Drug discovery is an expensive and lengthy process. Among the different phases, drug discovery and preclinical trials play an important role as only 5–10 of all drugs that begin preclinical tests proceed to clinical trials. Indeed, current high-throughput screening technologies are very expensive, as they are unable to dispense small liquid volumes in an accurate and quick way. Moreover, despite being simple and fast, drug screening assays are usually performed under static conditions, thus failing to recapitulate tissue-specific architecture and biomechanical cues present in vivo even in the case of 3D models. On the contrary, microfluidics might offer a more rapid and cost-effective alternative. Although considered incompatible with high-throughput systems for years, technological advancements have demonstrated how this gap is rapidly reducing. In this Review, we want to further outline the role of microfluidics in high-throughput drug screening applications by looking at the multiple strategies for cell seeding, compartmentalization, continuous flow, stimuli administration (e.g., drug gradients or shear stresses), and single-cell analyses.

Opportunely arranged micro/nano-scaled fibers represent an extremely attractive architecture for tissue engineering, as they offer an intrinsically porous structure, a high available surface, and an ideal microtopography for guiding cell migration. When fibers are made with naturally occurring polymers, matrices that closely mimic the architecture of the native extra-cellular matrix and offer specific chemical cues can be obtained. Along this track, electrospinning of collagen or gelatin is a typical and effective combination to easily prepare fibrous scaffolds with excellent properties in terms of biocompatibility and biomimicry, but an appropriate cross-linking strategy is required. Many common protocols involve the use of swelling solvents and can result in significant impairment of fibrous morphology and porosity. As a consequence, the efforts for processing gelatin into a fiber network can be vain, as a film-like morphology will be eventually presented to cells. However, this appears to be a frequently overlooked aspect. Here, the effect on fiber morphology of common cross-linking protocols was analyzed, and different strategies to improve the final morphology were evaluated (including alternative solvents, cross-linker concentration, mechanical constraint, and evaporation conditions). Finally, an optimized, fiber-preserving protocol based on carbodiimide (EDC) chemistry was defined.

Cancer is one of the most life-threatening diseases worldwide. Despite the huge efforts, the failure rate of therapies remains high due to cells heterogeneity, so physiologically relevant models are strictly necessary. Bioprinting is a technology able to form highly complex 3D tissue models and enables the creation of large-scale constructs. In cancer research, Matrigel^® is the most widely used matrix, but it is hardly bioprinted pure, without the use of any other bioink as reinforcement. Its complex rheological behavior makes the control with a standard bioprinting process nearly impossible. In this work, we present a customized bioprinting strategy to produce pure Matrigel^® scaffolds with good shape fidelity. To this aim, we realized a custom-made volumetric dispensing system and performed printability evaluations. To determine optimal printing parameters, we analyzed fibers spreading ratio on simple serpentines. After identifying an optimal flow rate of 86.68 ± 5.77 µL/min and a printing speed of 10 mm/min, we moved forward to evaluate printing accuracy, structural integrity and other key parameters on single and multi-layer grids. Results demonstrated that Matrigel^® was able to maintain its structure in both simple and complex designs, as well as in single and multilayer structures, even if it does not possess high mechanical strength. In conclusion, the use of volumetric dispensing allowed printing pure Matrigel^® constructs with a certain degree of shape fidelity on both single and multiple layers.

Sound is a crucial factor in everyday life, since it impacts on people feelings and reactions to circumstances. Therefore, classifying sounds based on experiential, physiological, and behavioral responses becomes a key factor in the under-standing of the relationship between sound and emotions. As reported in the literature, since using a single type of analysis results to be only partially reliable, there is an increasing demand of integration among the various kind of analyses. The aim of this study is to integrate both physiological and self-reported outcomes, in order to provide a more accurate information about the emotions induced by pleasant and unpleasant audio stimuli. In particular, several indices were extracted from physiological signals, which were matched with self-reported outcomes. The results of this study show that cardiac response in terms of sympathetic activation is significantly different for the two types of acoustic artefacts. To further support the physiological responses results, statistical analysis of Loudness, Roughness and Sharpness values was performed. The two types of stimuli seemed to be characterized by significantly different levels of Loudness and Roughness, which were found higher for unpleasant stimuli. Therefore, it is possible to conclude that only one type of measure is not always sufficient to characterize the emotional response to sounds and more than one measure is needed as indicator of listener emotions.

Current clinical strategies to repair peripheral nerve injuries draw on different approaches depending on the extent of lost tissue. Nerve guidance conduits (NGCs) are considered to be a promising, off-the-shelf alternative to autografts when modest gaps need to be repaired. Unfortunately, to date, the implantation of an NGC prevents the sacrifice of a healthy nerve at the price of suboptimal clinical performance. Despite the significant number of materials and fabrication strategies proposed, an ideal combination has not been yet identified. Validation and comparison of NGCs ultimately requires in vivo animal testing due to the lack of alternative models, but in the spirit of the 3R principles, a reliable in vitro model for preliminary screening is highly desirable. Nevertheless, more traditional in vitro tests, and direct cell seeding on the material in particular, are not representative of the actual regeneration scenario. Thus, we have designed a very simple set-up in the attempt to appreciate the relevant features of NGCs through in vitro testing, and we have verified its applicability using electrospun NGCs. To this aim, neural cells were encapsulated in a loose fibrin gel and enclosed within the NGC membrane. Different thicknesses and porosity values of two popular polymers (namely gelatin and polycaprolactone) were compared. Results indicate that, with specific implementation, the system might represent a useful tool to characterize crucial NGC design aspects.

Three-dimensional (3D) cell culture systems rely on the manipulation of a biologically derived matrix, typically soluble Basement Membrane Extract (sBME), in which cells or cellular aggregates, such as organoids, are suspended. This matrix provides mechanobiological support, promoting cellular processes. However, the handling of sBME-based matrices containing cellular constructs poses significant challenges due to their rheological properties. We developed an integrated bioprinting system to surpass the conventional pipetting, seeding and culture in multiwell plates. The system combines a fluidic cartridge with innovative 3D-printed biocompatible culture tools designed to host and preserve high-throughput microcultures of Patient-Derived Organoids (PDOs) in sBME. The miniaturized hanging-drop configuration enables extended culture periods and high-throughput imaging screenings. This comprehensive approach overcomes common issues associated with sBME, including sedimentation of cellular aggregates, premature gelation, and structural collapse, which negatively impact culture quality and reproducibility throughout the entire 3D culture workflow, from seeding to culture maintenance, and post-culture analyses.