Evaluating hASCs Attachment and Morphology on Biomaterials

The attachment of hASCs onto biomaterials surface, as well as the morphology and growth pattern of cultures were determined after 120 h of cells propagation. The microscopic evaluation of hASCs on biomaterials surface was performed using scanning electron microscope (SEM, Auriga 60, Zeiss, Oberkochen, Germany) and confocal microscope Cell Observer SD (Zeiss, Oberkochen, Germany) as described previously [36 (link),37 (link),38 (link)]. To prepare samples for observations the cultures were fixed with 4% paraformaldehyde. The specimens were either dehydrated in graded ethanol series (concentrations ranges from 50% to 100%, every 10%) for SEM analysis or stained with fluorescent dyes for confocal microscope observations. To visualize the nuclei and cytoskeleton the cultures were stained with diamidino-2-phenylindole (DAPI; 1:800) and atto-488-labeled phalloidin (1:800). The staining was performed using well established protocols [8 (link),26 (link),30 (link)]. All reagents used during this protocol derived from Sigma Aldrich, Poznan, Poland.