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1.
Int J Nanomedicine ; 15: 6327-6338, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32922004

RESUMO

Purpose: To construct a three-dimensional (3D) culture model of adenovirus in vitro using the nanoself-assembling peptide RADA16-I as a 3D cell culture scaffold combined with virology experimental technology to provide a novel research method for virus isolation and culture, pathogenesis research, antiviral drug screening and vaccine preparation. Methods: The nanoself-assembling peptide RADA16-I was used as a 3D scaffold material for 293T cell culture, and adenovirus was cultured in the cells. The growth, morphological characteristics and pathological effects of 3D-cultured 293T cells after adenovirus infection were observed with an inverted microscope and MTS. The proliferation of adenovirus in 293T cells was observed by TEM and detected by qPCR. The levels of TNF-α and IL-8 secreted by adenovirus-infected 293T cells in the RADA16-I 3D culture system were detected by ELISA. Results: The 293T cells grew well in the RADA16-I 3D culture system for a prolonged period of time. The adenovirus infection persisted for a long time with multiple proliferation peaks, which closely resembled those of in vivo infections. The adenovirus virions amplified in the 3D system remained infectious. There were multiple secretion peaks of TNF-α and IL-8 secretion levels in adenovirus-infected 293T cells cultured in 3D culture systems. Conclusion: The nanoself-assembling peptide RADA16-I can be used as a 3D scaffold for adenovirus isolation, culture and research. The 3D culture system shows more realistic in vivo effects than two-dimensional (2D) culture.


Assuntos
Infecções por Adenoviridae/virologia , Adenoviridae/fisiologia , Técnicas de Cultura de Células/métodos , Nanopartículas/química , Peptídeos/química , Adenoviridae/crescimento & desenvolvimento , Adenoviridae/ultraestrutura , Proliferação de Células/efeitos dos fármacos , Citocinas/metabolismo , Células HEK293 , Humanos , Vírion/ultraestrutura
2.
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
3.
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
4.
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
5.
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
6.
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
7.
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
8.
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
9.
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
10.
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
11.
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
12.
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
13.
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
14.
PLoS One ; 15(7): e0236191, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32673365

RESUMO

Paclitaxel is an effective natural-source chemotherapeutic agent commonly applied to treat a vast range of cancers. In vitro Corylus avellana culture has been reported as a promising and inexpensive system for paclitaxel production. Fungal elicitors have been made known as the most efficient strategy for the biosynthesis induction of secondary metabolites in plant in vitro culture. In this research, C. avellana cell suspension culture (CSC) was exposed to cell extract (CE) and culture filtrate (CF) derived from Camarosporomyces flavigenus, either individually or combined treatment, in mid and late log phase. There is no report on the use of whole fungal elicitors (the combined treatment of CE and CF) for the elicitation of secondary metabolite biosynthesis in plant in vitro culture. The combined treatment of CE and CF significantly led to more paclitaxel biosynthesis and secretion than the individual use of them. Also, multivariate statistical approaches including stepwise regression (SR), ordinary least squares regression (OLSR), principal component regression (PCR) and partial least squares regression (PLSR) were used to model and predict paclitaxel biosynthesis and secretion. Based on value account for (VAF), root mean square error (RMSE), coefficient of determination (R2), mean absolute percentage error (MAPE) and relative percent difference (RPD) can be concluded that mentioned regression models effectively worked only for modeling and predicting extracellular paclitaxel portion in C. avellana cell culture.


Assuntos
Ascomicetos/fisiologia , Corylus/citologia , Paclitaxel/biossíntese , Ascomicetos/química , Ascomicetos/isolamento & purificação , Técnicas de Cultura de Células/métodos , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Corylus/metabolismo , Corylus/microbiologia , Análise dos Mínimos Quadrados , Modelos Biológicos , Paclitaxel/análise , Paclitaxel/química , Análise de Componente Principal
15.
J Vis Exp ; (161)2020 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-32716390

RESUMO

The limited reserve of mature, fertilizable oocytes represents a major barrier for the success of assisted reproduction in mammals. Considering that during the reproductive life span only about 1% of the oocytes in an ovary mature and ovulate, several techniques have been developed to increase the exploitation of the ovarian reserve to the growing population of non-ovulatory follicles. Such technologies have allowed interventions of fertility preservation, selection programs in livestock, and conservation of endangered species. However, the vast potential of the ovarian reserve is still largely unexploited. In cows, for instance, some attempts have been made to support in vitro culture of oocytes at specific developmental stages, but efficient and reliable protocols have not yet been developed. Here we describe a culture system that reproduce the physiological conditions of the corresponding follicular stage, defined to develop in vitro growing oocytes collected from bovine early antral follicles to the fully-grown stage, corresponding to the medium antral follicle in vivo. A combination of hormones and a phosphodiesterase 3 inhibitor was used to prevent untimely meiotic resumption and to guide oocyte's differentiation.


Assuntos
Técnicas de Cultura de Células/métodos , Oócitos/fisiologia , Reserva Ovariana/fisiologia , Reprodução/fisiologia , Animais , Bovinos , Ciclo Celular/fisiologia , Diferenciação Celular/fisiologia , Feminino , Oogênese/fisiologia , Folículo Ovariano/fisiologia , Ovário/citologia , Ovário/fisiologia
16.
J Vis Exp ; (160)2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32658206

RESUMO

Hepatitis E virus is the leading cause of liver cirrhosis and liver failure with increasing prevalence worldwide. The single-stranded RNA virus is predominantly transmitted by blood transfusions, inadequate sanitary conditions and contaminated food products. To date the off-label drug ribavirin (RBV) is the treatment of choice for many patients. Nonetheless, a specific HEV treatment remains to be identified. So far, the knowledge about the HEV life cycle and pathogenesis has been severely hampered because of the lack of an efficient HEV cell culture system. A robust cell culture system is essential for the study of the viral life cycle which also includes the viral pathogenesis. With the method described here one can produce viral titers of up to 3 x 106 focus forming unit/mL (FFU/mL) of non-enveloped HEV and up to 5 x 104 FFU/mL of enveloped HEV. Using these particles, it is possible to infect a variety of cells of diverse origins including primary cells and human, as well as animal cell lines. The production of infectious HEV particles from plasmids poses an infinite source, which makes this protocol exceedingly efficient.


Assuntos
Técnicas de Cultura de Células/métodos , Vírus da Hepatite E/patogenicidade , Hepatite E/virologia , Testes Imunológicos/métodos , Animais , Modelos Animais de Doenças , Hepatite E/patologia , Humanos
17.
Nat Commun ; 11(1): 2713, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32483127

RESUMO

Despite their rapidly-expanding therapeutic potential, human pluripotent stem cell (hPSC)-derived cell therapies continue to have serious safety risks. Transplantation of hPSC-derived cell populations into preclinical models has generated teratomas (tumors arising from undifferentiated hPSCs), unwanted tissues, and other types of adverse events. Mitigating these risks is important to increase the safety of such therapies. Here we use genome editing to engineer a general platform to improve the safety of future hPSC-derived cell transplantation therapies. Specifically, we develop hPSC lines bearing two drug-inducible safeguards, which have distinct functionalities and address separate safety concerns. In vitro administration of one small molecule depletes undifferentiated hPSCs >106-fold, thus preventing teratoma formation in vivo. Administration of a second small molecule kills all hPSC-derived cell-types, thus providing an option to eliminate the entire hPSC-derived cell product in vivo if adverse events arise. These orthogonal safety switches address major safety concerns with pluripotent cell-derived therapies.


Assuntos
Técnicas de Cultura de Células/métodos , Diferenciação Celular/genética , Edição de Genes/métodos , Células-Tronco Pluripotentes/metabolismo , Transplante de Células-Tronco/métodos , Animais , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Tacrolimo/análogos & derivados , Tacrolimo/farmacologia , Teratoma/genética , Teratoma/metabolismo , Teratoma/prevenção & controle
18.
Viruses ; 12(6)2020 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-32521706

RESUMO

Clinical samples collected in coronavirus disease 19 (COVID-19), patients are commonly manipulated in biosafety level 2 laboratories for molecular diagnostic purposes. Here, we tested French norm NF-EN-14476+A2 derived from European standard EN-14885 to assess the risk of manipulating infectious viruses prior to RNA extraction. SARS-CoV-2 cell-culture supernatant and nasopharyngeal samples (virus-spiked samples and clinical samples collected in COVID-19 patients) were used to measure the reduction of infectivity after 10 minute contact with lysis buffer containing various detergents and chaotropic agents. A total of thirteen protocols were evaluated. Two commercially available formulations showed the ability to reduce infectivity by at least 6 log 10, whereas others proved less effective.


Assuntos
Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , Inativação de Vírus/efeitos dos fármacos , Animais , Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , Betacoronavirus/fisiologia , Técnicas de Cultura de Células/métodos , Chlorocebus aethiops , Contenção de Riscos Biológicos/métodos , Contenção de Riscos Biológicos/normas , Humanos , Nasofaringe/virologia , Pandemias , RNA Viral/isolamento & purificação , Manejo de Espécimes/métodos , Células Vero , Carga Viral/métodos
19.
PLoS One ; 15(6): e0234441, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32525941

RESUMO

Human iPSC-derived hepatocytes hold great promise as a cell source for cell therapy and drug screening. However, the culture method for highly-quantified hepatocytes has not yet been established. Herein, we have developed an encapsulation and 3D cultivation method for iPSC-hepatocytes in core-shell hydrogel microfibers (a.k.a. cell fiber). In the fiber-shaped 3D microenvironment consisting of abundant extracellular matrix (ECM), the iPSC-hepatocytes exhibited many hepatic characteristics, including the albumin secretion, and the expression of the hepatic marker genes (ALB, HNF4α, ASGPR1, CYP2C19, and CYP3A4). Furthermore, we found that the fibers were mechanically stable and can be applicable to hepatocyte transplantation. Three days after transplantation of the microfibers into the abdominal cavity of immunodeficient mice, human albumin was detected in the peripheral blood of the transplanted mice. These results indicate that the iPSC-hepatocyte fibers are promising either as in vitro models for drug screening or as implantation grafts to treat liver failure.


Assuntos
Técnicas de Cultura de Células/métodos , Encapsulamento de Células/métodos , Hepatócitos/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Cavidade Abdominal/cirurgia , Alginatos/química , Animais , Técnicas de Cultura de Células/instrumentação , Diferenciação Celular , Encapsulamento de Células/instrumentação , Terapia Baseada em Transplante de Células e Tecidos/métodos , Perfilação da Expressão Gênica , Hepatócitos/transplante , Humanos , Hidrogéis/química , Falência Hepática/terapia , Masculino , Camundongos , Esferoides Celulares
20.
J Vis Exp ; (159)2020 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-32510508

RESUMO

Human pluripotent stem cells (hPSCs) have become a powerful tool for disease modeling and the study of human embryonic development in vitro. We previously presented a differentiation protocol for the derivation of autonomic neurons with sympathetic character that has been applied to patients with autonomic neuropathy. However, the protocol was built on Knock Out Serum Replacement (KSR) and feeder-based culture conditions, and to ensure high differentiation efficiency, cell sorting was necessary. These factors cause high variability, high cost, and low reproducibility. Moreover, mature sympathetic properties, including electrical activity, have not been verified. Here, we present an optimized protocol where PSC culture and differentiation are performed in feeder-free and chemically defined culture conditions. Genetic markers identifying trunk neural crest are identified. Further differentiation into postganglionic sympathetic neurons is achieved after 20 days without the need for cell sorting. Electrophysiological recording further shows the functional neuron identity. Firing detected from our differentiated neurons can be enhanced by nicotine and suppressed by the adrenergic receptor antagonist propranolol. Intermediate sympathetic neural progenitors in this protocol can be maintained as neural spheroids for up to 2 weeks, which allows expansion of the cultures. In sum, our updated sympathetic neuron differentiation protocol shows high differentiation efficiency, better reproducibility, more flexibility, and better neural maturation compared to the previous version. This protocol will provide researchers with the cells necessary to study human disorders that affect the autonomic nervous system.


Assuntos
Técnicas de Cultura de Células/métodos , Diferenciação Celular , Meios de Cultura/química , Gânglios Parassimpáticos/citologia , Neurônios/citologia , Células-Tronco Pluripotentes/citologia , Células Cultivadas , Humanos , Reprodutibilidade dos Testes
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