Effects of Substrata and Method of Tissue Dissociation on Adhesion, Cytoskeleton, and Growth of Chick Retinal Pigmented Epithelium in Vitro

In this report we compare attachment, morphology, and growth of retinal pigmented epithelial (RPE) cells isolated by either EDTA or dispase digestion and plated onto either uncoated substrata (plastic or glass) or substrata derivatized by covalent conjugation of proteins of reconstituted basement membrane gel. We show that the derivatized substrata promote better initial attachment and subsequent cell growth than the uncoated substrata. These effects are independent of the method of dissociation of cells from the tissue. Cell morphology, however, is strongly affected by the method used for tissue dispersion. The dispase-dissociated cells are very flat, display a circumferential arrangement of microfilaments and elaborate extensive arrays of vinculin-containing cell-to-cell junctions. In contrast, EDTA-dissociated cells are much less spread, display straight microfilament bundles criss-crossing the cytoplasm and have less extensive cell-to-cell junctions. The protein-derivatized substrata also promote maintenance of differentiated traits, such as pigmentation, by the RPE cells.