[show abstract][hide abstract] ABSTRACT: The current state-of-art in 3D microfluidic chemotaxis device (μFCD) is limited by the inherent coupling of the fluid flow
and chemical concentration gradients. Here, we present an agarose-based 3D μFCD that decouples these two important parameters,
in that the flow control channels are separated from the cell compartment by an agarose gel wall. This decoupling is enabled
by the transport property of the agarose gel, which—in contrast to the conventional microfabrication material such as polydimethylsiloxane
(PDMS)—provides an adequate physical barrier for convective fluid flow while at the same time readily allowing protein diffusion.
We demonstrate that in this device, a gradient can be pre-established in an agarose layer above the cell compartment (a gradient
buffer) before adding the 3D cell-containing matrix, and the dextran (10kDa) concentration gradients can be re-established
within 10min across the cell-containing matrix and remain stable indefinitely. We successfully quantified the chemotactic
response of murine dendritic cells to a gradient of CCL19, an 8.8kDa lymphoid chemokine, within a type I collagen matrix.
This model system is easy to set up, highly reproducible, and will benefit research on 3D chemoinvasion studies, for example
with cancer cells or immune cells. Because of its gradient buffering capacity, it is particularly suitable for studying rapidly
migrating cells like mature dendritic cells and neutrophils.
[show abstract][hide abstract] ABSTRACT: The migration of cells such as leukocytes, tumor cells, and fibroblasts through 3D matrices is critical for regulating homeostasis and immunity and for driving pathogenesis. Interstitial flow through the extracellular matrix, which can substantially increase during inflammation and in the tumor microenvironment, can influence cell migration in multiple ways. Leukocytes and tumor cells are heterogeneous in their migration responses to flow, yet most 3D migration studies use endpoint measurements representing average characteristics. Here we present a robust new microfluidic device for 3D culture with live imaging under well-controlled flow conditions, along with a comparison of analytical methods for describing the migration behavior of heterogeneous cell populations. We then use the model to provide new insight on how interstitial flow affects MDA-MB-231 breast cancer cell invasion, phenomena that are not seen from averaged or endpoint measurements. Specifically, we find that interstitial flow increases the percentage of cells that become migratory, and increases migrational speed in about 20% of the cells. It also increases the migrational persistence of a subpopulation (5-10% of cells) in the positive or negative flow direction. Cells that migrated upstream moved faster but with less directedness, whereas cells that migrated in the direction of flow moved at slower speeds but with higher directedness. These findings demonstrate how fluid flow in the tumor microenvironment can enhance tumor cell invasion by directing a subpopulation of tumor cells in the flow direction; i.e., towards the draining lymphatic vessels, a major route of metastasis.
[show abstract][hide abstract] ABSTRACT: Dendritic cell (DC) homing to the lymphatics and positioning within the lymph node is important for adaptive immunity, and is regulated by gradients of CCL19 and CCL21, ligands for CCR7. Despite the importance of DC chemotaxis, it is not well understood how DCs interpret gradients of these chemokines in a complex 3D microenvironment. Here, we use a microfluidic device that allows rapid establishment of stable gradients in 3D matrices to show that DC chemotaxis in 3D can respond to CCR7 ligand gradients as small as 0.4%, which helps explain how DCs sense lymphatic vessels in an environment where broadcast distance for chemokine diffusion is hindered by convective flows into the vessel. Interestingly, DCs displayed similar sensitivities to both chemokines at small gradients (≤ 60 nM/mm), but migrated more efficiently towards higher gradients of CCL21, which unlike CCL19 binds strongly to matrix proteoglycans and signals without the need for internalization. Furthermore, cells preferentially migrated towards CCL21 when exposed to equal and opposite gradients of CCL21 and CCL19 simultaneously, even when matrix-binding of CCL21 was prevented. Although these ligands have similar binding affinity to CCR7, our results demonstrate that, in a 3D environment, CCL21 is a more potent directional cue for DC migration than CCL19. These findings provide new quantitative insight into DC chemotaxis in a physiological 3D environment and suggest how CCL19 and CCL21 may signal differently to fine-tune DC homing and positioning within the lymphatic system. These results also have broad relevance to other systems of cell chemotaxis, which remain poorly understood in the 3D context.
Proceedings of the National Academy of Sciences 03/2011; 108(14):5614-9. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: The formation of neuronal networks, during development and regeneration, requires outgrowth of axons along reproducible paths toward their appropriate postsynaptic target cells. Axonal extension occurs at growth cones (GCs) at the tips of axons. GC advance and navigation requires the activity of their cytoskeletal networks, comprising filamentous actin (F-actin) in lamellipodia and filopodia as well as dynamic microtubules (MTs) emanating from bundles of the axonal core. The molecular mechanisms governing these two cytoskeletal networks, their cross-talk, and their response to extracellular signaling cues are only partially understood, hindering our conceptual understanding of how regulated changes in GC behavior are controlled. Here, we introduce Drosophila GCs as a suitable model to address these mechanisms. Morphological and cytoskeletal readouts of Drosophila GCs are similar to those of other models, including mammals, as demonstrated here for MT and F-actin dynamics, axonal growth rates, filopodial structure and motility, organizational principles of MT networks, and subcellular marker localization. Therefore, we expect fundamental insights gained in Drosophila to be translatable into vertebrate biology. The advantage of the Drosophila model over others is its enormous amenability to combinatorial genetics as a powerful strategy to address the complexity of regulatory networks governing axonal growth. Thus, using pharmacological and genetic manipulations, we demonstrate a role of the actin cytoskeleton in a specific form of MT organization (loop formation), known to regulate GC pausing behavior. We demonstrate these events to be mediated by the actin-MT linking factor Short stop, thus identifying an essential molecular player in this context.
[show abstract][hide abstract] ABSTRACT: Active regulation of dendritic cells (DC) by the lymphatics is just beginning to be explored . The importance of interstitial flow on CCR7 signaling has recently been demonstrated for breast cancer cells , which likely mimic DCs to home to lymph nodes. Both lymphatics and DCs secrete the CCR7 ligands CCL19 (which is not matrix-binding) and CCL21 (which is strongly matrix-binding), and both are important for lymph node homing of immune cells. Here we develop a dynamic computational model to be used in parallel with in vitro tests to describe the specific response of DCs to both CCR7 ligands and flow.
European Congress on Computational Methods in Applied Sciences and Engineering. 07/2008;
[show abstract][hide abstract] ABSTRACT: Dendrites represent arborising neurites in both vertebrates and invertebrates. However, in vertebrates, dendrites develop on neuronal cell bodies, whereas in higher invertebrates, they arise from very different neuronal structures, the primary neurites, which also form the axons. Is this anatomical difference paralleled by principal developmental and/or physiological differences? We address this question by focussing on one cellular model, motorneurons of Drosophila and characterise the compartmentalisation of these cells. We find that motorneuronal dendrites of Drosophila share with typical vertebrate dendrites that they lack presynaptic but harbour postsynaptic proteins, display calcium elevation upon excitation, have distinct cytoskeletal features, develop later than axons and are preceded by restricted localisation of Par6-complex proteins. Furthermore, we demonstrate in situ and culture that Drosophila dendrites can be shifted from the primary neurite to their soma, i.e. into vertebrate-like positions. Integrating these different lines of argumentation, we propose that dendrites in vertebrates and higher invertebrates have a common origin, and differences in dendrite location can be explained through translocation of neuronal cell bodies introduced during the evolutionary process by which arthropods and vertebrates diverged from a common urbilaterian ancestor. Implications of these findings for studies of dendrite development, neuronal polarity, transport and evolution are discussed.