Time at confluence for human RPE cells: effects on the adherens junction and in vitro wound closure

To determine how time at confluence affects the properties of cultured human retinal pigment epithelium (RPE) cells, with emphasis on the adherens junction.

Cultures were maintained at confluence without passage for intervals to several months. Adherens junction proteins (N-cadherin, E-cadherin, alpha-catenin, beta-catenin, plakoglobin, and actin) and the proliferation marker Ki-67 were localized in the cultures by fluorescence microscopy, and in vitro wound healing was compared. Adherens junctions were analyzed for protein solubility in detergent buffers and sensitivity to disruption by treatment with anti-cadherin antibodies and low calcium conditions.

Compared with cultures in early-confluence (2-3 days), postconfluent cultures (weeks) had more mature adherens junctions characterized by a circumferential (rather than linear) actin organization, and a zonular (rather than punctate) distribution of more detergent resistant cadherin and catenins. Postconfluent cultures also had fewer Ki-67-positive cells and a higher cell packing density. Early-confluence cells migrated into in vitro wounds as dissociated single cells, whereas postconfluent cells moved as contiguous sheets, retaining an intact junction during wound-induced cell migration and proliferation. Mature junctions were not disrupted by treatment of living cells with N-cadherin antibodies, which bound to and remained detectable at junctions for several days. Calcium withdrawal displaced N-cadherin from mature junctions and rendered it more soluble, but the dominant circumferential pattern of actin was stable. Restoration of medium calcium resulted in a rapid (hours) recovery of a nearly complete zonular pattern of insoluble N-cadherin.

Over long postconfluent periods, cultured RPE cells became more growth quiescent, and intercellular cadherin adhesions became more stable, exhibiting increased resistance to calcium removal and greater retention of junctional integrity during in vitro wound closure. Consideration should be given to whether the behavior of RPE cells in postconfluent cultures, where intercellular adhesions are more mature, more closely simulates RPE cells in situ than cells in early-confluence cultures, which are more commonly used for analysis.