An in vitro tissue culture bilayer model to examine early events in Mycobacterium tuberculosis infection

Abstract

A tissue culture bilayer system that mimics some aspects of early alveolar infection by Mycobacterium tuberculosis was developed. This model incorporates human lung epithelial type II pneumocyte (A549) (upper chamber) and endothelial cell (lower chamber) layers separated by a microporous membrane. This construction makes it possible to observe and quantify the passage of bacteria through the two layers, to observe the interaction of the bacteria with the various cell types, and to examine the basic mechanisms of immune cell recruitment to the site of infection. After 10(7) organisms were added to the upper chamber we microscopically observed large numbers of bacteria attached to and within the pneumocytes and we determined by viable-cell counting that a small percentage of the inoculum (0.02 to 0.43%) passed through the bilayer into the lower chamber. When peripheral blood mononuclear cells were added to the lower chamber, microscopic examination indicated a migration of the mononuclear cells through the bilayer to the apical surface, where they were seen associated with the mycobacteria on the pneumocytes. The added complexity of the bilayer system offers an opportunity to define more precisely the roles of the various lung cell types in the pathogenesis of early tuberculosis.

FIG. 1

Bilayer system. A microporous membrane separates a monolayer of A549 lung epithelial cells on the apical surface and a monolayer of HULEC on the basal surface.

FIG. 2

Bilayer 24 h after infection with M. tuberculosis Erdman: Bacteria (arrow) are seen closely associated with the pneumocytes. Magnification, ×1,000.

FIG. 3

Electron micrograph of bilayer after 48-h infection. Bacteria (arrow) are seen within pneumocytes (P) on the apical side of the membrane (M) with the HULEC layer (H) on the basal side. Magnification, ×7,000.

FIG. 4

Bilayers 4 h (a) and 24 h (b) after infection with M. tuberculosis Erdman. Mononuclear cells (arrows) have migrated from the lower chamber to the apical surface, where they are seen in association with bacilli. Magnification, ×1,000.