Caspase-9, a cysteine-aspartate protease traditionally associated with intrinsic apoptosis, has recently emerged as having non-apoptotic roles, including influencing cell migration-an aspect that has received limited attention in existing studies. In our investigation, we aimed to explore the impact of caspase-9 on the migration and invasion behaviors of MDA-MB-231, a triple-negative breast cancer (TNBC) cell line known for its metastatic properties. We established a stable cell line expressing an inducible caspase-9 (iC9) in MDA-MB-231 and assessed their metastatic behavior using both monolayer and the 3D organotypic model in co-culture with human Foreskin fibroblasts (HFF). Our findings revealed that caspase-9 had an inhibitory effect on migration and invasion in both models. In monolayer culture, caspase-9 effectively suppressed the migration and invasion of MDA-MB-231 cells, comparable to the anti-metastatic agent panitumumab (Pan). Notably, the combination of caspase-9 and Pan exhibited a significant additional effect in reducing metastatic behavior. Interestingly, caspase-9 demonstrated superior efficacy compared to Pan in the organotypic model. Molecular analysis showed down regulation of epithelial-mesenchymal transition and migratory markers, in caspase-9 activated cells. Additionally, flow cytometry analysis indicated a cell cycle arrest. Moreover, pre-treatment with activated caspase-9 sensitized cells to the chemotherapy of doxorubicin, thereby enhancing its effectiveness. In conclusion, the anti-metastatic potential of caspase-9 presents avenues for the development of novel therapeutic approaches for TNBC/metastatic breast cancer. Although more studies need to figure out the exact involving mechanisms behind this behavior.

Signaling pathways are involved in key cellular functions from embryonic development to pathological conditions, with a pivotal role in tissue homeostasis and transformation. Although most signaling pathways have been intensively examined, most studies have been carried out in murine models or simple cell culture. We describe the dissection of the TGF-β signaling pathway in human tissue using CRISPR-Cas9 genetically engineered human keratinocytes (N/TERT-1) in a 3D organotypic skin model combined with quantitative proteomics and phosphoproteomics mass spectrometry. The use of human 3D organotypic cultures and genetic engineering combined with quantitative proteomics and phosphoproteomics is a powerful tool providing insight into signaling pathways in a human setting. The methods are applicable to other gene targets and 3D cell and tissue models. Key features • 3D organotypic models with genetically engineered human cells. • In-depth quantitative proteomics and phosphoproteomics in 2D cell culture. • Careful handling of cell cultures is critical for the successful formation of the organotypic cultures. • For complete details on the use of this protocol, please refer to Ye et al. 2022.

Uterine leiomyoma (UL) is a prevalent benign tumor in women that frequently gives rise to a multitude of reproductive complications. The use of suicide gene therapy has been proposed as a highly promising method for treating UL. To achieve successful gene therapy, it is essential to develop carriers that can efficiently transport nucleic acids into targeted cells and tissues. The instability of polyplexes in blood and other biological fluids is a crucial factor to consider when using non-viral carriers. In this study, we present serum-resistant and cRGD-modified DNA complexes for targeted delivery genes to UL cells. Ternary polyplexes were formed by incorporating cystine-cross-linked polyglutamic acid modified with histidine residues. We employed two techniques in the production of cross-linked polyanionic coating: matrix polymerization and oxidative polycondensation. In this study, we investigated the physicochemical properties of ternary DNA complexes, including the size and zeta-potential of the nanoparticles. Additionally, we evaluated cellular uptake, toxicity levels, transfection efficiency and specificity in vitro. The study involved introducing the HSV-TK gene into primary UL cells as a form of suicide gene therapy modeling. We have effectively employed ternary peptide-based complexes for gene delivery into the UL organtypic model. By implementing in situ suicide gene therapy, the increase in apoptosis genes expression was detected, providing conclusive evidence of apoptosis occurring in the transfected UL tissues. The results of the study strongly suggest that the developed ternary polyplexes show potential as a valuable tool in the implementation of suicide gene therapy for UL.

As part of the development of new approach methodologies (NAMs), numerous in vitro methods are being developed to characterize the potential toxicity of inhalable xenobiotics (gases, volatile organic compounds, polycyclic aromatic hydrocarbons, particulate matter, nanoparticles). However, the materials and methods employed are extremely diverse, and no single method is currently in use. Method standardization and validation would raise trust in the results and enable them to be compared. This four-part review lists and compares biological models and exposure methodologies before describing measurable biomarkers of exposure or effect. The first section emphasizes the importance of developing alternative methods to reduce, if not replace, animal testing (3R principle). The biological models presented are mostly to cultures of epithelial cells from the respiratory system, as the lungs are the first organ to come into contact with air pollutants. Monocultures or cocultures of primary cells or cell lines, as well as 3D organotypic cultures such as organoids, spheroids and reconstituted tissues, but also the organ(s) model on a chip are examples. The exposure methods for these biological models applicable to airborne compounds are submerged, intermittent, continuous either static or dynamic. Finally, within the restrictions of these models (i.e. relative tiny quantities, adhering cells), the mechanisms of toxicity and the phenotypic markers most commonly examined in models exposed at the air-liquid interface (ALI) are outlined.

Osteomyelitis is a pathological condition of the bone, frequently associated with the presence of infectious agents - namely Staphylococcus aureus - that induce inflammation and tissue destruction. Recent advances in the understanding of its pathophysiology and the identification of innovative therapeutic approaches were gathered from experimental in vitro and in vivo systems. However, cell culture models offer limited representativeness of the cellular functionality and the cell-cell and cell-matrix interactions, further failing to mimic the three-dimensional tissue organization; and animal models allow for limited mechanistic assessment given the complex nature of systemic and paracrine regulatory systems and are endorsed with ethical constraints. Accordingly, this study aims at the establishment and assessment of a new ex vivo bone infection model, upon the organotypic culture of embryonic chicken femurs colonized with S. aureus, highlighting the model responsiveness at the molecular, cellular, and tissue levels. Upon infection with distinct bacterial inoculums, data reported an initial exponential bacterial growth, followed by diminished metabolic activity. At the tissue level, evidence of S. aureus-mediated tissue destruction was attained and demonstrated through distinct methodologies, conjoined with decreased osteoblastic/osteogenic and increased osteoclastic/osteoclastogenic functionalities-representative of the osteomyelitis clinical course. Overall, the establishment and characterization of an innovative bone tissue infection model that is simple, reproducible, easily manipulated, cost-effective, and simulates many features of human osteomyelitis, further allowing the maintenance of the bone tissue\'s three-dimensional morphology and cellular arrangement, was achieved. Model responsiveness was further demonstrated, showcasing the capability to improve the research pipeline in bone tissue infection-related research.

BACKGROUND: Tissue fusion is a mechanism involved in the development of the heart, iris, genital tubercle, neural tube, and palate during embryogenesis. Failed fusion of the palatal shelves could result in cleft palate (CP), a common birth defect. Organotypic models constructed of human cells offer an opportunity to investigate developmental processes in the human. Previously, our laboratory developed an organoid model of the human palate that contains human mesenchyme and epithelial progenitor cells to study the effects of chemicals on fusion.
METHODS: Here, we developed an organoid model more representative of the embryonic palate that includes three cell types: mesenchyme, endothelial, and epithelial cells. We measured fusion by a decrease in epithelial cells at the contact point between the organoids and compared the effects of CP teratogens on fusion and toxicity in the previous and current organoid models. We further tested additional suspect teratogens in our new model.
RESULTS: We found that the three-cell-type model is more sensitive to fusion inhibition by valproic acid and inhibitors of FGF, BMP, and TGFβRI/II. In this new model, we tested other suspect CP teratogens and found that nocodazole, topiramate, and Y27632 inhibit fusion at concentrations that do not induce toxicity.
CONCLUSIONS: This sensitive human three-cell-type organotypic model accurately evaluates chemicals for cleft palate teratogenicity.

Bacterial pleural infections are associated with high mortality. Treatment is complicated due to biofilm formation. A common causative pathogen is Staphylococcus aureus (S. aureus). Since it is distinctly human-specific, rodent models do not provide adequate conditions for research. The purpose of this study was to examine the effects of S. aureus infection on human pleural mesothelial cells using a recently established 3D organotypic co-culture model of pleura derived from human specimens. After infection of our model with S. aureus, samples were harvested at defined time points. Histological analysis and immunostaining for tight junction proteins (c-Jun, VE-cadherin, and ZO-1) were performed, demonstrating changes comparable to in vivo empyema. The measurement of secreted cytokine levels (TNF-α, MCP-1, and IL-1β) proved host-pathogen interactions in our model. Similarly, mesothelial cells produced VEGF on in vivo levels. These findings were contrasted by vital, unimpaired cells in a sterile control model. We were able to establish a 3D organotypic in vitro co-culture model of human pleura infected with S. aureus resulting in the formation of biofilm, including host-pathogen interactions. This novel model could be a useful microenvironment tool for in vitro studies on biofilm in pleural empyema.

The oral cavity is inhabited by abundant microbes which continuously interact with the host and influence the host\'s health. Such host-microbe interactions (HMI) are dynamic and complex processes involving e.g. oral tissues, microbial communities and saliva. Due to difficulties in mimicking the in vivo complexity, it is still unclear how exactly HMI influence the transition between healthy status and disease conditions in the oral cavity. As an advanced approach, three-dimensional (3D) organotypic oral tissues (epithelium and mucosa/gingiva) are being increasingly used to study underlying mechanisms. These in vitro models were designed with different complexity depending on the research questions to be answered. In this review, we summarised the existing 3D oral HMI models, comparing designs and readouts, discussing applications as well as future perspectives.

The emergence of new SARS-CoV-2 variants and the replacement of preceding isolates have been observed through B.1.1.7, B.1.351, B.1.617.2, and B.1.1.529 lineages (corresponding to alpha, beta, delta, and omicron variants of concern (VoC), respectively). However, there is still a lack of biological evidence to which extent those VoC differ from the ancestral lineages. By exploiting human airway epithelial cell (HAEC) cultures, which closely resemble the human airway architecture and physiology, we report distinctive SARS-CoV-2 tropism in different respiratory tissues. In general, SARS-CoV-2 VoC predominantly infect and replicate in HAEC better than the progenitor USA-WA1 isolate or the BavPat1 isolate, which contains the D614G mutation, even though there is little to no difference between variants regarding their infectivity (i.e., virion-per-vRNA copy ratio). We also observe differential tissue-specific innate immunity activation between the upper and lower respiratory tissues in the presence of the virus. Our study provides better comprehension of the behavior of the different VoC in this physiologically relevant ex vivo model.

Cellular technologies are one of the most promising areas of biomedicine, which is based on the isolation of cells of various types, followed by their cultivation and use, or the use of their metabolic products, for medical purposes. Today, a significant part of biomedical research is carried out in vitro. On the other hand, organotypic culture can be used as a powerful model system and can complement cell culture and in vivo studies in different biomedical applications. Uterine leiomyoma (UL) is a very common benign tumor and often leads to many reproductive complications. Herein we describe a fast and reliable method of isolation and UL primary cells culturing along with the development of a UL organotypic model. We propose the usage of UL primary cells in experimental work at a first passage to prevent loss of driver mutations in MED12 and HMGA2 genes. New optimized conditions for the growth and maintenance of 2D and 3D models of uterine leiomyoma in vitro are suggested.