Epithelial contact guidance on well-defined micro- and nanostructured substrates

Ana I. Teixeira, George A. Abrams, Paul J. Bertics, Christopher J. Murphy and Paul F. Nealey

Journal of Cell Science 116, 1881-1892. 2003

http://jcs.biologists.org/cgi/reprint/116/10/1881

Teixeira et al. were interested in the effects of topographical nanoscale patterning of a substrate on the cell behavior of a epithelial cell culture. Previous studies had shown tendencies for cells to align along groove and ridge topographical features on the order of 1 micron in width as opposed to cell behavior on smooth surfaces which did not demonstrate alignment but rather remained round. This alignment of cells along topographical features is known as contact guidance. While previous studies had gone as small as 500 nm ridges and grooves, no one had yet demonstrated this phenomenon on substrates with 70 nm features, which is the biomimetic size for the felt-like surfaces of the corneal basement membrane. A better understanding of topographical signals to cell morphology would lead to clearer design requirements for cell cultures and tissue engineering.

This article is of particular interest to our team, the “Itsy Bitsy Tiny Weeny Yellow Polka Dot Collagen Fibers Group (IBTWYPDCFG)”, because it focuses on two key elements that we plan to further explore and develop in our project. First of all it focuses on micropatterning very thin topographical features and characterizes the effect on a specific cell functionality (alignment). Secondly it demonstrates successful cell adhesion to a Silicon Oxide substrate which we were concerned might be a problem. The difference between their micropatterning methods and ours is that they used a photoresist mask and Silicon etching procedure to achieve 70 nm groves with 600 nm depths consisting of pure silicon whereas we plan to use an electrospining technique of a polymer-collagen mixture to deposit 100 nm fibers onto a silicon wafer.

Other notable features in their experiment that we plan to deviate from include:

• They focused only on single cell attachment and alignment of corneal epithelial cells. We plan to culture many fibroblast cells in contact with each other and measure their proliferation and production of collagen as a function of the substrate pattern that they grow on.
• They measured their cell functionality based on cell alignment using both light microscopy as well as SEM and possessing the data in ImageJ. Our cell functionality measurements will be based more on techniques that we’ve practiced in lab that allow us to measure cell viability and the production of collagen by our seeded cells (Live/Dead assays, Western Blotting and RT-PCR).
• They were able to perform time lapse microscopy to measure the mobility of a seeded cell over a 12 hour period. We will not do that.

Features that worked for them that we plan to emulate if possible include:

• The use of serum in the culture medium enhanced the cell functionality (specifically alignment). It would be useful to compare the results of two substrates one with serum, one without.
• The processing and cleaning of the silicon wafer followed a standard protocol similar to that used in Bekeley’s EE143 microprocessing lab. In particular close attention was paid to the removal of native oxide using Phiranha solution and the sterilization of the wafer prior to being used for tissue culture.
• The production of focal adhesions by the cell was measured using a mouse anti human vinculin primary antibody followed by a donkey anti-mouse immunoglobulin secondary antibody.

In the end Teixeira et al. were able to determine that the most dominant feature for cell alignment in their substrate was the groove depth, specifically that the highest percentage of alignment occurred when the groove depth was 600 nm. In addition they were able to show that cells had much higher rates of elongation on all patterned substrates (specifically the 70 nm biomimetic sized grooves and ridges) when compared to the blank Silicon Oxide substrates. This experiment was pretty cool and is hopefully useful in helping us prepare for ours. Yeah Science!