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1.
PLoS Comput Biol ; 16(5): e1007611, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32379821

RESUMO

Modeling cancer cells is essential to better understand the dynamic nature of brain tumors and glioma cells, including their invasion of normal brain. Our goal is to study how the morphology of the glioma cell influences the formation of patterns of collective behavior such as flocks (cells moving in the same direction) or streams (cells moving in opposite direction) referred to as oncostream. We have observed experimentally that the presence of oncostreams correlates with tumor progression. We propose an original agent-based model that considers each cell as an ellipsoid. We show that stretching cells from round to ellipsoid increases stream formation. A systematic numerical investigation of the model was implemented in [Formula: see text]. We deduce a phase diagram identifying key regimes for the dynamics (e.g. formation of flocks, streams, scattering). Moreover, we study the effect of cellular density and show that, in contrast to classical models of flocking, increasing cellular density reduces the formation of flocks. We observe similar patterns in [Formula: see text] with the noticeable difference that stream formation is more ubiquitous compared to flock formation.


Assuntos
Neoplasias Encefálicas/patologia , Biologia Computacional/métodos , Glioma/patologia , Contagem de Células/métodos , Movimento Celular/fisiologia , Forma Celular/fisiologia , Humanos , Modelos Biológicos , Modelos Teóricos , Simulação de Dinâmica Molecular
2.
PLoS One ; 15(4): e0231473, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32315325

RESUMO

The aim of this study was to determine the effect of autologous serum (AS) eye drops on the density of human leucocyte antigen (HLA)-DR-positive epithelial cells and Langerhans cells on the ocular surface of patients with bilateral severe dry eye disease (DED) due to graft-versus-host disease (GvHD) or Sjögren's syndrome (SS). The study was conducted on 24 patients (48 eyes). AS was applied 6-10 times daily for 3 months together with regular artificial tear therapy. HLA-DR-positive cells were detected by direct immunocytochemistry on upper bulbar conjunctiva imprints obtained before and after treatment. The application of AS drops led to a statistically significant increase in the mean density of aberrant HLA-DR-positive conjunctival epithelial cells (p < 0.05) and HLA-DR-positive Langerhans cells (p < 0.05) in the GvHD group. Aberrant HLA-DR-positive epithelial cells in the SS group were decreased non-significantly. All patients reported a significant decrease in the Ocular Surface Disease Index (p < 0.01), which indicates improvement of the patient's subjective feelings after therapy. There was an expected but non-significant decrease of aberrant HLA-DR-positive conjunctival epithelial cells in the SS group only. However, the increased density of HLA-DR-positive cells, indicating slight subclinical inflammation, does not outweigh the positive effect of AS in patients with DED from GvHD.


Assuntos
Túnica Conjuntiva/metabolismo , Epitélio/metabolismo , Doença Enxerto-Hospedeiro/tratamento farmacológico , Doença Enxerto-Hospedeiro/metabolismo , Antígenos HLA-DR/metabolismo , Soro/metabolismo , Síndrome de Sjogren/metabolismo , Adulto , Idoso , Contagem de Células/métodos , Túnica Conjuntiva/efeitos dos fármacos , Síndromes do Olho Seco/tratamento farmacológico , Síndromes do Olho Seco/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Humanos , Imuno-Histoquímica/métodos , Masculino , Pessoa de Meia-Idade , Soluções Oftálmicas/uso terapêutico , Síndrome de Sjogren/tratamento farmacológico
3.
Am J Hematol ; 95(5): 456-464, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31990387

RESUMO

The complement system is an innate immune defense cascade that can cause tissue damage when inappropriately activated. Evidence for complement over activation has been reported in small cohorts of patients with sickle cell disease (SCD). However, the mechanism governing complement activation in SCD has not been elucidated. Here, we observe that the plasma concentration of sC5b-9, a reliable marker for terminal complement activation, is increased at steady state in 61% of untreated SCD patients. We show that greater complement activation in vitro is promoted by SCD erythrocytes compared to normal ones, although no significant differences were observed in the regulatory proteins CD35, CD55, and CD59 in whole blood. Complement activation is positively correlated with the percentage of dense sickle cells (DRBCs). The expression levels of CD35, CD55, and CD59 are reduced in DRBCs, suggesting inefficient regulation when cell density increases. Moreover, the surface expression of the complement regulator CD46 on granulocytes was inversely correlated with the plasma sC5b-9. We also show increased complement deposition in cultured human endothelial cells incubated with SCD serum, which is diminished by the addition of the heme scavenger hemopexin. Treatment of SCD patients with hydroxyurea produces substantial reductions in complement activation, measured by sC5b-9 concentration and upregulation of CD46, as well as decreased complement activation on RBCs in vitro. In conclusion, complement over activation is a common pathogenic event in SCD that is associated with formation of DRBCs and hemolysis. And, it affects red cells, leukocytes and endothelial cells. This complement over activation is partly alleviated by hydroxyurea therapy.


Assuntos
Anemia Falciforme/terapia , Contagem de Células/métodos , Ativação do Complemento/genética , Hemólise/fisiologia , Hidroxiureia/uso terapêutico , Adolescente , Adulto , Feminino , Humanos , Hidroxiureia/farmacologia , Pessoa de Meia-Idade , Adulto Jovem
4.
Am J Physiol Lung Cell Mol Physiol ; 318(1): L200-L211, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31747308

RESUMO

Stem cell factor (SCF) and its receptor c-kit have been implicated in inflammation, tissue remodeling, and fibrosis. Ingenuity Integrated Pathway Analysis of gene expression array data sets showed an upregulation of SCF transcripts in idiopathic pulmonary fibrosis (IPF) lung biopsies compared with tissue from nonfibrotic lungs that are further increased in rapid progressive disease. SCF248, a cleavable isoform of SCF, was abundantly and preferentially expressed in human lung fibroblasts and fibrotic mouse lungs relative to the SCF220 isoform. In fibroblast-mast cell coculture studies, blockade of SCF248 using a novel isoform-specific anti-SCF248 monoclonal antibody (anti-SCF248), attenuated the expression of COL1A1, COL3A1, and FN1 transcripts in cocultured IPF but not normal lung fibroblasts. Administration of anti-SCF248 on days 8 and 12 after bleomycin instillation in mice significantly reduced fibrotic lung remodeling and col1al, fn1, acta2, tgfb, and ccl2 transcript expression. In addition, bleomycin increased numbers of c-kit+ mast cells, eosinophils, and ILC2 in lungs of mice, whereas they were not significantly increased in anti-SCF248-treated animals. Finally, mesenchymal cell-specific deletion of SCF significantly attenuated bleomycin-mediated lung fibrosis and associated fibrotic gene expression. Collectively, these data demonstrate that SCF is upregulated in diseased IPF lungs and blocking SCF248 isoform significantly ameliorates fibrotic lung remodeling in vivo suggesting that it may be a therapeutic target for fibrotic lung diseases.


Assuntos
Fibrose Pulmonar Idiopática/metabolismo , Pulmão/metabolismo , Isoformas de Proteínas/metabolismo , Fator de Células-Tronco/metabolismo , Animais , Bleomicina/farmacologia , Contagem de Células/métodos , Células Cultivadas , Técnicas de Cocultura/métodos , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibrose/tratamento farmacológico , Fibrose/metabolismo , Humanos , Fibrose Pulmonar Idiopática/tratamento farmacológico , Pulmão/efeitos dos fármacos , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
5.
Biosens Bioelectron ; 148: 111833, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31733465

RESUMO

More recently, detection of circulating tumor cells (CTCs) has been considered as an appealing prognostic and diagnostic approach for cancer patients. CTCs as a type of tumor-derived cells are secreted by the tumor and released into the blood circulation. Since the migration of CTCs is an early event in cancer progression, patients who still have tumor-free lymph nodes have to be well examined for the CTCs presence in their blood circulation. Nowadays, there is a broad range of detection methods available to identify CTCs. As artificial RNA oligonucleotides or single-stranded DNA with receptor and catalytic characteristics, aptamers have been standing out, owing to their target-induced conformational modifications, elevated stability, and target specificity to be implemented in biosensing techniques. To date, several sensitivity-enhancement methods alongside smart nanomaterials have been used for the creation of new aptasensors to address the limit of detection (LOD), and improve the sensitivity of numerous analyte identification methods. The present review article supports a focused overview of the recent studies in the identification and quantitative determination of CTCs by aptamer-based biosensors and nanobiosensors.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , Neoplasias/diagnóstico , Células Neoplásicas Circulantes/patologia , Animais , Contagem de Células/métodos , Separação Celular/métodos , Técnicas Eletroquímicas/métodos , Humanos , Medições Luminescentes/métodos , Neoplasias/patologia , Células Neoplásicas Circulantes/química
6.
Ann Lab Med ; 40(2): 122-130, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31650728

RESUMO

BACKGROUND: Automated cellular analyzers are expected to improve the analytical performance in body fluid (BF) analysis. We evaluated the analytical performance of three automated cellular analyzers and established optimum reflex analysis guidelines. METHODS: A total of 542 BF samples (88 cerebrospinal fluid [CSF] samples and 454 non-CSF samples) were examined using manual counting and three automated cellular analyzers: UniCel DxH 800 (Beckman Coulter), XN-350 (Sysmex), and UF-5000 (Sysmex). Additionally, 2,779 BF analysis results were retrospectively reviewed. For malignant cell analysis, the receiver operating characteristic (ROC) curve was used, and the detection of high fluorescence-BF cells (HF-BFs) using the XN-350 analyzer was compared with cytology results. RESULTS: All three analyzers showed good agreement for total nucleated cell (TNC) and red blood cell (RBC) counts, except for the RBC count in CSF samples using the UniCel DxH 800. However, variable degrees of differences were observed during differential cell counting. For malignant cell analysis, the area under the curve was 0.63 for the XN-350 analyzer and 0.76 for manual counting. We established our own reflex analysis guidelines as follows: HF-BFs <0.7/100 white blood cells (WBCs) is the criterion for quick scans with 100× magnification microscopic examination as a rule-out cut-off, while HF-BFs >83.4/100 WBCs or eosinophils >3.8% are the criteria for mandatory double check confirmation with 1,000× magnification examination. CONCLUSIONS: The three automated analyzers showed good analytical performances. Application of reflex analysis guidelines is recommended for eosinophils and HF-BFs, and manual confirmation is warranted.


Assuntos
Líquidos Corporais/citologia , Contagem de Células/métodos , Área Sob a Curva , Automação , Contagem de Células/instrumentação , Citometria de Fluxo , Humanos , Limite de Detecção , Curva ROC , Reprodutibilidade dos Testes
7.
Biomed Res Int ; 2019: 7878906, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31687397

RESUMO

It has been reported that paclitaxel administration could cause sensorineural hearing loss, and Wnt activation is important for the development and cell protection of mouse cochlea. However, the effect of Wnt signaling in spiral ganglion neurons (SGNs) damage induced by paclitaxel has not yet been elucidated. In this study, we explored the effect of paclitaxel on SGNs in the mouse cochlea and the neuroprotective effects of Wnt signaling pathway against paclitaxel-induced SGN damage by using Wnt agonist/antagonists in vitro. We first found that paclitaxel treatment resulted in a degenerative change and reduction of cell numbers in SGNs and induced caspase-mediated apoptosis in SGNs. The expression levels of ß-catenin and C-myc were increased, thus indicating Wnt signaling was activated in SGNs after paclitaxel treatment. The activation of Wnt signaling pathway protected against SGN loss after exposure to paclitaxel, whereas the suppression of Wnt signaling in SGNs made them more vulnerable to paclitaxel treatment. We also showed that activation of Wnt signaling in SGNs inhibited caspase-mediated apoptosis. Our findings demonstrated that Wnt signaling had an important role in protecting SGNs against paclitaxel-induced damage and thus might be an effective therapeutic target for the prevention of paclitaxel-induced SGN death.


Assuntos
Cóclea/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Paclitaxel/farmacologia , Gânglio Espiral da Cóclea/efeitos dos fármacos , Via de Sinalização Wnt/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Caspases/metabolismo , Contagem de Células/métodos , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cóclea/metabolismo , Citoproteção/efeitos dos fármacos , Camundongos , Neurônios/metabolismo , beta Catenina/metabolismo
8.
Prev Vet Med ; 172: 104803, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31634754

RESUMO

The objective of this study was to investigate the new differential somatic cell count (DSCC) as a supplementary indicator to SCC for the identification of intramammary infection (IMI) in dairy cows at the end of the lactation period. Different approaches for identification of cows with IMI (i.e. often based on SCC) and targeted antimicrobial treatment of those rather than of all cows have been developed (i.e. selective dry cow treatment). Recently, DSCC representing the proportion of polymorphonuclear neutrophils and lymphocytes, has been introduced as an additional indicator for the presence of IMI. We used the last dairy herd improvement (DHI) samples taken within 42 d prior to dry-off as well as hand-stripped samples collected within 5 days prior to dry-off to measure DSCC and SCC. The bacteriological status was determined using quarter foremilk samples collected close to drying off. In total, 582 cows were dried off during our study but not all of them could be included in the data analysis for different reasons (e.g. incomplete data, samples too old for reliable determination of SCC and DSCC, contamination). Eventually, the final data set comprised of 310 cows of which 64 and 149 were infected with major and minor pathogens, respectively, and 97 were uninfected. The area under receiver-operating characteristics curves (AUC) were calculated to compare the diagnostic abilities of the different parameters. The AUC for identification of IMI by major pathogens when using the combination of DSCC and SCC was 0.64 compared to 0.62 for SCC alone and 0.62 for DSCC alone. The different parameters were further compared based on test characteristics and predictive values. For example, classifying cows as infected based on a cut-off of 200,000 cells/ml for SCC alone and in terms of using DSCC combined with SCC based on either >60% and/or >200,000 cells/ml, the sensitivity changed from 47 to 66% and the specificity from 74 to 54%. At the same time, the negative predictive value changed from 84 to 86% and the positive predictive value from 32 to 27%. Test characteristics and predictive values of the parameters DSCC and SCC were similar using DHI and hand-stripped samples. In conclusion, our study provides first indications on test characteristics and predictive values for the combination of DSCC and SCC. However, more work on this subject and the actual practical application is needed.


Assuntos
Doenças dos Bovinos/diagnóstico , Contagem de Células/veterinária , Glândulas Mamárias Animais/microbiologia , Mastite Bovina/diagnóstico , Animais , Bovinos , Doenças dos Bovinos/microbiologia , Contagem de Células/métodos , Feminino , Mastite Bovina/microbiologia , Sensibilidade e Especificidade
9.
Acta Neurobiol Exp (Wars) ; 79(3): 302-308, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31587022

RESUMO

Preclinical studies have suggested that increased adult neurogenesis in the hippocampus might have potential therapeutic effects for Alzheimer's disease and depression; therefore, it is a target for the treatment of some brain diseases. In this technical communication, we propose a cell-based fluorescence assay to study the neurogenesis of adult hippocampal progenitor cells that can be used for high-throughput screening of drugs promoting neurogenesis. Three fluorescent dyes (DAPI, Alexa Fluor 488, and Alexa Fluor 594) and a fluorescence spectrophotometry reader were used, which confirmed that the mutual interference of the three fluorescent dyes is very low. We used this cell-based fluorescence assay to evaluate the effects of three neurotrophic factors, ciliary neurotrophic factor (CNTF), insulin-like growth factor 1 (IGF-1), and IGF-2 on the promotion of neurogenesis in adult hippocampal neural progenitor cells. The fluorescence intensity ratio of the neuronal marker, class III ß-tubulin, to the housekeeping protein, glyceraldehyde 3-phosphate dehydrogenase, or nuclear staining dye, DAPI, in CNTF-treated cells was significantly higher than in control cells. The ratios in IGF-1 and IGF-2-treated cells were slightly higher under higher cell density conditions. These results are consistent with those in previous reports; therefore, this report proved the efficacy of this method. Taken together, the results showed that this simple, rapid, and economical cell-based immunofluorescence assay could be a powerful tool for the rapid screening of drugs that promote adult neurogenesis.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Hipocampo/patologia , Células-Tronco Neurais/citologia , Neurogênese/efeitos dos fármacos , Neurônios/citologia , Animais , Contagem de Células/métodos , Diferenciação Celular/fisiologia , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Imunofluorescência , Hipocampo/efeitos dos fármacos , Humanos , Fatores de Crescimento Neural/farmacologia , Células-Tronco Neurais/efeitos dos fármacos , Neurogênese/fisiologia , Neurônios/efeitos dos fármacos
10.
Cells ; 8(9)2019 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-31480740

RESUMO

High-content and high-throughput digital microscopes have generated large image sets in biological experiments and clinical practice. Automatic image analysis techniques, such as cell counting, are in high demand. Here, cell counting was treated as a regression problem using image features (phenotypes) extracted by deep learning models. Three deep convolutional neural network models were developed to regress image features to their cell counts in an end-to-end way. Theoretically, ensembling imaging phenotypes should have better representative ability than a single type of imaging phenotype. We implemented this idea by integrating two types of imaging phenotypes (dot density map and foreground mask) extracted by two autoencoders and regressing the ensembled imaging phenotypes to cell counts afterwards. Two publicly available datasets with synthetic microscopic images were used to train and test the proposed models. Root mean square error, mean absolute error, mean absolute percent error, and Pearson correlation were applied to evaluate the models' performance. The well-trained models were also applied to predict the cancer cell counts of real microscopic images acquired in a biological experiment to evaluate the roles of two colorectal-cancer-related genes. The proposed model by ensembling deep imaging features showed better performance in terms of smaller errors and larger correlations than those based on a single type of imaging feature. Overall, all models' predictions showed a high correlation with the true cell counts. The ensembling-based model integrated high-level imaging phenotypes to improve the estimation of cell counts from high-content and high-throughput microscopic images.


Assuntos
Aprendizado Profundo , Processamento de Imagem Assistida por Computador/métodos , Neoplasias/patologia , Contagem de Células/métodos , Contagem de Células/normas , Humanos , Processamento de Imagem Assistida por Computador/normas , Células Tumorais Cultivadas
11.
Respir Res ; 20(1): 185, 2019 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-31420048

RESUMO

BACKGROUND: We previously reported cryobiopsy (Cryo) with endobronchial ultrasonography-guide sheath (EBUS-GS) for peripheral pulmonary lesions (PPLs) provides significantly larger tissues than transbronchial biopsy (TBB) and provides high quantity and quality DNA for gene analysis by next generation sequencing. However, the tumor cell yields and programmed death ligand 1 (PD-L1) expression between each approach have not been compared. Here, we assessed the tumor cell numbers and PD-L1 expression for Cryo with EBUS-GS for PPLs and TBB in patients with lung cancer. METHODS: Sixteen patients were enrolled in this prospective study from June to November 2017 at Tokyo Women's Medical University Hospital. The number of tumor cells from a single biopsy, total number of tumor cells, average number of tumor cells, and 22C3 PD-L1 expression (≥ 50% and ≥ 1%) were compared between Cryo and TBB. RESULTS: The numbers of tumor cells from a single biopsy, total numbers of tumor cells, and average numbers of tumor cells obtained by Cryo were significantly larger than those obtained by TBB (Cryo [means ± standard errors of the means]: 1321 ± 303.7, 1981 ± 411.7, and 1406 ± 310.3; TBB: 208.8 ± 38.24, 1044 ± 189.0, and 208.8 ± 37.81; P < 0.0001, P = 0.0474, P = 0.0006, respectively). PD-L1 ≥ 50% and ≥ 1% patients for Cryo were 18.8 and 56.3%, respectively, whereas those for TBB were 12.5 and 37.5%, respectively. The sensitivity, specificity, positive predictive value, negative predictive value, concordance, and κ coefficient based on Cryo for TBB were 66.7, 100, 100, 92.9, 93.8%, and 0.7647, respectively, for PD-L1 ≥ 50%; and 44.4, 71.4, 66.7, 50, 56.3%, and 0.1515, respectively, for PD-L1 ≥ 1%. CONCLUSION: Cryo with EBUS-GS may be a useful diagnostic approach for lung cancer, with advantages over TBB for gene analysis and whole exon sequencing. Particularly, it could contribute to patients taking pembrolizumab as first-line therapy when PD-L1 was negative by evaluating TBB specimens. It could also provide ample tissue for PD-L1 expression analysis in addition to accurate diagnosis and gene analysis.


Assuntos
Antígeno B7-H1/biossíntese , Brônquios/metabolismo , Brônquios/patologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Ultrassonografia de Intervenção/métodos , Idoso , Idoso de 80 Anos ou mais , Antígeno B7-H1/genética , Biópsia/métodos , Brônquios/diagnóstico por imagem , Contagem de Células/métodos , Criocirurgia/métodos , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos
12.
Sensors (Basel) ; 19(15)2019 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-31370234

RESUMO

This paper proposes a simple approach to optimize the operating frequency band of a lab-on-a-chip based on bio-impedance cytometry for a single cell. It mainly concerns applications in low-conductivity media. Bio-impedance allows for the characterization of low cell concentration or single cells by providing an electrical signature. Thus, it may be necessary to perform impedance measurements up to several tens of megahertz in order to extract the internal cell signature. In the case of single cells, characterization is performed in a very small volume down to 1 pL. At the same time, measured impedances increase from tens of kilo-ohms for physiological liquids up to several mega-ohms for low conductivity media. This is, for example, the case for water analysis. At frequencies above hundreds of kilohertz, parasitic effects, such as coupling capacitances, can prevail over the impedance of the sample and completely short-circuit measurements. To optimize the sensor under these conditions, a complete model of a cytometry device was developed, including parasitic coupling capacitances of the sensor to take into account all the impedances. It appears that it is possible to increase the pass band by optimizing track geometries and placement without changing the sensing area. This assumption was obtained by measuring and comparing electrical properties of yeast cells in a low-conductivity medium (tap water). Decreased coupling capacitance by a factor higher than 10 was obtained compared with a previous non-optimized sensor, which allowed for the impedance measurement of all electrical properties of cells as small as yeast cells in a low-conductivity medium.


Assuntos
Técnicas Biossensoriais , Contagem de Células/métodos , Dispositivos Lab-On-A-Chip , Saccharomyces cerevisiae/isolamento & purificação , Capacitância Elétrica , Condutividade Elétrica , Impedância Elétrica , Citometria por Imagem , Técnicas Analíticas Microfluídicas/métodos , Saccharomyces cerevisiae/fisiologia , Análise de Célula Única
13.
J Biotechnol ; 305: 23-26, 2019 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-31442502

RESUMO

Cell counting and viability assessment is an integral part of mammalian cell line development. While manual counting with a hemocytometer is still the gold standard method, its subjectivity and high labor intensity has resulted in its reduced use in favor of automated systems. In addition, some of these automated systems offer multiwell plate based high throughput cell count, which is an asset for biopharmaceutical companies generating hundreds of high-performance cell lines per year. In this study, we used Chinese Hamster Ovary (CHO)-K1 cells cultured in suspension in order to evaluate two automated viable-cell counters, the Guava® easyCyte HT and the CytoFLEX®, for their performance in monitoring Viable Cell Density (VCD) and viability. Our results show that specificity, accuracy, precision and repeatability was comparable between the two systems and when compared to manual counting, thus providing efficient alternatives particularly when analyzing high sample numbers in a daily mode.


Assuntos
Contagem de Células/métodos , Técnicas de Cultura de Células/métodos , Ensaios de Triagem em Larga Escala/métodos , Animais , Células CHO , Sobrevivência Celular , Cricetulus
14.
PLoS Biol ; 17(8): e3000399, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31381560

RESUMO

Most models of cancer cell population expansion assume exponential growth kinetics at low cell densities, with deviations to account for observed slowing of growth rate only at higher densities due to limited resources such as space and nutrients. However, recent preclinical and clinical observations of tumor initiation or recurrence indicate the presence of tumor growth kinetics in which growth rates scale positively with cell numbers. These observations are analogous to the cooperative behavior of species in an ecosystem described by the ecological principle of the Allee effect. In preclinical and clinical models, however, tumor growth data are limited by the lower limit of detection (i.e., a measurable lesion) and confounding variables, such as tumor microenvironment, and immune responses may cause and mask deviations from exponential growth models. In this work, we present alternative growth models to investigate the presence of an Allee effect in cancer cells seeded at low cell densities in a controlled in vitro setting. We propose a stochastic modeling framework to disentangle expected deviations due to small population size stochastic effects from cooperative growth and use the moment approach for stochastic parameter estimation to calibrate the observed growth trajectories. We validate the framework on simulated data and apply this approach to longitudinal cell proliferation data of BT-474 luminal B breast cancer cells. We find that cell population growth kinetics are best described by a model structure that considers the Allee effect, in that the birth rate of tumor cells increases with cell number in the regime of small population size. This indicates a potentially critical role of cooperative behavior among tumor cells at low cell densities with relevance to early stage growth patterns of emerging and relapsed tumors.


Assuntos
Contagem de Células/métodos , Proliferação de Células/fisiologia , Neoplasias/metabolismo , Linhagem Celular Tumoral , Ecossistema , Humanos , Cinética , Modelos Biológicos , Modelos Teóricos
15.
Methods Cell Biol ; 153: 69-92, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31395385

RESUMO

Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disorder characterized by bilateral fluid-filled cysts, renal inflammation and extensive fibrosis, leading to the progressive decline in kidney function. Renal cyst formation begins in utero from aberrant proliferation of tubule epithelial cells; however, the mechanisms for cystogenesis remain unclear. Cell proliferation and Cl--dependent fluid secretion, which drives the accumulation of cyst fluid, are responsible for inexorable growth of cysts and the remarkable appearance of massively enlarged ADPKD kidneys. Investigators have used in vitro assays to explore cellular and molecular mechanisms involved in ADPKD cyst epithelial cell proliferation and Cl--dependent fluid secretion in experimentally controlled environments. These assays have been used to evaluate potential therapeutic approaches to inhibit cellular pathways involved in cyst growth. This chapter discusses methods for measuring ADPKD cell proliferation, transepithelial Cl- secretion, and net fluid transport across cyst epithelial cell monolayers.


Assuntos
Proliferação de Células , Cloretos/metabolismo , Células Epiteliais/fisiologia , Rim Policístico Autossômico Dominante/patologia , Cultura Primária de Células/métodos , Transporte Biológico/fisiologia , Contagem de Células/instrumentação , Contagem de Células/métodos , Humanos , Rim/citologia , Rim/metabolismo , Rim/patologia , Cultura Primária de Células/instrumentação
16.
Biotechniques ; 67(3): 98-109, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31347927

RESUMO

Poloxamers are water-soluble polymers that are widely used in cell culture bioprocessing to protect cells against shearing forces. Use of poor-quality poloxamers may lead to a drastic reduction in cell growth, viabilities and productivities in cell culture-based manufacturing. In order to evaluate poloxamer quality and promote more consistent performance, a rapid cell membrane adhesion to hydrocarbon assay was developed based on the adhesive properties of cell membranes to selective hydrocarbons. The assay can identify a poor-performing poloxamer characterized by significant drop in viable cell density and percent viability. The assay was verified across multiple good and bad poloxamer lots, and the results were in agreement with established cell growth and high-performance liquid chromatography assays.


Assuntos
Técnicas de Cultura de Células/métodos , Sobrevivência Celular/efeitos dos fármacos , Citoproteção/efeitos dos fármacos , Poloxâmero/farmacologia , Substâncias Protetoras/farmacologia , Animais , Células CHO , Contagem de Células/métodos , Técnicas de Cultura de Células/economia , Proliferação de Células/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão/métodos , Cricetulus , Células HEK293 , Humanos , Poloxâmero/análise , Substâncias Protetoras/análise , Fatores de Tempo
17.
PLoS One ; 14(7): e0220132, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31344132

RESUMO

Retinal fundus imaging is a non-invasive method that allows visualizing the structure of the blood vessels in the retina whose features may indicate the presence of diseases such as diabetic retinopathy (DR) and glaucoma. Here we present a novel method to analyze and quantify changes in the retinal blood vessel structure in patients diagnosed with glaucoma or with DR. First, we use an automatic unsupervised segmentation algorithm to extract a tree-like graph from the retina blood vessel structure. The nodes of the graph represent branching (bifurcation) points and endpoints, while the links represent vessel segments that connect the nodes. Then, we quantify structural differences between the graphs extracted from the groups of healthy and non-healthy patients. We also use fractal analysis to characterize the extracted graphs. Applying these techniques to three retina fundus image databases we find significant differences between the healthy and non-healthy groups (p-values lower than 0.005 or 0.001 depending on the method and on the database). The results are sensitive to the segmentation method (manual or automatic) and to the resolution of the images.


Assuntos
Algoritmos , Fundo de Olho , Processamento de Imagem Assistida por Computador/métodos , Vasos Retinianos/diagnóstico por imagem , Vasos Retinianos/patologia , Estudos de Casos e Controles , Contagem de Células/métodos , Bases de Dados Factuais , Retinopatia Diabética/diagnóstico , Retinopatia Diabética/patologia , Diagnóstico Diferencial , Glaucoma/diagnóstico , Glaucoma/patologia , Humanos , Oftalmoscopia , Retina/diagnóstico por imagem , Retina/patologia , Sensibilidade e Especificidade
18.
Cancer Immunol Immunother ; 68(8): 1341-1350, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31324947

RESUMO

Gastric cancer (GC) is the most common malignant tumor in digestive organs, and the prognosis of GC patients who have undergone surgery remains poor because of frequent recurrence. Therefore, the identification of new markers to predict the outcome of these patients is needed. Monocyte count is a negative prognostic factor associated with inflammation. We investigated the relationship between peripheral monocytes in the peri-operative period and prognosis in GC patients. A high pre-operative monocyte count was identified as a prognostic factor in a retrospective analysis of 278 stage II and III GC patients who underwent curative gastrectomy. In contrast, an increased post-operative monocyte count compared to the pre-operative monocyte count was a marker of poor prognosis, particularly for early relapse. In a prospective analysis of 75 GC patients, a subset of the increased post-operative monocytes was similar to CD14+ HLA-DR- CD11b+ CD33+ cells by flow cytometry, and these monocytes produced IDO and arginase and suppressed T cell functions; therefore, we classified these cells as monocytic myeloid-derived suppressive cells (M-MDSCs). Peri-operative neutrophils and C-reactive protein (CRP), which are also related to inflammation, did not affect the prognosis of GC patients, and a neutrophil immunosuppressive function was not observed. These results suggest that peripheral monocytes in the peri-operative period in GC patients are a useful marker for the prognosis of GC patients, and a subset of increased post-operative monocytes may be characterized as M-MDSCs.


Assuntos
Biomarcadores Tumorais , Contagem de Células/métodos , Monócitos/patologia , Células Supressoras Mieloides/patologia , Neoplasias Gástricas/diagnóstico , Idoso , Células Cultivadas , Feminino , Citometria de Fluxo , Gastrectomia , Humanos , Masculino , Recidiva Local de Neoplasia , Estadiamento de Neoplasias , Período Perioperatório , Prognóstico , Estudos Retrospectivos , Neoplasias Gástricas/imunologia , Neoplasias Gástricas/mortalidade , Análise de Sobrevida
19.
PLoS One ; 14(7): e0219524, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31291327

RESUMO

OBJECTIVES: The current study aims to identify markers that would reflect the number of Leydig cells present in the testis, to help determine whether labour-intensive methods such as stereology are necessary. We used our well-characterised Sertoli cell ablation model in which we have empirically established the size of the Leydig cell population, to try to identify transcriptional biomarkers indicative of population size. RESULTS: Following characterisation of the Leydig cell population after Sertoli cell ablation in neonatal life or adulthood, we identified Hsd3b1 transcript levels as a potential indicator of Leydig cell number with utility for informing decision-making on whether to engage in time-consuming stereological cell counting analysis.


Assuntos
Células Intersticiais do Testículo , Complexos Multienzimáticos/genética , Progesterona Redutase/genética , Esteroide Isomerases/genética , Animais , Animais Recém-Nascidos , Biomarcadores/metabolismo , Contagem de Células/métodos , Perfilação da Expressão Gênica , Masculino , Camundongos , Complexos Multienzimáticos/metabolismo , Progesterona Redutase/metabolismo , Esteroide Isomerases/metabolismo
20.
Anal Chim Acta ; 1077: 216-224, 2019 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-31307712

RESUMO

We designed a smartphone based field-portable cell counter combining the smartphone microscope for bright-field image recording and the smartphone application for automatically cell recognition, counting and analysis. To our best knowledge, it is the first time that a smartphone based cell counter can distinguish and count both live and dead cells simultaneously. Compared to the results obtained by hemocytometer, commercial cell counter and flow cytometer, the proposed device was proved to detect cell concentration and viability accurately within the application range between 105 cells/mL and 107 cells/mL. Though multiple fields of view were measured to increase the sampling amount for error reduction, the whole operations including image recording and processing can still be finished rapidly. Moreover, the proposed device is cost-effective with small size of 170 mm × 113 mm × 168 mm containing a built-in power supply. Considering its advantages as high accuracy, fast speed, low cost, long battery life and compact configuration, it is believed the proposed device is a potential tool applied in on-site cell analysis.


Assuntos
Contagem de Células/métodos , Smartphone , Animais , Contagem de Células/instrumentação , Chlorocebus aethiops , Desenho de Equipamento , Software , Células Vero
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