Culture Into Perfusion-Assisted Bioreactor Promotes Valve-Like Tissue Maturation of Recellularized Pericardial Membrane

Figures

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  Figure 1. Dynamics of pericardium recellularization process. (A) The upper part of the figure describes the alternate motion of the culture medium flow in the chamber of the perfusion bioreactor. The lower part indicates time-dependent representation of the flow during the cell seeding phase (left graph) occurring for the first 3 days and the maturation phase (right graph). (B) MTT staining of pericardial patches at different recellularization times shows effective growing of valve cells inside the pericardial matrix up to 21 days of culture. (C) Imaging of transversally sectioned recellularized pericardial samples at different times of culture stained with trichrome Masson's (top) and hematoxylin/eosin (bottom). As a comparison, sectioned native aortic valve tissue is included, where a distinction between the three layers, the Fibrosa (Fib), the Spongiosa (Sp), and the Ventricularis (Ve), is shown. Images in the bottom are magnifications of the insets highlighted by blue boxes in the middle panels.

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  Figure 2. Proteomic analysis of native valves and recellularized pericardial samples. (A) Principal component (PC) analysis of protein content in the analyzed samples. Three independent pericardia were recellularized with pig-derived VICs and analyzed at each time point (Samples #1- #3). Each of these samples is represented by experimental triplets evidenced by time-specific color code, for a total of nine samples per time point. Three porcine aortic valves tissues were analyzed in parallel (also in triplets) and are also indicated in the PC analysis. As shown, the alignment of the PC1 recellularized samples at day 21 with the PC1 of the valve tissue reveals a partial restoring of the native protein content by VICs. (B) Clusterization analysis of proteins differentially expressed at day 3 and day 21 vs. the native tissue (see Table S1 for description of the proteins and Figure S1 for the analysis including day 14 samples). Three protein clusters (each highlighted by a different color) were identified. A first cluster (characterized by the blue color) contains proteins that were expressed at higher levels in the native valves vs. the recellularized samples independently of time. A “red”…

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  Figure 3. (A–C) Example of graphic representation of selected proteins belonging to the three clusters identified by the analysis of proteomic data. Statistical analysis was performed by one way ANOVA with Newman-Keuls post-hoc on the normalized abundance of proteins identified by mass-spectrometry (see Table S1). * represents P < 0.05 (n = 3). (D) Clusterization analysis of proteins with functions in ECM structure/remodeling. Treatment of this data in a separate analysis helped to assess in better details the representation of matrix-related protein distribution during the course of the recellularization procedure. Although separate ECM protein clusters were still identified for the three tissue types, the heatmap clearly shows a higher separation between the day 3 samples and the day 21/native valve tissues, and the higher similarity of the day 21 samples to native valves. This last evidence suggests a trend of the day 21 samples to evolve toward a valve-like tissue.

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  Figure 4. Cellular phenotype and dynamics inside recellularized pericardia. (A–C) Immunofluorescence staining showing expression of αSMA, PCNA, and YAP (all in green fluorescence) in conjunction with Vimentin (in red fluorescence) inside the recellularized pericardial tissue at day 3 and 21 of culturing. In each panel the lower micrographs show the magnification of the boxed insets in the higher photographs. (D) Quantification of total number of cells by DAPI-stained nuclei counting (upper bar graph), PCNA+ cells (middle bar graph) and cells with nuclear localized YAP (nYAP+ cells, low bar graph) at all the considered time points. Statistical analysis was performed with one-way ANOVA with Newman Keuls post-hoc; * indicates P < 0.05 (n = 3).