Fig. 3

Evaluation of recellularization and biocompatibility of the decellularized scaffolds. Red fluorescent protein (RFP)-labelled mesenchymal stem cells were used for recellularization. The cells recolonized large areas of the ovarian scaffolds, including the deeper layers of all scaffold types. However, the cortical area of the scaffolds accommodated more cells than the medulla regions (low power magnification a-c; high magnification (d-i). All the detected cells were double-labeled with RFP (red) and DAPI (blue; a-f), and indicated that all cells originated from the recellularization episode and were not the result of an incomplete decellularization. Many cells were positive for the proliferation marker Ki67 (red, arrows point out examples of positive cells), while the cells remained negative for the apoptotic marker cleaved caspase-3 (green; g-i) after 2 weeks in vitro. The surface of the scaffolds was densely populated with cells in all scaffold types, as confirmed by both immunocytochemistry (a-i) and scanning electron microscopy (j-k; arrows indicate the cell structures in a recellularized construct after the application of the decellularization protocol 1 (P1). It was more difficult to visualize cells in cross sectioned recellularized scaffolds with SEM. However, cells that were morphologically unaffected by the sampling preparation could be visualized in the hollow- and porous structures of the ovarian scaffolds, including the deeper scaffold compartments (k; the arrows indicate cell structures). Green color (a-f) represents auto fluorescence from the ovarian tissue and was included in the images to enable the visualization of the extracellular matrix (ECM) structure