Ordered, Random, Monotonic and Non-Monotonic Digital Nanodot Gradients

Ongo, Grant; Ricoult, Sébastien G.; Kennedy, Timothy E.; Juncker, David

doi:10.1371/journal.pone.0106541

Cited by 4 publications

(9 citation statements)

References 31 publications

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“…A variation of this printing process was used to pattern digital nanodot gradients (DNGs) of surface bound proteins. DNGs are gradients made up of discrete 200 × 200 nm 2 protein dots, spaced according to algorithmically generated density functions that can mimic biological noise by superimposing sinusoidal waves, as well as randomizing the position of individual nanodots . DNGs were previously used to investigate C2C12 myoblast migration on gradients of netrin-1 …”

Section: Introductionmentioning

confidence: 99%

Nanocontact Printing of Proteins on Physiologically Soft Substrates to Study Cell Haptotaxis

et al. 2016

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Surface bound guidance cues and gradients are vital for directing cellular processes during development and repair. In vivo, these cues are often presented within a soft extracellular matrix with elastic moduli E < 10 kPa, but in vitro haptotaxis experiments have been conducted primarily on hard substrates with elastic moduli in the MPa to GPa range. Here, a technique is presented for patterning haptotactic proteins with nanometer resolution on soft substrates with physiological elasticity. A new nanocontact printing process was developed that circumvented the use of plasma activation that was found to alter the mechanical properties of the substrate. A dissolvable poly(vinyl alcohol) film was first patterned by lift-off nanocontact printing, and in turn printed onto the soft substrate, followed by dissolution of the film in water. An array of 100 unique digital nanodot gradients (DNGs), consisting of millions of 200 × 200 nm protein nanodots, was patterned in less than 5 min with with <5% average deviation from the original gradient design. DNGs of netrin-1, a known protein guidance cue, were patterned, and the unpatterned surface was backfilled with a reference surface consisting of 75% polyethylene glycol grafted with polylysine and 25% poly-d-lysine. Haptotaxis of C2C12 myoblasts demonstrated the functionality of the DNGs patterned on soft substrates. In addition, high densities of netrin-1 were observed to induce cell spreading, while live imaging of sinusoidal control gradients highlighted cell migration and navigation by "inching". The nanopatterning technique developed here paves the way for studying haptotactic responses to diverse digital nanodot patterns on surfaces covering the full range of physiological elasticity, and is expected to be applicable to the study of both culture and primary cells, such as neutrophils and neurons.

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Section: Introductionmentioning

confidence: 99%

Nanocontact Printing of Proteins on Physiologically Soft Substrates to Study Cell Haptotaxis

et al. 2016

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“…We conclude that for the range studied, cell haptotaxis decisions are dictated by the overall protein surface concentrations experienced by the entire cell. The ultimate limit for nanodot size is single-protein nanodots, and further experiments are needed to determine whether for a fixed protein density, there is a threshold for cell response based on a minimal size, or whether nanodot order and distribution could have an effect 28 , 29 . Quantitative molecular data from nanodot arrays could be obtained using live cell microscopy, which could be used to quantify focal adhesion size across the different nanodot sizes along with cell migration velocities and thus contribute to unraveling the relationship between nanodot size and focal adhesion formation.…”

Section: Discussionmentioning

confidence: 99%

“…Our group introduced low-cost digital nanodot gradients (DNGs) to study cell migration 27 and designed a set of 100 DNGs formed of 200 × 200 nm 2 nanodots. DNGs with ordered and random nanodot distributions, with different ranges of linear and exponential slopes, and with different noise patterns were designed with a large dynamic range of up to 3.86 orders of magnitude 27 , 28 . These DNGs reflect the noncontinuity and large dynamic range characteristic of in vivo gradients 11 and were used in cell migration studies 29 .…”

Section: Introductionmentioning

confidence: 99%

Combinatorial nanodot stripe assay to systematically study cell haptotaxis

2020

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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.

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“…Using these new designs, the gradients reached an unprecedented dynamic range of 3.85 OM (Ongo et al, 2014 ), which could more accurately represent the expected dynamic range of gradients in vivo . More complex algorithms were also developed to introduce noise into the gradients at the nanoscale by pseudo-randomly distributing dots within a row of constant density and compensating for dot overlap based on the probability of overlap at the given density.…”

Section: Methods To Create Substrate-bound Protein Gradientsmentioning

confidence: 99%

Substrate-Bound Protein Gradients to Study Haptotaxis

Ricoult

Kennedy

Juncker

2015

Front. Bioeng. Biotechnol.

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Cells navigate in response to inhomogeneous distributions of extracellular guidance cues. The cellular and molecular mechanisms underlying migration in response to gradients of chemical cues have been investigated for over a century. Following the introduction of micropipettes and more recently microfluidics for gradient generation, much attention and effort was devoted to study cellular chemotaxis, which is defined as guidance by gradients of chemical cues in solution. Haptotaxis, directional migration in response to gradients of substrate-bound cues, has received comparatively less attention; however, it is increasingly clear that in vivo many physiologically relevant guidance proteins – including many secreted cues – are bound to cellular surfaces or incorporated into extracellular matrix and likely function via a haptotactic mechanism. Here, we review the history of haptotaxis. We examine the importance of the reference surface, the surface in contact with the cell that is not covered by the cue, which forms a gradient opposing the gradient of the protein cue and must be considered in experimental designs and interpretation of results. We review and compare microfluidics, contact printing, light patterning, and 3D fabrication to pattern substrate-bound protein gradients in vitro. The range of methods to create substrate-bound gradients discussed herein makes possible systematic analyses of haptotactic mechanisms. Furthermore, understanding the fundamental mechanisms underlying cell motility will inform bioengineering approaches to program cell navigation and recover lost function.

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Ordered, Random, Monotonic and Non-Monotonic Digital Nanodot Gradients

Cited by 4 publications

References 31 publications

Nanocontact Printing of Proteins on Physiologically Soft Substrates to Study Cell Haptotaxis

Nanocontact Printing of Proteins on Physiologically Soft Substrates to Study Cell Haptotaxis

Combinatorial nanodot stripe assay to systematically study cell haptotaxis

Substrate-Bound Protein Gradients to Study Haptotaxis

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