Evaluation of endothelial cell migration with a novel in vitro assay system.
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 alpha (TNFalpha). 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 mm(2) and 5.3 mm(2) for TNFalpha-treated and untreated HUVEC, respectively. HUVEC migration on FN/collagen was slightly greater - 6.4 mm(2) and 6.5 mm(2) with and without TNFalpha 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-alpha 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.
Cai
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Cardeza Foundation for Hematologic Research, Jefferson Medical College of Thomas Jefferson University, Philadelphia, PA 19107, USA.
Lian
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J
Shapiro
S S
SS
Beacham
D A
DA
D002465:Cell Movement / Q000502:physiology*; D003094:Collagen / Q000737:chemistry; D004727:Endothelium / Q000166:cytology* / Q000502:physiology*; D005109:Extracellular Matrix / Q000737:chemistry* / Q000502:physiology*; D005353:Fibronectins / Q000737:chemistry; D006801:Humans; D066298:In Vitro Techniques; D008899:Mineral Oil; D015203:Reproducibility of Results; D014409:Tumor Necrosis Factor-alpha; D014471:Umbilical Veins; D014841:von Willebrand Factor / Q000737:chemistry
D005353:Fibronectins; D014409:Tumor Necrosis Factor-alpha; D014841:von Willebrand Factor; D008899:Mineral Oil; D003094:Collagen