PREPARATION OF EXPLANT AND ORGAN-CULTURES AND SINGLE CELLS FROM AIRWAY EPITHELIUM

In this chapter, the methods for preparing explant and organ cultures as well as the enzymatic isolation of cells are described. The inaccessibility of the ciliated epithelium of the mammalian respiratory tract has hampered in vivo studies of its structure and function, including those regarding the regulatory mechanisms of mucociliary transport. Consequently, several in vitro approaches have been developed to maintain and study airway tissues in culture. These include explant cultures, where pieces of tissue are seeded on a substrate to encourage the spreading and outgrowth of the epithelial cells; organ cultures, in which the tissue is maintained to preserve its original structural and functional form; monolayer cell cultures, where enzymatically dissociated cells are plated onto a noncellular substrate; and feeder layer cultures, where isolated epithelial cells are grown on a cellular substrate such as treated fibroblasts. Explant cultures have been utilized for the measurement of ciliary beat frequency, including the timings of the phases of the beat cycle, of single and multiple cells, as well as for the analysis of intercellular Ca2+ signaling. Isolated epithelial cells have been used in patch–clamp studies and in assays to measure inositol trisphosphate levels. There is description of preparation of rat tail collagen, of collagen-coated coverslips, and tracheal epithelial explant culture—the materials to be used and the procedure. The technique is sterile. There is description of dissection of rabbit trachea, technique is aseptic. Details of preparation of organ culture and preparation of enzymatically isolated cells have been covered. In organ culture, the tracheal mucosa is supported at a liquid-air interface to mimic in vivo conditions of nutrient and gas exchange. Isolated cell preparations are obtained either from freshly dissected tissues or from tissues that had previously been placed in organ culture. © 1995, Academic Press Inc.