Abstract Strategies are needed to improve repopulation of decellularized lung scaffolds with stromal and functional epithelial cells. We demonstrate that decellularized mouse lungs recellularized in a dynamic low fluid shear suspension bioreactor, termed the rotating wall vessel (RWV), contained more cells with decreased apoptosis, increased proliferation and enhanced levels of total RNA compared to static recellularization conditions. These results were observed with two relevant mouse cell types: bone marrow-derived mesenchymal stromal (stem) cells (MSCs) and alveolar type II cells (C10). In addition, MSCs cultured in decellularized lungs under static but not bioreactor conditions formed multilayered aggregates. Gene expression and immunohistochemical analyses suggested differentiation of MSCs into collagen I-producing fibroblast-like cells in the bioreactor, indicating enhanced potential for remodeling of the decellularized scaffold matrix. In conclusion, dynamic suspension culture is promising for enhancing repopulation of decellularized lungs, and could contribute to remodeling the extracellular matrix of the scaffolds with subsequent effects on differentiation and functionality of inoculated cells.

Citation: Crabbé A, Liu Y, Sarker SF, Bonenfant NR, Barrila J, Borg ZD, et al. (2015) Recellularization of Decellularized Lung Scaffolds Is Enhanced by Dynamic Suspension Culture. PLoS ONE 10(5): e0126846. https://doi.org/10.1371/journal.pone.0126846 Academic Editor: Monica Soncini, Politecnico di Milano, ITALY Received: October 27, 2014; Accepted: April 8, 2015; Published: May 11, 2015 Copyright: © 2015 Crabbé et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited Data Availability: All relevant data are within the paper and its Supporting Information files. Funding: This work was funded by the National Institutes of Health (National Institutes of Health grants RC4HL106625 [DJW and CAN], NHLBI R21HL108689 [DJW], NHLBI R21HL094611 [DJW], NIGMS COBRE P30 GM103532 [C Irvin PI]), the National Aeronautics and Space Administration (NASA grants NNX10AO52G [CAN] and NNX13AM01G [CAN]) and by a Mayo Clinic-ASU Seed grant (AC, CAN, JJL). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction Chronic obstructive pulmonary disease (COPD) affects over 64 million people worldwide and is predicted by the World Health Organization to become the third leading cause of mortality by 2030. While allogeneic lung transplantation is the only definitive treatment for the growing number of patients with end-stage lung disease, only one out of four patients on the organ waiting list undergoes transplantation, given the limited availability of donor organs. Moreover, the clinical success of lung transplantation is hampered by lifelong immunosuppression and chronic rejection, reflected in a 10–20% survival rate 10 years post-transplantation [1]. A promising option to increase the donor organ pool is to use allogeneic or xenogeneic decellularized lungs as a scaffold to engineer functional lung tissue ex vivo [2–7]. Decellularization of mouse, rat, goat, sheep, pig, non-human primate and human lung tissue has been accomplished with several detergent-based approaches as well as with freeze-thaw cycles, and resulted in three-dimensional (3-D) acellular scaffolds that are generally devoid of detectable residual DNA and nuclei [2–17]. While the decellularized lung scaffolds produced by different methods generally retain major extracellular matrix (ECM) proteins, several ECMs can be less abundant (e.g., collagen I, collagen IV), fragmented (fibronectin) or largely absent (elastin) as compared to the native tissue [2,5,8]. Repopulation of decellularized lungs has been reported using a number of different cell types, including transformed cell lines such as A549 lung adenocarcinoma cells, fetal lung cells, endothelial cells, embryonic stem cells (ESC), fibroblasts, induced pluripotent stem cells (iPSC), primary or immortalized airway and alveolar epithelial cells, and bone marrow or adipose-derived mesenchymal stem cells (MSCs) [2–17]. However, apart from use of fetal lung homogenates in combination with A549 and vascular endothelial cells, only partial recellularization of alveoli, airways and pulmonary vasculature has been achieved [2,8,15,18]. One potential approach to improve recellularization of decellularized lung scaffolds is to use the dynamic rotating wall vessel (RWV) bioreactor, which has been shown to promote growth and differentiation of stem and/or epithelial cells (in the presence or absence of a growth substrate) [19–21]. The RWV is an optimized form of continuous suspension culture wherein cells are cultured in horizontally rotating bioreactors that are completely filled with media [22]. The bioreactor rotation offsets sedimentation, creating a constant, gentle fall of cells and their growth substrate/scaffolds through the culture medium under conditions of physiological fluid shear [23–25], such as those encountered in the interstitium [26]. Under these conditions, gentle media mixing and excellent mass diffusion are obtained [22]. The RWV technology has been used to generate differentiated lung tissue culture models, with important applications in the fields of infectious disease and regenerative medicine [7,22,27,28–31]. Specifically, tumorigenic A549 lung epithelial cells cultured alone and in combination with functional macrophages on the surface of extracellular matrix-coated porous microcarrier beads exhibited phenotypic reversion towards a more normal differentiated phenotype [27,28,31]. Moreover; when challenged with respiratory pathogens and their toxins, these 3-D lung models exhibited a more in vivo-like response as compared to conventional 2-D monolayers of the same cell line [27,28]. In the field of lung regenerative medicine, the RWV has been used to differentiate mESCs into multiple distal respiratory epithelial cell types using conditioned medium, which could not be obtained using static culture [29]. Cortiella et al used the RWV for repopulating various natural and synthetic ECM scaffolds with ESCs, including decellularized lung scaffolds [7]. While this study demonstrated enhanced cell differentiation and viability of ESCs on decellularized scaffolds compared to other ECM scaffolds in the RWV, the authors focused on the role of different ECM scaffolds (and not on RWV growth conditions) as identical static controls were not included. Therefore, it remained unclear whether the standard RWV culture conditions (~20 RPM) that enhanced cellular differentiation and growth in previous reports [27–29,31], could be utilized to improve recellularization efficiency and stem cell differentiation in decellularized lung scaffolds. In this study, we examined whether the use of the RWV would (i) enhance recellularization efficacy using two relevant mouse cell types, i.e. bone marrow derived MSCs and alveolar type II epithelial cells (C10), and (ii) affect differentiation of the inoculated MSCs in the decellularized lungs.

Materials and Methods Cells, media and growth conditions MSCs from bone marrow of adult C57BL/6 mice were obtained from the NCRR/NIH Center for Preparation and Distribution of Adult Stem cells at Texas A&M University [32]. C10 cells are a non-transformed alveolar type II epithelial cell line derived from normal BALB/c mouse lungs [33], and were kindly provided by Matthew Poynter (University of Vermont). MSCs were cultured in Iscove’s Modification of Dulbecco’s Medium (IMDM, basal medium), according to previous reports using this cell type [2,3,15,18], supplemented with 10% fetal bovine serum (Invitrogen), 10% horse serum (Invitrogen), 2 mM L-glutamine (Sigma), 100 μg/mL primocin (InvivoGen), and 100 U/mL penicillin and 100 μg/mL streptomycin (Sigma). MSCs were grown in T175 aerated flasks to 70% confluency and were used up to passage 7. C10 cells were cultured in a basal cell culture medium GTSF-2 [34], based on previous reports on alveolar epithelial cell culturing in the RWV bioreactor [27,28], modified by replacing fungizone with 100 μg/mL primocin and supplementing with 1.5 g/L NaHCO 3 to support growth in a 5% CO 2 incubator at 37°C. Decellularization of lungs Heart-lung blocs of adult male and female C57BL/6 mice (8–24 weeks, Jackson Laboratories) were decellularized using an SDS-Triton based detergent approach as described previously [2,6]. This study was approved by the University of Vermont (UVM) Institutional Animal Care and Use Committee (IACUC), and animals were maintained at UVM in accordance with IACUC standards and review (UVM IACUC 11–003 “Bioengineering New Lungs from Cadaveric Scaffolds”). Euthanasia was performed under standards of the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) and UVM IACUC using lethal intraperitoneal injection of sodium pentobarbital followed by removal of the heart lung bloc. Animals experienced only momentary distress during the injection. Recellularization of decellularized lungs in static and RWV bioreactor conditions An overview of the experimental set-up is provided in Fig 1. For recellularization in both static and RWV conditions, 4 x 106 MSCs or C10 cells grown as monolayers were suspended in 3 mL IMDM or GTSF-2 medium, respectively and injected in the decellularized lung scaffold through the cannulated trachea. The cell number injected per lung is in the range of previous reports [2,15,18]. Any liquid that was not retained in the lungs upon the first injection was re-injected up to five times, until more than 99% of the initially added cells remained in the scaffolds. Each seeded heart-lung bloc was transferred to a 50 mL conical tube containing 15 mL of IMDM (for MSCs) or GTSF-2 (for C10 cells) medium and incubated in a 37°C, 5% CO 2 incubator. Lungs for static recellularization were kept in these conditions for the duration of the experiment, and medium was changed every 2–3 days. Lungs for RWV bioreactor recellularization were maintained statically for 4 days to allow adequate initial adherence of cells to the matrix, after which the cannula was removed and the heart-lung bloc subsequently transferred to a 50 mL RWV bioreactor. The need for static incubation prior to bioreactor incubation was determined empirically, since immediate transfer of lungs to the bioreactor precluded effective cell attachment. A rotation speed of 20 rpm was adopted to keep the lungs in suspension culture, and lungs were incubated for 3, 10, and 24 additional days in the RWV (total incubation of 7, 14, and 28 days respectively) for MSCs, or 7 and 10 additional days in the RWV for C10 cells (total incubation of 11 and 14 days respectively). Bioreactors were inspected daily for potential formation of air bubbles, removed if necessary and culture media was changed weekly given the larger bioreactor volume (50 mL) compared to static conditions (15 mL). PPT PowerPoint slide

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larger image TIFF original image Download: Fig 1. Overview of experimental set-up used to recellularize decellularized lung scaffolds with MSCs or C10 cells in static and bioreactor conditions. MSCs or C10 cells were introduced in the decellularized lung scaffolds through the cannulated trachea. Next, lungs were statically incubated for 4 days, regardless of the subsequent test condition. Culture medium for MSCs was IMDM and for C10 cells GTSF-2. Different time points were tested to assess recellularization with MSCs (3, 10, 24 days) or C10 cells (7, 10 days) in static or bioreactor conditions. https://doi.org/10.1371/journal.pone.0126846.g001 Fixation of static and RWV-recellularized lungs for mRNA expression and immunohistochemistry Per heart-lung bloc, one lung was assessed for total RNA contents and for mRNA expression by quantitative real time PCR (qRT-PCR), and the other lung was fixed for immunohistochemistry. The lungs for RNA analysis were incubated overnight at 4°C in RNA Protect (Qiagen) and were then stored at -80°C until processed for RNA extraction and purification. The lungs for immunohistochemistry were fixed using 4% paraformaldehyde (PFA) (Electron Microscopy Services) for 30 min and were stored at 4°C in sterile PBS until further processing. mRNA expression analysis The total RNA of fixed lung samples was isolated using TRIzol reagent (Life Technologies) followed by purification with the RNeasy kit (Qiagen), according to the manufacturer’s instructions. RNA quantity and quality was assessed using a Nanodrop spectrophotometer. RNA was converted to cDNA using the Monsterscript 1st-strand cDNA synthesis kit (Epicenter Biotechnologies). Quantitect SYBR Green Master mix (Qiagen) was used to assess differential gene expression with qRT-PCR, according to the manufacturer’s protocol. Potential differentiation of MSCs was assessed through expression of markers typically (but not solely) expressed by (i) different types of lung epithelial cells (tight junctional marker ZO-1, water channel marker AQP5, and SPD), specific lung cell populations (CCSP and Scgb3a2 for Club cells—formerly named Clara cells; SPC, SPB, SPA for type II alveolar epithelial cells; MUC5AC and Spdef for goblet cells; Trp63 for basal cells) [35], early lung differentiation markers (TTF-1 or NKX2.1, and FoxJ1) [35], (ii) adipocytes (adiponectin), (iii) osteocytes (osteopontin), (iv) cartilage (Col2a1), (v) fibroblasts and other mesenchymal cells (FAP, Fsp1, Col1a1, FN, ∞-SMA), (vi) a cytokine (TGF-β), (vii) tumor-associated MSCs (stanniocalcin, MMP3) [36,37], and (viii) multipotent cells (endoglin, Sca-1, CD106) (abbreviations and primers are defined in S1 Table). Primer specificity was tested on DNA from MSCs grown as monolayers (extracted using the DNeasy kit, Qiagen) and on cDNA from (i) MSCs grown as monolayers and (ii) mouse lungs (pooled RNA from 25 lungs of 25 week old male mice, Amsbio). The qRT-PCR reactions were performed using an Eppendorf Mastercycler EP RealPlex 2S system. A melting curve was run at the end of each reaction to test for the presence of a single PCR product. The qRT-PCR reaction product was run on a 3% agarose gel in the presence of a low molecular weight DNA ladder (New England BioLabs), to assess for potential non-specific binding and primer dimerization. C T values were exported using the Eppendorf Database tool, whereafter the delta delta C T method [38] was adopted to determine relative gene expression between different test conditions. An average of six housekeeping genes was used for normalization (β-actin, GAPDH, Yhwaz, Sdha, Tbp, and Pkg) [39,40], [41]. The gene expression of MSCs grown under static or bioreactor conditions was compared to the expression of MSCs grown as conventional monolayers. Histology Following PFA fixation (see above), recellularized lungs were paraffin-embedded, and 5 μm slices were mounted on positively charged glass slides. Paraffin-embedded normal mouse lung slices (Amsbio) were included as a histology control and were processed the same way. Staining with hematoxylin/eosin was performed using standard protocols [2]. Alizarin red staining was performed to assess potential differentiation of MSCs along the osteoblastic lineage in the different test conditions, and was done using standard protocols [2]. For Alizarin red staining, a negative control (decellularized lungs without cells) and positive control (MSC monolayers differentiated along the osteoblastic lineage) was included as well. For this positive Alizarin red control, MSC monolayers were exposed to osteogenic differentiation medium, comprised of IMDM supplemented with 1 nM dexamethasone, 50 μM L-ascorbic acid-2 phosphate, and 20 mM β-glycerol phosphate, for 3 weeks [42], where after they were processed for immunohistochemistry as described above. Stained slices were imaged by standard bright field microscopy (100x and 400x) (Zeiss AxioVert A1), and at least 10 regions in each of 5 tissue slices per sample were analyzed. Immunofluorescence staining and imaging Paraffin-embedded slices from recellularized lung scaffolds and control mouse lung slices (Amsbio) were deparaffinized by sequential incubation in three baths (150 mL/bath) of xylene for 5 min, one bath of 50% xylene 50% absolute ethanol for 2 min, two baths of absolute ethanol for 2 min, one bath of 95% ethanol for 2 min, one bath of 70% ethanol for 2 min, followed by rinsing with MilliQ water. Then, antigen retrieval was performed by heating tissue slices in sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) (Dako) at 100°C for 20 min and cooling to room temperature prior to immunofluorescence staining. Tissue sections were stained with specific antibodies as described previously [27]. The following primary antibodies were used: CC10 or uteroglobin (Abcam, ab40873, 1:1600), osteopontin (Abcam, ab8448, 1:100), collagen I (Abcam, ab292, 1:100), Fsp1 (Abcam, ab41532, 1:200), Annexin V (Abcam, ab14196, 1:500), and PCNA (Abcam, ab29, 1:1000). Secondary antibodies used in this study were: goat anti-mouse Alexa Fluor 488 or 555 (Invitrogen), goat anti-rabbit Alexa Fluor 488 or 555 (Invitrogen) at a 1:500 dilution. Cell nuclei were stained with DAPI (Prolong Gold with DAPI mounting solution, Invitrogen). Stained tissue sections were imaged using a Zeiss LSM 510 Duo laser scanning microscope or Zeiss Axiovert A1. Images were acquired using a Plan-Neofluar 40x or 63x objective and were analyzed with the Zeiss LSM software package or Zen Lite. Axiovision 4.7 or Zen 2011 softwares from Carl Zeiss were used to further process collected images. Statistical analysis All studies were conducted at least in biological triplicate. Statistical significance (α = 0.05) was determined using a two-sample Student’s t-test on the biological replicates. To determine the suitability of the housekeeping genes and their average for normalization of the qRT-PCR data, the coefficient of variation was calculated. Since the coefficient of variation for target, housekeeping genes and the average of housekeeping genes was comparable (S1 Fig), the adopted normalization approach did not influence the gene expression data.

Discussion A promising approach for ex vivo lung engineering is the repopulation of decellularized lung scaffolds with autologous stem and/or progenitor cells derived from the actual transplant recipient. The decellularized scaffolds also provide a novel tool for studying cell-matrix interactions and other aspects of lung biology. While decellularized lung scaffolds derived from rodents, sheep, pigs, primates, and humans have been repopulated with a variety of different cell types [2–10,12–15,17,18,43], improved culture techniques are needed to enhance the full recellularization of the lung surface area with viable, differentiated cells. In addition, as critical cell binding epitopes on the ECM proteins remaining in the scaffold may be affected by the decellularization process [2,3], restoring the normal ECM content and function of the decellularized scaffolds is of importance to support cell growth and differentiation. In this study, we demonstrated that recellularization efficiency (i.e. number of cells adhering to scaffolds) and viability were improved when decellularized lung scaffolds were cultured in a dynamic suspension bioreactor system (RWV) as specifically compared to static conditions. We speculate that the biomechanical force of fluid shear in the RWV and associated mass transfer result in differential oxygenation, nutrient availability, waste dispersal, some or all of which may have contributed directly or indirectly towards our phenotypic and molecular genetic observations [22]. In agreement with our results, growth stimulation of progenitor cells in the RWV bioreactor has been reported previously [19,44]. Cortiella et al previously demonstrated that decellularized lungs repopulated with ESCs in the RWV retained more viable cells and induced higher levels of apparent lung-specific lineages in response to defined differentiation agents, compared to other synthetic scaffolds and 2-D ESC monolayers [7]. In our study, the enhanced attachment of MSCs to the decellularized scaffolds in the bioreactor could, in part, be potentially explained by induced TGF-β expression, which has been shown to enhance attachment of MSCs to collagen I through induction of integrin subunits [45]. The well-spread polygonal cell shape predominantly observed in bioreactor conditions is an indicator of healthy MSCs [46,47]. On the other hand, this characteristic cell shape could also imply a lower multipotentiality and higher commitment towards differentiation as compared to the rounder phenotype in static conditions [48–50]. In line with this hypothesis is (i) the induced expression of the multipotency markers endoglin and CD106 in static but not bioreactor conditions, and (ii) the absence of MSC cell clusters in bioreactor growth conditions, which could possibly be a characteristic of multilineage-differentiating stress-enduring (Muse) cells that have the ability to differentiate into endoderm, ectoderm and mesoderm in vitro and in vivo [51,52]. Alternatively, it is possible that the stacked cell clusters observed in static conditions could reflect an abnormal cell phenotype. Our collective data suggest that a portion of the bioreactor-cultured MSCs may have differentiated into fibroblast-like cells (expression of Fsp1, FAP, osteopontin, collagen I). Fsp1 is specifically expressed in fibroblasts but at very low levels in epithelium and MSCs [53–55] and has been correlated with collagen deposition and lung remodeling in bleomycin-treated mice [54]. Similarly, FAP and osteopontin are involved in lung tissue remodeling [56,57]. Since fluid shear triggers tissue remodeling and extracellular matrix deposition [58,59], enhanced fluid shear levels in the bioreactor could potentially have caused the enhanced generation of fibroblasts. Hence, the induction of fibroblast markers in bioreactor culture conditions could contribute to the regeneration and/or remodeling of decellularized lung scaffolds. Specifically, since decellularized lung scaffolds are depleted of key ECM proteins that could play a role in cell growth and differentiation [2,5,8], we speculate that potential restoration of the native ECM scaffolds by fibroblast-like cells could be beneficial for ex vivo lung tissue generation. In a similar fashion, mESCs recellularizing the lung scaffolds in the RWV bioreactor secreted ECM components that were depleted in the acellular lung [7]. Growth of MSCs on decellularized lung scaffolds in both static and bioreactor test conditions in basal medium induced the expression of mRNAs encoding two markers that are expressed in lung epithelial cells (as well as other cell types), AQP5 and CCSP. Recent studies suggest basal expression of lung epithelial markers in some preparations of MSCs, and in response to lung differentiation media [13,60,61]. However, CCSP expression could not be confirmed at the protein level in our study. Discrepancies between qRT-PCR and immunofluorescence results have been reported previously for MSCs on decellularized lung scaffolds, and could potentially be explained by differences in sensitivity of both methods [13] or by potential posttranscriptional modifications [62]. An alternative explanation is the inability of the growth conditions used in this study to support phenotypic expression of these markers. In a similar fashion as for MSCs, bioreactor conditions gave rise to lung scaffolds populated with a higher number of viable C10 cells as compared to static conditions. Previous studies described abundant apoptosis of C10 cells when grown in decellularized whole lungs statically for 7 to 14 days, or during continuous vascular perfusion [3,10,18]. When C10 cells were grown on decellularized lung slices, only limited apoptosis was observed up to 28 days of culture [15]. Collectively, these data indicate that C10 cells could be sensitive to the growth conditions inside whole decellularized lungs. We and others have observed shrinking and collapsing (atelectasis) of the scaffolds as a function of time [7], which could result in oxygen and nutrient limitation and waste product accumulation. Therefore, the continuous movement of the lung scaffolds in the medium during recellularization in the RWV bioreactor, which results in differential oxygenation, nutrient provision and waste dispersal [22], may have provided more optimal culture conditions for this particular cell type as compared to static culture. In conclusion, we demonstrated that the RWV bioreactor confers advantages for recellularization of decellularized lung scaffolds, with regards to cell growth, cell health, and differentiation as compared to static conditions. Since MSCs have been suggested to serve as stroma for repopulating decellularized scaffolds with other cell types (such as endogenous progenitors and induced pluripotent stem cells) [63], further investigations into whether the enhanced expression of genes involved in ECM remodeling in the RWV bioreactor resulted in reconstituting and/or remodeling of the native ECM scaffold is of interest. Furthermore, this study could have downstream implications for studying tissue homeostasis and identifying the underlying factors that contribute to the transition of normal to fibrotic lung phenotypes.

Supporting Information S1 Fig. Coefficient of variation for target genes, housekeeping genes and average of housekeeping genes for qRT-PCR gene expression data at 7 days (A) and 14 days (B) of culture in the different test conditions. https://doi.org/10.1371/journal.pone.0126846.s001 (TIF) S1 Table. Primers used for qRT-PCR analysis. https://doi.org/10.1371/journal.pone.0126846.s002 (DOCX)

Acknowledgments We are grateful to Jenny Pattengill (Mayo Clinic Histology Core Facility) for processing samples for histology. We thank Phillip Stafford (The Biodesign Institute, Innovations in Medicine) for his expertise in bioinformatics data analysis for this work. We thank Mark Ott and his team at the Microbiology Laboratory at the NASA Johnson Space Center for their support of data analysis. We thank John Wallis for helpful suggestions on recellularizing decellularized lung scaffolds. This work was funded by the National Institutes of Health (NIH grants RC4HL106625 (DJW and CAN), NHLBI R21HL108689 (DJW), NHLBI R21HL094611 (DJW), NIGMS COBRE P30 GM103532 (C Irvin PI)), the National Aeronautics and Space Administration (NASA grants NNX10AO52G (CAN) and NNX13AM01G (CAN)) and by a Mayo Clinic-ASU Seed grant (AC, CAN, JJL).

Author Contributions Conceived and designed the experiments: AC DJW CAN. Performed the experiments: AC YL SFS NRB JB ZDB. Analyzed the data: AC YL JJL DJW CAN. Wrote the paper: AC DJW CAN.