Sean Campbell

Biological length scale topography enhances cell-substratum adhesion of human corneal epithelial cells

The basement membrane possesses a rich 3-dimensional nanoscale topography that provides a physical stimulus, which may modulate cell-substratum adhesion. We have investigated the strength of cell-substratum adhesion on nanoscale topographic features of a similar scale to that of the native basement membrane. SV40 human corneal epithelial cells were challenged by well-defined fluid shear, and cell detachment was monitored. We created silicon substrata with uniform grooves and ridges having pitch dimensions of 400-4000 nm using X-ray lithography. F-actin labeling of cells that had been incubated for 24 hours revealed that the percentage of aligned and elongated cells on the patterned surfaces was the same regardless of pitch dimension. In contrast, at the highest fluid shear, a biphasic trend in cell adhesion was observed with cells being most adherent to the smaller features. The 400 nm pitch had the highest percentage of adherent cells at the end of the adhesion assay. The effect of substratum topography was lost for the largest features evaluated, the 4000 nm pitch. Qualitative and quantitative analyses of the cells during and after flow indicated that the aligned and elongated cells on the 400 nm pitch were more tightly adhered compared to aligned cells on the larger patterns. Selected experiments with primary cultured human corneal epithelial cells produced similar results to the SV40 human corneal epithelial cells. These findings have relevance to interpretation of cell-biomaterial interactions in tissue engineering and prosthetic design.

Non-toxic thermotropic liquid crystals for use with mammalian cells

This paper reports the results of a study that aimed to identify thermotropic liquid crystals that are not toxic to mammalian cells. Mesogenic compounds were mixed to create eight liquid crystalline phases, each with a unique set of functional groups. We investigated the toxicity of each liquid crystalline phase using two mammalian cell lines—3T3 fibroblast and SV-40 transformed human corneal epithelial (HCEC) cells. Using dual fluorescent staining assays based on calcein acetoxymethylester (Calcein-AM) and ethidium homodimer, we measured correspondingly the number of viable and dead cells following immersion of the cells in the liquid crystals. It was found that most of the liquid crystals, such as commonly used 5CB and E7, caused cell death after contact with cells for four hours. However, we identified a class of liquid crystals containing fluorophenyl groups that possess minimal or no toxicity (as indicated by results of assays based on Calcein-AM and ethidium homodimer) to cells. Following immersion in fluorophenyl liquid crystals for four hours, the cells were observed to proliferate in culture medium at rates similar to control cells exposed to phosphate-buffered saline (PBS) for four hours. In contrast, treatment of cells for 24 hours with either PBS or liquid crystals (media containing no nutrients and growth factors) resulted in cell death.

Effect of norepinephrine on proliferation, migration, and adhesion of SV-40 transformed human corneal epithelial cells

Purpose To determine the ability of norepinephrine to modulate proliferation, adhesion, and migration of SV-40 transformed human corneal epithelial cells. Methods Assays were performed using SV-40 transformed human corneal epithelial cells. For proliferation assays, cells were plated in 96-well plates coated with fibronectin and collagen (FNC). A dose-response curve was generated for norepinephrine in concentrations of 100 n M-100 μ M. The cell number in each well was evaluated using the fluorochrome Calcein AM (an intracellular esterase cleavage substrate), and fluorescence was determined using an automated fluorescent plate reader. For cell adhesion, 25×103 cells were plated onto FNC-coated 96-well plates, incubated in 10 n M-100 μ M norepinephrine for 90 min, gently irrigated, and the remaining adherent cells quantitated. Cell migration was measured using blind-well migration chambers with a 10-μm pore size and FNC-coated filters. Cells (250×103) were added to the upper chamber, incubated for 18 h in the presence of factors, after which time the cells that had migrated through the filter were quantitated. The toxicity of norepinephrine was evaluated using a standard Live/Dead assay employing the combined fluorochromes of ethidium homodimer (to indicate dead cells) and Calcein AM (to indicate viable cells). Varying concentrations of norepinephrine were added, and the cells incubated for 3 h and the fluorometric assay performed. Results Norepinephrine stimulated corneal epithelial cell proliferation and migration over a wide range of concentrations. It did not modulate cell adhesion and demonstrated cell toxicity only at the highest (supraphysiologic) concentration tested. Conclusions Norepinephrine is normally found in the cornea and may be important in the maintenance of normal corneal homeostasis and in wound-healing processes.