Microfluidic-based analysis of 3d cell migration under different biophysical and chemical gradients

Resumen

Several mechanochemical factors are involved in cell migration, fundamental to establish and maintain the proper organization of multicellular organisms. The alteration of migratory patterns of cells could be related to the development of several pathologies. Focusing this work on tissue regeneration, more specifically wound healing, bone regeneration and blood vessel formation, the main aim of this work is to advance in the understanding of how chemical or physical factors present in the cell niche can regulate the cell movement (fibroblasts, osteoblasts and endothelial cells respectively). In an effort to understand what mechanisms are involved, it has been seen that both the extracellular matrix surrounding tissue cells and the biomolecules present in the cellular microenvironment can affect the behavior of cells [1–3]. In turn, interstitial fluid flow, defined as the convective transport of liquids through the extracellular matrix of tissue, is also capable of altering the morphology and cellular movement. Similarly, biomolecules, such as growth factors or drugs, modify the migration pattern. The main mechanisms studied throughout this thesis have been chemotaxis, durotaxis and rheotaxis. The biological processes for which these analyses have been performed were angiogenesis, wound healing and bone regeneration respectively. For the in vitro study of these variables, and making use of novel microfabrication techniques such as microfluidics, new platforms for 3D cell culture have been developed [4,5]. The microfluidic chips used allow replication of the ex vivo tissue microenvironment through the use of hydrogels and the generation of concentration gradients and controlled fluid flows. It should be noted that the versatility of this technology has allowed us to simultaneously study several microenvironmental factors, such as chemical gradients and matrix stiffness applied to fibroblast culture to understand its behavior in the wound area. In addition, these types of systems allow the visualization and/or monitoring of the cellular response in real time, being able to quantify the cellular migration. For the application of fluid flow, a novel system was designed to avoid the rupture of the hydrogels, allowing to obtain a stable interstitial flow inside the chip chamber. Throughout this thesis, it has been seen that there are several factors involved in 3D cell migration. Not only variables such as the chemical gradient (studied in endothelial cells and fibroblasts) or the rigidity of the extracellular matrix (analyzed in fibroblasts and osteoblasts) affect cells [6,7]. The architecture of the matrix, more specifically the disposition of the fibers that conform this matrix, has been identified as playing an important role in cell migration, also altering the morphology of cells, in this case osteoblasts. [1] N. Movilla, C. Borau, C. Valero, J.M. García-Aznar, Degradation of extracellular matrix regulates osteoblast migration : a microfluidic-based study, Bone. 107 (2018) 10–17. [2] O. Moreno-Arotzena, C. Borau, N. Movilla, M. Vicente-Manzanares, J.M. García-Aznar, Fibroblast Migration in 3D is Controlled by Haptotaxis in a Non-muscle Myosin II-Dependent Manner, Ann. Biomed. Eng. (2015). doi:10.1007/s10439-015-1343-2. [3] O. Moreno-Arotzena, G. Mendoza, M. Cóndor, T. Rüberg, J.M. García-Aznar, Inducing chemotactic and haptotactic cues in microfluidic devices for three-dimensional in vitro assays, Biomicrofluidics. 64122 (2014). doi:10.1063/1.4903948. [4] W.A. Farahat, L.B. Wood, I.K. Zervantonakis, A. Schor, S. Ong, D. Neal, R.D. Kamm, H.H. Asada, Ensemble analysis of angiogenic growth in three-dimensional microfluidic cell cultures., PLoS One. 7 (2012) e37333. doi:10.1371/journal.pone.0037333. [5] Y. Shin, S. Han, J.S. Jeon, K. Yamamoto, I.K. Zervantonakis, R. Sudo, R.D. Kamm, S. Chung, Microfluidic assay for simultaneous culture of multiple cell types on surfaces or within hydrogels, Nat Protoc. 7 (2012) 1247–1259. doi:10.1038/nprot.2012.051. [6] C. Del Amo, C. Borau, R. Gutiérrez, J. Asín, J.M. García-Aznar, Quantification of angiogenic sprouting under different growth factors in a microfluidic platform, J. Biomech. 49 (2016) 1340–1346. doi:10.1016/j.jbiomech.2015.10.026. [7] C. Del Amo, C. Borau, N. Movilla, J. Asín, J.M. Garcia-Aznar, Quantifying 3D chemotaxis in microfluidic-based chips with step gradients of collagen hydrogel concentrations, Integr. Biol. (2017) 1–27. doi:10.1039/C7IB00022G.