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INTRODUCTION: Cell-matrix mechanical interactions modulate cell fate decisions and multicellular organization and as such are important for tissue engineering. We have developed optical imaging procedures and computational algorithms to quantify cellular forces in 3D multicellular culture systems relevant for tissue engineering. Here we show their importance for the study of angiogenesis. METHODS: 3D in vitro assays of vascular endothelial invasion in collagen hydrogels were optimized for live cell optical imaging by means of either confocal fluorescence microscopy or selective plane illumination microscopy (SPIM). SPIM provides fast 3D imaging of relatively large, multicellular systems with reduced phototoxicity, making it ideal for live cell imaging. Cell-matrix mechanical interactions during vascular invasion were quantified by means of novel traction force microscopy (TFM) algorithms. First, non-rigid image registration was performed to resolve 3D matrix deformations around invading sprouts. Second, cellular forces that were responsible for the measured deformations were quantified using an iterative, finite element based solution scheme that makes use of a novel physics-based regularization method. RESULTS: We previously demonstrated the superior accuracy and robustness of non-rigid image registration for 3D matrix deformation calculation, based on the use of fiducial markers as well as collagen network imaging (by means of Second Harmonic Generation) [1]. Applying our novel TFM methods to invading angiogenic sprouts, we observe robust patterns of radially pulling forces around sprout protrusions. When interfering with actin turnover (by adding cytochalasin D), forces are diminished, in turn inhibiting vascular invasion. Time lapse imaging data revealed strong correlations between matrix deformations and protrusion dynamics. Figure 1: Cell-induced 3D matrix deformations around angiogenic sprout (shown in white) in collagen hydrogel. Arrows are colour-coded for displacement magnitude (0-9 µm). CONCLUSION: Our 3D TFM methods enable to study cellular forces in 3D multicellular systems relevant for tissue engineering. ACKNOWLEDGEMENTS: ERC StG n°308223, FWO (G.0821.13, G0B9615N, G087018N) and KU Leuven (C14/17/111). REFERENCES [1] Jorge-Peñas A et al. Biomaterials. 2017; 136: 86-97