Sébastien G. Ricoult

Generation of microisland cultures using microcontact printing to pattern protein substrates

The capacity to isolate small numbers of neurons in vitro is an essential tool to study the cell biology of synapses and the development of neuronal networks by specific cell types. Microisland culture assays allow for single neurons, or simple neural networks, to be isolated on islands of glial cells; however, the techniques commonly used to produce microisland substrates are expensive, challenging to control, and typically result in many discarded substrates. Here, we used microcontact printing to pattern a glass surface with islands of extracellular matrix proteins known to support neural cell growth and differentiation. To promote segregation of the cells to the islands, the substrate surrounding the islands was backfilled with polyethylene glycol (PEG), forming a relatively non-permissive surface on which cell attachment is limited. Astrocytes, and subsequently hippocampal neurons, were then seeded onto the islands of patterned protein. Using this method, readily reproducible patterns of protein islands were produced that permit cell attachment, differentiation, and growth. The technique is a rapid, inexpensive, and reliable means to generate patterned substrates appropriate for microisland cultures.

Tuning cell–surface affinity to direct cell specific responses to patterned proteins

Interactions with local extracellular cues direct cell migration. A versatile method to study cell response to a protein consists of patterning the protein cue on a substrate and quantifying the distribution of cells between patterned and non-patterned areas. Here, we define the concepts of (i) cell-surface affinity to describe cell choices, and of (ii) reference surface (RS) to clarify that the choice is made relative to a reference. Furthermore, we report a method to systematically tune the RS and show that it can dominate the experimental cell response to a protein cue. The cell response to a cue can be switched from strong preference to strong aversion by only changing the RS. Using microcontact printing, we patterned the extracellular matrix proteins fibronectin or netrin-1 adjacent to a series of RSs with different ratios of poly-D-lysine (PDL) and polyethylene glycol (PEG), which are of high affinity and of low-affinity for cells, respectively. C2C12 myoblasts or primary neurons seeded on substrates with a high affinity RS (high % PDL) did not respond to a printed protein of interest, and conversely on RSs of low affinity (high % PEG) the cells preferred the printed protein even in the absence of a specific interaction. However, when testing cell response to a standardized series of RSs varying from high to low affinity, a specific response curve was obtained that was unique to each cell type-protein pair. Importantly, for intermediate RSs with moderate affinity, the cell response to the cue was dependent on the activation of biologically relevant protein-specific biochemical signal transduction pathways. Our results establish that choices made by cells in response to a surface-bound cue must take into account, and be interpreted in the context of, the RS. The use of a series of RSs with varying cell-surface affinity reveals specific response curves of cells to a cue that can be compared quantitatively and that may help gain new insights into cellular responses to extracellular proteins.

Substrate-bound protein gradients to study haptotaxis

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.

Large Dynamic Range Digital Nanodot Gradients of Biomolecules Made by Low-Cost Nanocontact Printing for Cell Haptotaxis

A novel method is introduced for ultrahigh throughput and ultralow cost patterning of biomolecules with nanometer resolution and novel 2D digital nanodot gradients (DNGs) with mathematically defined slopes are created. The technique is based on lift-off nanocontact printing while using high-resolution photopolymer stamps that are rapidly produced at a low cost through double replication from Si originals. Printed patterns with 100 nm features are shown. DNGs with varying spacing between the dots and a record dynamic range of 4400 are produced; 64 unique DNGs, each with hundreds of thousands of dots, are inked and printed in 5.5 min. The adhesive response and haptotaxis of C2C12 myoblast cells on DNGs demonstrated their biofunctionality. The great flexibility in pattern design, the massive parallel ability, the ultra low cost, and the extreme ease of polymer lift-off nanocontact printing will facilitate its use for various biological and medical applications.

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

Cell navigation is directed by inhomogeneous distributions of extracellular cues. It is well known that noise plays a key role in biology and is present in naturally occurring gradients at the micro- and nanoscale, yet it has not been studied with gradients in vitro. Here, we introduce novel algorithms to produce ordered and random gradients of discrete nanodots – called digital nanodot gradients (DNGs) – according to monotonic and non-monotonic density functions. The algorithms generate continuous DNGs, with dot spacing changing in two dimensions along the gradient direction according to arbitrary mathematical functions, with densities ranging from 0.02% to 44.44%. The random gradient algorithm compensates for random nanodot overlap, and the randomness and spatial homogeneity of the DNGs were confirmed with Ripleys K function. An array of 100 DNGs, each 400×400 µm2, comprising a total of 57 million 200×200 nm2 dots was designed and patterned into silicon using electron-beam lithography, then patterned as fluorescently labeled IgGs on glass using lift-off nanocontact printing. DNGs will facilitate the study of the effects of noise and randomness at the micro- and nanoscales on cell migration and growth.

Spatially Selective Dissection of Signal Transduction in Neurons Grown on Netrin-1 Printed Nanoarrays via Segmented Fluorescence Fluctuation Analysis

Axonal growth cones extend during neural development in response to precise distributions of extracellular cues. Deleted in colorectal cancer (DCC), a receptor for the chemotropic guidance cue netrin-1, directs F-actin reorganization, and is essential for mammalian neural development. To elucidate how the extracellular distribution of netrin-1 influences the distribution of DCC and F-actin within axonal growth cones, we patterned nanoarrays of substrate bound netrin-1 using lift-off nanocontact printing. The distribution of DCC and F-actin in embryonic rat cortical neuron growth cones was then imaged using total internal reflection fluorescence (TIRF) microscopy. Fluorescence fluctuation analysis via image cross-correlation spectroscopy (ICCS) was applied to extract the molecular density and aggregation state of DCC and F-actin, identifying the fraction of DCC and F-actin colocalizing with the patterned netrin-1 substrate. ICCS measurement of spatially segmented images based on the substrate nanodot patterns revealed distinct molecular distributions of F-actin and DCC in regions directly overlying the nanodots compared to over the reference surface surrounding the nanodots. Quantifiable variations between the populations of DCC and F-actin on and off the nanodots reveal specific responses to the printed protein substrate. We report that nanodots of substrate-bound netrin-1 locally recruit and aggregate DCC and direct F-actin organization. These effects were blocked by tetanus toxin, consistent with netrin-1 locally recruiting DCC to the plasma membrane via a VAMP2-dependent mechanism. Our findings demonstrate the utility of segmented ICCS image analysis, combined with precisely patterned immobilized ligands, to reveal local receptor distribution and signaling within specialized subcellular compartments.

Microfluidic Probes to Process Surfaces, Cells, and Tissues

Over the last two decades, microfluidics has made significant contributions in cell biology research and tissue engineering by providing novel approaches and methods. Microfluidics is defined as the manipulation of fluidics at the micro-scale, where the physics of fluid behavior is dominated by different phenomena than at the macroscale, yet predictable and controllable. In this chapter, we briefly discuss the use of microfluidic devices in biological research, and highlight their use in processing surfaces, cells, and tissues. We explain the advantages that microfluidic devices can bring to biological experiments in comparison to the conventional methods such as Petri dishes, flasks, and microtiter plates (Fig. 1(a)). We also describe the limitations and challenges that are associated with microfluidic devices, mainly because of their closed-channel configuration. A microfluidic device can be described as a network of closed channels and chambers where at least one of the dimensions is

Digital nanodot gradients and adjustable reference surfaces to investigate axonal turning on substrate-bound protein gradients

complexity, valence) including the age in which these scripts were acquired (i.e., “when did you first learn to perform this behaviour”). In two fMRI studies, participants processed both the procedural (“how”) and teleological (“why”) aspects of late-acquired and earlyacquired scripts. In both studies, and controlling for the various possible confounds and reaction times, processing late-acquired (vs. early-acquired) scripts activated the “default mode network”. These findings suggest that the default network is associated with mature cognitive processing, and thus join previous work in arguing against theories that ascribe very basic, early-acquired functions to the default network. We note three main interpretations of our data: (i) a relatively uncontroversial argument according to which the on-line processing of late-acquired (vs. early acquired) scripts recruited a different set of cognitive functions; (ii) a stronger claim according to which the differences in on-line patterns of activation stem from differences that existed at the time of script acquisition; (iii) a radical claim according to which age of script acquisition is one of the organizing principles of the cortex.