An immunohistochemical study of lens epithelial cell growth on the capsule in culture

Purpose. To investigate cytoskeletal structure in lens epithelial cells during growth in an in vitro capsular bag model under a variety of culture conditions. Methods. Following sham cataract operations on human donor eyes capsular bags were incubated under various conditions including +/- an IOL, +/- serum and +/- the Ca²⁺ATPase inhibitor thapsigargin. The capsular bags, as well as whole capsule/epithelium mounts from uncultured donor eyes, were fixed and prepared by immunohistochemical staining for investigation by epifluorescence microscopy. Results. Epithelial cells were observed to be growing on three surfaces of the capsular bags and also on IOLs. All cells had a normal configuration of vimentin filaments and microtubules, which ran throughout the cytoplasm. Cells at the leading edge of growth on the posterior capsule showed polarised actin stress fibres at right angles to the ruffled membranes, polygonal arrays of actin filaments and cell-cell contacts. Confluent cells were more cuboid with a differentiated actin skeleton of stress-type fibres basally and an apical mat of polygonal arrays, as in native epithelia. These structures were not altered by the presence of an IOL or by serum-free culture. In the presence of thapsigargin the actin cytoskeleton depolymerised and the vimentin was reorganised into thick filaments prior to degradation. Conclusion. Growth of cells on three surfaces of the capsular bag and the IOL could be monitored by immunofluorescent staining. After reaching confluency the cells closely resembled native epithelial cells in all aspects studied, and thus provide an excellent model for investigations. The presence of thapsigargin lead to rearrangement of intermediate filaments and loss of the actin cytoskeleton.