Microporosity of the substratum regulates differentiation of MDCK cells in vitro

Abstract

We have analyzed the ability of the physical substratum to modulate both the ultrastructural and protein synthetic characteristics of the Madin-Darby canine kidney (MDCK) renal cell line. When MDCK cells were seeded on Millipore Millicell CM microporous membrane cell culture inserts they demonstrated a more columnar organization with an increase in cell density sixfold greater than the same cells seeded on conventional plastic substrata. After 1 wk postseeding on the microporous membrane a partial basal lamina was noted, with a contiguous basement membrane being apparent after 2 wk. One-dimensional sodium dodecyl sulfate gel electrophoresis was used to analyze detergent-solubilized proteins from MDCK cells maintained on plastic substrata vs. microporous membranes. When proteins were pulse-labeled with [35S]methionine, a 55 kDa protein was evident in the cytosolic extract of cells grown on collagen, laminin, and nontreated plastic substrata; but this labeled protein was not evident in similar extracts from cells grown on collagen and laminin-coated microporous membranes. To test if the polarized, basement-membrane secreting phenotype of the MDCK cells could be generated on a microporous membrane without pretreatment with any extracellular matrix (ECM) components, cells were seeded on the Millipore Millicell HA (cellulosic) microporous membrane. This type of substrata does not need a coating of ECM components for cell attachment. A partial basement membrane was formed below cells where the basal surface of the cell was planar, but not in areas where the cell formed large cytoplasmic extensions into the filter. This led us to the conclusion that the microporous nature of the substrata can dictate both ultrastructural and protein synthetic activities of MDCK cells. Furthermore, we suggest that both the planar nature of the basal surface and the microporosity of the substrate are corequisites for the deposition of the basement membrane.

Persistent adhesion of epithelial tissue is sensitive to polymer topography

“…This result was consistent with those published for MDCK cells grown on solid plastic surfaces, 19,29,30 but it was a little unexpected in the light of our previous findings 8 that showed some interfacial deposition of ECM proteins involved with these adhesive structures on this surface. Overall, this suggested that while at least some of the component proteins were present on the nonporous surface, they were unable to assemble to form recognizable adhesive structures.…”

“…Clearly this warrants separate consideration now that we have identified the size of the discontinuity that interrupts the formation of adhesive structures. In the light of our findings, it is interesting to reflect on the earlier work by Cook,19 who proposed that substratum porosity and the planar nature of the basal cell surface in MDCK cells were important factors in the deposition of basement membrane components. These results differ from those of Clark,7,12 who demonstrated the significance of groove depth over groove pitch in the guidance of MDCK cells on a solid surface.…”

“…Microporosity of the substratum is considered to be essential to the differentiation, ECM deposition, and formation of stable adhesive structures in epithelial cells. 19 Largely, the role of topographical cues associated with the presence of pores has not been the focus of previous studies although it has been recognized that discontinuities associated with topographic features of a surface may trigger changes in protein adsorption and conformation. 20 Data from our laboratories have shown that the deposition of ECM proteins associated with basement membrane and adhesion complex formation by corneal epithelial tissue was affected by a polymer surface topography created by pores.…”

Modulation of corneal epithelial stratification by polymer surface topography

“…This result is consistent with a previous study in which the polarity in MDCK epithelial cells was examined on porous surfaces and microporosity was found to influence both the polarized phenotype and the deposition of basement membrane components. 22 In our study, the nonporous surface did not score highly in terms of tissue stratification, reflecting the fact that basal and suprabasal cells showed few signs of morphological differentiation. This was expected, since the porosity of a substratum has been shown to have a profound effect on the morphology of MDCK epithelial cell lines.…”

“…Porosity promotes the polarity of various cell types in vitro, including cells of epithelial origin. 22,23 Topography and/or porosity created by the presence of pores in a material affects corneal epithelial tissue outgrowth in vitro.…”