Unique morphology of HeLa cell attachment, spreading and detachment from microcarrier beads covalently coated with a specific and non-specific substratum

Abstract

A SEM and TEM evaluation of adhesion of HeLa-S3 cells to suspensions of culture microcarriers coated with various substrata revealed two unique cell morphologies. One is similar to that for cells attaching to culture dishes and the other one only appeared with microcarriers stirred under high shear conditions. The usual appearance of a spreading cell is to change from a sphere to the shape of a 'fried egg'. This proceeded in HeLa cells by a radial extension of the filopodia in between which the cytoplasm subsequently filled. Fluorescent antibody staining of actin suggested that more actin was present at the periphery of the spreading edges of the cell than inwards. The above morphology was characteristic of HeLa cell attachment to gelatin-coated microcarriers. However, the morphology of the attachment to microcarriers coated with non-biological substances such as negatively charged sulfonate groups or positively charged polyethyleneimine or even with the attachment protein laminin was quite different. Here the cells attached and began to spread as with gelatin-microcarriers, however, the spreading was not radial but occurred from one or two major regions of the cell periphery. The cell then appeared to constrict with the formation of a substratum attached pedestal upon which the cell body was perched. With time the cell pinched-off from pedestal. Evidence indicated that the pedestal was quite fragile. Furthermore, fluorescent antiactin staining indicated that the initial spreading region contained abundant actin which was depleted upon pedestal formation and detachment. The above in addition to previous kinetic measurements provided the information to classify cell substrate attachment materials into two distinct types. One is specific substrata which promote normal attachment and spreading and appear to interact with specific cell surface proteins. The other is non-specific substrata which in high shear conditions induces pedestal formation followed by pinching-off of the cells. Had previous attachment assays been done under high shear as done with the microcarriers and HeLa cells it is likely that substrata classified as specific might be reclassified into non-specific.