Detachment of epithelial structures from underlying basement membrane (BM) represents an important component of a number of human disease processes e.g. airway and alveolar diseases, gastrointestinal ulceration, and retinal diseases. This study describes a method of evaluating human epithelial cell detachment from BM that is simple, rapid, inexpensive, quantifiable and which, because it utilizes BM rather than tissue culture plastic, more closely mimics the in vivo situation than other methods. In this model human amnionic epithelial cells attached to their underlying BM are isolated from fresh placentae and mounted in a multi-well chemotaxis assembly. These membranes can be studied with the epithelial cell monolayer intact. Protease-induced detachment of the epithelial cells from the underlying BM was readily quantifiable using light microscopy and spectroscopy. Following removal of the native amnionic epithelial cells, immunoperoxidase staining for the BM attachment proteins laminin, fibronectin, and type IV collagen demonstrated that these molecules remain intact. The BM could also be used as an attachment surface to reconstitute other epithelial cell monolayers. Cultured human amnionic cells and human respiratory epithelial cells were both able to attach to the denuded BM in the absence of serum (% attachment = 85 +/- 15% and 92 +/- 8% respectively, P = 0.8). Natural BM was a better substrate for epithelial cell attachment than tissue culture plastic in that, in the absence of serum, cultured epithelial cell attachment to tissue culture plastic was 20 +/- 4% of the value for BM (P less than 0.05). Furthermore, cells attached to plastic adhered less effectively than to BM in that trypsin concentrations required to induce 50% cell detachment were 0.72 +/- 0.4 for plastic and 62 +/- 13 BAEE U/ml for BM (P less than 0.001). In view of the complex protein interactions known to be involved in the anchorage of human epithelial cells to BM, it is likely this model will be a useful tool for evaluating the mechanisms underlying human epithelial cell attachment and detachment in a variety of normal and disease situations.
Amnion Basement Membrane