RESOURCES

Abstract
Abstract Although being invaluable tools in biomedical research, traditional 2D cell culture models often fail in recapitulating complex environments, limiting their predictive power. To address this limitation, it have developed an open-source, low-cost system that combines long-term 3D cell culture under controlled perfusion conditions with live-cell imaging. By integrating bioprinted extracellular matrix-based scaffolds, this study mimics mechanical and biochemical cues experienced by cells within complex tissue contexts. Here, this system is used to generate a model of the cerebrospinal fluid-filled subarachnoid space to study responses of resident cell types such as meningothelial cells to altered fluid flow conditions. Using fluorescent biosensors, it demonstrates that meningothelial cells respond to modulated fluid flow by differentially activating focal adhesion kinase, a key mechanosensor. The model thus not only provides a powerful platform for investigating the impact of mechanical and other cues on cellular responses, but also bears great potential for the generation of impactful cell biological and pathophysiological models.

Abstract
The demand for high-throughput and scalable cell expansion platforms that can accommodate diverse cell types remains a critical requirement across various biomedical fields. Fibronectin (Fn), an essential component of the extracellular matrix (ECM), has been used as a conformal surface coating for two-dimensional (2D) cell culture systems. However, the soluble, globular Fn used for 2D coatings differs structurally from the native Fn, which possesses a three-dimensional (3D) fibrillar structure. Herein, a large-scale engineered ECM (EECM) cell expansion platform based on a 3D fibrillar Fn network spanning over centimeters is presented. Extended fibrillar networks are formed by shearing dilute Fn solutions over tessellated polymeric scaffolds, which are conveniently prepared by 3D printing. The structure and size of the Fn-based 3D EECM scaffold are optimized by evaluating the proliferation of a colorectal tumor cell line, CT26, commonly used in the in vivo tumor immunotherapy models. The 3D EECM scaffolds support a fourfold more efficient tumor cell expansion than a conventional 2D culture system, demonstrating the potential efficacy in supporting the robust expansion of cancer cells ex vivo with an eye on cancer immunotherapy.

Abstract
Human mesenchymal stem cells (HMSCs) are important for cell-based therapies. However, the success of HMSC therapy requires large-scale in vitro expansion of these multipotent cells. The traditional expansion of HMSCs on tissue-culture-treated stiff polystyrene induces significant changes in their shape, multipotency, and secretome, leading to early senescence and subdued paracrine activity. To enhance their therapeutic potential, here, we have developed two-dimensional soft hydrogels with imprinted microscale aligned grooves for use as HMSC culture substrates. We showed that, depending on the dimensions of the topographical features, these substrates led to lower cellular spreading and cytoskeletal tension, maintaining multipotency and osteogenic and adipogenic differentiate potential, while lowering cellular senescence. We also observed a greater capacity of HMSCs to produce anti-inflammatory cytokines after short-term priming on these hydrogel substrates. Overall, these soft hydrogels with unique surface topography have shown great promise as in vitro culture substrates to maximize the therapeutic potential of HMSCs.

Abstract
Compared to standard 2D culture systems, new methods for 3D cell culture of adipocytes could provide more physiologically accurate data and a deeper understanding of metabolic diseases such as diabetes. By resuspending living cells in a bioink of nanocellulose and hyaluronic acid, we were able to print 3D scaffolds with uniform cell distribution. After one week in culture, cell viability was 95%, and after two weeks the cells displayed a more mature phenotype with larger lipid droplets than standard 2D cultured cells. Unlike cells in 2D culture, the 3D bioprinted cells did not detach upon lipid accumulation. After two weeks, the gene expression of the adipogenic marker genes PPAR γ and FABP4 was increased 2.0- and 2.2-fold, respectively, for cells in 3D bioprinted constructs compared with 2D cultured cells. Our 3D bioprinted culture system produces better adipogenic differentiation of mesenchymal stem cells and a more mature cell phenotype than conventional 2D culture systems.