MDCK cysts: An in vitro model of epithelial cyst formation and growth

Abstract

SUMMARY: MDCK cells dissociated from monolayer culture were either dispersed within medium-hydrated collagen gel or seeded atop a collagen substrate which was immediately overlaid with collagen gel. Individual cells exhibited clonal growth in three dimensions to form spherical cysts in which a simple epithelium surrounded a fluid-filled lumen. The ceils of MDCK cysts were polarized with apical surface bordering the lumen. MDCK cysts increased in diameter with continued culture. Maximum cyst size was dependent on seeding density and was influenced by medium composition. MDCK cysts could be isolated from the collagen substrate by digestion with collagenase. Also, collagen gel could be dissected from the cyst wall to give unrestricted access to regions of the basolateral cell surface. This novel method of renal cell culture provides a study system to model the influence of the extracellular matrix on kidney epithelial cell structure and function. It also offers an in vitro model of general application to the study of epithelial cyst formation and growth.

Genetically Encoded Sender–Receiver System in 3D Mammalian Cell Culture

“…For 3D MDCK culture, 40 cells were trypsinized and resuspended to 3 × 10 4 cells/ml in a type I collagen solution containing PureCol (3 mg/mL), Advanced BioMatrix 5005-B, 10× MEM, NaHCO 3 (2.35 mg/mL), l -glutamine (29.2 mg/mL), and 1 M HEPES (pH 7.6). Cells in suspension were added to wells containing a cell-free collagen mix (polymerized previously at 37 °C) and were incubated at 37 °C (without CO2) for 20–30 min.…”

Synthetic matrices reveal contributions of ECM biophysical and biochemical properties to epithelial morphogenesis

“…Bissell, Mostov, and others have pioneered the use of 3D collagen and laminin (i.e., Matrigel) gels in organotypic cultures that recreate the epithelial morphogenetic developmental program ( O’Brien et al, 2002 ; Mroue and Bissell, 2013 ). In these natural matrices, epithelial cells, such as MDCK cells, proliferate from single cells to form multicellular, hollow spherical monolayer structures (cysts) within 10 d that bear the hallmarks of epithelial polarity, recapitulating the morphogenetic program for the formation of a rudimentary epithelial organ ( McAteer et al, 1986 ). These 3D cultures have revealed that interactions between integrin adhesion receptors and secreted laminin and the density of ECM, which impacts the gel mechanical properties, regulate the establishment of cell polarity and morphogenesis as well as tumorigenesis ( Barcellos-Hoff et al, 1989 ; O’Brien et al, 2001 ; Wozniak et al, 2003 ; Levental et al, 2009 ; Provenzano et al, 2009 ).…”

Synthetic hydrogels mimicking basement membrane matrices to promote cell-matrix interactions

“…Studies using 3D collagen and laminin gel cultures have demonstrated that the epithelial morphogenesis developmental program is regulated by biochemical and mechanical signals that result from cell-BM interactions (O'Brien et al, 2002; Yu et al, 2005; Mroue and Bissell, 2013). In these studies, single-cell encapsulation of epithelial cells, such as MDCK cells, results in cell proliferation into a multicellular aggregate and further development of cysts composed of a polarized epithelial monolayer that recreates the morphogenesis of epithelial organs (McAteer et al, 1986). Although these natural matrices have been able to recapitulate the epithelial morphogenesis program of MDCK cells, they do not offer the capacity to study individual contributions of the mechanical and biochemical properties of the ECM towards epithelial morphogenesis.…”