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Evaluation of endothelial cell migration with a novel in vitro assay system

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Methods in Cell Science

Abstract

In this study we introduce a novel in vitro 'oil-drop' assay system for the measurement of endothelial cell (EC) migration, based on the original concept of the Teflon fence assay (Pratt et al., 1984; Am. J. Pathol. 117: 349–354). An aliquot of 15–20,000 human umbilical vein EC (HUVEC) is pipetted through a layer of mineral oil. The cells readily attach, spread and migrate on the surface of a matrix-coated tissue culture dish as a confluent circular monolayer. Migration is measured as the net increase in the total area covered at 24 hours. We have used this system to quantify EC migration on matrices composed of a mixture of type I collagen and either von Willebrand factor (vWF) or fibronectin (FN) in the presence or absence of tumor necrosis factor α (TNFα). Plating efficiency on both vWF/collagen and FN/collagen, measured by counting cells after attachment and spreading, is about 80%. With this method, migration on vWF/collagen was about 6.4 mm2 and 5.3 mm2 for TNFα-treated and untreated HUVEC, respectively. HUVEC migration on FN/collagen was slightly greater – 6.4 mm2 and 6.5 mm2 with and without TNFα treatment, respectively. During the 24 hour time period, HUVEC numbers increased 30–40% on vWF/collagen, and 60–80% on FN/collagen, with increased proliferation observed with TNF-α treatment. EC proliferation could be completely inhibited by 2 mM hydroxyurea. This assay system has proven useful in our studies to quantify cell migration and proliferation.

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Authors
  1. Guoping Cai
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  2. Jie Lian
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  3. Sandor S. Shapiro
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  4. Dorothy A. Beacham
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Cai, G., Lian, J., Shapiro, S.S. et al. Evaluation of endothelial cell migration with a novel in vitro assay system. Methods Cell Sci 22, 107–114 (2000). https://doi.org/10.1023/A:1009864613566

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