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1.
mSphere ; 5(5)2020 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-32878932

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected millions within just a few months, causing severe respiratory disease and mortality. Assays to monitor SARS-CoV-2 growth in vitro depend on time-consuming and costly RNA extraction steps, hampering progress in basic research and drug development efforts. Here, we developed a simplified quantitative real-time PCR assay that bypasses viral RNA extraction steps and can monitor SARS-CoV-2 growth from a small amount of cell culture supernatants. In addition, we show that this approach is easily adaptable to numerous other RNA and DNA viruses. Using this assay, we screened the activities of a number of compounds that were predicted to alter SARS-CoV-2 entry and replication as well as HIV-1-specific drugs in a proof-of-concept study. We found that E64D (inhibitor of endosomal proteases cathepsin B and L) and apilimod (endosomal trafficking inhibitor) potently decreased the amount of SARS-CoV-2 RNA in cell culture supernatants with minimal cytotoxicity. Surprisingly, we found that the macropinocytosis inhibitor ethylisopropylamiloride (EIPA) similarly decreased SARS-CoV-2 RNA levels in supernatants, suggesting that entry may additionally be mediated by an alternative pathway. HIV-1-specific inhibitors nevirapine (a nonnucleoside reverse transcriptase inhibitor [NNRTI]), amprenavir (a protease inhibitor), and allosteric integrase inhibitor 2 (ALLINI-2) modestly inhibited SARS-CoV-2 replication, albeit the 50% inhibitory concentration (IC50) values were much higher than that required for HIV-1. Taking the data together, this simplified assay will expedite basic SARS-CoV-2 research, be amenable to mid-throughput screening assays (i.e., drug, CRISPR, small interfering RNA [siRNA], etc.), and be applicable to a broad number of RNA and DNA viruses.IMPORTANCE Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of the coronavirus disease 2019 (COVID-19) pandemic, is continuing to cause immense respiratory disease and social and economic disruptions. Conventional assays that monitor SARS-CoV-2 growth in cell culture rely on costly and time-consuming RNA extraction procedures, hampering progress in basic SARS-CoV-2 research and development of effective therapeutics. Here, we developed a simple quantitative real-time PCR assay to monitor SARS-CoV-2 growth in cell culture supernatants that does not necessitate RNA extraction and that is as accurate and sensitive as existing methods. In a proof-of-concept screen, we found that E64D, apilimod, EIPA, and remdesivir can substantially impede SARS-Cov-2 replication, providing novel insight into viral entry and replication mechanisms. In addition, we show that this approach is easily adaptable to numerous other RNA and DNA viruses. This simplified assay will undoubtedly expedite basic SARS-CoV-2 and virology research and be amenable to use in drug screening platforms to identify therapeutics against SARS-CoV-2.


Assuntos
Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/crescimento & desenvolvimento , Técnicas de Cultura de Células/métodos , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Betacoronavirus/genética , Betacoronavirus/patogenicidade , Pandemias , RNA Viral/análise , RNA Viral/isolamento & purificação , Replicação Viral/efeitos dos fármacos
2.
Hell J Nucl Med ; 23 Suppl: 8-14, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32860390

RESUMO

On December 2019, a new coronavirus disease (COVID-19) emerged in China and spread worldwide, causing acute severe respiratory syndrome. Due to the increased transmission rate of the virus, it became of great importance the early diagnosis of the disease. The coronavirus pandemic led to the development of numerous tests in order to mass screening population for active viral load and for the identification of antibodies for epidemiological purposes. This review summarizes the different diagnostic tests available to the clinicians for the diagnosis and follow up of the SARS COV-2 infections.


Assuntos
Infecções por Coronavirus/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Pneumonia Viral/diagnóstico , Radiografia Torácica/métodos , Técnicas de Cultura de Células/métodos , Infecções por Coronavirus/sangue , Infecções por Coronavirus/diagnóstico por imagem , Humanos , Imunoensaio/métodos , Imunoensaio/normas , Técnicas de Diagnóstico Molecular/normas , Pandemias , Pneumonia Viral/sangue , Pneumonia Viral/diagnóstico por imagem , Radiografia Torácica/normas , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Sensibilidade e Especificidade , Testes Sorológicos/métodos , Testes Sorológicos/normas
3.
J Med Life ; 13(2): 241-248, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32742521

RESUMO

Cell culture is one of the most commonly used techniques in the production of biological products. Many physical and chemical parameters may affect cell growth and proliferation. This study was conducted to investigate the effect of chemical components as supplements using the experimental design method, which aimed at reducing the number of experiments. For this purpose, supplements including chemical components using four levels, with three replications in suspension and batch culture conditions, were examined for 72 hours using the Taguchi experimental design method. From the experiments, it was concluded that the culture media composition had a significant impact on final cell count and pH. High concentrations of different media composition alone were insufficient to ensure higher cell count. According to the results, this insufficiency was associated with an increase of 20% in the number of final cells. In the majority of cultures, the number of final cells at 48 hours increased relative to the number of final cells at 24 hours after culturing the cells.


Assuntos
Técnicas de Cultura de Células/métodos , Vírus da Febre Aftosa/imunologia , Rim/citologia , Vacinas Virais/imunologia , Aminoácidos/farmacologia , Animais , Contagem de Células , Células Cultivadas , Cricetinae , Vírus da Febre Aftosa/efeitos dos fármacos , Glucose/farmacologia , Concentração de Íons de Hidrogênio , Polietilenoglicóis/química , Proteínas/farmacologia , Vitaminas/farmacologia
4.
PLoS One ; 15(8): e0237541, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32834007

RESUMO

There is growing interest in exploring the chickens' intestinal microbiota and understanding its interactions with the host. The objective is to optimize this parameter in order to increase the productivity of farm animals. With the goal to isolate candidate probiotic strains, specific culturomic methods were used in our study to culture commensal bacteria from 7-days old chicks raised in two farms presenting long history of high performance. A total of 347 isolates were cultured, corresponding to at least 64 species. Among the isolates affiliated to the Firmicutes, 26 had less than 97% identity of their partial 16S sequence with that of the closest described species, while one presented less than 93% identity, thus revealing a significant potential for new species in this ecosystem. In parallel, and in order to better understand the differences between the microbiota of high-performing and low-performing animals, caecal contents of animals collected from these two farms and from a third farm with long history of low performance were collected and sequenced. This compositional analysis revealed an enrichment of Faecalibacterium-and Campylobacter-related sequences in lower-performing animals whereas there was a higher abundance of enterobacteria-related sequences in high-performing animals. We then investigated antibiosis activity against C. jejuni ATCC 700819 and C. jejuni field isolate as a first phenotypic trait to select probiotic candidates. Antibiosis was found to be limited to a few strains, including several lactic acid bacteria, a strain of Bacillus horneckiae and a strain of Escherichia coli. The antagonist activity depended on test conditions that mimicked the evolution of the intestinal environment of the chicken during its lifetime, i.e. temperature (37°C or 42°C) and oxygen levels (aerobic or anaerobic conditions). This should be taken into account according to the stage of development of the animal at which administration of the active strain is envisaged.


Assuntos
Infecções por Campylobacter/microbiologia , Infecções por Campylobacter/veterinária , Campylobacter/isolamento & purificação , Ceco/microbiologia , Galinhas/microbiologia , Microbioma Gastrointestinal , Doenças das Aves Domésticas/epidemiologia , Envelhecimento , Animais , Campylobacter/genética , Técnicas de Cultura de Células/métodos , Fazendas , Doenças das Aves Domésticas/microbiologia
5.
PLoS One ; 15(8): e0237478, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32853208

RESUMO

Paclitaxel as a microtubule-stabilizing agent is widely used for the treatment of a vast range of cancers. Corylus avellana cell suspension culture (CSC) is a promising strategy for paclitaxel production. Elicitation of paclitaxel biosynthesis pathway is a key approach for improving its production in cell culture. However, optimization of this process is time-consuming and costly. Modeling of paclitaxel elicitation process can be helpful to predict the optimal condition for its high production in cell culture. The objective of this study was modeling and forecasting paclitaxel biosynthesis in C. avellana cell culture responding cell extract (CE), culture filtrate (CF) and cell wall (CW) derived from endophytic fungus, either individually or combined treatment with methyl-ß-cyclodextrin (MBCD), based on four input variables including concentration levels of fungal elicitors and MBCD, elicitor adding day and CSC harvesting time, using adaptive neuro-fuzzy inference system (ANFIS) and multiple regression methods. The results displayed a higher accuracy of ANFIS models (0.94-0.97) as compared to regression models (0.16-0.54). The great accordance between the predicted and observed values of paclitaxel biosynthesis for both training and testing subsets support excellent performance of developed ANFIS models. Optimization process of developed ANFIS models with genetic algorithm (GA) showed that optimal MBCD (47.65 mM) and CW (2.77% (v/v)) concentration levels, elicitor adding day (16) and CSC harvesting time (139 h and 41 min after elicitation) can lead to highest paclitaxel biosynthesis (427.92 µg l-1). The validation experiment showed that ANFIS-GA method can be a promising tool for selecting the optimal conditions for maximum paclitaxel biosynthesis, as a case study.


Assuntos
Técnicas de Cultura de Células/métodos , Corylus/química , Paclitaxel/biossíntese , Algoritmos , Corylus/metabolismo , Fungos/química , Fungos/metabolismo , Modelos Lineares , Células Vegetais/química , Células Vegetais/metabolismo , beta-Ciclodextrinas/química
6.
Nature ; 584(7822): 535-546, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32848221

RESUMO

Substantial research over the past two decades has established that extracellular matrix (ECM) elasticity, or stiffness, affects fundamental cellular processes, including spreading, growth, proliferation, migration, differentiation and organoid formation. Linearly elastic polyacrylamide hydrogels and polydimethylsiloxane (PDMS) elastomers coated with ECM proteins are widely used to assess the role of stiffness, and results from such experiments are often assumed to reproduce the effect of the mechanical environment experienced by cells in vivo. However, tissues and ECMs are not linearly elastic materials-they exhibit far more complex mechanical behaviours, including viscoelasticity (a time-dependent response to loading or deformation), as well as mechanical plasticity and nonlinear elasticity. Here we review the complex mechanical behaviours of tissues and ECMs, discuss the effect of ECM viscoelasticity on cells, and describe the potential use of viscoelastic biomaterials in regenerative medicine. Recent work has revealed that matrix viscoelasticity regulates these same fundamental cell processes, and can promote behaviours that are not observed with elastic hydrogels in both two- and three-dimensional culture microenvironments. These findings have provided insights into cell-matrix interactions and how these interactions differentially modulate mechano-sensitive molecular pathways in cells. Moreover, these results suggest design guidelines for the next generation of biomaterials, with the goal of matching tissue and ECM mechanics for in vitro tissue models and applications in regenerative medicine.


Assuntos
Elasticidade , Matriz Extracelular/metabolismo , Substâncias Viscoelásticas , Materiais Biocompatíveis/química , Materiais Biocompatíveis/metabolismo , Técnicas de Cultura de Células , Forma Celular , Matriz Extracelular/química , Humanos , Mecanotransdução Celular , Células-Tronco Mesenquimais/citologia , Modelos Biológicos , Medicina Regenerativa
7.
Chem Biol Interact ; 330: 109178, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32738201

RESUMO

The capsaicin (vanilloid) receptor, TRPV1, is a heat-activated cation channel modulated by inflammatory mediators and contributes to acute and chronic pain. TRPV1 channel is one of the most researched and targeted mechanisms for the development of novel analgesics. Over the years, natural products have contributed enormously to the development of important therapeutic drugs used currently in modern medicine. A literature review was conducted using Medline, Google Scholar, and PubMed. Searching the literature resulted in listing 136 natural compounds that interacted with TRPV1 channel. These compounds were phytochemicals that belong to different chemical groups including vanilloids, flavonoids, alkaloids, terpenoids, terpenyl phenols, fatty acids, cannabinoids, sulfur_containing compounds, etc. Other natural TRPV1 modulators were of animal, fungal or bacterial origin. Some natural products were small agonists or antagonists of TRPV1. Others were protein venoms. Most in vitro studies utilized electrophysiological or calcium imaging techniques to study calcium flow through the channel using primary cultures of rat dorsal root and trigeminal ganglia. Other studies used hTRPV1 or rTRPV1 expressed in HEK239, CHO cells or Xenopus oocytes. In vivo studies concentrated on different pain models conducted mainly in mice and rats. In conclusion, natural products are highly diverse in their modulatory action on TRPV1. Many gaps in natural product research are present in distinguishing modality-specific from polymodal antagonists. Species' differences in TRPV1 functionality must be taken into account in any future study. Proceeding into clinical trials needs more efforts to discover potent TRPV1 antagonists devoid of hyperthermia, the main side effect.


Assuntos
Analgésicos/farmacologia , Produtos Biológicos/farmacologia , Canais de Cátion TRPV/metabolismo , Analgésicos/efeitos adversos , Analgésicos/uso terapêutico , Animais , Produtos Biológicos/efeitos adversos , Cálcio/metabolismo , Técnicas de Cultura de Células , Febre/etiologia , Humanos , Dor/complicações , Dor/tratamento farmacológico , Canais de Cátion TRPV/agonistas , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/efeitos dos fármacos , Canais de Cátion TRPV/genética
8.
Chem Biol Interact ; 330: 109227, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32818478

RESUMO

The use of 3D models in various scientific applications is becoming more popular to replace traditional monolayers models. In this work, we used a three-dimensional in-house model of epidermis using HaCaT immortalized cells to evaluate the dermal toxicity induced by Basic Blue 99 and Basic Red 51, both present in commercial hair dye formulations. Our data show that cells cultured in the 3D model respond differently to those cultured in monolayer. Basic Red 51 dye induces apoptosis an DNA breaks in both models, however, these effects is more pronounced in cells cultured in monolayer. The toxic mode of action of Basic Blue 99 seems to be the induction of cell death, without genotoxic effects, but while the necrotic pathway is observed in HaCaT monolayer cell culture, was apoptosis seen in the Equivalent Human Epidermis (EHE) model. We could also confirm that cells in EHE model, an environment that could better mimic human effects, react differently to chemical stressors than the cells cultivated in 2D.


Assuntos
Técnicas de Cultura de Células/métodos , Epiderme/efeitos dos fármacos , Tinturas para Cabelo/toxicidade , Apoptose/efeitos dos fármacos , Compostos Azo/toxicidade , Técnicas de Cultura de Células/normas , Linhagem Celular , Dano ao DNA/efeitos dos fármacos , Tinturas para Cabelo/análise , Humanos , Naftoquinonas/toxicidade , Necrose/induzido quimicamente , Compostos de Amônio Quaternário/toxicidade
9.
Ecotoxicol Environ Saf ; 204: 111058, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32739676

RESUMO

Skeletal fluorosis causes growth plate impairment and growth retardation during bone development. However, the mechanism of how fluoride impairs chondrocyte is unclear. To explore the effect of fluoride on chondrocyte differentiation and the regulation of circadian clock signaling pathway during chondrogenesis, we treated ATDC5 cells with fluoride and carried out a series of experiments. 10-3 M fluoride inhibited cell viability and significantly decreased the expression of Sox9 and Col2a1 (P < 0.05). Fluoride inhibited proteoglycan synthesis and decreased significantly the expression of Aggrecan, Ihh and Col10a1 (P < 0.05). Meanwhile, fluoride significantly inhibited the expression of Bmal1 and disrupted circadian clock signaling pathway (P < 0.05). Furthermore, fluoride disrupted the time-dependent expression of circadian clock molecules and stage-specific differentiation markers. Overexpression of Bmal1 by lentivirus reversed the adverse effects of fluoride on chondrogenesis. These results suggested that fluoride inhibited chondrocyte viability and delayed chondrocyte differentiation. Fluoride delayed chondrogenesis partly via interfering with Bmal1 and circadian clock signaling pathway. Nevertheless, the specific mechanism of circadian clock in fluoride-induced cartilage damage needs to be further studied.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Condrogênese/efeitos dos fármacos , Relógios Circadianos , Poluentes Ambientais/toxicidade , Fluoretos/toxicidade , Animais , Técnicas de Cultura de Células , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Condrócitos/citologia , Condrócitos/metabolismo , Condrogênese/fisiologia , Colágeno Tipo X/genética , Colágeno Tipo X/metabolismo , Lâmina de Crescimento/efeitos dos fármacos , Lâmina de Crescimento/metabolismo , Camundongos , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Transdução de Sinais
10.
PLoS One ; 15(8): e0236397, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32756566

RESUMO

Self-contained imaging systems are versatile instruments that are becoming a staple in cell culture laboratories. Many of these machines possess motorized stages and on-stage incubators that permit programmable imaging of live cells that make them a sensible tool for high-throughput applications. The EVOS imaging system is such a device and is capable of scanning multi-well dishes and stitching together multiple adjacent fields to produce coherent individual images of each well. Automated batch analysis and quantification of these tiled images does however require off-loading files to other software platforms. Our initial attempts to quantify tiled images captured on an EVOS device was plagued by some expected-and other unforeseeable-issues that arose at nearly every stage of analysis. These included: high background, illumination and stitching artifacts, low contrast, noise, focus inconsistencies, and image distortion-all of which negatively impacted processing efficiency. We have since overcome these obstacles and have created a rigorous cell counting pipeline for analyzing images captured by the EVOS scan function. We present development and optimization of this automated pipeline and submit it as an effective and facile tool for accurately counting cells from tiled images.


Assuntos
Técnicas de Cultura de Células/métodos , Rastreamento de Células/métodos , Processamento de Imagem Assistida por Computador/métodos , Software , Humanos , Células MCF-7 , Imagem Óptica/métodos
11.
Curr Protoc Stem Cell Biol ; 54(1): e118, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32640120

RESUMO

The normal development of the pulmonary system is critical to transitioning from placental-dependent fetal life to alveolar-dependent newborn life. Human lung development and disease have been difficult to study due to the lack of an in vitro model system containing cells from the large airways and distal alveolus. This article describes a system that allows human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) to differentiate and form three-dimensional (3D) structures that emulate the development, cytoarchitecture, and function of the lung ("organoids"), containing epithelial and mesenchymal cell populations, and including the production of surfactant and presence of ciliated cells. The organoids can also be invested with mesoderm derivatives, differentiated from the same human pluripotent stem cells, such as alveolar macrophages and vasculature. Such lung organoids may be used to study the impact of environmental modifiers and perturbagens (toxins, microbial or viral pathogens, alterations in microbiome) or the efficacy and safety of drugs, biologics, and gene transfer. © 2020 Wiley Periodicals LLC. Basic Protocol: hESC/hiPSC dissection, definitive endoderm formation, and lung progenitor cell induction.


Assuntos
Infecções por Coronavirus/patologia , Pulmão/citologia , Organoides/citologia , Pneumonia Viral/patologia , Infecções Respiratórias/patologia , Betacoronavirus , Técnicas de Cultura de Células , Diferenciação Celular , Infecções por Coronavirus/terapia , Endoderma/citologia , Células-Tronco Embrionárias Humanas/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Pulmão/crescimento & desenvolvimento , Pulmão/fisiologia , Modelos Biológicos , Pandemias , Modelagem Computacional Específica para o Paciente , Pneumonia Viral/terapia , Infecções Respiratórias/terapia , Imagem com Lapso de Tempo
12.
PLoS One ; 15(7): e0235356, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32628693

RESUMO

As a new class of cancer therapeutic agents, oncolytic viruses (OVs) have gained much attention not only due to their ability to selectively replicate in and lyse tumor cells, but also for their potential to stimulate antitumor immune responses. As a result, there is an increasing need for in vitro modeling systems capable of recapitulating the 3D physiological tumor microenvironment. Here, we investigated the potential of our recently developed microphysiological system (MPS), featuring a vessel-like channel to reflect the in vivo tumor microenvironment and serving as culture spaces for 3D multicellular tumor spheroids (MCTSs). The MCTSs consist of cancer A549 cells, stromal MRC5 cells, endothelial HUVECs, as well as the extracellular matrix. 3D MCTSs residing in the MPS were infected with oncolytic VSV expressing GFP (oVSV-GFP). Post-infection, GFP signal intensity increased only in A549 cells of the MPS. On the other hand, HUVECs were susceptible to virus infection under 2D culture and IFN-ß secretion was quite delayed in HUVECs. These results thus demonstrate that OV antitumoral characteristics can be readily monitored in the MPS and that its behavior therein somewhat differs compared to its activity in 2D system. In conclusion, we present the first application of the MPS, an in vitro model that was developed to better reflect in vivo conditions. Its various advantages suggest the 3D MCTS-integrated MPS can serve as a first line monitoring system to validate oncolytic virus efficacy.


Assuntos
Neoplasias/terapia , Terapia Viral Oncolítica , Vírus Oncolíticos/imunologia , Vesiculovirus/imunologia , Células A549 , Técnicas de Cultura de Células/métodos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Matriz Extracelular , Células Endoteliais da Veia Umbilical Humana , Humanos , Neoplasias/imunologia , Vírus Oncolíticos/genética , Esferoides Celulares , Vesiculovirus/genética
13.
Int J Nanomedicine ; 15: 4625-4637, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32636622

RESUMO

Purpose: Besides the tumor cells themselves, solid tumors are comprised of numerous cell types including infiltrating immune cells such as tumor-associated macrophages (TAMs). TAMs are vital stromal components of host immune system and play a critical role in the development of cancer. TAMs can be divided into two subtypes: M1 tumor-suppressive macrophage and M2 tumor-supportive macrophage. To better address the observations of TAMs functional performance, we describe an in vitro system that mimics the populations of TAMs infiltrated into the tumor mass by using our disintegrable supramolecular gelatin (DSG) hydrogels, which are physically crosslinked by host-guest complexations. Materials and Methods: The host-guest interaction was adopted between the aromatic groups of gelatin and the photocrosslinkable acrylated ß-cyclodextrins (Ac-ß-CDs) to form the DSG hydrogels. The convenient macrophage/endometrial cancer cells heterospheroid 3D model was set up by DSG hydrogels. RT-PCR and Western blot assays were developed to evaluate the efficiencies of inducers on the macrophages. The ELISA and oxygen saturation assays were performed to measure the secretion of VEGF and consumption of oxygen of tumor and/or macrophages, respectively. To determine the antitumor effects of M2 reprogrammed macrophages in vitro and in vivo, migration assay and tumor xenograft model were used, respectively. Results: The host-guest complexations of DSG hydrogels were controllably broken efficiently by soaking into the solution of competitive guest monomers 1-adamantanamine hydrochloride. The DSG hydrogels help IFN-γ reprogram the M2 to M1 and then decrease the tumor/M2 reprogrammed macrophage cells heterospheroid secretion of VEGF and increase the relative oxygen saturation. Significantly, the co-cultural tumor/M2 reprogrammed group from the disintegrated DSG hydrogels reduced the migration of cancer cells in vitro and the tumor growth in vivo. Conclusion: We obtain a TAMs/tumor microenvironment-responsive 3D model based on the novel DSG hydrogels, and will be of utility in cancer therapy and drug discovery.


Assuntos
Neoplasias do Endométrio/patologia , Gelatina/química , Hidrogéis/química , Macrófagos/citologia , Macrófagos/transplante , Animais , Técnicas de Cultura de Células , Neoplasias do Endométrio/terapia , Matriz Extracelular/patologia , Feminino , Gelatina/farmacocinética , Humanos , Hidrogéis/farmacocinética , Camundongos Endogâmicos BALB C , Esferoides Celulares/citologia , Microambiente Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto
14.
PLoS One ; 15(7): e0235827, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32667933

RESUMO

Homogenization of the initial cell distribution is essential for effective cell development. However, there are few previous reports on efficient cell seeding methods, even though the initial cell distribution has a large effect on cell proliferation. Dense cell regions have an inverse impact on cell development, known as contact inhibition. In this study, we developed a method to homogenize the cell seeding density using secondary flow, or Ekman transportation, induced by orbital movement of the culture dish. We developed an orbital shaker device that can stir the medium in a 35-mm culture dish by shaking the dish along a circular orbit with 2 mm of eccentricity. The distribution of cells in the culture dish can be controlled by the rotational speed of the orbital shaker, enabling dispersion of the initial cell distribution. The experimental results indicated that the cell density became most homogeneous at 61 rpm. We further evaluated the cell proliferation after homogenization of the initial cell density at 61 rpm. The results revealed 36% higher proliferation for the stirred samples compared with the non-stirred control samples. The present findings indicate that homogenization of the initial cell density by Ekman transportation contributes to the achievement of higher cell proliferation.


Assuntos
Técnicas de Cultura de Células/instrumentação , Mioblastos/citologia , Animais , Contagem de Células , Técnicas de Cultura de Células/economia , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Desenho de Equipamento , Camundongos
15.
Proc Natl Acad Sci U S A ; 117(30): 17796-17807, 2020 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-32651268

RESUMO

Fluctuation in signal transduction pathways is frequently observed during mammalian development. However, its role in regulating stem cells has not been explored. Here we tracked spatiotemporal ERK MAPK dynamics in human epidermal stem cells. While stem cells and differentiated cells were distinguished by high and low stable basal ERK activity, respectively, we also found cells with pulsatile ERK activity. Transitions from Basalhi-Pulselo (stem) to Basalhi-Pulsehi, Basalmid-Pulsehi, and Basallo-Pulselo (differentiated) cells occurred in expanding keratinocyte colonies and in response to differentiation stimuli. Pharmacological inhibition of ERK induced differentiation only when cells were in the Basalmid-Pulsehi state. Basal ERK activity and pulses were differentially regulated by DUSP10 and DUSP6, leading us to speculate that DUSP6-mediated ERK pulse down-regulation promotes initiation of differentiation, whereas DUSP10-mediated down-regulation of mean ERK activity promotes and stabilizes postcommitment differentiation. Levels of MAPK1/MAPK3 transcripts correlated with DUSP6 and DUSP10 transcripts in individual cells, suggesting that ERK activity is negatively regulated by transcriptional and posttranslational mechanisms. When cells were cultured on a topography that mimics the epidermal-dermal interface, spatial segregation of mean ERK activity and pulses was observed. In vivo imaging of mouse epidermis revealed a patterned distribution of basal cells with pulsatile ERK activity, and down-regulation was linked to the onset of differentiation. Our findings demonstrate that ERK MAPK signal fluctuations link kinase activity to stem cell dynamics.


Assuntos
Diferenciação Celular , Células Epidérmicas/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células-Tronco/metabolismo , Animais , Técnicas de Cultura de Células , Proliferação de Células , Ativação Enzimática , Células Epidérmicas/citologia , Queratinócitos/metabolismo , Mamíferos , Camundongos , Fosfoproteínas Fosfatases/metabolismo , Transdução de Sinais , Células-Tronco/citologia
16.
PLoS One ; 15(7): e0235798, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32673324

RESUMO

During the course of the asexual erythrocytic stage of development, Plasmodium spp. parasites undergo a series of morphological changes and induce alterations in the host cell. At the end of this stage, the parasites egress from the infected cell, after which the progeny invade a new host cell. These processes are rapid and occur in a time-dependent manner. Of particular importance, egress and invasion of erythrocytes by the parasite are difficult to capture in an unsynchronized culture, or even a culture that has been synchronized within a window of one to several hours. Therefore, precise synchronization of parasite cultures is of paramount importance for the investigation of these processes. Here we describe a method for synchronizing Plasmodium falciparum and Plasmodium knowlesi asexual blood stage parasites with ML10, a highly specific inhibitor of the cGMP-dependent protein kinase (PKG) that arrests parasite growth approximately 15 minutes prior to egress. This inhibitor allows parasite cultures to be synchronized so that all parasites are within a window of development of several minutes, with a simple wash step. Furthermore, we show that parasites remain viable for several hours after becoming arrested by the compound and that ML10 has advantages, owing to its high specificity and low EC50, over the previously used PKG inhibitor Compound 2. Here, we demonstrate that ML10 is an invaluable tool for the study of Plasmodium spp. asexual blood stage biology and for the routine synchronization of P. falciparum and P. knowlesi cultures.


Assuntos
Eritrócitos/parasitologia , Malária/parasitologia , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium knowlesi/crescimento & desenvolvimento , Técnicas de Cultura de Células/métodos , Humanos , Malária Falciparum/parasitologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium knowlesi/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Fatores de Tempo
17.
Virol Sin ; 35(3): 311-320, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32602046

RESUMO

The mechanism of how SARS-CoV-2 causes severe multi-organ failure is largely unknown. Acute kidney injury (AKI) is one of the frequent organ damage in severe COVID-19 patients. Previous studies have shown that human renal tubule cells could be the potential host cells targeted by SARS-CoV-2. Traditional cancer cell lines or immortalized cell lines are genetically and phenotypically different from host cells. Animal models are widely used, but often fail to reflect a physiological and pathogenic status because of species tropisms. There is an unmet need for normal human epithelial cells for disease modeling. In this study, we successfully established long term cultures of normal human kidney proximal tubule epithelial cells (KPTECs) in 2D and 3D culture systems using conditional reprogramming (CR) and organoids techniques. These cells had the ability to differentiate and repair DNA damage, and showed no transforming property. Importantly, the CR KPTECs maintained lineage function with expression of specific transporters (SLC34A3 and cubilin). They also expressed angiotensin-converting enzyme 2 (ACE2), a receptor for SARS-CoV and SARS-CoV-2. In contrast, cancer cell line did not express endogenous SLC34A3, cubilin and ACE2. Very interestingly, ACE2 expression was around twofold higher in 3D organoids culture compared to that in 2D CR culture condition. Pseudovirion assays demonstrated that SARS-CoV spike (S) protein was able to enter CR cells with luciferase reporter. This integrated 2D CR and 3D organoid cultures provide a physiological ex vivo model to study kidney functions, innate immune response of kidney cells to viruses, and a novel platform for drug discovery and safety evaluation.


Assuntos
Betacoronavirus/metabolismo , Técnicas de Cultura de Células/métodos , Infecções por Coronavirus/virologia , Coronavirus/metabolismo , Células Epiteliais/virologia , Rim/virologia , Pneumonia Viral/virologia , Animais , Betacoronavirus/patogenicidade , Linhagem Celular , Coronavirus/patogenicidade , Dano ao DNA , Modelos Animais de Doenças , Humanos , Organoides , Pandemias , Peptidil Dipeptidase A/metabolismo , Receptores de Superfície Celular/metabolismo , Vírus da SARS/metabolismo , Vírus da SARS/patogenicidade , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIc/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo
18.
Curr Res Transl Med ; 68(3): 105-110, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32616467

RESUMO

The relative ease of isolation of mesenchymal stem cells (MSCs) from different tissues coupled with their culture expansion in vitro and their differentiation capacity to mesodermal, endodermal and ectodermal lineages have made these cells attractive for a large number of therapeutic applications. In recent years, there has been remarkable progress in the utilization of MSCs in diverse clinical indications both in animal models and human clinical trials. However, the potential of MSCs to control or treat viral diseases is still in its infancy. In this study, we report quantitative data on the MSC-based clinical trials over the last ten years as they appear on the online database of clinical research studies from US National Institutes of Health. In particular, we provide comprehensive review of either completed or ongoing clinical trials using MSCs for virus-associated diseases focusing on HIV, hepatitis B virus and COVID-19 virus.


Assuntos
Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/fisiologia , Viroses/terapia , Fenômenos Fisiológicos Virais , Animais , Betacoronavirus/fisiologia , Técnicas de Cultura de Células , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/terapia , Infecções por Coronavirus/virologia , HIV/fisiologia , Vírus da Hepatite B/fisiologia , Humanos , Transplante de Células-Tronco Mesenquimais/métodos , Transplante de Células-Tronco Mesenquimais/tendências , Pandemias , Pneumonia Viral/imunologia , Pneumonia Viral/terapia , Pneumonia Viral/virologia , Viroses/epidemiologia , Viroses/imunologia , Vírus/patogenicidade
19.
Theranostics ; 10(16): 7034-7052, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32641977

RESUMO

This review provides an update for the international research community on the cell modeling tools that could accelerate the understanding of SARS-CoV-2 infection mechanisms and could thus speed up the development of vaccines and therapeutic agents against COVID-19. Many bioengineering groups are actively developing frontier tools that are capable of providing realistic three-dimensional (3D) models for biological research, including cell culture scaffolds, microfluidic chambers for the culture of tissue equivalents and organoids, and implantable windows for intravital imaging. Here, we review the most innovative study models based on these bioengineering tools in the context of virology and vaccinology. To make it easier for scientists working on SARS-CoV-2 to identify and apply specific tools, we discuss how they could accelerate the discovery and preclinical development of antiviral drugs and vaccines, compared to conventional models.


Assuntos
Antivirais/isolamento & purificação , Antivirais/farmacologia , Betacoronavirus , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/prevenção & controle , Pandemias/prevenção & controle , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/prevenção & controle , Vacinas Virais/isolamento & purificação , Vacinas Virais/farmacologia , Betacoronavirus/química , Betacoronavirus/genética , Betacoronavirus/imunologia , Bioengenharia/métodos , Bioengenharia/tendências , Reatores Biológicos , Técnicas de Cultura de Células , Simulação por Computador , Infecções por Coronavirus/imunologia , Descoberta de Drogas/métodos , Descoberta de Drogas/tendências , Avaliação de Medicamentos/métodos , Avaliação de Medicamentos/tendências , Farmacorresistência Viral , Interações entre Hospedeiro e Microrganismos/genética , Interações entre Hospedeiro e Microrganismos/imunologia , Humanos , Modelos Biológicos , Organoides/citologia , Organoides/virologia , Pneumonia Viral/imunologia , Nanomedicina Teranóstica
20.
Artigo em Inglês | MEDLINE | ID: mdl-32667389

RESUMO

Blastocystis sp. is a protist commonly found in stool samples of humans and animals. Biological and genetic factors of this organism remain controversial. The present study aimed to develop and implement the Blastocystis in vitro culture of Brazilian human isolates for routine use. The fecal isolates (n = 20) were maintained in our laboratory by several passages in Pavlova's medium. Cultures were monitored every 72 h by light microscopy. Genomic DNA was extracted to identify the subtypes (STs). In most isolates, the vacuolar form was prevalent. The amoeboid, granular and cystic forms were observed during in vitro cultivation. STs 1, 2, 3, 4 and 7 were identified. Our preliminary results show the generation time and forms present in the in vitro culture of Blastocystis subtypes isolated from Brazilian human isolates. Therefore, we emphasize the use of in vitro culture as a tool in future studies for the better understanding of the biological aspects of Blastocystis sp.


Assuntos
Infecções por Blastocystis/parasitologia , Blastocystis/genética , Técnicas de Cultura de Células/métodos , Fezes/parasitologia , Microscopia/métodos , Reação em Cadeia da Polimerase/métodos , Animais , Blastocystis/citologia , Blastocystis/isolamento & purificação , Infecções por Blastocystis/diagnóstico , Brasil , Humanos , Prevalência
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