We present a study on modification of culture conditions in serially cultured human bronchial epithelial cells (HBEC), necessary to achieve bronchial epithelial cells similar to the native epithelium. Cells were obtained from bronchial biopsies and serially cultured using a previously described method (In Vitro Cell. Dev. Biol. 1993; 29A:379-387). At the air-liquid interface, the second and the subsequent passages of HBEC cultures were grown 7 to 31 days, in medium containing fetal calf serum, using de-epidermized dermis or collagen discs as substratum. Scanning and transmission electron microscopy revealed ciliogenesis after 7 days and maturation of the cilia up to 31 days, irrespective of whether de-epidermized dermis or collagen membrane was used. The transmission electron microscopy of the developing cilia showed fibrogranular masses, procentrioles, basal bodies, and in the mature cilia a normal ultrastructure of the axoneme, the nine doublets, the central pair, radial spokes, and dynein arms in the ciliary shaft. In contrast, the submerged cultures showed no signs of ciliogenesis in the same time course. Results of experiments, in which cell seeding density, the substrate used, and the manner of nutrient supplementation were modulated, revealed that the air-exposure of the cultured HBEC is a necessary requirement for the ciliogenesis. The development pathway of ciliated cells in air-exposed HBEC cultures was similar to the differentiation and maturation pattern in human fetal tracheal cells. The in vitro model of human bronchial epithelial cells derived from biopsies obtained by fiberoptic bronchoscopy offers an attractive model for future studies on the function of human bronchial epithelial cells under normal and pathologic conditions.

In the present study we describe the establishment of serial cultures of human bronchial epithelial cells derived from biopsies obtained by fiberoptic bronchoscopy. The cell cultures were initiated from small amounts of material (2 mm forceps biopsies) using either explants or epithelial cell suspensions in combination with a feeder-layer technique. The rate of cell proliferation and the number of passages (up to 8 passages) achieved were similar, irrespective of whether the explants or dissociated cells were used. To modulate the extent of differentiation, the bronchial epithelial cells were cultured either under submerged, low calcium (0.06 mM) (proliferating), normal calcium (1.6 mM) (differentiation enhancing) conditions, or at the air-liquid interface. Characterization of the bronchial epithelial cell cultures was assessed on the basis of cell morphology, cytokeratin expression, and ciliary activity. The cells cultured under submerged conditions formed a multilayer consisting of maximally three layers of polygonal-shaped, small cuboidal cells, an appearance resembling the basal cells in vivo. In the air-exposed cultures, the formed multilayer consisted of three to six layers exhibiting squamous metaplasia. The cytokeratin profile in cultured bronchial epithelial cells was similar in submerged and air-exposed cultures and comparable with the profile found in vivo. In addition to cytokeratins, vimentin was co-expressed in a fraction of the subcultured cells. The ciliary activity was observed in primary culture, irrespective of whether the culture had been established from explants or from dissociated cells. This activity was lost upon subculturing and it was not regained by prolongation of the culture period. In contrast to submerged cultures and despite the squamous metaplasia appearance, the cells showed a reappearance of cilia when cultured at the air-liquid interface. Human bronchial epithelial cell cultures can be a representative model for controlling the mechanisms of regulation of bronchial epithelial cell function.

A bronchioloalveolar carcinoma presented as a large cavitatory lesion with an irregular lining in a 28 year old man.Bronchioloalveolar carcinoma is a malignancy that may appear as a radiologically well circumscribed, solitary peripheral nodule, in which cavitation is extremely rare. The clinical and pathological features of a cavitating bronchioloalveolar carcinoma in a young man are discussed. Case reportA 28 year old man was admitted for evaluation ofacute right sided pleuritic chest pain. He denied cough, dyspnoea, anorexia, loss of weight, or fever. No recent journeys abroad had been made. He had been smoking 20 cigarettes a day for 10 years. Because of haemoptysis a chest radiograph had been taken at another institution one year previously. This showed a sharply circumscribed, thin walled cavitary lesion (4-5-5 cm in diameter) in the right lower lobe (fig 1). No further action was taken at that time and the haemoptysis did not recur. Other previous chest radiographs were not available.At presentation we saw a young man in apparent good health. Physical examination indicated nothing abnormal apart from a right basal pleural rub. Routine laboratory investigations showed no abnormalities except for increased serum lactate dehydrogenase activity (397 (normal < 160) U/1). Notably the erythrocyte sedimentation rate, white blood cell count, and total eosinophil count were normal. He was tuberculin negative and had negative sputum cultures. A chest radiograph and tomogram showed that the cavitary lesion in the posterobasal segment of the right lower lobe had grown. The originally smooth inner lining of the lesion now showed an irregular, nodular contour. An aortogram showed no evidence of lung sequestration. Bronchoscopy showed no abnormalities, and bronchial washings did not disclose acid fast bacilli or malignant cells.Over the next few days a high fever (39-5°C) developed and the patient started to cough, producing purulent sputum that in culture yielded Staphylococcus aureus. On the chest radiograph the cavity was partly filled with fluid (fig 2). The patient was treated with flucloxacillin for 10 days and underwent thoracotomy once the fever had subsided.Address for reprint requests: Dr P M de Jong, Department of Pulmonology, University Hospital, Rynsburgerweg 10, 2333 AA Leiden, The Netherlands. Accepted 2 December 1988At thoracotomy a smooth surfaced and partly necrotic tumour mass (11 cm in diameter) was found, fixed to the diaphragm. Lobectomy with total removal ofthe tumour was performed. Microscopically this was seen to be a bronchioloalveolar carcinoma. Lymph nodes were not affected. DiscussionBronchioloalveolar carcinoma is known as a malignancy with an insidious onset.' It accounts for 1-6 5% of primary lung carcinomas and commonly appears as a localised, well circumscribed, solitary peripheral nodule in the lung parenchyma.23 Cavitation must be very rare, for in several large series with a total of 358 patients this radiographic appearance was never mentioned,'4 though a recent textbook stat...