Quantification of untreated biofilms of the six bacterial species on candidate selective media compared to a general medium.

<p><b>(A)</b><i>P</i>. <i>aeruginosa</i> AA2 <b>(B)</b> <i>S</i>. <i>aureus</i> SP 123 <b>(C)</b> <i>S</i>. <i>anginosus</i> LMG 14696 <b>(D)</b> <i>A</i>. <i>xylosoxidans</i> LMG 26680 <b>(E)</b> <i>R</i>. <i>mucilaginosa</i> DSM 20476 <b>(F)</b> <i>G</i>. <i>haemolysans</i> LMG 18984. PIA = Pseudomonas Isolation Agar; LB tricl = LB agar supplemented with triclosan in various concentrations (μg/mL); LB NaCl = LB supplemented with 7.5% NaCl; McA5 = McConkey agar supplemented with 5 μg/mL aztreonam; NMC = nutrient agar supplemented with 5 μg/mL mupirocin and 10 μg/mL colistin sulphate; CA + co32 = Columbia agar with 32/6.4 μg/mL co-trimoxazole. Graphs show mean recovery and standard deviations. * p < 0.05, n ≥ 3.</p

Quantification of biofilms after antibiotic treatment on selective media compared to a general medium.

<p><b>(A)</b><i>P</i>. <i>aeruginosa</i> AA2 <b>(B)</b> <i>S</i>. <i>aureus</i> SP 123 <b>(C)</b> <i>S</i>. <i>anginosus</i> LMG 14696 <b>(D)</b> <i>A</i>. <i>xylosoxidans</i> LMG 26680 <b>(E)</b> <i>R</i>. <i>mucilaginosa</i> DSM 20476 <b>(F)</b> <i>G</i>. <i>haemolysans</i> LMG 18984. PIA = Pseudomonas Isolation Agar; LB tricl = LB agar supplemented with triclosan in various concentrations (μg/mL); LB NaCl = LB supplemented with 7.5% NaCl; McA5 = McConkey agar supplemented with 5 μg/mL aztreonam; NMC = nutrient agar supplemented with 5 μg/mL mupirocin and 10 μg/mL colistin sulphate; CA + co32 = Columbia agar with 32/6.4 μg/mL co-trimoxazole. Graphs show mean recovery and standard deviations. * p < 0.05, n ≥ 3.</p

Composition of optimized selective media.

<p>Composition of optimized selective media.</p

Overview of strains used in this study and isolation sites.

<p>Overview of strains used in this study and isolation sites.</p

Overview of antibiotic concentrations used for quantitative recovery experiments.

<p>Overview of antibiotic concentrations used for quantitative recovery experiments.</p

Relative expression of target genes in MSCs grown on decellularized lung scaffolds in static and bioreactor conditions as compared to monolayers.

<p>* P < 0.08</p><p>** P < 0.05</p><p>*** P < 0.01</p><p>Bold = upregulated compared to MSC monolayer; Italic = downregulated compared to MSC monolayer; White empty cell: P > 0.08 and/or fold-change < 1.5; While cell with ∞: Present in ML, absent in ≥ 50% of samples; White cell with Ф: Not expressed in ML and sample, but expressed in lung.</p><p>Relative expression of target genes in MSCs grown on decellularized lung scaffolds in static and bioreactor conditions as compared to monolayers.</p

A: Immunohistochemical profiling of decellularized lung scaffolds recellularized with MSCs in static and bioreactor conditions for 14 days.

<p>Profiling of whole normal mouse lung tissue and MSC monolayers was performed as well. Cell nuclei are labeled in blue; markers of interest are labeled in green and are Fsp1 (panel A to E), collagen I (panel a to e), and osteopontin (panel aa to ee). White arrows point to multilayered cell aggregates, observed in static recellularization conditions and for this test condition profiling for both aggregates (A, a, aa) and single cells (B, b, bb) is presented. Since collagen I-positive cells showed higher signal intensity compared to that of the collagen I-positive scaffold, the background scaffold signal in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126846#pone.0126846.g005" target="_blank">Fig 5Ab and 5Ac</a> was removed for clarity. To demonstrate the collagen I-positive scaffolds, the background signal is only shown in Fig 5Aa. Magnifications are 400x or 630x. B: Alizarin red staining of decellularized lung scaffolds recellularized with MSCs in static (panels A, B) and bioreactor (panel C) conditions for 14 days. As a negative control, decellularized lungs that were not seeded with cells are presented (panel D). MSC monolayers differentiated along the osteoblastic lineage are included as positive control (panel E). Magnification is 200x or 630x. For each condition, images are representative of the entire lung.</p

Overview of experimental set-up used to recellularize decellularized lung scaffolds with MSCs or C10 cells in static and bioreactor conditions.

<p>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.</p

Recellularization of Decellularized Lung Scaffolds Is Enhanced by Dynamic Suspension Culture

<div><p>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.</p></div

Total RNA levels of decellularized lungs recellularized with MSCs (A) or C10 cells (B) in static versus bioreactor conditions.

<p>For each condition, mean RNA levels +/- standard deviation for the left lung is presented. * p < 0.05, ** p < 0.01.</p