Lumen formation by epithelial cell lines in response to collagen overlay: a morphogenetic model in culture.

Abstract

Two cell lines-Madin-Darby canine kidney (MDCK) and normal murine mammary gland (NMuMG)-growing as monolayers on collagen gels were overlaid with another collagen gel. The cells responded to the overlay by undergoing reorganization resulting in the creation of lumina. MDCK cells formed lumina that coalesced to form large cavities comparable in size with a tubule. NMuMG cells formed clusters surrounding small lumina, which appeared similar to acini of glandular tissue. The characteristic arrangements, described here by light and electron microscopy, resembled the morphology of the tissues of cell line origin. MDCK cells, grown in the presence of serum, formed lumina whether or not serum was removed at the time of overlay, whereas NMuMG cells required either a nondialyzable component of serum or hormonal supplements in serum-free defined media. Lumen formation was delayed by MDCK cells in the presence of the glutamine analog 6-diazo-5-oxo-L-norleucine, but this compound did not affect NMuMG lumen formation. In both cell lines, lumen formation was unaffected by the absence of sulfate, the presence of an inhibitor of sulfated glycosaminoglycan synthesis, or an inhibitor of collagen synthesis. DNA synthesis accompanied lumen formation but was not required.The living stroma is typically separated from the exterior by a delimiting interface of epithelial cells. Epithelial cells can exist as stratified layers that may directly face the exterior, as for example in epidermis, or as a monolayer that may be folded into circular acini or ducts containing lumina continuous with the exterior. Certain specialized epithelial cells, such as those in the thyroid or cornea, border internal fluid-filled cavities that are closed to the exterior. The epithelial cell is functionally and morphologically polarized with respect to the two environments it separates. The basal cell surface is attached to extracellular matrix (ECM) material specialized as a layered basal lamina adjacent to the ECM of the stroma. In most epithelia, the opposite apical surface is free from an apposed extracellular layer. The rim of the apical surface is girdled by tight junctions to adjacent cells, so that the cell layer serves as a boundary to molecular diffusion. From the histological organization of epithelium, it appears that attachment to the ECM has a function in directing the polarity. This is strongly reinforced by the histology ofinvasive tumors. Metastatic epithelial tumor cells secrete hydrolytic enzymes that degrade the basal lamina before they invade the stroma (1). Tumor cells that have degraded the basal lamina lose their polarity, but if they maintain contact with undegraded basal lamina, they remain polar (2).The purpose of the work described here is to establish morphogenetic models in culture in which ECM material, in this case type I collagen, induces epithelial rearrangement and thus provide a system for analysis of the biochemical steps involvedThe publication costs ofthis article were defrayed in part by page charge payment. Thi...

Dual-Purpose Bioreactors to Monitor Noninvasive Physical and Biochemical Markers of Kidney and Liver Scaffold Recellularization

“…35-010-CV, Corning) and 1% penicillin/streptomycin. Bioreactors were transferred to an incubator and perfused at 5 mL/min for at least 1 h. For seeding, perfusion was halted, and 40 million immortalized Madin-Darby canine kidney (MDCK) cells 21,22 or 6.25 million primary human renal papillae-derived CD133/1 + cells from anonymous cadaveric donors 23 were loaded into a syringe (2 mL volume) and manually injected at *2 mL/min…”

“…They are characterized by a polygonal appearance in two-dimensional culture, typical of renal epithelial cells, and become polarized to form primary cilia. 21,22 Before ureteral seeding, vacuum was applied to the chamber, lowering the gauge pressure to -40 mmHg. After injection into the ureter, cells were allowed to attach for 10 min before flow through the bioreactor circuit was resumed at 4 mL/min under normal atmospheric pressure.…”

The microenvironmental determinants for kidney epithelial cyst morphogenesis

“…Although earlier studies found that ECM is important for epithelial cyst formation, our study for the first time clearly concluded that an ECM 3D environment is essential for cystogenesis. MDCK cysts formed in a 3D ECM environment are properly polarized (Hall et al, 1982;O'Brien et al, 2002;Kroschewski, 2004; this study). However, MDCK cells form aberrantly polarized cysts when cultured in suspension (Wang et al, 1990a, b).…”

“…Multicellular, spherical, polairzed cyst formation of MDCK cells in collagen gel has been used to study epithelial polarization and morphogenesis (Hall et al, 1982;McAteer et al, 1987;Warren and Nelson, 1987;Mangoo-Karim et al, 1989;Wang et al, 1990a, b;Montesano et al, 1991a;Rahikkala et al, 2001;Yu et al, 2005). Because of the simplicity of its morphogenetic process, MDCK cyst formation is an ideal model for determining the cellular events critical for epithelial morphogenesis and the molecular mechanism(s) governing it.…”

“…For example, attachment of epithelial cells to the ECM is required for the establishment of cellular polarity and subsequent morphogenesis (Nelson, 1992;Rodriguez-Boulan and Powell, 1992). After being exposed to the ECM 3D environment, MDCK epithelial cells typically undergo cystogenesis (Hall et al, 1982). Among various ECM proteins, collagen and laminin are expressed in the early developmental stage of organs such as kidney (Ekblom, 1989).…”