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1.
Eur J Pharm Biopharm ; 145: 85-95, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31639418

RESUMO

The RPMI 2650 cell line has been a subject of evaluation as a physiological and pharmacological model of the nasal epithelial barrier. However, its suitability for drug permeability assays has not yet been established on a sufficiently large set of model drugs. We investigated two RPMI 2650 cell models (air-liquid and liquid-liquid) for nasal drug permeability determination by adopting the most recent regulatory guidelines on showing suitability of in vitro permeability methods for drug permeability classification. The permeability of 23 model drugs and several zero permeability markers across the cell models was assessed. The functional expression of two efflux transporters P-glycoprotein (P-gp) and Breast Cancer Resistant Protein (BCRP) was shown to be negligible by bidirectional transport studies using appropriate transporter substrates and inhibitors. The model drug permeability determined in the two RPMI 2650 cell models was correlated with the fully differentiated nasal epithelial model (MucilAir™). Additionally, correlations between the drug permeability in the investigated cell models and the ones determined in the Caco-2 cells and isolated rat jejunum were established. In conclusion, the air-liquid RPMI 2650 cell model is a promising pharmacological model of the nasal epithelial barrier and is much more suitable than the liquid-liquid model for nasal drug permeability prediction.


Assuntos
Técnicas de Cultura de Células/métodos , Mucosa Nasal/metabolismo , Preparações Farmacêuticas/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Células CACO-2 , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Permeabilidade , Ratos
2.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi ; 33(11): 1433-1438, 2019 Nov 15.
Artigo em Chinês | MEDLINE | ID: mdl-31650762

RESUMO

Objective: To isolate cancer stem cells (CST) from human breast cancer cell line (MCF-7) and study their sensitivity toward oxidative stress. Methods: MCF-7 cells were cultured in serum-free suspension culture medium to identify cells forming the sphere phenotype. The morphological changes of MCF-7 cells were observed by inverted phase contrast microscope (compared with MCF-7 cells cultured in serum-free suspension culture medium). The expression of CST marker CD133 was detected by immunocytochemical staining in CST cell spheres (experimental group) with a diameter of 100 µm and MCF-7 cells (control group) with a fusion degree of 70%. The positive rate of CD133 was detected by flow cytometry in the third generation of tumor cells with diameter of 150 µm. The second generation of tumor globular cells (experimental group) with diameter of 150 µm and corresponding MCF-7 cells (control group) were taken to be damaged by 50 mol/L H 2O 2 for 120 minutes. The expression of DNA damage marker histone H2AX phosphorylation (γH2AX) was detected by immunocytochemical staining. Results: Inverted phase contrast microscopy showed that MCF-7 cells grew initially in a single-cell adherent state, then aggregated and grew in serum-free suspension culture medium, and finally formed CST cell spheres, while the control MCF-7 cells cultured in MCF-7 cell culture medium grew extensively and could not grow in suspension. Fluorescence microscopy showed that the expression of CD133 in MCF-7 cells of control group was negative, while that in experimental group was positive. Flow cytometry showed that CD133 was positive in CST cells, and the positive rate was 92%. Inverted fluorescence microscopy showed that the expression of γH2AX in CST tumor spheres of experimental group was significantly lower than that in MCF-7 cells of control group after 120 minutes of H 2O 2 injury. Conclusion: Serum-free suspension culture medium can produce globular CST cells from MCF-7 tumor cell line, which have strong antioxidant damage.


Assuntos
Técnicas de Cultura de Células , Células-Tronco Neoplásicas , Animais , Linhagem Celular Tumoral , Citometria de Fluxo , Humanos
3.
An Acad Bras Cienc ; 91(3): e20180487, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31618408

RESUMO

Authentication of cell lines is of paramount importance to validate the results from their use in biomedical research. Although isoenzyme polymorphism is the standard method, molecular methods based on mitochondrial DNA (mtDNA) have been developed to replace it. The aim of this study was the improvement of our isoenzyme electrophoretic analysis and the validation of one molecular technique targeted at mtDNA for the authentication of our animal cell lines. The combined method of cellular lysing through osmotic shock, followed by freezing-thawing in N2 to obtain isoenzyme extracts, and with 42 × 106 cells maintained the best efficiency. The superior electrophoretic conditions were PAGE run at 200 V. All cell lines had isoenzymatic mobility corresponding to their species to lactate dehydrogenase, malate-dehydrogenase, and glucose-6-phosphate dehydrogenase isoenzymes, and could be distinguished from each other. Two molecular techniques based on mtDNA were tested, one on the cytochrome b gene and other on cytochrome c oxidase I subunit gene. Due to difficulties in distinguishing all cell lines using only one these techniques, we merged the primers of two methods in such a way that there was a sufficient differentiation of all DNA fragments. The sequencing of these PCR products was also performed to validate these data.


Assuntos
Técnicas de Cultura de Células/métodos , DNA Mitocondrial/genética , Isoenzimas/análise , Animais , Linhagem Celular , Eletroforese , Glucosefosfato Desidrogenase/análise , L-Lactato Desidrogenase/análise , Malato Desidrogenase/análise , Reação em Cadeia da Polimerase
4.
Nat Biotechnol ; 37(10): 1198-1208, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31501559

RESUMO

Harnessing the potential of human embryonic stem cells to mimic normal and aberrant development with standardized models is a pressing challenge. Here we use micropattern technology to recapitulate early human neurulation in large numbers of nearly identical structures called neuruloids. Dual-SMAD inhibition followed by bone morphogenic protein 4 stimulation induced self-organization of neuruloids harboring neural progenitors, neural crest, sensory placode and epidermis. Single-cell transcriptomics unveiled the precise identities and timing of fate specification. Investigation of the molecular mechanism of neuruloid self-organization revealed a pulse of pSMAD1 at the edge that induced epidermis, whose juxtaposition to central neural fates specifies neural crest and placodes, modulated by fibroblast growth factor and Wnt. Neuruloids provide a unique opportunity to study the developmental aspects of human diseases. Using isogenic Huntington's disease human embryonic stem cells and deep neural network analysis, we show how specific phenotypic signatures arise in our model of early human development as a consequence of mutant huntingtin protein, outlining an approach for phenotypic drug screening.


Assuntos
Ectoderma/fisiologia , Células-Tronco Embrionárias/fisiologia , Doença de Huntington , Neurulação/fisiologia , Telencéfalo/crescimento & desenvolvimento , Técnicas de Cultura de Células , Diferenciação Celular/fisiologia , Linhagem Celular , Humanos , Neurogênese , Telencéfalo/fisiologia
5.
Biomed Environ Sci ; 32(8): 592-601, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31488235

RESUMO

OBJECTIVE: To investigate the development and characterizations of the hepatocytes isolated from fetal ovine and to determine the effect of hypoxia on their growth and metabolism. METHODS: Fresh hepatocytes were isolated from the liver of fetal ovine at late gestation, cultured in specific media, and exposed to normoxia (21% O2) or hypoxia (2% O2). The cellular characteristics and population purity were identified by immunocytochemistry and flow cytometry (FCM). The effects of hypoxia on cell cycle and apoptosis of the hepatocytes were evaluated by FCM, whereas the cellular ultrastructure changes were examined with a transmission electron microscope. RESULTS: The cell purity of hepatocytes was over 95%. Under hypoxia exposure, the hepatocytes showed a gradual increase in proportion at the S phase and in proliferative index, followed with a compatible increase in apoptosis and progressively decreased cell viability. Additionally, the organelles of the hepatocytes demonstrated dramatic changes, including swelling of mitochondria, disorder in cristae arrangement, expansion of endoplasmic reticulum, and a large number of circular lipid droplets emerging in the cytoplasm. CONCLUSION: Fetal ovine hepatocytes could be primarily cultured in a short-term culture system with a high purity of over 95% and with their preserved original characteristics. Hypoxia could induce changes in ultrastructural and inhibit the proliferation of cultured fetal ovine hepatocytes through apoptotic mechanisms.


Assuntos
Feto/fisiologia , Hepatócitos/fisiologia , Oxigênio/análise , Ovinos/fisiologia , Anaerobiose , Animais , Técnicas de Cultura de Células
6.
Nat Protoc ; 14(10): 2781-2817, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31492957

RESUMO

The application of tissue-engineering approaches to human induced pluripotent stem (hiPS) cells enables the development of physiologically relevant human tissue models for in vitro studies of development, regeneration, and disease. However, the immature phenotype of hiPS-derived cardiomyocytes (hiPS-CMs) limits their utility. We have developed a protocol to generate engineered cardiac tissues from hiPS cells and electromechanically mature them toward an adult-like phenotype. This protocol also provides optimized methods for analyzing these tissues' functionality, ultrastructure, and cellular properties. The approach relies on biological adaptation of cultured tissues subjected to biomimetic cues, applied at an increasing intensity, to drive accelerated maturation. hiPS cells are differentiated into cardiomyocytes and used immediately after the first contractions are observed, when they still have developmental plasticity. This starting cell population is combined with human dermal fibroblasts, encapsulated in a fibrin hydrogel and allowed to compact under passive tension in a custom-designed bioreactor. After 7 d of tissue formation, the engineered tissues are matured for an additional 21 d by increasingly intense electromechanical stimulation. Tissue properties can be evaluated by measuring contractile function, responsiveness to electrical stimuli, ultrastructure properties (sarcomere length, mitochondrial density, networks of transverse tubules), force-frequency and force-length relationships, calcium handling, and responses to ß-adrenergic agonists. Cell properties can be evaluated by monitoring gene/protein expression, oxidative metabolism, and electrophysiology. The protocol takes 4 weeks and requires experience in advanced cell culture and machining methods for bioreactor fabrication. We anticipate that this protocol will improve modeling of cardiac diseases and testing of drugs.


Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Miocárdio , Engenharia Tecidual/métodos , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Coração/fisiologia , Humanos , Miocárdio/citologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/fisiologia
7.
Adv Exp Med Biol ; 1158: 183-196, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31452141

RESUMO

The term 'mitochondrial dynamics' is commonly used to refer to ongoing fusion and fission of mitochondrial structures within a living cell. A growing number of diseases, from Charcot Marie Tooth Type 2a neuropathies to cancer, is known to be associated with the dysregulation of mitochondrial dynamics, leading to irregularities of mitochondrial network morphology that are associated with aberrant metabolism and cellular dysfunction. Studying these phenomena, and potential pharmacological interventions to correct them, in cultured cells is a powerful approach to developing treatments or cures. Appropriately designed experiments and quantitative approaches for characterizing mitochondrial morphology and function are essential for furthering our understanding. In this chapter, we discuss the importance of cell incubation conditions, choices around imaging modalities, and data analysis tools with respect to experimental outcomes and the interpretation of results from studies of mitochondrial dynamics. We focus primarily on the quantitative analysis of mitochondrial morphology, providing an overview of the available tools and approaches currently being used and discussing some of the strengths and weaknesses associated with each. Finally, we discuss how the ongoing development of imaging and analysis tools continues to improve our ability to study normal and aberrant mitochondrial physiology in vitro and in vivo.


Assuntos
Mitocôndrias , Dinâmica Mitocondrial , Proteínas Mitocondriais , Técnicas de Cultura de Células , Linhagem Celular , Doença de Charcot-Marie-Tooth/fisiopatologia , Humanos , Mitocôndrias/patologia , Mitocôndrias/fisiologia , Dinâmica Mitocondrial/fisiologia , Proteínas Mitocondriais/metabolismo , Neoplasias/fisiopatologia
8.
Adv Exp Med Biol ; 1152: 413-427, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31456197

RESUMO

For decades 2D culture has been used to study breast cancer. In recent years, however, the importance of 3D culture to recapitulate the complexity of human disease has received attention. A breakthrough for 3D culture came as a result of a Nature editorial 'Goodbye Flat Biology' (Anonymous, Nature 424:861-861, 2003). Since then scientists have developed and implemented a range of different and more clinically relevant models, which are used to study breast cancer. In this chapter multiple different 3D models will be discussed including spheroids, microfluidic and bio-printed models and in silico models.


Assuntos
Neoplasias da Mama/patologia , Técnicas de Cultura de Células , Feminino , Humanos , Microfluídica , Modelos Anatômicos , Impressão Tridimensional
9.
Braz Oral Res ; 33: e058, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31432925

RESUMO

Cementum is the mineralized tissue covering the tooth root that functions in tooth attachment and post-eruptive adjustment of tooth position. It has been reported to be highly similar to bone in several respects but remains poorly understood in terms of development and regeneration. Here, we investigate whether cementocytes, the residing cells in cellular cementum, have the potential to be protagonist in cementum homeostasis, responding to endocrine signals and directing local cementum metabolism. Cells from healthy erupted human teeth were isolated using sequential collagenase/EDTA digestions, and maintained in standard cell culture conditions. A cementocyte-like cell line was cloned (HCY-23, for human cementocyte clone 23), which presented a cementocyte compatible gene expression signature, including the expression of dentin matrix protein 1 ( DMP1 ), sclerostin ( SOST ), and E11/gp38/podoplanin ( E11 ). In contrast, these cells did not express the odontoblast/dentin marker dentin sialoprotein ( DSPP ). HCY-23 cells produced mineral-like nodules in vitro under differentiation conditions, and were highly responsive to inorganic phosphate (Pi). Within the limits of the present study, it can be concluded that cementocytes are phosphate-responsive cells, and have the potential do play a key role in periodontal homeostasis and regeneration.


Assuntos
Técnicas de Cultura de Células/métodos , Cemento Dentário/citologia , Adolescente , Adulto , Análise de Variância , Proteínas Morfogenéticas Ósseas/análise , Proteínas Morfogenéticas Ósseas/genética , Linhagem Celular , Cemento Dentário/metabolismo , Proteínas da Matriz Extracelular/análise , Proteínas da Matriz Extracelular/genética , Feminino , Imunofluorescência , Expressão Gênica , Marcadores Genéticos/genética , Humanos , Masculino , Glicoproteínas de Membrana/análise , Glicoproteínas de Membrana/genética , Dente Molar/citologia , Fosfatos/farmacologia , Fosfoproteínas/análise , Fosfoproteínas/genética , Sialoglicoproteínas/análise , Sialoglicoproteínas/genética , Fatores de Tempo , Adulto Jovem
10.
Nat Cell Biol ; 21(8): 1041-1051, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31371824

RESUMO

Endometrial disorders represent a major gynaecological burden. Current research models fail to recapitulate the nature and heterogeneity of these diseases, thereby hampering scientific and clinical progress. Here we developed long-term expandable organoids from a broad spectrum of endometrial pathologies. Organoids from endometriosis show disease-associated traits and cancer-linked mutations. Endometrial cancer-derived organoids accurately capture cancer subtypes, replicate the mutational landscape of the tumours and display patient-specific drug responses. Organoids were also established from precancerous pathologies encompassing endometrial hyperplasia and Lynch syndrome, and inherited gene mutations were maintained. Endometrial disease organoids reproduced the original lesion when transplanted in vivo. In summary, we developed multiple organoid models that capture endometrial disease diversity and will provide powerful research models and drug screening and discovery tools.


Assuntos
Avaliação Pré-Clínica de Medicamentos , Neoplasias do Endométrio/patologia , Organoides/patologia , Doenças Uterinas/patologia , Técnicas de Cultura de Células/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Neoplasias do Endométrio/tratamento farmacológico , Neoplasias do Endométrio/metabolismo , Endométrio/patologia , Feminino , Humanos , Organoides/efeitos dos fármacos , Organoides/metabolismo , Doenças Uterinas/tratamento farmacológico , Doenças Uterinas/metabolismo
11.
Cell Prolif ; 52(5): e12668, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31379046

RESUMO

OBJECTIVES: Reproducing human hair follicles in vitro is often limited by various reasons such as the lack of a systematic approach to culture distinct hair follicle cell types to reproduce their spatial relationship. Here, we reproduce hair follicle-like constructs resembling the spatial orientation of different cells in vivo, to study the role of keratinocytes in maintaining cellular compartmentalization among hair follicle-related cells. MATERIALS AND METHODS: Dermal papilla (DP) cells, HaCaT keratinocytes and human dermal fibroblast (HDF) cells were seeded sequentially into three-dimensional (3D) microwells fabricated from polyethylene glycol diacrylate hydrogels. Quantitative polymerase chain reaction was used to compare inductive gene expression of 3D and two-dimensional (2D) DP. DP and HaCaT cells were transfected with green fluorescent protein and red fluorescent protein lentivirus, respectively, to enable cell visualization using confocal microscopy. RESULTS: The 3D DP cultures showed significantly enhanced expression of essential DP genes as compared 2D cultures. Core-shell configurations containing keratinocytes forming the outer shell and DP forming the core were observed. Migratory polarization was mediated by cell-cell interaction between the keratinocytes and HDF cells, while preserving the aggregated state of the DP cells. CONCLUSIONS: Keratinocytes may play a role in maintaining compartmentalization between the DP and the surrounding HDF residing in the dermis, and therefore maintains the aggregative state of the DP cells, necessary for hair follicle development and function.


Assuntos
Técnicas de Cultura de Células/métodos , Derme/citologia , Fibroblastos/citologia , Queratinócitos/citologia , Células Cultivadas , Derme/metabolismo , Fibroblastos/metabolismo , Humanos , Hidrogéis/química , Queratinócitos/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal
12.
Tumour Biol ; 41(8): 1010428319866369, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31402761

RESUMO

Gaining a better understanding of the biological properties of cell-free DNA constitutes an important step in the development of clinically meaningful cell-free DNA-based tests. Since the in vivo characterization of cell-free DNA is complicated by the immense heterogeneity of blood samples, an increasing number of in vitro cell culture experiments, which offer a greater level of control, are being conducted. However, cell culture studies are currently faced with three notable caveats. First, the concentration of cell-free DNA in vitro is relatively low. Second, the median amount and size of cell-free DNA in culture medium varies greatly between cell types. Third, the amount and size of cell-free DNA in the culture medium of a single cell line fluctuates over time. Although these are interesting findings, it can also be a great source of experimental confusion and emphasizes the importance of method optimization and standardization. Therefore, in this study, we compared five commonly used cell-free DNA quantification methods, including quantitative polymerase chain reaction, Qubit Double-Stranded DNA High Sensitivity assay, Quant-iT PicoGreen Assay, Bioanalyzer High Sensitivity DNA assay, and NanoDrop Onec. Analysis of the resulting data, along with an interpretation of theoretical values (i.e. the theoretical detection and quantification limits of the respective methods), enables the calculation of optimal conditions for several important preanalytical steps pertaining to each quantification method and different cell types, including the (1) time-point at which culture medium should be collected for cell-free DNA extraction, (2) amount of cell culture supernatant from which to isolate cell-free DNA, (3) volume of elution buffer, and (4) volume of cell-free DNA sample to use for quantification.


Assuntos
Ácidos Nucleicos Livres/química , Meios de Cultura/química , Técnicas de Cultura de Células , Corantes Fluorescentes/química , Humanos , Compostos Orgânicos/química
13.
Sheng Wu Gong Cheng Xue Bao ; 35(8): 1374-1381, 2019 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-31441608

RESUMO

As one of the top 10 breakthrough and emerging technologies in the world in 2018, cultured meat has attracted extensive attention due to its advantages of traceable origin, food safety and green sustainable development. Europe and the United States have invested a lot of resources to focus on research about cultured meat, which will affect our domestic meat and food market in the future. At present, the challenge of cultured meat production is how to efficiently simulate the growth environment of animal muscle tissue and realize large-scale production in bioreactor. Although cell tissue engineering has been deeply studied and achieved varying successful application, it is still difficult to obtain large-scale cultured meat production due to the high cost and technical requirements. Therefore, the development of efficient and safe cell culture technology is an urgent problem for large-scale cultured meat production, which can effectively reduce costs and achieve industrial application. In this review, we summarize the research progress of animal cell tissue culture technology used for cultured meat, and highlighted the current challenges and possible strategies in further applications.


Assuntos
Técnicas de Cultura de Células , Carne , Animais , Reatores Biológicos , Engenharia Tecidual , Estados Unidos
14.
Carbohydr Polym ; 222: 114974, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31320071

RESUMO

Stable hydrogels with a mechanically strong matrix microenvironment are favorable biomaterials for three-dimensional cell culture. Acidic collagen solution is commonly combined with chemical crosslinkers for rapid network formation. Herein, dialdehyde carboxymethyl cellulose (DCMC) was selected as an optimal crosslinking reagent for its excellent biocompatibility and suitable chemical reactivity. Both shielding of electrostatic attractions between these two oppositely charged biomaterials and obtaining concentrated collagen solution were achieved using a novel biphasic acetic acid /1-ethyl-3-methylimidazolim acetate (AA/[EMIM][Ac]) solvent system. Hydrogel composites containing more crosslinks were obtained by increasing collagen concentrations (5-25 mg/mL), as confirmed by the improved mechanical properties, thermal denaturation temperature, anti-enzymatic ability and compact microstructure. Moreover, cell proliferation assay demonstrated that all the obtained DCMC-crosslinked collagen hydrogel composites ensures commendable biocompatibility. This study provides a promising strategy for manipulating stable and biocompatible hydrogel composites by blending concentrated collagen solution with DCMC in a biphasic solvent system.


Assuntos
Materiais Biocompatíveis/química , Carboximetilcelulose Sódica/química , Técnicas de Cultura de Células/métodos , Celulose/análogos & derivados , Colágeno/química , Hidrogéis/química , Animais , Fenômenos Biomecânicos , Bovinos , Células Cultivadas , Celulose/química , Reagentes para Ligações Cruzadas/química , Pele/metabolismo , Solventes/química
15.
Zhonghua Gan Zang Bing Za Zhi ; 27(6): 424-429, 2019 Jun 20.
Artigo em Chinês | MEDLINE | ID: mdl-31357757

RESUMO

Objective: To explore the effect of substrate mechanical microenvironment and cell-cell interaction on differentiation of bone marrow mesenchymal stem cells (BMSCs), intrahepatic cellular function and phenotype. Methods: Bone marrow mesenchymal stem cells (BMSCs)-hepatocytes (HCs) and BMSCs-hepatic stellate cells (HSCs) were co-cultured on polyvinyl alcohol (PVA) hydrogel substrates at different stiffness (4.50 ± 0.47 kPa, 19.00 ± 3.51 kPa and 37.00 ± 2.09 kPa) by non-contact co-culture method. Furthermore, the effect of substrate mechanical microenvironment on BMSCs, HCs and HSCs and the activation and proliferation of HCs under different co-cultured condition was studied. A Student's t-test was used to compare the two groups. Results: The expression ofα-smooth muscle actin (α-SMA) and collagenα1- I (Col1A1) in BMSCs and HSCs cultured on its own increased with increase of substrate stiffness. After 72 h, the expression of albumin (ALB) of HCs on three stiff substrates was significantly higher than that of 24 and 48 h. Moreover, the expression of ALB of HCs increased with the increase of substrate stiffness. During the co-culture of BMSCs and HSCs, BMSCs of all three stiffness substrates promoted the expression ofα-SMA, Col1A1 in HSCs, but reduced the expression of PPARγin HSCs cells, thererby promoted the activation of HSCs, with apparent stiffness at 37 kPa. HSCs promoted the expression of ABL in BMSCs at three stiff substrates, but inhibited the expression of alpha-SMA and Col1A1 in BMSCs at 37 kPa, suggesting that co-culture had inhibited the differentiation of BMSCs myofibroblasts, and promoted the differentiation of hepatocyte-like cells, especially at high stiff substrates. In the co-culture of BMSCs and hepatic parenchymal cells, BMSCs had promoted the proliferation of hepatic parenchymal cells at 4.5 kPa. Further, hepatic parenchymal cells had inhibited the expression ofα-SMA in BMSCs, and promoted the expression of Alb, with inhibition of BMSCs differentiation towards myofibroblasts. Conclusion: The differentiation of BMSCs affects the substrate mechanical microenvironment, co-culture of HCs and HSCs. Simultaneously, affecting the function of hepatocytes in relation to the mechanical state of the substrates.


Assuntos
Células da Medula Óssea , Comunicação Celular , Técnicas de Cultura de Células , Diferenciação Celular , Células Estreladas do Fígado , Células-Tronco Mesenquimais , Animais , Células da Medula Óssea/citologia , Comunicação Celular/fisiologia , Técnicas de Cultura de Células/métodos , Células Cultivadas , Microambiente Celular/fisiologia , Células Estreladas do Fígado/citologia , Humanos , Células-Tronco Mesenquimais/citologia , Ratos , Ratos Sprague-Dawley
16.
Biol Res ; 52(1): 39, 2019 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-31358053

RESUMO

In the growth condition(s) of plants, numerous secondary metabolites (SMs) are produced by them to serve variety of cellular functions essential for physiological processes, and recent increasing evidences have implicated stress and defense response signaling in their production. The type and concentration(s) of secondary molecule(s) produced by a plant are determined by the species, genotype, physiology, developmental stage and environmental factors during growth. This suggests the physiological adaptive responses employed by various plant taxonomic groups in coping with the stress and defensive stimuli. The past recent decades had witnessed renewed interest to study abiotic factors that influence secondary metabolism during in vitro and in vivo growth of plants. Application of molecular biology tools and techniques are facilitating understanding the signaling processes and pathways involved in the SMs production at subcellular, cellular, organ and whole plant systems during in vivo and in vitro growth, with application in metabolic engineering of biosynthetic pathways intermediates.


Assuntos
Reguladores de Crescimento de Planta/metabolismo , Fenômenos Fisiológicos Vegetais , Metabolismo Secundário/fisiologia , Estresse Fisiológico/fisiologia , Técnicas de Cultura de Células , Regulação da Expressão Gênica de Plantas/fisiologia , Raízes de Plantas/metabolismo , Brotos de Planta/metabolismo , Plantas/metabolismo , Transdução de Sinais
17.
Adv Exp Med Biol ; 1140: 531-539, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31347069

RESUMO

Stable isotope labeling by amino acids in cell culture (SILAC) is a powerful approach for high-throughput quantitative proteomics. SILAC allows highly accurate protein quantitation through metabolic encoding of whole cell proteomes using stable isotope labeled amino acids. Since its introduction in 2002, SILAC has become increasingly popular. In this chapter we review the methodology and application of SILAC, with an emphasis on three research areas: dynamics of posttranslational modifications, protein-protein interactions, and protein turnover.


Assuntos
Aminoácidos/química , Técnicas de Cultura de Células , Marcação por Isótopo , Proteômica/métodos , Proteoma
18.
Adv Exp Med Biol ; 1140: 575-583, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31347072

RESUMO

The global measurement of assembly and turnover of protein containing complexes within cells has advanced with the development of pulse stable isotope labelled amino acid approaches. Stable isotope labeling with amino acids in cell culture (SILAC) allows the incorporation of "light" 12-carbon amino acids or "heavy" 13-carbon amino acids into cells or organisms and the quantitation of proteins and peptides containing these amino acid tags using mass spectrometry. The use of these labels in pulse or pulse-chase scenarios has enabled measurements of macromolecular dynamics in cells, on time scales of several hours. Here we review advances with this approach and alternative or parallel strategies. We also examine the statistical considerations impacting datasets detailing mitochondrial assembly, to highlight key parameters in establishing significance and reproducibility.


Assuntos
Aminoácidos/química , Técnicas de Cultura de Células , Marcação por Isótopo , Espectrometria de Massas , Proteínas/análise , Reprodutibilidade dos Testes
19.
Anticancer Res ; 39(7): 3413-3418, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31262863

RESUMO

One aim of cell-based in vitro assays is to identify the best drug candidate to develop using the best tumor cell model. This is challenging in every anticancer drug discovery process. Briefly, we summarize the parameters to be taken into account when performing in vitro cell assays, in order to obtain reliable and reproducible results, which was fundamentally discussed by lecturers at the educational course on preclinical and early-phase clinical pharmacology studies, at the 40th Winter Meeting of the Pharmacology and Molecular Mechanisms Group of the European Organization for Research and Treatment of Cancer. Moreover, specific cellular sensitivity tests are described. In addition to monolayer in vitro cell models for the screening of new potential candidate drugs, three-dimensional tumor/cell tissue models are emerging as new pre-clinical tools that more closely reflect the in vivo microenvironment. Therefore, the use of different in vitro models for drug screening can enhance the predictability and reliability of pre-clinical drug-discovery phases and target validation.


Assuntos
Avaliação Pré-Clínica de Medicamentos , Farmacologia Clínica/métodos , Bioensaio , Técnicas de Cultura de Células , Humanos
20.
APMIS ; 127(12): 737-745, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31273832

RESUMO

Currently, adoptive immunotherapy is considered as one of the leading treatments in cancer. Successful adoptive immunotherapy depends on producing large numbers of desired T cells ex vivo for infusion. This requires an effective protocol for maximum functional T-cell expansion while keeping the time and costs to a minimum. Current T-cell expansion protocols are diverse in their methodology, and a universal protocol of expansion is wanting. Also, new findings regarding T-cell biology, signaling, and activation have reshaped the strategies of T-cell propagation over the years, introducing new ways to expand T cells. Here, we reviewed different conditions for blood-derived polyclonal T-cell expansion so as to elucidate the influential factors of T-cell expansion and their efficacy.


Assuntos
Imunoterapia Adotiva , Ativação Linfocitária/imunologia , Linfócitos T/imunologia , Técnicas de Cultura de Células , Proliferação de Células/efeitos dos fármacos , Meios de Cultura , Humanos , Interleucinas/farmacologia , Neoplasias/terapia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo
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