Figure 3
Creation and characterization of the smooth muscle cell (SMC)-endothelial progenitor cell (EPC) bi-level cell sheet. (A) The diagram shows the methods for engineering the cell sheet. Cell-sheet is harvested from the temperature-responsive culture dish. (B) A round-shaped scaffold-free SMC-EPC bi-level cell sheet in a 35-mm dish. (C) The bi-level cell sheet maintained CD31 positive EPC and SM22-α positive SMC in separate layers. Green indicates CD31; red, SM22-α. The cellular nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar = 50 μm. (D-F) Representative scanning electron microscope (SEM) images of SMC-EPC bi-level cell sheet. (D) The examination of the surface (EPC layer) of the SMC-EPC bi-level cell sheet suggested that EPCs form a smooth confluent layer with cell borders in close contact with each other, thereby forming a thin film-like layer on top of the SMC. (E) The examination of the bottom (SMC layer) of the SMC-EPC bi-level cell sheet indicated that SMC morphology varies regionally from spherical to flattened, suggesting that SMCs differentiated into an elongated and spindle-shaped dense sheet-like construct with cellular extensions forming a densely adherent layer. (F) The interface between the two layers of the SMC-EPC bi-level cell sheet (SMC-EPC border-zone) showed the development of a cross-linked structure.
