Figure 1
From: A Simulated Microgravity Environment Causes a Sustained Defect in Epithelial Barrier Function
HT-29.cl19a cells attach and form 3-D monolayers on individual microcarrier beads. HT-29.cl19a intestinal epithelial cells were cultured with microcarrier beads for 18 days under the conditions of simulated microgravity in a rotating wall vessel (RWV) at 16–17.8 rpm. (A,B) Samples were removed from the RWV for observation under an inverted light microscope (Panel A,: day 1, panel B, day 11) and embedded in LR White and stained with Toluidine Blue for EM imaging (Panels C,D, day 1, day 18). Panels A and C show a bead lacking cell attachment. By comparison, panels B and D show that cells attached and grew around individual beads. (D) Cells in panel D were able to take up the vital dye, Toluidene Blue, thus demonstrating viability of cells in situ on the microcarrier bead. Panels C,D shown in cross-section with the center of the hollow bead indicated by an asterisk. (E,F) whole cell-bead aggregates were stained for nuclei (DAPI) and the tight junction protein, occludin, showing an intact layer around the surface of the bead and tight junction formation. (G,H) Cross-section shows that cells grew as a unicellular layer around the outer surface of the bead. (I) Staining of cellular junction formation on cell-bead aggregates shows apico-lateral staining of (J) occludin and (K) actin with (I) nuclei stained by DAPI and (L) showing an overlay image of all three stains. Arrow indicates occludin staining at a cell-cell junction.
