Hamster tracheal organ culture in serum-free media: A quantitative comparison of in vitro epithelial morphology with that of in vivo controls

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Abstract
The epithelial morphology of the hamster trachea in serum-free organ culture was compared with that of age-matched in vivo control tissues by collecting and statistically analyzing several quantifiable parameters. By this technique it was possible to detect both subtle and dramatic epithelial alterations. Midtracheal tissues from 6-wk-old male Syrian golden hamsters were used as the explants. Explants were placed on Gelfoam sponges and cultured for 1, 2, and 3 wk in CMRL 1066 alone and in CMRL 1066 to which seven factors were added: insulin and transferrin (5 μg/ml); hydrocortisone (5×10−7 M); epidermal growth facotr (5 ng/ml); bovine pituitary extract (0.5%); and phosphoethanolamine and ethanolamine (5×10−5 M). The following data were collected and statistically analyzed for each tracheal ring: number of epithelial cells; proportion and number of each cell type; basement membrane length; linear density of epithelial cells; epithelial height; and mitotic index. Compared to controls, ciliated cells decreased by 52% during washes in Leibovitz (L15) medium and tissue manipulation performed before culture and this loss persisted after cutlure for 1 wk. Explants culturedwithout the factors showed marked changes after 2 and 3 wk including epithelial thickening and folding, which was associated with increased linear density. Many cells in these specimens could not be categorized by type (22% were unidentifiable after 3 wk). Epithelial migration onto the outside of the explant was inhibited. In contrast, explants culturedwith the factors maintained a morphology similar to controls at 2 and 3 wk and epithelial migration onto the outside of the explant was supported. This study shows that explants in CMRL 1066 with the seven factors provide a useful biological model for the in vitro study of the mucociliary respiratory epithelium.

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