Abstract
Haptotaxis is critical to cell guidance and development and has been studied in vitro using either gradients or stripe assays that present a binary choice between full and zero coverage of a protein cue. However, stripes offer only a choice between extremes, while for gradients, cell receptor saturation, migration history, and directional persistence confound the interpretation of cellular responses. Here, we introduce nanodot stripe assays (NSAs) formed by adjacent stripes of nanodot arrays with different surface coverage. Twenty-one pairwise combinations were designed using 0, 1, 3, 10, 30, 44 and 100% stripes and were patterned with 200 × 200, 400 × 400 or 800 × 800 nm2 nanodots. We studied the migration choices of C2C12 myoblasts that express neogenin on NSAs (and three-step gradients) of netrin-1. The reference surface between the nanodots was backfilled with a mixture of polyethylene glycol and poly-d-lysine to minimize nonspecific cell response. Unexpectedly, cell response was independent of nanodot size. Relative to a 0% stripe, cells increasingly chose the high-density stripe with up to ~90% of cells on stripes with 10% coverage and higher. Cell preference for higher vs. lower netrin-1 coverage was observed only for coverage ratios >2.3, with cell preference plateauing at ~80% for ratios ≥4. The combinatorial NSA enables quantitative studies of cell haptotaxis over the full range of surface coverages and ratios and provides a means to elucidate haptotactic mechanisms.
Highlights
Cell migration is essential for many biological processes including angiogenesis[1], immune responses[2], tissue repair[3], cancer metastasis[4], and embryonic development[5]
nanodot stripe assays (NSAs) design and fabrication The NSA we introduce consists of stripes of ordered nanodot arrays patterned in a square lattice with seven surface coverages of 0, 1, 3, 10, 30, 44 and 100% (Fig. 1a)
A combinatorial array of NSAs was used to systematically study cellular haptotactic choices between lower and higher protein surface densities across seven different coverages and three nanodot sizes resulting in 63 different binary concentration tests
Summary
Cell migration is essential for many biological processes including angiogenesis[1], immune responses[2], tissue repair[3], cancer metastasis[4], and embryonic development[5]. Studies of haptotactic signaling in vitro have monitored cellular responses to proteins patterned as either stripes or surface gradients. These surface-bound protein patterns can be formed using different techniques including microfluidic serial diluters and gradient generators, microfluidic probes, hydrogel stamp diffusion, dip-pen nanolithography, laser-assisted protein adsorption by photobleaching (LAPAP), porous capillary filters, and Dlamini et al Microsystems & Nanoengineering (2020)6:114 microcontact printing[19]. Ricoult et al.[21], using classical stripe assays, demonstrated that haptotaxis is potently modulated by the cell-surface affinity of the reference surface (RS)—the area between the patterned protein cue—which in many cases dictated the cellular response. Ricoult et al introduced an RS made of a mixture of poly-D-lysine (PDL, with cell adhesive properties) and polyethylene glycol (PEG, cell repellent), whereby the ratio of PDL and PEG was tuned to maximize cell response to the guidance cues[21]
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