共查询到20条相似文献,搜索用时 15 毫秒
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Kevin J. Zwezdaryk Jessica A. Warner Heather L. Machado Cindy A. Morris Kerstin H?ner zu Bentrup 《Journal of visualized experiments : JoVE》2012,(59)
The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines1 support our understanding of invasion of the uterine wall2 and remodeling of uterine spiral arteries3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu13.We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations.
In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS. 相似文献
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Jack Litwin 《Applied microbiology》1970,20(6):899-906
Human embryonic diploid lung fibroblasts grown in Eagle's medium were exposed continually to a variety of environmental conditions over a large number of passages to observe how these conditions affected the growth and longevity of these cells in vitro. The cells grew well at temperatures between 34 and 37 C and some cells could be adapted to grow at 40 C. Very limited growth occurred at 30 to 31 C; however, confluent monolayers of cells could be maintained for months at 30 C and still give rise to actively growing cultures. Increasing the amino acid concentration in Eagle's medium or the calf serum concentration above 10% had no effect on the growth rate or longevity. One per cent calf serum could not support prolonged active growth. Trypsin concentrations between 1 and 0.1% and crystalline trypsin at 50 μg/ml showed no influence on cell growth. Ethylenediaminetetraacetic acid treatment and scraping, however, destroyed many of the cells, and the survivors grew poorly. The clonal morphology varied with age. Young cells frequently gave rise to densely packed clones, whereas older cells gave rise to clones with widely scattered cells. The cloning efficiency was high when the cells were young but decreased rapidly with successive passage. It was relatively constant from the 7th to 20th passage at about 15%. 相似文献
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Cleo Goyvaerts Jozef Dingemans Kurt De Groeve Carlo Heirman Ellen Van Gulck Guido Vanham Patrick De Baetselier Kris Thielemans Geert Raes Karine Breckpot 《Journal of virology》2013,87(20):11304-11308
Antigen-presenting cells are a heterogeneous group of cells that are characterized by their functional specialization. Consequently, targeting specific antigen-presenting cell subsets offers opportunities to induce distinct T cell responses. Here we report on the generation and use of nanobodies (Nbs) to target lentivectors specifically to human lymph node-resident myeloid dendritic cells, demonstrating that Nbs represent a powerful tool to redirect lentivectors to human antigen-presenting cell subsets. 相似文献
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Ernesto Méndez Claudia Mu?oz-Ya?ez Claudia Sánchez-San Martín Gabriela Aguirre-Crespo M. del Rocio Ba?os-Lara Michelle Gutierrez Rafaela Espinosa Yunuén Acevedo Carlos F. Arias Susana López 《Journal of virology》2014,88(5):2452-2460
Human astroviruses (HAstV) are a frequent cause of gastroenteritis in young children and immunocompromised patients. To understand the early steps of HAstV infection in the highly permissive Caco-2 cell line, the binding and entry processes of the virus were characterized. The half-time of virus binding to the cell surface was about 10 min, while virus decapsidation took around 130 min. Drugs affecting clathrin-mediated endocytosis, endosome acidification, and actin filament polymerization, as well as those that reduce the presence of cholesterol in the cell membrane, decreased the infectivity of the virus. The infection was also reduced by silencing the expression of the clathrin heavy chain (CHC) by RNA interference or by overexpression of dominant-negative mutants of dynamin 2 and Eps15. Furthermore, the entry of HAstV apparently depends on the maturation of endosomes, since the infection was reduced by silencing the expression of Rab7, a small GTPase involved in the early- to late-endosome maturation. Altogether, our results suggest that HAstV enters Caco-2 cells using a clathrin-dependent pathway and reaches late endosomes to enter cells. Here, we have characterized the mechanism used by human astroviruses, important agents of gastroenteritis in children, to gain entry into their host cells. Using a combination of biochemical and genetic tools, we found that these viruses enter Caco-2 cells using a clathrin-dependent endocytic pathway, where they most likely need to travel to late endosomes to reach the cytoplasm and begin their replication cycle. 相似文献
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Variation in the size of cells, and the DNA they contain, is a basic feature of multicellular organisms that affects countless aspects of their structure and function. Within humans, cell size is known to vary by several orders of magnitude, and differences in nuclear DNA content among cells have been frequently observed. Using published data, here we describe how the quantity of nuclear DNA across 19 different human cell types increases with cell volume. This observed increase is similar to intraspecific relationships between DNA content and cell volume in other species, and interspecific relationships between diploid genome size and cell volume. Thus, we speculate that the quantity of nuclear DNA content in somatic cells of humans is perhaps best viewed as a distribution of values that reflects cell size distributions, rather than as a single, immutable quantity.Our understanding of the complexity of multicellular organisms, and the diverse cells of which they are comprised, has dramatically increased over the past several decades. Yet, we still lack an understanding of some of the most basic features of the cells that constitute multicellular organisms. For example, the number of different cell types in an organism, or the rate at which different cells grow, divide, and die, remain poorly understood (see Niklas 2015). But perhaps most important, we lack an understanding of the size and abundance of cells that constitute an organism (see Amodeo and Skotheim 2015). Cell size, in particular, affects virtually all structural and functional attributes of multicellular organisms, from the molecular level to the whole organism level.One key feature of organisms that may vary with cell size is the amount of nuclear DNA. Across species, genome size has long been known to correlate positively with cell and nuclear volume (Price et al. 1973; Szarski 1976; Olmo 1983). But within species, too, the nuclear DNA content of somatic cells has been shown in a few instances to increase with cell size in species such as Daphnia (Beaton and Hebert 1989) and Arabidopsis (Jovtchev et al. 2006). Such increases in nuclear DNA content can have important consequences for cell function, in general, and gene expression, in particular (Hancock et al. 2008; Lee et al. 2009; De Veylder et al. 2011; Marguerat and Bähler 2012).In the case of humans, substantial differences in DNA content have been observed in many human cell types. Indeed, since Watson and Crick described the structure of DNA, studies of healthy human tissues have reported the presence of polyploid cells (Winkelmann et al. 1987; Biesterfeld et al. 1994). The cell types in which this has been observed appear to have little in common, except that they are generally stable, fully differentiated cells (Winkelmann et al. 1987). Still, these observations have done little to change the traditional view that all healthy somatic cells in the human body hold the same characteristic quantity of DNA (∼7 billion base pairs) based on the long-standing principle of DNA constancy (Mirsky and Ris 1949). Deviations from the diploid quantity of DNA in humans, like other animals, are still often viewed as exceptional, tissue-specific, or indicative of pathology. A more synthetic view of differences in nuclear DNA content across human cell types may provide some clarity on these and other issues.In this review, we compile and analyze published data to examine the extent to which nuclear DNA content varies across diverse human cell types, and whether such variation is correlated with cell size. We then compare these results with previously reported relationships between nuclear DNA content and cell size within four other species. Finally, we compare these results with the relationships between diploid genome size and cell size observed across species in several broad taxonomic groups. These analyses suggest that systematic variation in nuclear DNA content is a more ubiquitous phenomenon in human cells than was previously appreciated. However, as we later discuss, the mechanisms underlying these patterns remain in question. 相似文献
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Fabrizio D’Anselmi Maria Grazia Masiello Alessandra Cucina Sara Proietti Simona Dinicola Alessia Pasqualato Giulia Ricci Gabriella Dobrowolny Angela Catizone Alessandro Palombo Mariano Bizzarri 《PloS one》2013,8(12)
The microenvironment drives mammary gland development and function, and may influence significantly both malignant behavior and cell growth of mammary cancer cells. By restoring context, and forcing cells to properly interpret native signals from the microenvironment, the cancer cell aberrant behavior can be quelled, and organization re-established. In order to restore functional and morphological differentiation, human mammary MCF-7 and MDA-MB-231 cancer cells were allowed to grow in a culture medium filled with a 10% of the albumen (EW, Egg White) from unfertilized chicken egg. That unique microenvironment behaves akin a 3D culture and induces MCF-7 cells to produce acini and branching duct-like structures, distinctive of mammary gland differentiation. EW-treated MDA-MB-231 cells developed buds of acini and duct-like structures. Both MCF-7 and MDA-MB-231 cells produced β-casein, a key milk component. Furthermore, E-cadherin expression was reactivated in MDA-MB-231 cells, as a consequence of the increased cdh1 expression; meanwhile β-catenin – a key cytoskeleton component – was displaced behind the inner cell membrane. Such modification hinders the epithelial-mesenchymal transition in MDA-MB-231 cells. This differentiating pathway is supported by the contemporary down-regulation of canonical pluripotency markers (Klf4, Nanog). Given that egg-conditioned medium behaves as a 3D-medium, it is likely that cancer phenotype reversion could be ascribed to the changed interactions between cells and their microenvironment. 相似文献
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Jing Luo Petr Baranov Sherrina Patel Hong Ouyang John Quach Frances Wu Austin Qiu Hongrong Luo Caroline Hicks Jing Zeng Jing Zhu Jessica Lu Nicole Sfeir Cindy Wen Meixia Zhang Victoria Reade Sara Patel John Sinden Xiaodong Sun Peter Shaw Michael Young Kang Zhang 《The Journal of biological chemistry》2014,289(10):6362-6371
Cell transplantation is a potential therapeutic strategy for retinal degenerative diseases involving the loss of photoreceptors. However, it faces challenges to clinical translation due to safety concerns and a limited supply of cells. Human retinal progenitor cells (hRPCs) from fetal neural retina are expandable in vitro and maintain an undifferentiated state. This study aimed to investigate the therapeutic potential of hRPCs transplanted into a Royal College of Surgeons (RCS) rat model of retinal degeneration. At 12 weeks, optokinetic response showed that hRPC-grafted eyes had significantly superior visual acuity compared with vehicle-treated eyes. Histological evaluation of outer nuclear layer (ONL) characteristics such as ONL thickness, spread distance, and cell count demonstrated a significantly greater preservation of the ONL in hRPC-treated eyes compared with both vehicle-treated and control eyes. The transplanted hRPCs arrested visual decline over time in the RCS rat and rescued retinal morphology, demonstrating their potential as a therapy for retinal diseases. We suggest that the preservation of visual acuity was likely achieved through host photoreceptor rescue. We found that hRPC transplantation into the subretinal space of RCS rats was well tolerated, with no adverse effects such as tumor formation noted at 12 weeks after treatment. 相似文献
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Endocrine Cell Clustering During Human Pancreas Development 总被引:1,自引:0,他引:1
Jongmin Jeon Mayrin Correa-Medina Camillo Ricordi Helena Edlund Juan A. Diez 《The journal of histochemistry and cytochemistry》2009,57(9):811-824
The development of efficient, reproducible protocols for directed in vitro differentiation of human embryonic stem (hES) cells into insulin-producing β cells will benefit greatly from increased knowledge regarding the spatiotemporal expression profile of key instructive factors involved in human endocrine cell generation. Human fetal pancreases 7 to 21 weeks of gestational age, were collected following consent immediately after pregnancy termination and processed for immunostaining, in situ hybridization, and real-time RT-PCR expression analyses. Islet-like structures appear from approximately week 12 and, unlike the mixed architecture observed in adult islets, fetal islets are initially formed predominantly by aggregated insulin- or glucagon-expressing cells. The period studied (7–22 weeks) coincides with a decrease in the proliferation and an increase in the differentiation of the progenitor cells, the initiation of NGN3 expression, and the appearance of differentiated endocrine cells. The present study provides a detailed characterization of islet formation and expression profiles of key intrinsic and extrinsic factors during human pancreas development. This information is beneficial for the development of efficient protocols that will allow guided in vitro differentiation of hES cells into insulin-producing cells. (J Histochem Cytochem 57:811–824, 2009) 相似文献
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GLYNIS SCOTT JAMES EWING DANIEL RYAN CAMILLE ABBOUD 《Pigment cell & melanoma research》1994,7(1):44-51
Stem cell factor (SCF) is hypothesized to play a critical role in the migration of melanocytes during embryogenesis because mutations in either the SCF gene, or its ligand, c-kit, result in defects in coat pigmentation in mice and in skin pigmentation in humans. In this report we directly show that SCF alters the adhesion and migration of human melanocytes to extracellular matrix (ECM) ligands and regulates integrin expression at the protein level. SCF decreased adhesion of neonatal and fetal cells to collagen IV, and increased attachment of fetal cells to laminin. Attachment of fetal cells to fibronectin was decreased, but was unchanged in neonatal cells. Flow cytometry analysis of neonatal melanocytes showed that SCF down-regulated the expression of the α2 receptor, and up-regulated the expression of the α3, α5 and β1 integrin receptors. SCF down-regulated expression of α2, α5 and β1 integrins by fetal melanocytes, and up-regulated expression of the αv and α3 integrin receptors. Analysis of melanocyte migration using time-lapse videomicroscopy showed that SCF significantly increased migration of neonatal, but not fetal, melanocytes on fibronectin (FN). We conclude that SCF regulates integrin expression at the protein level and that SCF has pleiotropic effects on melanocyte attachment and migration on ECM ligands. We suggest that this may be one mechanism by which SCF regulates melanocyte migration during development of the skin. 相似文献
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人胰腺细胞培养及胰岛素的分泌王石泉,汤国枝,张鹤云,李敏意,金以丰(南京大学生物化学系,南京210093)胰岛β细胞的体外培养获得胰岛素已有报道,但大多采用新生大鼠胰腺,且β细胞成活率低,分泌量少,还处在研究阶段[1-4].本实验采用人胰腺细胞做较大... 相似文献
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重组人干细胞因子的纯化 总被引:2,自引:0,他引:2
干细胞因子 (SCF)是一种重要的造血生长因子 ,它在自体造血干细胞动员、肿瘤放疗化疗的辅助治疗、贫血和其它血液病的治疗上具有良好的应用前景 .为建立一种实用的rhSCF制备工艺 ,从表达rhSCF的工程菌分离包涵体 ,用尿素变性 ,低浓度尿素溶液稀释复性 .复性液中的rhSCF经离子交换层析吸附、浓缩 ,凝胶排阻层析分离去除共价二聚体和疏水层析精纯化 ,获得纯化的rhSCF .纯化的rhSCF纯度大于 95 % ,回收率为 4 7% ,可促进人红白血病细胞TF - 1的生长 ,比活性为 0 .9× 10 6U mg ,与英国NIBSC的rhSCF标准品相似 .上述结果表明 ,所建立的制备高纯度rhSCF工艺高效简便 ,产物具有良好的生物活性 相似文献
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The microassay for human and chick interferon described in this paper required much smaller amounts of samples and reagents and considerably less time and effort than the plaque reduction assay while yielding comparable results. 相似文献
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Although Merkel cell carcinoma of the eyelid is reported frequently in the literature, only limited information exists about the distribution of Merkel cells in this tissue. Therefore, serial sections of 18 human cadaver eyelids (donors ages ranging between 63 and 97 years) were stained for cytokeratin 20 in various planes. The overall appearance of Merkel cells in these samples was low and mainly located in the outer root layer of the cilia hair follicles. Merkel cells were more frequent in the middle, and almost not detectable at the nasal and temporal edges. The localization is in accordance with that of Merkel cell carcinoma, but concerning the scarce appearance within this adulthood group, a specific physiological role of these cells in the eyelid is difficult to establish.Key words: eyelid, human, cytokeratin 20, Merkel cell 相似文献