Anisotropic tissue structures provide guidance for navigating neurons in vitro and in vivo. Here we optimized the generation of comparable anisotropic monolayers of astrocytes, endothelial cells, and Schwann cells as a first step toward determining which properties of anisotropic cells are sufficient for nerve guidance. The statistical experimental design method Design of Experiments (DOE) and the experimental analysis method Response Surface Methodology (RSM) were applied to improve efficiency and utility. Factors investigated included dimensions of microcontact printed protein patterns, cell density, and culture duration. Protein patterning spacing had the strongest influence. When cells initially aligned at borders and proliferated to fill in spaces, space between stripes was most effective when it was comparable to cell size. Maximizing the area of adhesive molecule coverage was also important for confluence of these types of cells. When cells adhered and aligned over the width of a stripe and broadened to fill spaces, space width about half the cell width was most effective. These findings suggest that if the mechanism of alignment, alignment at borders or over the width of the stripe, is predetermined and the cell size determined, the optimal size of the micropatterning for aligned monolayers of other cell types can be predicted. This study also demonstrates the effective use of DOE and RSM to probe cellular responses to various and multiple factors toward determination of optimal conditions for a desired cellular response.
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The authors would like to thank Christina Johnson and Grace Li for helpful discussion of the initial experimental set-up. They would also like to thank Carmichael Ong, Liane Livi, Cameron Rementer, and Jesse Thon for assistance with imaging and analysis. Si wafers were fabricated at the BioMEMS Resource Center with the generous assistance of Octavio Hurtado. This work was funded by an NSF Career Award and NIH R01 EB005722-01A2 to DHK and an AAUW Selected Professions dissertation fellowship to CK.
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Kofron, C.M., Hoffman-Kim, D. Optimization by Response Surface Methodology of Confluent and Aligned Cellular Monolayers for Nerve Guidance. Cel. Mol. Bioeng. 2, 554 (2009) doi:10.1007/s12195-009-0087-1
- Design of Experiments (DOE)
- Schwann cell
- Endothelial cell