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Mayumi Kaida, Feng Cao, Christine M. B. Skumatz, Pamela E. Irving, Janice M. Burke; Time at Confluence for Human RPE Cells: Effects on the Adherens Junction and In Vitro Wound Closure. Invest. Ophthalmol. Vis. Sci. 2000;41(10):3215-3224.
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purpose. To determine how time at confluence affects the properties of
cultured human retinal pigment epithelium (RPE) cells, with emphasis on
the adherens junction.
methods. Cultures were maintained at confluence without passage for intervals to
several months. Adherens junction proteins (N-cadherin, E-cadherin,α
-catenin, β-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
results. 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.
conclusions. 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
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