Advanced materials technologies to unravel mechanobiological phenomena.

Advancements in materials-driven mechanobiology have yielded significant progress. Mechanobiology explores how cellular and tissue mechanics impact development, physiology, and disease, where extracellular matrix (ECM) dynamically interacts with cells. Biomaterial-based platforms emulate synthetic ECMs, offering precise control over cellular behaviors by adjusting mechanical properties. Recent technological advances enable in vitro models replicating active mechanical stimuli in vivo. These models manipulate cellular mechanics even at a subcellular level. In this review we discuss recent material-based mechanomodulatory studies in mechanobiology. We highlight the endeavors to mimic the dynamic properties of native ECM during pathophysiological processes like cellular homeostasis, lineage specification, development, aging, and disease progression. These insights may inform the design of accurate in vitro mechanomodulatory platforms that replicate ECM mechanics.

3D engineered tissue models for studying human-specific infectious viral diseases.

Viral infections cause damage to various organ systems by inducing organ-specific symptoms or systemic multi-organ damage. Depending on the infection route and virus type, infectious diseases are classified as respiratory, nervous, immune, digestive, or skin infections. Since these infectious diseases can widely spread in the community and their catastrophic effects are severe, identification of their causative agent and mechanisms underlying their pathogenesis is an urgent necessity. Although infection-associated mechanisms have been studied in two-dimensional (2D) cell culture models and animal models, they have shown limitations in organ-specific or human-associated pathogenesis, and the development of a human-organ-mimetic system is required. Recently, three-dimensional (3D) engineered tissue models, which can present human organ-like physiology in terms of the 3D structure, utilization of human-originated cells, recapitulation of physiological stimuli, and tight cell-cell interactions, were developed. Furthermore, recent studies have shown that these models can recapitulate infection-associated pathologies. In this review, we summarized the recent advances in 3D engineered tissue models that mimic organ-specific viral infections. First, we briefly described the limitations of the current 2D and animal models in recapitulating human-specific viral infection pathology. Next, we provided an overview of recently reported viral infection models, focusing particularly on organ-specific infection pathologies. Finally, a future perspective that must be pursued to reconstitute more human-specific infectious diseases is presented.

Microtumor Spheroids Provide a Model for Studying Molecules Involved in Vascular Organization: An Illustrative Study for VE-cadherin.

BACKGROUND/AIM: A growing body of research is contributing to the development of three-dimensional (3D) tissue models to close the gap between two-dimensional (2D) cell culture and animal models. Here, we report fundamental studies to confirm the modification of vascular endothelial (VE)-cadherin by a tumor microenvironment using 2D and 3D in vitro models of triple-negative breast cancer cells co-cultured with endothelial cells. MATERIALS AND METHODS: Breast cancer cells were cultivated as a monolayer (2D) on plates for 5 days or as microtumor spheroids (3D) with endothelial cells for up to 6 days. Phosphotyrosine-containing protein panels were analyzed in both cell types and upon co-culture. Microtumor spheroid size was evaluated via phase contrast microscopy. The content of VE-cadherin and phospho-VE-cadherin was determined. The effect of microtumor spheroid on the capillary network formed by endothelial cells was quantified by ImageJ Angiogenesis Analyzer. Sunitinib was used to determine drug efficacy in this model. RESULTS: The activity of signaling pathways in endothelial cells, including phosphorylation of Y685-VE-cadherin, was increased by the presence of breast cancer cells. In the 3D co-culture system, we established a ratio of the two cell types which allowed viability for 6 days. As a proof-of-concept of the 3D co-culture system for the process of drug discovery and development, we used the system to quantify the efficacy of sunitinib on the phosphorylation of VE-cadherin. CONCLUSION: In summary, we established 2D and 3D breast cancer-endothelial cell test systems compatible for detection of minimally tyrosine-phosphorylated proteins including VE-cadherin. The systems are capable of quantifying the effect of drugs on a tissue model of angiogenesis. This is a step towards developing tools for drug-efficacy testing that do not rely on live animals.

Exploring metal availability in the natural niche of Streptococcus pneumoniae to discover potential vaccine antigens.

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite for pneumococcal transmission and disease. Current vaccines protect only against disease and colonization caused by a limited number of serotypes, consequently allowing serotype replacement and transmission. Therefore, the development of a broadly protective vaccine against colonization, transmission and disease is desired but requires a better understanding of pneumococcal adaptation to its natural niche. Hence, we measured the levels of free and protein-bound transition metals in human nasal fluid, to determine the effect of metal concentrations on the growth and proteome of S. pneumoniae. Pneumococci cultured in medium containing metal levels comparable to nasal fluid showed a highly distinct proteomic profile compared to standard culture conditions, including the increased abundance of nine conserved, putative surface-exposed proteins. AliA, an oligopeptide binding protein, was identified as the strongest protective antigen, demonstrated by the significantly reduced bacterial load in a murine colonization and a lethal mouse pneumonia model, highlighting its potential as vaccine antigen.

A Microfluidic Tumor-on-a-Chip for Assessing Multifunctional Liposomes' Tumor Targeting and Anticancer Efficacy.

Two principal methods for cancer drug testing are widely used, namely, in vitro 2D cell monolayers and in vivo animal models. In vitro 2D culture systems are simple and convenient but are unable to capture the complexity of biological processes. Animal models are costly, time-consuming, and often fail to replicate human activity. Here a microfluidic tumor-on-a-chip (TOC) model designed for assessing multifunctional liposome cancer targeting and efficacy is presented. The TOC device contains three sets of hemispheric wells with different sizes for tumor spheroid formation and evaluation of liposomes under a controlled flow condition. There is good agreement between time-elapsed tumor targeting of fluorescent liposomes in the TOC model and in in vivo mouse models. Evaluation of the anticancer efficacy of four PTX-loaded liposome formulations shows that compared to 2D cell monolayers and 3D tumor spheroid models, the TOC model better predicts the in vivo anticancer efficacy of targeted liposomes. Lastly, the TOC model is used to assess the effects of flow rates and tumor size on treatment outcome. This study demonstrates that the TOC model provides a convenient and powerful platform for rapid and reliable cancer drug evaluation.

Establishment of Microfluidic Spheroid Cultures for Biomedical Applications.

Multicellular spheroid is a three-dimensional (3D) cell culture model that mimics cancer tumor environment. Its widespread use for anticancer therapy evaluation is nowadays limited by accessibility of 3D compatible assays. Here, a microfluidic system for spheroid formation, culture and analysis is presented. The system is compatible with standard microplate readers. The microfluidic chip enables long-term 3D cell culture and in situ monitoring of cellular viability. Moreover, design of the assay enables observation of delayed type of toxicity or application of repeated doses of a drug.

Evaluation of assays for drug efficacy in a three-dimensional model of the lung.

BACKGROUND: The focus of the outlined work is the establishment of a three-dimensional lung model for various drug-screening applications. METHODS: The non-small cell lung cancer (NSCLC) cell line Colo699 was cultivated as monolayer (2D) on plates for 5 days or as microtissues (3D) using a hanging-drop system for 5 and 10 days. Cells and microtissues were treated with afatinib (10-80 µM), cisplatin (100-800 µM) or vinorelbine (25-200 µM) for 24 or 48 hours (h). Cell proliferation and viability were analysed by intra-cellular adenosine triphosphate (ATP) and lactate dehydrogenase release (LDH) assays, annexin V/propidium iodide (PI) staining, and cell cycle determination. Microtissue morphology and size, as well as cell death were evaluated via phase contrast microscopy. RESULTS: Our results demonstrate the valid determination of viability and cell death using established assays in the 3D system for drug testing. The comparison of ATP, LDH and cytometry data showed moderate (0.40) to very strong (0.99) correlations. Thereby, we observed partially significant differences in drug efficacy between microtissues and 2D cultures dependent from the applied treatment and read-out method. Altogether, microtissues developed resistance to cisplatin and vinorelbine; but remained more vulnerable to afatinib. These findings were confirmed with microscopy. CONCLUSION: In summary, we established an NSCLC 3D test system with multiple assays compatible for drug-testing applications of substances with different mechanisms of action. In addition, our data support the usage of microtissues as more accurate tools for drug-efficacy testing with the possibility of long-term cultivation and treatment.