3D multi-chamber fluidic systems for analysis of angiogenesis in biomaterials: development, characterization and applications

The cellular processes leading to capillary morphogenesis depend on external stimuli, in particular flow and morphogen gradients. Several devices have been designed to study these interactions in vitro. However, most of these devices allow a single experiment at a time, making it difficult to compare multiple conditions, which is needed for most biology experiments. Furthermore, it can be of great interest to use the same set of cells. Here we present two multi-chamber interstitial flow systems for side-by-side culture with the capability to follow the dynamics of individual cells. While a 9-chamber system was designed for cell culture or co-culture under radial flow, a multi-chamber migration assay allows studying cell migration into biomaterials under a constant flow profile. The effects of flow on lymphatic and blood endothelial cell morphology, as well as the extent of organization induced by VEGF, were observed. In addition, we demonstrated the feasibility of studying cell-cell interactions as well as cell-matrix interactions in a fibroblast and tumor cell co-culture. The two systems presented will allow for the study of interstitial flow and its effects

Effects of dynamic environments on extracellular morphogen gradients

Mark Fleury

Tissue morphogenesis and remodeling is often orchestrated by cell-secreted proteins, referred to as morphogens that can trigger cellular responses in addition to modifying the local extracellular matrix (ECM). Of specific interest is the distribution of cell secreted proteins in the cell microenvironment and how this distribution can lead to varied responses. Morphogens often provide directional cues, and as such their specific distribution is critical to the signaling that they induce, in other words cells are sensitive to morphogen gradients and not just absolute amounts. Natural cellular environments in native tissue involve three dimensional ECM and are typically subjected to dynamic conditions such as interstitial flow (IF). Despite the importance of the extracellular environment in signaling, very little is known regarding the effects of mechanical stresses and subtle interstitial flows. This thesis examines the role of IF on morphogen gradients using mass transport models to elucidate mechanisms responsible for several specific biological phenomena including in vitro capillary formation and lymphatic metastasis of tumor cells. In vitro capillary formation had been shown in our lab to be separately enhanced by bound VEGF alone and slow IF alone, with the combination of these two conditions resulting in marked increase in organization. Using our computational model we were able to propose a novel mechanistic model to explain this synergy. Specifically showed how, through the combined effects of IF and matrix-bound growth factors, cells could create their own biased chemokine gradients without recourse to internal amplification methods. This process, which we have termed "autologous chemotaxis," drives directional signaling and migration in the direction of flow. This can occur even at very low Peclet numbers, when diffusion still dominates the overall transport, but convection changes the shape of the gradient, which is what a cell responds to. We then examined the effects of IF on tumor cell migration to explore a novel mechanism of lymphatic metastasis. Tumors often produce high amounts of proteoglycans creating a microenvironment is rich in morphogen binding sites, and tumors are also a source of interstitial flow due to their leaky vasculature. The lymphatic system drains interstitial fluid, therefore IF is always directed toward lymphatic capillaries. The lymphatic system is also a common route of tumor metastasis, which is what lead us to study the role of IF mediated chemotaxis. A computational model of an in vitro tumor invasion assay was constructed that predicted tumor cell migration that was in very good agreement with the in vitro experimental results. This phenomenon was further studied by modeling an in vivo geometry and examining the factors controlling autologous gradient formation in a physiological setting. In conclusion, we have demonstrated the importance of convective mass transport at very low flow velocities in a biological context. While these velocities are low enough that the convective effects would typically be ignored, their subtle effects on pericellular mass transport can be translated into relevant and observable morphogenic responses.

EPFL2007

Autologous chemotaxis as a mechanism of tumor cell homing to lymphatics via interstitial flow and autocrine CCR7 signaling

Mark Fleury, Melody Swartz, Alice Anna Tomei, Carolyn Yong Pullin

CCR7 is implicated in lymph node metastasis of cancer, but its role is obscure. We report a mechanism explaining how interstitial flow caused by lymphatic drainage directs tumor cell migration by autocrine CCR7 signaling. Under static conditions, lymphatic endothelium induced CCR7-dependent chemotaxis of tumor cells through 3D matrices. However, interstitial flow induced strong increases in tumor cell migration that were also CCR7 dependent, but lymphatic independent. This autologous chemotaxis correlated with metastatic potential in four cell lines and was verified by visualizing directional polarization of cells in the flow direction. Computational modeling revealed that transcellular gradients of CCR7 ligand were created under flow to drive this response. This illustrates how tumor cells may be guided to lymphatics during metastasis.

Elsevier2007

Dendritic cell chemotaxis in 3D

Ulrike Haessler

Dendritic cells (DCs) are crucial for the onset of adaptive immunity. Their outstanding migratory capacities in combination with their antigen presenting properties make them perfectly suited to pick up antigens in the periphery and transport them to the lymph nodes, where antigen presentation to T-cells is most efficient [1]. Their migration from the periphery to the lymph nodes, called DCs cell homing, is involved in both mounting effective adaptive immune responses as well as maintaining tolerance to self-antigens [2]. Both of them need to be balanced to maintain a healthy organism. So far two alternative mechanisms have been proposed to guide DCs from the interstitium through the extracellular matrix to the proximal lymphatic vessel [3]. One of these mechanisms is based on paracrine chemotaxis towards a CCL21 gradient created by the proximal lymphatic vessel. The second mechanism, called autologous chemotaxis, describes how cells can follow an autocrine gradient, created by the biasing of self-secreted chemokine by interstitial flow [4, 5]. To answer the question of how these two mechanisms contribute to DC homing, a quantitative understanding of how cells respond to gradients and interstitial flow present in their physiological microenvironment is needed [6]. To date, appropriate tools to study DC migration under defined gradients and in the presence of tunable interstitial flow rates in a 3D environment have been lacking. Within this thesis, we present two micro-scale devices that allow live cell observation on an inverted microscope. The first was optimized to expose cells to a steady-state chemokine gradient within a 3D microenvironment and is based on a microfluidic agarose scaffold which functions as a gradient buffer (Chapter 3). The novelty of a gradient buffer allows us to quickly establish stable gradients, which is an important feature in order to create stable and precisely defined gradients during tracking of fast-moving cells like DCs. We then performed an in-depth study of DC cell chemotactic responses towards the CCR7 ligands (CCL19 and CCL21) in 3D using this new agarose chamber (Chapter 4). With the help of an integrated computational model, we could precisely predict the gradient formation of matrix-bound CCL21 versus soluble CCL19 in the microcellular environment and found that DC answer differently to the two chemokines in response to gradient steepness. Furthermore, CCL21 was more chemoattractive than CCL19 when cells were exposed to competing gradients of both chemokines, and this was independent of the CCL21-binding properties. This represents the first quantitative study of CCR7-mediated DC chemotaxis in 3D in response to well-defined ligand gradients. The second device we developed for studying the effects of the physiological flow environment, which cells experience in capillary-fed tissues, on cell migration [6]. A 2-gel strategy allowed us to flexibly fine tune interstitial flow velocities and to precisely determine the actual flow rate for each position imaged on a microscope. We exposed DCs to a wide range of flow rates and observed an increase in persistence under flow compared to static (Chapter 6). In conclusion, this work presents quantitative insights into DC migration within precisely controlled 3D-microenvironments using new devices. It also highlights the role of a quantitative understanding of biological processes to distinguish between chemotactic mechanisms of DC cells.