Transepithelial transport in cell culture. A theoretical and experimental analysis of the biophysical properties of domes

Abstract

Dissociated cells of transporting epithelia, when cultured on an impermeant substratum, form polarized monolayers frequently characterized by the presence of domes. If the assumption is made that the monolayer exhibits a uniform stretch modulus of elasticity and tension of cell-dish adhesion, Ta, then biophysical properties of the epithelium can be predicted. We have shown that for such epithelia, domes should (a) have circular bases, (b) be sections of spheres with a constant height to radius, h/r, ratio, (c) have a dome-wall tension, Tw, that is constant, and (d) have a dome volume that is a function of radius alone. Additionally, a Laplace equation derived for this geometry predicted the hydrostatic pressure from within to outside domes as a decreasing function of radius alone. By microscopy, domes had predominantly circular bases and were found to be sections of spheres with a constant height, h, to radius, r, ratio of 0.684. Using the Laplace equation derived for this geometry and measurements of delta P and r, the tension of cell-dish adhesion, Ta, and dome-wall tension, Tw, were found to be constants of 6.60 and 7.08 torr, respectively. Combining the constants for Ta and h/r ratio, and the fact that domes are sections of spheres, delta P and dome volume were shown to be known functions of radius alone. In addition, the modulus of elasticity of the epithelium was calculated to be 4.82 X 10(3) dyn/cm2.

Identification of riboflavin transport by MDCK cells using quantitative fluorescence video microscopy

“…These values were used to calculate the volume and surface area of the dome, assuming it was a symmetrical right hemisphere. It has been reported (Tanner, Framach & Misfeldt, 1983) and confirmed by our own preliminary results that this is an accurate method for the measurement of dome volume. The focal plane was adjusted to the base of the dome, and an initial fluorescent background image was recorded.…”

RasMutation Impairs Epithelial Barrier Function to a Wide Range of Nonelectrolytes

“…As shown in Table 1, the Ras trans- fectant shows a statistically greater number of domes, which are 50% smaller. The relationship of domes to fluid transport, and barrier function, properties described in Table 3, has been discussed previously (Leighton et al, 1970;Lever, 1979;Tanner et al, 1983).Also evidenced in the Ras transfectant were occasional multilayered foci of cells (Figure 2, right, white arrow). In thick section microscopy, this sporadic tendency by the Ras transfectant to multilayer is even better illustrated, and contrasts with the monolayer phenotype of LLC-PK 1 and pBabe (Figure 3).…”

“…As shown in Table 1, the Ras trans- fectant shows a statistically greater number of domes, which are 50% smaller. The relationship of domes to fluid transport, and barrier function, properties described in Table 3, has been discussed previously (Leighton et al, 1970;Lever, 1979;Tanner et al, 1983).…”

Quantification of dome formation by image analysis in LLC-PK1 renal cells: A sensitive method for the assessment of in vitro nephrotoxicity

“…Dissociated epithelial cells cultured on nonporous substratum (culture dish) reach confluency and form an epithelial sheet with fusion of cellular membranes at the tight junction level (18). This epithelial tissue which is able to perform transepithelial transport (19) may be distorted by the formation of water-filled hemicysts (domes) (18) directly linked to the basolateral Na +, K + ATPase activity, an enzyme currentl)~ evaluated in nephrotoxicity studies (11).…”

“…This epithelial tissue which is able to perform transepithelial transport (19) may be distorted by the formation of water-filled hemicysts (domes) (18) directly linked to the basolateral Na +, K + ATPase activity, an enzyme currentl)~ evaluated in nephrotoxicity studies (11).…”