EVALUATION OF CELL MORPHOLOGY BY VIDEO RECORDING AND COMPUTER-ASSISTED IMAGE-ANALYSIS

The aim of this study was to evaluate the effects of various cell cult ure conditions on cell morphology, Cell morphology was estimated by me ans of video recording and computer-assisted image analysis. Cell cont ours from the stored images of either Live cells or fixed and stained cells were determined automatically, and cellular area, form factor, a nd average cell brightness were calculated. Using the mouse fibroblast oid L 929 cell line (L-cells) and the rat glioma BT4C cell line, it wa s found that a number of methodological parameters strongly affected c ell morphology. These included confluency of cells before dissociation , dissociation procedure, cell seeding density, cultivation time, and culture substratum. The substratum, particularly collagen type I and f ibronectin, profoundly affected cell morphology. Using drugs affecting cytoskeletal organization or cell substratum interactions, it was sho wn that average cell brightness was a valuable parameter for estimatio n of cellular attachment. Cytochalasin D, which impairs actin filament s, caused a dramatic increase in the average cell brightness in both c ell Lines, Nocodazole, which depolymerizes microtubules, mainly affect ed the L-cells, whereas the BT4C-cells were largely unaffected, indica ting that microtubules were morphological determinants for the former cell line but not for the latter, When cells were grown on fibronectin , an RGD-peptide only affected L-cell attachment, indicating that BT4C -cells only expressed low (if any) amounts of RGD recognizing integrin s, The interassay precision of the employed procedure depended on cult ure substratum; the coefficients of variation ranged from 7-24%. Lowes t variations in area determination were found for cells grown on fibro nectin. The coefficient of variation of form factor determinations was generally around 20%, independent of substrata and culture time. (C) 1997 Wiley-Liss, Inc.