L Fjellbirkeland, R Bjerkvig, SK Steinsvag and OD Laerum
Department of Pathology, Gade Institute, Haukeland Hospital, Bergen, Norway.

The supply of fresh bronchial tissue from human donors for in vitro culture is limited. Routine fiberoptic bronchoscopy offers a safe and easy procedure for obtaining minor biopsies and we wanted to see if the material provided could be used for organ culture by using a simple liquid overlay technique. Bronchial biopsies were cut into fragments 400-500 microns and kept immersed in a standard serum-supplemented medium for 40 days. An agar base prevented adhesion of the tissue. By light and electron microscopy it was shown that the tissue fragments had a differentiated epithelium at their surface throughout the culture period. An outgrowth of epithelial cells on the scaffold of the exposed stroma, covering the surface of the whole fragment, occurred within the first 5 days of culture. This epithelium was partly ciliated, 2-4 cell layers thick with squamous and cuboidal cells and expressed epithelial markers (cytokeratin and Ber-Ep4). The amount of cilia increased during the first 15 days of culture. The epithelium rested on a neosynthesized basement membrane as visualized by electron microscopy and immunohistochemistry with antibodies directed against collagen IV, laminin, and fibronectin. The central stroma consisted of loose connective tissue with fibroblasts. This simple tissue culture model combines maintenance and neoformation of bronchial epithelium on top of a living natural substrate, thus enabling direct biological studies on clinical biopsy material under perfectly viable conditions.

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