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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.
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A three-dimensional (3D) cell culture can more precisely mimic tissues architecture and functionality, being a promising alternative model to study disease pathophysiology and drug screening. Chagas disease (CD) is a neglected parasitosis that affects 7 million people worldwide. Trypanosoma cruzi's (T. cruzi) mechanisms of invasion/persistence continue to be elucidated. Benznidazole (BZ) and Nifurtimox (NF) are trypanocidal drugs with few effects on the clinical manifestations of the chronic disease. Chronic Chagas cardiomyopathy (CCC) is the main manifestation of CD due to its frequency and severity. The development of fibrosis and hypertrophy in cardiac tissue can lead to heart failure and sudden death. Thus, there is an urgent need for novel therapeutic options. Our group has more than fifteen years of expertise using 3D primary cardiac cell cultures, being the first to reproduce fibrosis and hypertrophy induced by T. cruzi infection in vitro. These primary cardiac spheroids exhibit morphological and functional characteristics that are similar to heart tissue, making them an interesting model for studying CD cardiac fibrosis. Here, we aim to demonstrate that our primary cardiac spheroids are great preclinical models which can be used to develop new insights into CD cardiac fibrosis, presenting advances already achieved in the field, including disease modeling and drug screening.
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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].
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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.
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Compostos de Cálcio , Sobrevivência Celular , Resinas Epóxi , Teste de Materiais , Materiais Restauradores do Canal Radicular , Silicatos , Materiais Restauradores do Canal Radicular/farmacologia , Humanos , Compostos de Cálcio/farmacologia , Silicatos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cultura de Células em Três Dimensões/métodos , Mediadores da Inflamação/metabolismo , Microscopia de Fluorescência , Osteoblastos/efeitos dos fármacosRESUMO
Human Adipose-Derived Mesenchymal Stem/Stromal Cells (hAD-MSCs) have great potential for tissue regeneration. Since transplanted hAD-MSCs are likely to be placed in a hypoxic environment, culturing the cells under hypoxic conditions might improve their post-transplantation survival and regenerative performance. The combination of hAD-MSCs and PCL-nHA nanofibers synergically improves the contribution of both components for osteoblast differentiation. In this work, we hypothesized that this biomaterial constitutes a hypoxic environment for hAD-MSCs. We studied the cellular re-arrangement and the subcellular ultrastructure by Transmission Electron Microscopy (TEM) of hAD-MSCs grown into PCL-nHA nanofibers, and we compared them with the same cells grown in two-dimensional cultures, over tissue culture-treated plastic, or glass coverslips. Among the most evident changes, PCL-nHA grown cells showed enlarged mitochondria, and accumulation of glycogen granules, consistent with a hypoxic environment. We observed a 3.5 upregulation (p = 0.0379) of Hypoxia Inducible Factor (HIF)-1A gene expression in PCL-nHA grown cells. This work evidences for the first time intra-cellular changes in three-dimensional compared to two-dimensional cultures, which are adaptive responses of the cells to an environment more closely resembling that of the in vivo niche after transplantation, thus PCL-nHA nanofibers are adequate for hAD-MSCs pre-conditioning.
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Células-Tronco Mesenquimais , Nanofibras , Humanos , Alicerces Teciduais/química , Durapatita/química , Durapatita/metabolismo , Poliésteres/química , Materiais Biocompatíveis/química , Diferenciação Celular , Nanofibras/química , Engenharia Tecidual/métodosRESUMO
Spheroids are in vitro three-dimensional multicellular microstructures able to mimic the biological microenvironment, including the complexity of tumor architecture. Therefore, results closer to those expected for in vivo organisms can be reached using spheroids compared to the cell culture monolayer model. Inorganic nanoparticles (NPs) have also been playing relevant roles in the comprehension of biological processes. Moreover, they have been probed as novel diagnostic and therapeutical nanosystems. In this context, in this review, we present applications, published in the last five years, which show that spheroids can be versatile models to study and evaluate biological interactions involving inorganic NPs. Applications of spheroids associated with (i) basic studies to assess the penetration profile of nanostructures, (ii) the evaluation of NP toxicity, and (iii) NP-based therapeutical approaches are described. Fundamentals of spheroids and their formation methods are also included. We hope that this review can be a reference and guide future investigations related to this interesting three-dimensional biological model, favoring advances to Nanobiotechnology.
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Nanopartículas , Nanoestruturas , Neoplasias , Humanos , Esferoides Celulares , Nanopartículas/química , Técnicas de Cultura de Células , Microambiente TumoralRESUMO
PURPOSE: There is growing evidence of an association between physical activity and a reduced risk of cancer and cancer recurrence. The aim of this study was to assess the effects of exercise-conditioned human serum (HS) effects on the proliferative and tumorigenic potential of triple-negative breast cancer (TNBC) and prostate cancer (PC) cells. Moreover, modulated mechanisms and several physiological factors that can predict exercise effects were investigated. METHODS: Thirty healthy sedentary subjects were recruited for the study. The subjects performed two high-intensity endurance cycling (HIEC) sessions before and after a nine-week period of high-intensity interval training (HIIT). Cell tumorigenic capacity affected by HS collected before (t0), immediately after (t1), 4 h (t2), and 24 h (t3) after the HIEC sessions was evaluated by in vitro three-dimensional colony formation. The modulation of molecular pathways was analyzed by western blotting and qPCR in TNBC and PC cells, and in TNBC xenografts in exercised mice. RESULTS: All of the HIEC-conditioned HS (t1, t2, and t3) markedly impacted the proliferative and the microtumor-forming capacity of both TNBC and PC cell lines, while the HS collected from the subjects at rest did not. Modulation of the Hippo and Wnt/ß-catenin pathways by HIEC-conditioned HS before and after the period of HIIT was shown. Multiple linear regression analysis showed relationships between the effects of HIEC-conditioned HS in PC cells, lactate threshold and VO2max. CONCLUSIONS: These results highlight the potential of HIEC bouts in tumor progression control and the importance of optimizing an approach to identify physiological predictors of the effects of acute exercise in tertiary cancer prevention.
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Ciclismo/fisiologia , Proliferação de Células/fisiologia , Treinamento Intervalado de Alta Intensidade , Neoplasias da Próstata/patologia , Neoplasias de Mama Triplo Negativas/patologia , Animais , Linhagem Celular Tumoral , Meios de Cultivo Condicionados , Progressão da Doença , Feminino , Glicogênio Sintase Quinase 3 beta/metabolismo , Via de Sinalização Hippo , Humanos , Masculino , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Neoplasias da Próstata/prevenção & controle , Proteínas Serina-Treonina Quinases/metabolismo , Distribuição Aleatória , Análise de Regressão , Comportamento Sedentário , Prevenção Terciária , Fatores de Tempo , Neoplasias de Mama Triplo Negativas/prevenção & controle , Ensaio Tumoral de Célula-Tronco/métodos , Via de Sinalização Wnt , Adulto JovemRESUMO
Development of biomaterials' substitutes and/or equivalents to mimic normal tissue is a current challenge in tissue engineering. Thus, three-dimensional cell culture using type I collagen as a polymeric matrix cell support designed to promote cell proliferation and differentiation was employed to create a dermal equivalent in vitro, as well to evaluate the photobiomodulation using red light. Polymeric matrix cell support was prepared from porcine serous collagen (1.1%) hydrolyzed for 96 h. The biomaterial exhibited porosity of 95%, a median pore of 44 µm and channels with an average distance between the walls of 78 ± 14 µm. The absorption of culture medium was 95%, and the sponge showed no cytotoxicity to Vero cells, a non-tumor cell line. Additionally, it was observed that irradiation with light at 630 nm (fluency 30 J cm-2) leads to the cellular photobiomodulation in both monolayer and human dermal equivalent (three-dimensional cell culture system). It was also verified that the cells cultured in the presence of the polymeric matrix cell support, allows differentiation and extracellular matrix secretion. Therefore, the results showed that the collagen sponge used as polymeric matrix cell support and the photobiomodulation at 630 nm are efficient for the production of a reconstructed human dermal equivalent in vitro.
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Colágeno/química , Derme/citologia , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Animais , Materiais Biocompatíveis/química , Células Cultivadas , Chlorocebus aethiops , Humanos , Luz , Porosidade , Pele Artificial , Suínos , Células VeroRESUMO
Environmental conditions experienced during early embryonic development influence growth, metabolism, and gene expression of the embryo as well as the epigenetic profile of the offspring. The environment of the early embryo consists of the luminal fluid within the oviduct and uterus and the epithelial cells composing this fluid. Whether the embryo is able to shape its own microenvironment by interacting with the epithelial lining of the oviduct/uterus and which factors potentially interfere with or regulate these interactions remains to be elucidated. As early embryonic signals and the respective maternal responses are subtle and local events, it is challenging to study them in vivo. Therefore, adequate in vitro-models optimally mimicking the contact zone between the maternal reproductive tract and the early embryo are needed to a) elucidate basic mechanisms involved in early embryonic development and b) reduce the number of experimental animals used for such studies. Functional epithelial cells are generally defined by a polarized distribution of organelles and proteins. Proper polarization is tightly connected with physiological cell behavior and in vivo-like reactivity of the epithelium. Therefore, this review summarizes current strategies for in vitro preservation of epithelial cell polarity. It presents recent advances in 3D culture of female reproductive tract epithelia and embryo-epithelial cocultures. A special emphasis is set on compartmentalized culture systems, powerful tools for studying early embryo-maternal interactions in vitro. In such systems, cultured epithelial cells are manipulable from their basolateral as well as their apical cell pole, allowing concomitant application of embryonic as well as maternal effectors from the appropriate cellular compartment.
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Desenvolvimento Embrionário , Técnicas de Cultura Embrionária , Técnicas de Cultura Embrionária/veterinária , Endométrio/fisiologia , Modelos AnatômicosRESUMO
Environmental conditions experienced during early embryonic development influence growth, metabolism, and gene expression of the embryo as well as the epigenetic profile of the offspring. The environment of the early embryo consists of the luminal fluid within the oviduct and uterus and the epithelial cells composing this fluid. Whether the embryo is able to shape its own microenvironment by interacting with the epithelial lining of the oviduct/uterus and which factors potentially interfere with or regulate these interactions remains to be elucidated. As early embryonic signals and the respective maternal responses are subtle and local events, it is challenging to study them in vivo. Therefore, adequate in vitro-models optimally mimicking the contact zone between the maternal reproductive tract and the early embryo are needed to a) elucidate basic mechanisms involved in early embryonic development and b) reduce the number of experimental animals used for such studies. Functional epithelial cells are generally defined by a polarized distribution of organelles and proteins. Proper polarization is tightly connected with physiological cell behavior and in vivo-like reactivity of the epithelium. Therefore, this review summarizes current strategies for in vitro preservation of epithelial cell polarity. It presents recent advances in 3D culture of female reproductive tract epithelia and embryo-epithelial cocultures. A special emphasis is set on compartmentalized culture systems, powerful tools for studying early embryo-maternal interactions in vitro. In such systems, cultured epithelial cells are manipulable from their basolateral as well as their apical cell pole, allowing concomitant application of embryonic as well as maternal effectors from the appropriate cellular compartment.(AU)