Fully automated 3D fiber arrays for cell dynamic mechanical analysis – FibersForCells

We will develop a set of 3D grids of deformable fibers with regular meshes or gradients of spacing, stiffness or cross-linking, with the aim of reproducing in a simplified way a set of generic properties of the extracellular matrix, and in particular to have access to phenomena of exploration of 3D networks of deformable fibers, isotropic or not, or of migration according to gradients of stiffness or topographic gradients (durotaxis and topotaxis). These grids will be based on two types of chemistry: polyethylene glycol diacrylate fibers (fibronectin-coated) for an optimal modulation of mechanical properties, or collagen for its biomimetic and degradable character, with the possibility of composite assemblies already operational. Finally, one of the great assets of the system is the integrated detection of 3D forces exerted by the cells, from the automatic quantification of 3D deflections coupled to the characterization of mechanical properties by Atomic Force Microscopy and the development of a Finite Element Model. We will study in these grids the behavior of a range of cells labeled for actin and nucleus: fibroblasts, tumor cells with different invasive properties, and immune cells - macrophages and dendritic cells. The acquisition of image sequences by spinning disk or Lattice Light Sheet microscopy, and the development of image analysis based on artificial intelligence techniques, will allow us to systematically define descriptors of cell behavior, at the global scale (cell shape, migration and migration bias, shape and deformation of the nucleus) or at the local scale (local 3D forces, local degradation, cell protrusions). These results will allow us to get systematic information on optimal architectures for a given cell type and to have extensive insights on the effect of the different architectural parameters.

Our innovative method for quantifying 3D forces in our fiber networks is now in its final stage, with a publication in redaction.

The innovative integrated tool developed for measuring cell-fiber dynamic interactions will be central to quantify 3D forces in relation to cell or tissue motility, to protease degradation, and to the generation of specific protrusive events triggered by microtopography. We believe that the advances in microfabrication, the breakthrough in 3D force measurement, the automated analysis, and the derived systematic approach performed for several pathophysiological systems will be of general use for the community, and will pave the way to future applications in the domain of screening (drug screenings, cell differentiation…), as it allows to get direct mechanical outputs in a perfectly controlled and reproducible 3D context.
We will make the fully integrated 3D fiber arrays widely available for the community, where there is a large field of application for mechanobiological studies performed on many individual cells. Stem cells will be particularly interesting candidates, as their differentiation is known to tightly depend on stiffness and nanotopographies. For these different cell types, our parallelized assay would allow large possibilities of molecule screenings, targeting migration, traction forces or differentiation status. Beyond individual cells, monitoring the interactions between cells in controlled 3D fiber environments will pave the way to a wide range of applications, from immune recognition, myoblast differentiation and myotube formation, to angiogenesis or collective migration from tumor spheroids. So, our fully controllable system should be of general interest for cell and developmental biology as well as for screening and differentiation assays. Our study is based on the use of generic 3D grids with simplified architectures, but in addition the technological developments achieved for two-photon polymerization will provide other tools for approaches reconstituting the whole complexity of 3D matrix from in vivo images. At last, two-photon polymerized 3D structures have been proposed in regenerative medicine, and the possibility to build controlled 3D arrays of collagen fibers would open new perspectives of transfer to in vivo bioimplants.

Previous related publications:

1. Ucla P, Ju X, Demircioglu M, Baiz S, Muller L, Germain S, Monnot C, Semetey V, Coscoy S. (2022) Dynamics of endothelial engagement and filopodia formation in complex 3D microscaffolds. Int. J. Mol. Sci., 23(5), 2415. Special Issue 3D Printing and Biomaterials for Biological and Medical Application. doi.org/10.3390/ijms23052415

2. Coscoy S, Baiz S, Octon J, Rhoné B, Perquis L, Tseng Q, Amblard F, Semetey V. (2018) Microtopographies control the development of basal protrusions in epithelial sheets. Biointerphases. 13(4):041003. doi: 10.1116/1.5024601.

The project FibersForCells aims at building 3D fiber architectures perfectly controlled in a geometrical, chemical and mechanical point of view, in which cell dynamics and mechanics will be automatically analyzed in order to access cell behavior in reconstituted systems mimicking prototypical organizations of the extracellular matrix. We propose to use two-photon polymerization to build versatile fiber arrays in which migration and protrusive cell activities will be quantified, in link with 3D forces and cell-mediated remodeling and degradation. The project is based on the expertise of the consortium on chemistry, two-photon polymerization and cell dynamics, including automated image analysis, and on current innovative methodological developments to develop an original system for the measurement of 3D forces in such fiber arrays. We will overcome the challenges to produce tridimensional fiber arrays fitting the main matrix 3D architectures, extent our chemical developments on surface functionalization and protein photopolymerization, in particular to the polymerization of collagen or derivatives in link with study and control of their degradation, develop the analysis system for 3D force measurements, and implement additional mechanical stimulations and automated image detection and analysis. In practical terms, we will develop a set of 3D grids of deformable fibers with regular meshes or gradients of spacing, stiffness or cross-linking, with the aim of reproducing in a simplified way a set of generic properties of the extracellular matrix, and in particular to have access to phenomena of exploration of 3D networks of deformable fibers, isotropic or not, or of migration according to gradients of stiffness or topographic gradients (durotaxis and topotaxis). These grids will be based on two types of chemistry: polyethylene glycol diacrylate fibers (fibronectin-coated) for an optimal modulation of mechanical properties, or collagen for its biomimetic and degradable character, with the possibility of composite assemblies. Finally, one of the great assets of the system is the integrated detection of 3D forces exerted by the cells. We will study in these grids the behavior of a range of cells labeled for actin and nucleus: fibroblasts, tumor cells with different invasive properties, and immune cells - macrophages and dendritic cells. The acquisition of image sequences by spinning disk or Lattice Light Sheet microscopy, and the development of image analysis based on artificial intelligence techniques, will allow us to systematically define descriptors of cell behavior, at the global scale (cell shape, migration and migration bias, shape and deformation of the nucleus) or at the local scale (local 3D forces, local degradation, cell protrusions). These results will allow us to get systematic information on optimal architectures for a given cell type and to have extensive insights on the effect of the different architectural parameters. This interdisciplinary project, currently in a rapid development phase, relies on a large corpus of preliminary results from the consortium and on a strong collaborative network covering the fields of chemistry, microfabrication, biomechanics, imaging and cell biology. The development of this 3D fiber system will be of general interest to the biology community. The technological advances may subsequently benefit other types of studies aimed at reconstituting the full complexity of the extracellular matrix, or open future possibilities for the use of perfectly controlled collagen fibers in bio-implants. More generally, our fully controlled 3D fiber system will pave the way to future applications in the domain of screening (drug screenings, control of cell differentiation…), as it allows to get direct mechanical outputs in a perfectly controlled and reproducible 3D context.