UNASSIGNED: Accurate cell segmentation and classification in three-dimensional (3D) images are vital for studying live cell behavior and drug responses in 3D tissue culture. Evaluating diverse cell populations in 3D cell culture over time necessitates non-toxic staining methods, as specific fluorescent tags may not be suitable, and immunofluorescence staining can be cytotoxic for prolonged live cell cultures.
UNASSIGNED: We aim to perform machine learning-based cell classification within a live heterogeneous cell culture population grown in a 3D tissue culture relying only on reflectance, transmittance, and nuclei counterstained images obtained by confocal microscopy.
UNASSIGNED: In this study, we employed a supervised convolutional neural network (CNN) to classify tumor cells and fibroblasts within 3D-grown spheroids. These cells are first segmented using the marker-controlled watershed image processing method. Training data included nuclei counterstaining, reflectance, and transmitted light images, with stained fibroblast and tumor cells as ground-truth labels.
UNASSIGNED: Our results demonstrate the successful marker-controlled watershed segmentation of 84% of spheroid cells into single cells. We achieved a median accuracy of 67% (95% confidence interval of the median is 65-71%) in identifying cell types. We also recapitulate the original 3D images using the CNN-classified cells to visualize the original 3D-stained image\'s cell distribution.
UNASSIGNED: This study introduces a non-invasive toxicity-free approach to 3D cell culture evaluation, combining machine learning with confocal microscopy, opening avenues for advanced cell studies.

Three-dimensional (3D) bioprinting culture models capable of reproducing the pathological architecture of diseases are increasingly advancing. In this study, 3D scaffolds were created using extrusion-based bioprinting method with alginate, gelatin, and hyaluronic acid to investigate the effects of hyaluronic acid on the physical properties of the bioscaffold as well as on the formation of liver cancer spheroids. Conformational analysis, rheological characterization, and swelling-degradation tests were performed to characterize the scaffolds. After generating spheroids from hepatocellular carcinoma cells on the 3D scaffolds, cell viability and proliferation assays were performed. Flow cytometry and immunofluorescence microscopy were used into examine the expression of albumin, CD44, and E-cadherin to demonstrate functional capability and maturation levels of the spheroid-forming cells. The results show that hyaluronic acid in the scaffolds correlates with spheroid formation and provides high survival rates. It is also associated with an increase in CD44 expression and a decrease in E-cadherin, while there is no significant change in the albumin expression in the cells. Overall, the findings demonstrate that hyaluronic acid in a 3D hydrogel scaffold supports spheroid formation and may induce stemness. We present a promising 3D scaffold model for enhancing liver cancer spheroid formation and mimicking solid tumors. This model also has the potential for further studies to examine stem cell properties in 3D models.

Osteoporosis represents a widespread and debilitating chronic bone condition that is increasingly prevalent globally. Its hallmark features include reduced bone density and heightened fragility, which significantly elevate the risk of fractures due to the decreased presence of mature osteoblasts. The limitations of current pharmaceutical therapies, often accompanied by severe side effects, have spurred researchers to seek alternative strategies. Adipose-derived stem cells (ADSCs) hold considerable promise for tissue repair, albeit they encounter obstacles such as replicative senescence in laboratory conditions. In comparison, employing ADSCs within three-dimensional (3D) environments provides an innovative solution, replicating the natural extracellular matrix environment while offering a controlled and cost-effective in vitro platform. Moreover, the utilization of photobiomodulation (PBM) has emerged as a method to enhance ADSC differentiation and proliferation potential by instigating cellular stimulation and facilitating beneficial performance modifications. This literature review critically examines the shortcomings of current osteoporosis treatments and investigates the potential synergies between 3D cell culture and PBM in augmenting ADSC differentiation towards osteogenic lineages. The primary objective of this study is to assess the efficacy of combined 3D environments and PBM in enhancing ADSC performance for osteoporosis management. This research is notably distinguished by its thorough scrutiny of the existing literature, synthesis of recent advancements, identification of future research trajectories, and utilization of databases such as PubMed, Scopus, Web of Science, and Google Scholar for this literature review. Furthermore, the exploration of biomechanical and biophysical stimuli holds promise for refining treatment strategies. The future outlook suggests that integrating PBM with ADSCs housed within 3D environments holds considerable potential for advancing bone regeneration efforts. Importantly, this review aspires to catalyse further advancements in combined therapeutic strategies for osteoporosis regeneration.

Breast cancer is the most prevalent cancer among women worldwide. Therapeutic strategies to control tumors and metastasis are still challenging. Three-dimensional (3D) spheroid-type systems more accurately replicate the features of tumors in vivo, working as a better platform for performing therapeutic response analysis. This work aimed to characterize the epithelial-mesenchymal transition and doxorubicin (dox) response in a mammary tumor spheroid (MTS) model. We evaluated the doxorubicin treatment effect on MCF-7 spheroid diameter, cell viability, death, migration and proteins involved in the epithelial-mesenchymal transition (EMT) process. Spheroids were also produced from tumors formed from 4T1 and 67NR cell lines. MTSs mimicked avascular tumor characteristics, exhibited adherens junction proteins and independently produced their own extracellular matrix. Our spheroid model supports the 3D culturing of cells isolated from mice mammary tumors. Through the migration assay, we verified a reduction in E-cadherin expression and an increase in vimentin expression as the cells became more distant from spheroids. Dox promoted cytotoxicity in MTSs and inhibited cell migration and the EMT process. These results suggest, for the first time, that this model reproduces aspects of the EMT process and describes the potential of dox in inhibiting the metastatic process, which can be further explored.

Aim: This work aims to standardize the three-dimensional hydroxyethyl-alginate-gelatin (HAG) scaffold as a model to evaluate Aspergillus fumigatus biofilm and antifungal treatments. Methods: The scaffold was characterized by physical, rheological and microscopic analyses; the antibiofilm action was evaluated by determination of cfu and metabolic activity. Results: The scaffold was non-toxic showing stability in aqueous media, swelling capacity, elasticity and had homogeneously distributed pores averaging 190 μm. The A. fumigatus biofilm established itself very well on the scaffold and treatment with amphotericin B and voriconazole reduced viable cells and metabolic activity. Conclusion: The HAG scaffold proved to be a model to mimic lung parenchyma, suitable for establishing a 3D biofilm culture of A. fumigatus and evaluating the efficacy of antifungals.
[Box: see text].

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.

UNASSIGNED: The ability to observe and monitor cell density and morphology has been imperative for assessing the health of a cell culture and for producing high quality, high yield cell cultures for decades. Microcarrier-based cultures, used for large-scale cellular expansion processes, are not compatible with traditional visualization-based methods, such as widefield microscopy, due to their thickness and material composition.
UNASSIGNED: Here, we assess the optical imaging compatibilities of commercial polystyrene microcarriers versus custom-fabricated gelatin methacryloyl (gelMA) microcarriers for non-destructive and non-invasive visualization of the entire microcarrier surface, direct cell enumeration, and sub-cellular visualization of mesenchymal stem/stromal cells.
UNASSIGNED: Mie scattering and wavefront error simulations of the polystyrene and gelMA microcarriers were performed to assess the potential for elastic scattering-based imaging of adherent cells. A Zeiss Z.1 light-sheet microscope was adapted to perform light-sheet tomography using label-free elastic scattering contrast from planar side illumination to achieve optical sectioning and permit non-invasive and non-destructive, in toto, three-dimensional, high-resolution visualization of cells cultured on microcarriers.
UNASSIGNED: The polystyrene microcarrier prevents visualization of cells on the distal half of the microcarrier using either fluorescence or elastic scattering contrast, whereas the gelMA microcarrier allows for high fidelity visualization of cell morphology and quantification of cell density using light-sheet fluorescence microscopy and tomography.
UNASSIGNED: The combination of optical-quality gelMA microcarriers and label-free light-sheet tomography will facilitate enhanced control of bioreactor-microcarrier cell culture processes.

OBJECTIVE: The aim of the present study was to assess the cytocompatibility of epoxy resin-based AH Plus Jet (Dentsply De Trey, Konstanz, Germany), Sealer Plus (MK Life, Porto Alegre, Brazil), calcium silicate-based Bio-C Sealer (Angelus, Londrina, PR, Brazil), Sealer Plus BC (MK Life) and AH Plus BC (Dentsply) through a tridimensional (3D) culture model of human osteoblast-like cells.
METHODS: Spheroids of MG-63 cells were produced and exposed to fresh root canal sealers extracts by 24 h, and the cytotoxicity was assessed by the Lactate Dehydrogenase assay (LDH). The distribution of dead cells within the microtissue was assessed by fluorescence microscopy, and morphological effects were investigated by histological analysis. The secreted inflammatory mediators were detected in cell supernatants through flow luminometry (XMap Luminex).
RESULTS: Cells incubated with AH Plus Jet, AH Plus BC, Sealer Plus BC and Bio-C Sealer extracts showed high rates of cell viability, while the Sealer Plus induced a significant reduction of cell viability, causing reduction on the spheroid structure. Sealer Plus and Seaker Plus BC caused alterations on 3D microtissue morphology. The AH Plus BC extract was associated with the downregulation of secretion of pro-inflammatory cytokines IL-5, IL-7, IP-10 and RANTES.
CONCLUSIONS: The new AH Plus BC calcium silicate-based endodontic sealer did not reduce cell viability in vitro, while led to the downregulation of pro-inflammatory cytokines.
CONCLUSIONS: Choosing the appropriate endodontic sealer is a crucial step. AH Plus BC demonstrated high cell viability and downregulation of pro-inflammatory cytokines, appearing reliable for clinical use, while Sealer Plus presented lower cytocompatibility.

Cell culture meat is based on the scaled-up expansion of seed cells. The biological differences between seed cells from large yellow croakers in the two-dimensional (2D) and three-dimensional (3D) culture systems have not been explored. Here, satellite cells (SCs) from large yellow croakers (Larimichthys crocea) were grown on cell climbing slices, hydrogels, and microcarriers for five days to analyze the biological differences of SCs on different cell scaffolds. The results exhibited that SCs had different cell morphologies in 2D and 3D cultures. Cell adhesion receptors (Itgb1andsdc4) and adhesion spot markervclof the 3D cultures were markedly expressed. Furthermore, myogenic decision markers (Pax7andmyod) were significantly enhanced. However, the expression of myogenic differentiation marker (desmin) was significantly increased in the microcarrier group. Combined with the transcriptome data, this suggests that cell adhesion of SCs in 3D culture was related to the integrin signaling pathway. In contrast, the slight spontaneous differentiation of SCs on microcarriers was associated with rapid cell proliferation. This study is the first to report the biological differences between SCs in 2D and 3D cultures, providing new perspectives for the rapid expansion of cell culture meat-seeded cells and the development of customized scaffolds.

Caulerpa lentillifera is rich in polysaccharides, and its polysaccharides show a significant effect in different biological activities including anti-cancer activity. As an edible algae-derived polysaccharide, exploring the role of colon cancer can better develop the application from a dietary therapy perspective. However, more in-depth studies of C. lentillifera polysaccharide on anti-colon cancer activity and mechanism are needed. In this study, we found that Caulerpa lentillifera polysaccharides (CLP) showed potential anti-colon cancer effect on human colon cancer cell HT29 in monolayer (IC50 = 1.954 mg/mL) and spheroid (IC50 = 0.402 mg/mL). Transcriptomics and metabolomics analyses revealed that CLP had an inhibitory effect on HT29 3D spheroid cells by activating aminoacyl-tRNA biosynthesis as well as arginine and proline metabolism pathways. Furthermore, the anti-colon cancer effects of CLP were confirmed through other human colon cancer cell HCT116 and LoVo in monolayer cells (IC50 = 1.890 mg/mL and 1.437 mg/mL, respectively) and 3D spheroid cells (IC50 = 0.344 mg/mL and 0.975 mg/mL, respectively), and three patient-derived organoids with IC50 values of 6.333-8.780 mg/mL. This study provided basic data for the potential application of CLP in adjuvant therapeutic food for colon cancer on multiple levels, while further investigation of detailed mechanism in vivo was still required.