2.5 Mesenchymal stromal cells repopulation, cell viability and proliferation assays

Primary human Bone Marrow-Derived MSCs (PCS-500–012, ATCC) were cultured in MSCs Basal Medium (PCS-500–030, ATCC) following manufacturer’s instructions at 37°C, 5% CO₂ and 95% relative humidity.

Lungs from 20 animals (n = 5, young/aged and RV/FV) were decellularized. Then, ∼100 µm-thick scaffolds were washed with PBS and 5·10⁴ cells/cm² MSCs were seeded on top of the lung scaffolds. Control cultures were seeded on traditional culture plates (TCP). After 72 h, samples were stained using the LIVE/DEAD Viability/Cytotoxicity kit (L-3224, Invitrogen) (Bonenfant et al., 2013; ONeill et al., 2013; Syed et al., 2014). F-Actin (phalloidin, Thermo Fisher Scientific) and Ki67 (Thermo Fisher Scientific) were stained and visualized by a Nikon D-Eclipse Ci confocal microscope with a ×20 Plan Apo immersion oil objective (Nikon). Image quantification was performed using a custom MATLAB script. Initially, images in both Hoechst and Ki67 channels were converted to grayscale. User-defined thresholds were applied to distinguish cellular regions based on pixel intensity. Following the thresholding, region properties such as area, major axis length, and centroid were computed for individual cellular regions. To exclude artifacts and non-cellular elements, a minimum diameter filter was applied, removing any identified regions smaller than a pre-defined pixel size. To compensate for potential positional differences between the Hoechst and Ki67 staining, a dilation operation was performed on the Hoechst image. Ki67-positive cells were identified by overlaying the dilated Hoechst image with the Ki67 image, ensuring colocalization. Finally, the percentage of Ki67-positive nuclei relative to the total Hoechst-stained nuclei was computed to determine cellular proliferation.