*). 279x215mm (300 x 300 DPI)



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Figure 6. Implantation of acellular and recellularized rat lung scaffolds. (A) Decellularized left lung implanted orthotopically in an adult rat. Arrows indicate areas of the lung that are not perfused with blood. (B) Endothelial cell-seeded left lung implanted orthotopically. (C) Partial pressure of carbon dioxide (PaCO2) in the carotid artery before and after implantation of a decellularized lung scaffold. (D) Partial pressure of oxygen (PaO2) in the carotid artery of a recipient of a recellularized lung scaffold. (E) PaCO2 in the carotid artery with a decellularized lung scaffold. (F) PaO2 in the carotid artery of a recipient of a recellularized lung scaffold. For C-F, time after incision is indicated on the x-axis. 232x277mm (300 x 300 DPI)


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Figure 7. Hematoxylin and eosin histological images of left rat lung implants post-implantation. (A) Large vessels of explanted decellularized left lung after implant contain large intravascular clots, along with extravascular blood. (B) Areas of microvasculature in explanted decellularized left lung after implant contain relatively few red blood cells. (C) Large vessels in explanted endothelial cell-seeded left lungs after implant contain red blood cells; red blood cells are also located extravascularly. (D) Microvasculature of explanted endothelial cell-seeded left lung after implantation contains abundant red blood cells. Scale bars = 200 µm. 256x189mm (300 x 300 DPI)


Efficient and Functional Endothelial Repopulation ... - ACS Publications

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