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

A tissue culture bilayer system that mimics some aspects of early alveolar infection by Mycobacterium tuberculosis was developed. This model incorpora tes human lung epithelial type II pneumocyte (A549) (upper chamber) and end othelial cell (lower chamber) layers separated by a microporous membrane. T his construction makes it possible to observe and quantify the passage of b acteria 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 co unting that a small percentage of the inoculum (0.02 to 0.43%) passed throu gh the bilayer into the lower chamber. When peripheral blood mononuclear ce lls were added to the lower chamber, microscopic examination indicated a mi gration 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.