A micro-perfusion bioreactor for on line investigation of ECM remodeling under hydrodynamic and biochemical stimulation

Tissue-on-chip (TOC) systems aim at replicating complex biological dynamics in vitro with the potential either to improve the understanding of human biology or to develop more accurate therapeutic strategies. To replicate faithfully the intricate interrelationships between cells and their surrounding microenvironment, the three-dimensional (3D) tissue model must possess a responsive extracellular matrix (ECM). ECM remodeling plays a pivotal role in guiding cells and tissues functions and such aspect is somewhat denied during in vitro studies. For this purpose, we fabricated a micro-perfusion bioreactor capable to sustain the viability of 3D engineered tissue models recapitulating the process of the native ECM deposition and assembly. Engineered human dermis micro-tissue precursors (HD-mu TP) were used as building blocks to generate a final tissue. HD-mu TP were loaded in the perfusion space of the micro-perfusion bioreactor and, under the superimposition of different fluid dynamic regimes and biochemical stimulation, they synthesized new collagen proteins that were, then, assembled in the perfusion space forming a continuum of cells embedded in their own ECM. The micro-perfusion bioreactor was fabricated to allow the on-line monitoring of the oxygen consumption and the assembly of the newly formed collagen network via real time acquisition of the second harmonic generation (SHG) signal. The possibility to detect the collagen reorganization due to both fluid dynamic and biochemical stimulation, let us to define the optimal perfusion configuration in order to obtain a TOC system based on an endogenous and responsive ECM.