In vitro growth of Chlamydia trachomatis in conjunctival and corneal epithelium

Abstract

This study used primary cultures of conjunctival and corneal epithelial cells from rabbit, monkey and humans to investigate the infection process and host interactions of chlamydia. The epithelial cells were isolated from bulbar and palpebral conjunctivae and the cornea following incubation with EDTA or dispase and microdissection. The cells were trypsinized and grown in microtest wells or Rose Chambers (gelatin-coated glass substratum). The cell origin of the cultured epithelial cells was verified by immunofluorescence staining with anticytokeratins. The cells were nonreactive when stained with antivimentin, a fibroblast marker. The growth kinetics of the cultured epithelial cells were determined by morphological criteria. Cells reached confluency by 5-7 days, and remained as such for 18 days. The cells were maintained as long as 32 days in primary culture. After the cells had attained confluency (5-7 days), they were infected with ocular strains B/TW-5/OT or C/TW-3/OT C. trachomatis. Chlamydial inclusions were identified by light and electron microscopy. Direct FITC C. trachomatis staining (species-specific monoclonal antibody) of the inclusions was used to determine the infectivity of the various types of epithelial cells. The dynamics of the infection process was documented by time lapse photomicroscopy. Small inclusions were identified by 18 hr postinoculation (pi). By 48 hr pi, the inclusions had dramatically increased in size, occupying much of the host perikarya. The reticulate bodies were very active at this time. By 70 to 100 hr pi, the inclusions contained the highly infectious elementary bodies. At this time, rupture of the inclusion and lysis of the infected cells usually occurred.(ABSTRACT TRUNCATED AT 250 WORDS)