New method for preparing large surfaces of intact human basement membrane for tumor invasion studies

Abstract

A new method has been developed to prepare large surfaces of continuous native human basement membrane for in vitro studies of tumor invasion. Fresh amniotic membrane is dissected intact from human placenta. The membrane is composed of a single layer of epithelium attached to a continuous basement membrane overlying a thin avascular stroma. The epithelium is denuded using a brief treatment with 4% deoxycholate and the integrity of the basement membrane is verified by: (a) impermeability to carbon particles and labeled serum proteins; (b) continuity of the basement membrane glycoproteins by PAS staining; and (c) continuity of the lamina densa by electron microscopy and immunoperoxidase staining using antibodies against type IV (basement membrane) collagen. When metastatic tumor cells are cultured on the intact basement membrane surface, local dissolution of the lamina densa occurs at the region of cell attachment.