WIDE-RANGE COMPRESSION FORCES TO INVESTIGATE SINGLE-CELL IN-FLOW MOTIONS, MECHANOBIOLOGICAL RESPONSES AND INTRACELLULAR DELIVERY

The aim of the PhD work is to create a new microfluidic approach to finely tune applied in-flow forces in order to explore controlled single-cell deformation. In fact, we propose a microfluidic device based on compression forces arising from a viscoelastic fluid solution that firstly align cells and then deform them. By simply changing the rheological properties and the imposed fluid-flow conditions, our approach represents an easy-to-use and versatile tool to collect a comprehensive mapping of single-cell properties, investigating both biophysical and biomechanical characteristics. In a wide-range of applied compression, we observe how different degrees of deformation lead to cell-specific deformation-dependent in-flow dynamics, which correlate the classical deformation parameters (e.g. cell aspect-ratio), with dynamic quantities (e.g. revolution time of rotation during in-flow motion). Thus, a precise in-flow label-free cell phenotyping is achieved allowing the distinction of different cell classes. The observation of different degrees of deformation corresponding to variable compression, lead us to interrogate the inner cell structures possibly involved into the mechanical responses. We demonstrate that re-organization phenomena of actin cortex and microtubules as well as of nuclear envelope and chromatin content, occur. Also in this case, cell-specific responses are collected, allowing us to distinguish healthy from pathological cells depending on the structural mechanical reaction. Furthermore, by playing with the high levels of compression, we show preliminary results about the possibility to induce a nanoparticle intracellular delivery process by escaping physiological endocytosis. In fact, cells result to be able to incorporate nanoparticles into the cytoplasm, without involving a vesicle formation for the entry. These outcome open up new interesting scenarios about the possibility to use the microfluidic device as a platform for cell phenotyping and intracellular delivery, properly engineered for both diagnostic and therapeutic purposes

Advanced label-free cellular identification in flow by collaborative coherent imaging techniques

We investigated subclasses of living peripheral blood cells in a microfluidic-based system, with the aim to characterize their morphometric and optical properties, and to track their position in flow in a label-free modality. We employed two coherent imaging techniques: a scattering approach of precisely aligned single cells, and a digital holography approach to achieve optical cell reconstructions in flow. Cells were first 3D-aligned in round shaped capillary and subsequently measured in a following square shaped channel. Results were obtained at two fixed measurement positions, the first one was chosen close to the entrance of the measurement channel to ensure 3D cell alignment for scattering investigations; the second was placed 15 mm after to study additional cell properties by digital holography and to investigate possible variations of axial cell positions. First, the refractive index, ratio of the nucleus over cytoplasm, and cell dimension were investigated from scattering investigations. Further quantitative phase-contrast reconstructions by digital holography were employed to calculate surface area, dry mass, biovolume and positions of cells using the scattering outcomes as input parameters. The precise cell alignment at the first measurement position could be confirmed. At the second measurement position a full label-free characterization of cell classes in distinct vertical positions was realized and supported by applied microfluidic force calculations, which can be used to align, deform and/or separate cells. Our results confirm the possibility to differentiate cell classes in flow, thus avoiding chemical cell staining or labeling, which are nowadays used