Biomechanical Characterization at the Cell Scale: Present and Prospects

The rapidly growing field of mechanobiology demands for robust and reproducible characterization of cell mechanical properties. Recent achievements in understanding the mechanical regulation of cell fate largely rely on technological platforms capable of probing the mechanical response of living cells and their physico–chemical interaction with the microenvironment. Besides the established family of atomic force microscopy (AFM) based methods, other approaches include optical, magnetic, and acoustic tweezers, as well as sensing substrates that take advantage of biomaterials chemistry and microfabrication techniques. In this review, we introduce the available methods with an emphasis on the most recent advances, and we discuss the challenges associated with their implementation

YAP-TEAD1 control of cytoskeleton dynamics and intracellular tension guides human pluripotent stem cell mesoderm specification

The tight regulation of cytoskeleton dynamics is required for a number of cellular processes, including migration, division and differentiation. YAP-TEAD respond to cell-cell interaction and to substrate mechanics and, among their downstream effects, prompt focal adhesion (FA) gene transcription, thus contributing to FA-cytoskeleton stability. This activity is key to the definition of adult cell mechanical properties and function. Its regulation and role in pluripotent stem cells are poorly understood. Human PSCs display a sustained basal YAP-driven transcriptional activity despite they grow in very dense colonies, indicating these cells are insensitive to contact inhibition. PSC inability to perceive cell-cell interactions can be restored by tampering with Tankyrase enzyme, thus favouring AMOT inhibition of YAP function. YAP-TEAD complex is promptly inactivated when germ layers are specified, and this event is needed to adjust PSC mechanical properties in response to physiological substrate stiffness. By providing evidence that YAP-TEAD1 complex targets key genes encoding for proteins involved in cytoskeleton dynamics, we suggest that substrate mechanics can direct PSC specification by influencing cytoskeleton arrangement and intracellular tension. We propose an aberrant activation of YAP-TEAD1 axis alters PSC potency by inhibiting cytoskeleton dynamics, thus paralyzing the changes in shape requested for the acquisition of the given phenotype

Highly Tailorable and Monodisperse Porous Beads via Microfluidics

In tissue engineering practice, a scaffold is often needed to deliver cells to the desired body site needing to be repaired. Scaffolds supporting cells can be either implanted through a surgical operation or injected through a laparoscopic device. The latter option is a first-choice in cases where a small and irregularly shaped defect needs to be regenerated. In such circumstances, the cell carrier has to be miniaturised while maintaining the morphological features that make a scaffold an efficient cell culture support, i.e. a uniform and adequate porous texture in terms of pore and interconnect dimensions. In this work, we illustrate a novel and powerful method for the manufacturing of monodisperse porous beads of tailorable dimension (in the range ~ 300÷1500 m) and with an internal porous texture characterised by uniformly sized and fully interconnected pores. The fabrication method relies on the use of a flow-focusing microfluidic chip that generates a monodisperse oil-in-water emulsion (panel b). The aqueous phase of the emulsion contains a biopolymer and an appropriate surfactant. Here, we demonstrate that by extruding the emulsion through a needle immersed in a perfluorinated oil on top of which a coagulating aqueous bath is stratified and by applying a pulsed electrical field (panel a), it is possible to precisely control the size of the emulsion droplets detached from the needle. As soon as the emulsion droplets reach the interface between the perfluorinated oil and coagulating bath, they instantaneously solidify. An inverse relationship exist between intensity of the applied voltage and beads dimension (panels c,d,e). The presented process is very repeatable and brings about to beads rigorously monodisperse in size (panel f). Finally, such microbeads demonstrated to be a successful cell carrier

Quercetin and hydroxytyrosol as modulators of hepatic steatosis: A NAFLD-on-a-chip study

Organs-on-chip (OoCs) are catching on as a promising and valuable alternative to animal models, in line with the 3Rs initiative. OoCs enable the creation of three-dimensional (3D) tissue microenvironments with physiological and pathological relevance at unparalleled precision and complexity, offering new opportunities to model human diseases and to test the potential therapeutic effect of drugs, while overcoming the limited predictive accuracy of conventional 2D culture systems. Here, we present a liver-on-a-chip model to investigate the effects of two naturally occurring polyphenols, namely quercetin and hydroxytyrosol, on nonalcoholic fatty liver disease (NAFLD) using a high-content analysis readout methodology. NAFLD is currently the most common form of chronic liver disease; however, its complex pathogenesis is still far from being elucidated, and no definitive treatment has been established so far. In our experiments, we observed that both polyphenols seem to restrain the progression of the free fatty acid-induced hepatocellular steatosis, showing a cytoprotective effect due to their antioxidant and lipid-lowering properties. In conclusion, the findings of the present work could guide novel strategies to contrast the onset and progression of NAFLD

Neurovascular signals in amyotrophic lateral sclerosis

none8Sorrentino, Stefano; Polini, Alessandro; Arima, Valentina; Romano, Alessandro; Quattrini, Angelo; Gigli, Giuseppe; Mozetic, Pamela; Moroni, LorenzoSorrentino, Stefano; Polini, Alessandro; Arima, Valentina; Romano, Alessandro; Quattrini, Angelo; Gigli, Giuseppe; Mozetic, Pamela; Moroni, Lorenz

Pluronic F127 Hydrogel Characterization and Biofabrication in Cellularized Constructs for Tissue Engineering Applications

A new method for printing cellularised scaffolds from thermosensitive hydrogels was here proposed. Pluronic F127 solutions and hydrogels in water-based media (15-40%w/v) were investigated by rheological analysis and tube inverting test. Pluronic F127 hydrogel with 25%w/v concentration was selected as bioink due to its fast gelation at 37°C (5min), suitable viscoelastic properties (G’= 16500Pa at 37°C), pseudoplastic behaviour and fast viscosity recovery after shearing (approximately 5 s). Not cellularised and cellularised (with Balb/3T3 fibroblasts) scaffolds with a 0°/90° pattern were fabricated by additive manufacturing technique. Cells were well distributed along scaffold filaments and cell viability was preserved during printing