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Studying brain diseases and developing therapies requires versatile in vitro systems for long-term neuronal cultures. SH-SY5Y neuroblastoma cells are ideal for modeling neurodegenerative diseases. Although SH-SY5Y cells are commonly used in 2D cultures, 3D systems offer more physiologically relevant models. Studies have shown 3D culturing up to 7 days, but a simple, reproducible, and tunable system has yet to be identified. Cellulose holds potential to fulfill these needs. Cellulose and its derivatives are sustainable, cytocompatible, and ideal for synthesizing biocompatible hydrogels. Its abundance and ease of chemical modification make it a highly attractive biomaterial. This study explored nanocellulose-based hydrogels for promoting neuronal growth and morphogenesis. To enhance cell adhesion, a small amount of collagen was added to the hydrogel, and the resulting cell morphologies were analyzed and compared with those cultured in collagen and Matrigel. By chemically oxidizing cellulose and adjusting the blend, we developed composites that maintained neuronal viability for over 14 days in 3D cultures. Our findings show that nanocellulose-collagen composites offer superior cytocompatibility, promoting neuronal viability and neurite outgrowth more effectively than Matrigel and collagen. These tunable biomaterials support long-term 3D neuronal cultures, making them valuable for creating standardized models for disease research and drug development., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Mechanical signals are pivotal ingredients in how cells perceive and respond to their microenvironments, and to synthetic biomaterials that mimic them. In spite of increasing interest in mechanobiology, probing the effects of physical cues on cell behavior remains challenging for a cell biology laboratory without experience in fabrication of biocompatible materials. Hydrogels are ideal biomaterials recapitulating the physical cues that natural extracellular matrices (ECM) deliver to cells. Here, protocols are streamlined for the synthesis and functionalization of cell adhesive polyacrylamide-based (PAA-OH) and fully-defined polyethyleneglycol-based (PEG-RGD) hydrogels tuned at various rigidities for mechanobiology experiments, from 0.3 to >10 kPa.  The mechanosignaling properties of these hydrogels are investigated in distinct cell types by monitoring the activation state of YAP/TAZ. By independently modulating substrate stiffness and adhesiveness, it is found that although ECM stiffness represents an overarching mechanical signal, the density of adhesive sites does impact on cellular mechanosignaling at least at intermediate rigidity values, corresponding to normal and pathological states of living tissues. Using these tools, it is found that YAP/TAZ nuclear accumulation occurs when the projected area of the nucleus surpasses a critical threshold of approximatively 150 µm 2 . This work suggests the existence of distinct checkpoints for cellular mechanosensing., (© 2021 Wiley-VCH GmbH.)

In spite of tremendous advances made in the comprehension of mechanotransduction, implementation of mechanobiology assays remains challenging for the broad community of cell biologists. Hydrogel substrates with tunable stiffness are essential tool in mechanobiology, allowing to investigate the effects of mechanical signals on cell behavior. A bottleneck that slows down the popularization of hydrogel formulations for mechanobiology is the assessment of their stiffness, typically requiring expensive and sophisticated methodologies in the domain of material science. Here we overcome such barriers offering the reader protocols to set-up and interpret two straightforward, low cost and high-throughput tools to measure hydrogel stiffness: static macroindentation and micropipette aspiration. We advanced on how to build up these tools and on the underlying theoretical modeling. Specifically, we validated our tools by comparing them with leading techniques used for measuring hydrogel stiffness (atomic force microscopy, uniaxial compression and rheometric analysis) with consistent results on PAA hydrogels or their modification. In so doing, we also took advantage of YAP/TAZ nuclear localization as biologically validated and sensitive readers of mechanosensing, all in all presenting a suite of biologically and theoretically proven protocols to be implemented in most biological laboratories to approach mechanobiology. [ABSTRACT FROM AUTHOR]