Novel 3D human trophoblast culture to explore T. cruzi infection in the placenta.

Introduction: Human trophoblastic cell lines, such as BeWo, are commonly used in 2D models to study placental Trypanosoma cruzi infections. However, these models do not accurately represent natural infections. Three-dimensional (3D) microtissue cultures offer a more physiologically relevant in vitro model, mimicking tissue microarchitecture and providing an environment closer to natural infections. These 3D cultures exhibit functions such as cell proliferation, differentiation, morphogenesis, and gene expression that resemble in vivo conditions. Methods: We developed a 3D culture model using the human trophoblastic cell line BeWo and nonadherent agarose molds from the MicroTissues® 3D Petri Dish® system. Both small (12-256) and large (12-81) models were tested with varying initial cell numbers. We measured the diameter of the 3D cultures and evaluated cell viability using Trypan Blue dye. Trophoblast functionality was assessed by measuring ß-hCG production via ELISA. Cell fusion was evaluated using confocal microscopy, with Phalloidin or ZO-1 marking cell edges and DAPI staining nuclei. T. cruzi infection was assessed by microscopy and quantitative PCR, targeting the EF1-α gene for T. cruzi and GAPDH for BeWo cells, using three parasite strains: VD (isolated from a congenital Chagas disease infant and classified as Tc VI), and K98 and Pan4 (unrelated to congenital infection and classified as Tc I). Results: Seeding 1000 BeWo cells per microwell in the large model resulted in comparable cellular viability to 2D cultures, with a theoretical diameter of 408.68 ± 12.65 µm observed at 5 days. Functionality, assessed through ß-hCG production, exceeded levels in 2D cultures at both 3 and 5 days. T. cruzi infection was confirmed by qPCR and microscopy, showing parasite presence inside the cells for all three tested strains. The distribution and progression of the infection varied with each strain. Discussion: This innovative 3D model offers a simple yet effective approach for generating viable and functional cultures susceptible to T. cruzi infection, presenting significant potential for studying the placental microenvironment.

Enhancing scaffold-free spheroid models: 3D cell bioprinting method for metastatic HSC3-Oral squamous carcinoma cell line.

3D in vitro systems offer advantages over the shortcomings of two-dimensional models by simulating the morphological and functional features of in vivo-like environments, such as cell-cell and cell-extracellular matrix interactions, as well as the co-culture of different cell types. Nevertheless, these systems present technical challenges that limit their potential in cancer research requiring cell line- and culture-dependent standardization. This protocol details the use of a magnetic 3D bioprinting method and other associated techniques (cytotoxicity assay and histological analysis) using oral squamous cell carcinoma cell line, HSC3, which offer advantages compared to existing widely used approaches. This protocol is particularly timely, as it validates magnetic bioprinting as a method for the rapid deployment of 3D cultures as a tool for compound screening and development of heterotypic cultures such as co-culture of oral squamous cell carcinoma cells with cancer-associated fibroblasts (HSC3/CAFs).

Naphthoquinone-Quinolone Hybrids with Antitumor Effects on Breast Cancer Cell Lines-From the Synthesis to 3D-Cell Culture Effects.

Breast cancer stands as one of the foremost cause of cancer-related deaths globally, characterized by its varied molecular subtypes. Each subtype requires a distinct therapeutic strategy. Although advancements in treatment have enhanced patient outcomes, significant hurdles remain, including treatment toxicity and restricted effectiveness. Here, we explore the anticancer potential of novel 1,4-naphthoquinone/4-quinolone hybrids on breast cancer cell lines. The synthesized compounds demonstrated selective cytotoxicity against Luminal and triple-negative breast cancer (TNBC) cells, which represent the two main molecular types of breast cancer that depend most on cytotoxic chemotherapy, with potency comparable to doxorubicin, a standard chemotherapeutic widely used in breast cancer treatment. Notably, these derivatives exhibited superior selectivity indices (SI) when compared to doxorubicin, indicating lower toxicity towards non-tumor MCF10A cells. Compounds 11a and 11b displayed an improvement in IC50 values when compared to their precursor, 1,4-naphthoquinone, for both MCF-7 and MDA-MB-231 and a comparable value to doxorubicin for MCF-7 cells. Also, their SI values were superior to those seen for the two reference compounds for both cell lines tested. Mechanistic studies revealed the ability of the compounds to induce apoptosis and inhibit clonogenic potential. Additionally, the irreversibility of their effects on cell viability underscores their promising therapeutic utility. In 3D-cell culture models, the compounds induced morphological changes indicative of reduced viability, supporting their efficacy in a more physiologically relevant model of study. The pharmacokinetics of the synthesized compounds were predicted using the SwissADME webserver, indicating that these compounds exhibit favorable drug-likeness properties and potential as antitumor agents. Overall, our findings underscore the promise of these hybrid compounds as potential candidates for breast cancer chemotherapy, emphasizing their selectivity and efficacy.

A three-dimensional cell culture approach to investigate cytotoxic effects and production of inflammatory mediators by epoxy resin-based and calcium silicate-based endodontic sealer.

OBJECTIVES: 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. CLINICAL SIGNIFICANCE: 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.

Development of a Redox-Polymer-Based Electrochemical Glucose Biosensor Suitable for Integration in Microfluidic 3D Cell Culture Systems.

The inclusion of online, in situ biosensors in microfluidic cell cultures is important to monitor and characterize a physiologically mimicking environment. This work presents the performance of second-generation electrochemical enzymatic biosensors to detect glucose in cell culture media. Glutaraldehyde and ethylene glycol diglycidyl ether (EGDGE) were tested as cross-linkers to immobilize glucose oxidase and an osmium-modified redox polymer on the surface of carbon electrodes. Tests employing screen printed electrodes showed adequate performance in a Roswell Park Memorial Institute (RPMI-1640) media spiked with fetal bovine serum (FBS). Comparable first-generation sensors were shown to be heavily affected by complex biological media. This difference is explained in terms of the respective charge transfer mechanisms. Under the tested conditions, electron hopping between Os redox centers was less vulnerable than H2O2 diffusion to biofouling by the substances present in the cell culture matrix. By employing pencil leads as electrodes, the incorporation of these electrodes in a polydimethylsiloxane (PDMS) microfluidic channel was achieved simply and at a low cost. Under flow conditions, electrodes fabricated using EGDGE presented the best performance with a limit of detection of 0.5 mM, a linear range up to 10 mM, and a sensitivity of 4.69 µA mM-1 cm-2.

Three-dimensional cell cultures as preclinical models to assess the biological activity of phytochemicals in breast cancer.

The demand for the development of three-dimensional (3D) cell culture models in both/either drug screening and/or toxicology is gradually magnified. Natural Products derived from plants are known as phytochemicals and serve as resources for novel drugs and cancer therapy. Typical examples include taxol analogs (i.e., paclitaxel and docetaxel), vinca alkaloids (i.e., vincristine, vinblastine), and camptothecin analogs (topotecan, irinotecan). Breast cancer is the most frequent malignancy in women, with a 70% chance of patients being cured; however, metastatic disease is not considered curable using currently available chemotherapeutic options. In addition, phytochemicals present promising options for overcoming chemotherapy-related problems, such as drug resistance and toxic effects on non-target tissues. In the toxicological evaluation of these natural compounds, 3D cell culture models are a powerful tool for studying their effects on different tissues and organs in similar environments and behave as if they are in vivo conditions. Considering that 3D cell cultures represent a valuable platform for identifying the biological features of tumor cells as well as for screening natural products with antitumoral activity, the present review aims to summarize the most common 3D cell culture methods, focusing on multicellular tumor spheroids (MCTS) of breast cancer cell lines used in the discovery of phytochemicals with anticancer properties in the last ten years.

Generation of Urine-Derived Induced Pluripotent Stem Cells and Cerebral Organoids for Modeling Down Syndrome.

Down syndrome (DS, or trisomy 21, T21), is the most common genetic cause of intellectual disability. Alterations in the complex process of cerebral cortex development contribute to the neurological deficits in DS, although the underlying molecular and cellular mechanisms are not completely understood. Human cerebral organoids (COs) derived from three-dimensional (3D) cultures of induced pluripotent stem cells (iPSCs) provide a new avenue for gaining a better understanding of DS neuropathology. In this study, we aimed to generate iPSCs from individuals with DS (T21-iPSCs) and euploid controls using urine-derived cells, which can be easily and noninvasively obtained from most individuals, and examine their ability to differentiate into neurons and astrocytes grown in monolayer cultures, as well as into 3D COs. We employed nonintegrating episomal vectors to generate urine-derived iPSC lines, and a simple-to-use system to produce COs with forebrain identity. We observed that both T21 and control urine-derived iPSC lines successfully differentiate into neurons and astrocytes in monolayer, as well as into COs that recapitulate early features of human cortical development, including organization of neural progenitor zones, programmed differentiation of excitatory and inhibitory neurons, and upper-and deep-layer cortical neurons as well as astrocytes. Our findings demonstrate for the first time the suitability of using urine-derived iPSC lines to produce COs for modeling DS.

Opportunities involving microfluidics and 3D culture systems to the in vitro embryo production

Traditional methods of gamete handling, fertilization, and embryo culture often face limitations in efficiency, consistency, and the ability to closely mimic in vivo conditions. This review explores the opportunities presented by microfluidic and 3D culture systems in overcoming these challenges and enhancing in vitro embryo production. We discuss the basic principles of microfluidics, emphasizing their inherent advantages such as precise control of fluid flow, reduced reagent consumption, and high-throughput capabilities. Furthermore, we delve into microfluidic devices designed for gamete manipulation, in vitro fertilization, and embryo culture, highlighting innovations such as droplet-based microfluidics and on-chip monitoring. Next, we explore the integration of 3D culture systems, including the use of biomimetic scaffolds and organ-on-a-chip platforms, with a particular focus on the oviduct-on-a-chip. Finally, we discuss the potential of these advanced systems to improve embryo production outcomes and advance our understanding of early embryo development. By leveraging the unique capabilities of microfluidics and 3D culture systems, we foresee significant advancements in the efficiency, effectiveness, and clinical success of in vitro embryo production.(AU)

3D Cell Culture as Tools to Characterize Rheumatoid Arthritis Signaling and Development of New Treatments.

Rheumatoid arthritis (RA) is one of the most common autoimmune disorders affecting 0.5-1% of the population worldwide. As a disease of multifactorial etiology, its constant study has made it possible to unravel the pathophysiological processes that cause the illness. However, efficient and validated disease models are necessary to continue the search for new disease-modulating drugs. Technologies, such as 3D cell culture and organ-on-a-chip, have contributed to accelerating the prospecting of new therapeutic molecules and even helping to elucidate hitherto unknown aspects of the pathogenesis of multiple diseases. These technologies, where medicine and biotechnology converge, can be applied to understand RA. This review discusses the critical elements of RA pathophysiology and current treatment strategies. Next, we discuss 3D cell culture and apply these methodologies for rheumatological diseases and selected models for RA. Finally, we summarize the application of 3D cell culture for RA treatment.

Vitamin D3 and Salinomycin synergy in MCF-7 cells cause cell death via endoplasmic reticulum stress in monolayer and 3D cell culture.

1α, 25, dihydroxyvitamin D3 (1,25D), the active form of vitamin D3, has antitumor properties in several cancer cell lines in vitro. Salinomycin (Sal) has anticancer activity against cancer cell lines. This study aims to examine the cytotoxic and antiproliferative effect of Sal associated with 1,25D on MCF-7 breast carcinoma cell line cultured in monolayer (2D) and three-dimensional models (mammospheres). We also aim to evaluate the molecular mechanism of Sal and 1,25D-mediated effects. We report that Sal and 1,25D act synergistically in MCF-7 mammospheres and monolayer causing G1 cell cycle arrest, reduction of mitochondrial membrane potential (MMP), and reactive oxygen species (ROS) overproduction with a long-lasting cytotoxic response represented by clonogenic and mammosphere assay. We observed the induction of cell death by apoptosis with upregulation in mRNA levels of apoptosis-related genes (CASP7, CASP9, and BBC3). Extensive cytoplasmic vacuolization, a morphological characteristic found in paraptosis, was also seen and could be triggered by endoplasmic reticulum stress (ER) as we found transcriptional upregulation of genes related to ER stress (ATF6, GADD153, GADD45G, EIF2AK3, and HSPA5). Overall, Sal and 1,25D act synergistically, inhibiting cell proliferation by activating simultaneously multiple death pathways and may be a novel and promising luminal A breast cancer therapy strategy.

A Three-Dimensional Lung Cell Model to Leptospira Virulence Investigations.

Leptospirosis is a worldwide zoonosis and a serious public health threat in tropical and subtropical areas. The etiologic agents of leptospirosis are pathogenic spirochetes from the genus Leptospira. In severe cases, patients develop a pulmonary hemorrhage that is associated with high fatality rates. Several animal models were established for leptospirosis studies, such as rodents, dogs, and monkeys. Although useful to study the relationship among Leptospira and its hosts, the animal models still exhibit economic and ethical limitation reasons and do not fully represent the human infection. As an attempt to bridge the gap between animal studies and clinical information from patients, we established a three-dimensional (3-D) human lung cell culture for Leptospira infection. We show that Leptospira is able to efficiently infect the cell lung spheroids and also to infiltrate in deeper areas of the cell aggregates. The ability to infect the 3-D lung cell aggregates was time-dependent. The 3-D spheroids infection occurred up to 120 h in studies with two serovars, Canicola and Copenhageni. We standardized the number of bacteria in the initial inoculum for infection of the spheroids and we also propose two alternative culture media conditions. This new approach was validated by assessing the expression of three genes of Leptospira related to virulence and motility. The transcripts of these genes increased in both culture conditions, however, in higher rates and earlier times in the 3-D culture. We also assessed the production of chemokines by the 3-D spheroids before and after Leptospira infection, confirming induction of two of them, mainly in the 3-D spheroids. Chemokine CCL2 was expressed only in the 3-D cell culture. Increasing of this chemokine was observed previously in infected animal models. This new approach provides an opportunity to study the interaction of Leptospira with the human lung epithelium in vitro.

Three-dimensional cell culture for the study of nasal polyps.

OBJECTIVES: Three-dimensional (3D) cell cultures have many applications such as stem cell biology research, new drug discovery, cancer, and Chronic Rhinosinusitis with Nasal Polyps (CRSwNP). This disease is characterized by a significant impact on quality of life and productivity. The diversity of factors that act in the progression of CRSwNP point to the creation of a cell culture model that allows the integration of different cell types with extracellular matrix. This work aimed to create a cell culture model in 3 dimensions (spheroids) for the study of Nasal Polyposis. METHODS: Nasal polyp tissue from patients diagnosed with CRSwNP was mechanically dissociated using tweezers and a scalpel and the solution containing cells and small aggregates of nasal polyps was transferred to a Petri dish containing 5 mL of culture medium at the concentration of 106 cells/mL. RESULTS: The spheroids were cultivated for 20 days, fixed and analyzed using confocal microscopy. In a 3D culture environment, the spheroids were formed both by clustering cells and from small tissue fragments. In the cultures analyzed, the ciliary beat was present from the dissociation of the cells up to 20 days in culture. CONCLUSION: Our findings also point to these characteristics showing the environment generated in our study, the cells remained differentiated for a longer time and with ciliary beating. Thus, this work shows that nasal polyp-derived cells can be maintained in a 3D environment, enabling better strategies for understanding CRSwNP in situations similar to those found in vivo. LEVEL OF EVIDENCE: Laboratory studies.

Layer-by-Layer Biomimetic Microgels for 3D Cell Culture and Nonviral Gene Delivery.

Localized release of nucleic acid therapeutics is essential for many biomedical applications, including gene therapy, tissue engineering, and medical implant coatings. We applied the substrate-mediated transfection and layer-by-layer (LbL) technique to achieve an efficient local gene delivery. In the experiments presented herein, we embeded lipoplexes containing plasmid DNA encoding for enhanced green fluorescent protein (pEGFP) within polyelectrolyte alginate-based microgels composed of poly(allylamine hydrochloride) (PAH), chondroitin sulfate (CS), and poly-l-lysine (PLL) with diameters between 70 and 90 µm. Droplet-based microfluidics was used as the main process to produce the alginate (ALG)-based microgels with discrete size, shape, and low coefficient of variation. The physicochemical and morphological properties of the polyelectrolyte microgels were characterized via optical microscopy, scanning electron microscopy (SEM), and zeta potential analysis. We found that polyelectrolyte microgels provide low cytotoxicity and cell-material interactions (adhesion, spreading, and proliferation). In addition, the microsystem showed the ability to load lipoplexes and a loading efficiency equal to 83%, and it enabled in vitro surface-based transfection of MCF-7 cells. This approach provides a new suitable route for cell adhesion and local gene delivery.