Methods in laboratory investigation. Exclusion of trypan blue from microcarriers by endothelial cells: an in vitro barrier function test

Abstract

The introduction of microcarriers in cell culture work has offered new possibilities not least in the study of endothelial biology. This article describes the use of microcarriers for selection of endothelial cells from mixed cultures by using a simple migration-transfer technique. Colonies of either endothelial or smooth muscle cells were allowed to form on gelatin-coated dishes with mitomycin-treated feeder cells. Microcarriers were then seeded so that one or two of them attached to each colony. The cells populated the microspheres within 2 to 3 days. Microcarriers from pure endothelial cell colonies were selected for transfer and serial subcultivation. Furthermore, a method to study the barrier function of confluent microcarrier cultures of endothelium was outlined. This method utilizes the fact that intact confluent cells exclude the dye trypan blue from the matrix of the microspheres which otherwise binds the dye tightly. The rate of absorption of trypan blue from a standard buffer containing 0.2% dye and 1 mg/ml of serum albumin by a standard volume of packed cell covered microspheres was determined. It was shown that confluent endothelial cells provided a barrier for the dye for several hours and that the subcellular matrix contributed to the barrier but only to a small extent. Treatment of the cells with dicloxacilline, 0.1 gm/ml, and homocysteine, at 8 mM, also influenced the barrier markedly. Dicloxacilline caused a complete breakdown and homocysteine clearly increased the rate of absorption of the trypan blue. It is suggested that the barrier function of cultured cells may be studied by this technique or future modifications based on similar principles.