A three-dimensional organotypic culture of the human uterine exocervix for studying mucosal epithelial differentiation and migrating leukocytes

Abstract We report on a three-dimensional organotypic culture in vitro of explants from the human uterine exocervix. Exocervical fragments (2–3 mm 3 ) from pre-menopausal women were cultured on sponges submerged in Dulbecco's Modified Eagle's Medium containing p-nonylphenol and 10% fetal bovine serum for up to 3 weeks and the viability and cellular responses were assayed. The fragments were analyzed by immunohistochemistry for the expression and distribution of a broad spectrum of cellular markers: p63, Ki-67, involucrin, high molecular weight cytokeratins, estrogen receptor-α, vimentin, CD45, and CD31. The fragments preserved their tissue architecture and cellular heterogeneity comparable to that observed in exocervical tissue in vivo . Prior to culture, the original epithelium was composed of stratified multilayered keratinocytes with integrated monocyte/dendritic-like cells in the basal and suprabasal layers. The epithelium began to exfoliate in culture and within 4 days appeared to have lost its differentiated high-zone layers of keratinocytes. After 10 days a new epithelium, slightly different from the original one, was formed; it displayed an increasing prominence of basal and suprabasal keratinocyte layers, containing infiltrating leukocytes that had probably migrated from the submucosa. The epithelium subsequently lost its organization, concomitant with a progressive involution of the stroma. Subepithelial capillaries appeared to be well maintained throughout the culture period. Aside from the maintenance of cellular heterogeneity within the fragments of exocervix, these culture systems are a valuable tool for studying the mechanisms of epithelial regeneration, and may prove to be a useful model for studying mucosal immunity.