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Sample records for cultured beas-2b cells

  1. Culture medium type affects endocytosis of multi-walled carbon nanotubes in BEAS-2B cells and subsequent biological response.

    Science.gov (United States)

    Haniu, Hisao; Saito, Naoto; Matsuda, Yoshikazu; Tsukahara, Tamotsu; Maruyama, Kayo; Usui, Yuki; Aoki, Kaoru; Takanashi, Seiji; Kobayashi, Shinsuke; Nomura, Hiroki; Okamoto, Masanori; Shimizu, Masayuki; Kato, Hiroyuki

    2013-09-01

    We examined the cytotoxicity of multi-walled carbon nanotubes (MWCNTs) and the resulting cytokine secretion in BEAS-2B cells or normal human bronchial epithelial cells (HBEpCs) in two types of culture media (Ham's F12 containing 10% FBS [Ham's F12] and serum-free growth medium [SFGM]). Cellular uptake of MWCNT was observed by fluorescent microscopy and analyzed using flow cytometry. Moreover, we evaluated whether MWCNT uptake was suppressed by 2 types of endocytosis inhibitors. We found that BEAS-2B cells cultured in Ham's F12 and HBEpCs cultured in SFGM showed similar biological responses, but BEAS-2B cells cultured in SFGM did not internalize MWCNTs, and the 50% inhibitory concentration value, i.e., the cytotoxicity, was increased by more than 10-fold. MWCNT uptake was suppressed by a clathrin-mediated endocytosis inhibitor and a caveolae-mediated endocytosis inhibitor in BEAS-2B cells cultured in Ham's F12 and HBEpCs cultured in SFGM. In conclusion, we suggest that BEAS-2B cells cultured in a medium containing serum should be used for the safety evaluation of nanomaterials as a model of normal human bronchial epithelial cells. However, the culture medium composition may affect the proteins that are expressed on the cytoplasmic membrane, which may influence the biological response to MWCNTs.

  2. Cellular interactions and biological responses to titanium dioxide nanoparticles in HepG2 and BEAS-2B cells: role of cell culture media.

    Science.gov (United States)

    Prasad, Raju Y; Simmons, Steven O; Killius, Micaela G; Zucker, Robert M; Kligerman, Andrew D; Blackman, Carl F; Fry, Rebecca C; Demarini, David M

    2014-05-01

    We showed previously that exposure of human lung cells (BEAS-2B) to TiO2 nanoparticles (nano-TiO2 ) produced micronuclei (MN) only when the final concentration of protein in the cell-culture medium was at least 1%. Nanoparticles localize in the liver; thus, we exposed human liver cells (HepG2) to nano-TiO2 and found the same requirement for MN induction. Nano-TiO2 also formed small agglomerates in medium containing as little as 1% protein and caused cellular interaction as measured by side scatter by flow cytometry and DNA damage (comet assay) in HepG2 cells. Nano-TiO2 also increased the activity of the inflammatory factor NFkB but not of AP1 in a reporter-gene HepG2 cell line. Suspension of nano-TiO2 in medium containing 0.1% protein was sufficient for induction of MN by the nanoparticles in either BEAS-2B or HepG2 cells as long the final concentration of protein in the cell-culture medium was at least 1%.

  3. Signaling factors and pathways of α-particle irradiation induced bilateral bystander responses between Beas-2B and U937 cells

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    Fu, Jiamei; Wang, Juan; Wang, Xiangdong; Wang, Ping; Xu, Jinping; Zhou, Cuiping; Bai, Yang; Shao, Chunlin, E-mail: clshao@shmu.edu.cn

    2016-07-15

    Highlights: • Radiation damage of Beas-2B cells was enhanced by macrophage-mediated bilateral bystander responses. • Expressions of TNF-α and IL-8 in the α-irradiated Beas-2B cells were dependent on ERK and p38 pathways. • The neighboring U937 cells further increased the generation of TNF-α and IL-8 in the α-irradiated Beas-2B cells. • NF-κB dependent upregulation of TNF-α and IL-8 was induced in the bystander U937 cells. - Abstract: Although radiation induced bystander effects (RIBE) have been investigated for decades for their potential health risk, the underlying gene regulation is still largely unclear, especially the roles of immune system and inflammatory response in RIBE. In the present study, macrophage U937 cells and epithelial Beas-2B cells were co-cultured to disclose the cascades of bystander signaling factors and intercellular communications. After α-particle irradiation, both ERK and p38 pathways were activated in Beas-2B cells and were associated with the autocrine and paracrine signaling of TNF-α and IL-8, resulting in direct damage to the irradiated cells. Similar upregulation of TNF-α and IL-8 was induced in the bystander U937 cells after co-culture with α-irradiated Beas-2B cells. This upregulation was dependent on the activation of NF-κB pathway and was responsible for the enhanced damage of α-irradiated Beas-2B cells. Interestingly, the increased expressions of TNF-α and IL-8 mRNAs in the bystander U937 cells were clearly relayed on the activated ERK and p38 pathways in the irradiated Beas-2B cells, and the upregulation of TNF-α and IL-8 mRNAs in co-cultured Beas-2B cells was also partly due to the activated NF-κB pathway in the bystander U937 cells. With the pretreatment of U0126 (MEK1/2 inhibitor), SB203580 (p38 inhibitor) or BAY 11-7082 (NF-κB inhibitor), the aggravated damage in the α-irradiated Beas-2B cells could be largely alleviated. Our results disclosed novel signaling cascades of macrophage-mediated bilateral

  4. Cellular Interactions and Biological Responses to Titanium Dioxide Nanoparticles in HepG2 and BEAS-2B Cells: Role of Cell Culture Media

    Science.gov (United States)

    ABSTRACT We have shown previously that the composition of the biological medium used in vitro can affect the cellular interaction and biological response of titanium dioxide nanoparticles (nano-TiO2) in human lung epithelial cells. However, it is unclear if these effects are co...

  5. Cellular Interactions and Biological Responses to Titanium Dioxide Nanoparticles in HepG2 and BEAS-2B Cells: Role of Cell Culture Media

    Science.gov (United States)

    ABSTRACT We have shown previously that the composition of the biological medium used in vitro can affect the cellular interaction and biological response of titanium dioxide nanoparticles (nano-TiO2) in human lung epithelial cells. However, it is unclear if these effects are co...

  6. Propofol inhibits LPS-induced apoptosis in lung epithelial cell line, BEAS-2B.

    Science.gov (United States)

    Lv, Xiang; Zhou, Xuhui; Yan, Jia; Jiang, Jue; Jiang, Hong

    2017-03-01

    Lipopolysaccharide (LPS) plays an important role in lung endothelial apoptosis which is crucial for lung fibrogenesis in ARDS progression. Reactive oxygen species (ROS) has been reported to be involved in LPS-induced lung epithelial cell apoptosis. Propofol is a commonly used intravenous anesthetic agent in clinic and it could attenuate LPS-induced epithelial cells oxidation and apoptosis. However, the mechanisms are still obscure. In this study, we examined whether and how propofol attenuates LPS-induced oxidation and apoptosis in BEAS-2B cells. Compared with control group, LPS up-regulated Pin-1, phosphatase A2 (PP2A) expression, induced p66(Shc)-Ser(36) phosphorylation, and facilitated p66(Shc) mitochondrial translocation, thus leading to superoxide anion (O2(-)) generation, mitochondrial cytochrome c release, active caspase 3 over-expression and cell viability inhibition. Importantly, propofol was shown to down-regulate LPS-induced PP2A expression, limit p66(Shc) mitochondrial translocation, decrease O2(-) generation, inhibit mitochondrial cytochrome c release, reduce active caspase 3 expression, and recover cells viability, while propofol had no effects on LPS-induced Pin-1 expression and p66(Shc)-Ser(36) phosphorylation. Moreover, the protective effects of propofol on LPS-induced BEAS-2B cells apoptosis were similar to that of calyculin A, which is an inhibitor of PP2A. We also found that FTY720, which is an activator of PP2A, can effectively reverse the protective function of propofol. Our data illustrated that propofol could alleviate LPS-induced BEAS-2B cells oxidation and apoptosis through down-regulating PP2A expression, limiting p66(Shc)-Ser(36) dephosphorylation and p66(Shc) mitochondrial translocation, decreasing O2(-) generation, mitochondrial cytochrome c release, activating caspase 3 expression. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  7. Human bronchial epithelial BEAS-2B cells, an appropriate in vitro model to study heavy metals induced carcinogenesis

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    Park, Youn-hee; Kim, Donghern; Dai, Jin; Zhang, Zhuo

    2015-01-01

    Occupational and environmental exposure to arsenic (III) and chromium VI (Cr(VI)) have been confirmed to cause lung cancer. Mechanisms of these metals-induced carcinogenesis are still under investigation. Selection of cell lines to be used is essential for the mechanistic studies. Human bronchial epithelial BEAS-2B cells are the cells to be utilized by most of scientists. However, due to p53 missense mutation (CCG → TCG) at codon 47 and the codon 72 polymorphism (CGC → CCC) in BEAS-2B cells, its usage has frequently been questioned. The present study has examined activity and expression of 53 and its downstream target protein p21 upon acute or chronic exposure of BEAS-2B cells to arsenic and Cr(VI). The results show that short-term exposure of BEAS-2B cells to arsenic or Cr(VI) was able to activate both p53 and p21. Chronic exposure of BEAS-2B cells to these two metals caused malignant cell transformation and tumorigenesis. In arsenic-transformed BEAS-2B cells reductions in p53 promoter activity, mRNA expression, and phosphorylation of p53 at Ser392 were observed, while the total p53 protein level remained the same compared to those in passage-matched parent ones. p21 promoter activity and expression were decreased in arsenic-transformed cells. Cr(VI)-transformed cells exhibit elevated p53 promoter activity, mRNA expression, and phosphorylation at Ser15, but reduced phosphorylation at Ser392 and total p53 protein level compared to passage-matched parent ones. p21 promoter activity and expression were elevated in Cr(VI)-transformed cells. These results demonstrate that p53 is able to respond to exposure of arsenic or Cr(VI), suggesting that BEAS-2B cells are an appropriate in vitro model to investigate arsenic or Cr(VI) induced lung cancer. PMID:26091798

  8. Human bronchial epithelial BEAS-2B cells, an appropriate in vitro model to study heavy metals induced carcinogenesis

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    Park, Youn-hee; Kim, Donghern; Dai, Jin; Zhang, Zhuo, E-mail: zhuo.zhang@uky.edu

    2015-09-15

    Occupational and environmental exposure to arsenic (III) and chromium VI (Cr(VI)) have been confirmed to cause lung cancer. Mechanisms of these metals carcinogenesis are still under investigation. Selection of cell lines to be used is essential for the studies. Human bronchial epithelial BEAS-2B cells are the cells to be utilized by most of scientists. However, due to p53 missense mutation (CCG → TCG) at codon 47 and the codon 72 polymorphism (CGC → CCC) in BEAS-2B cells, its usage has frequently been questioned. The present study has examined activity and expression of 53 and its downstream target protein p21 upon acute or chronic exposure of BEAS-2B cells to arsenic and Cr(VI). The results show that short-term exposure of BEAS-2B cells to arsenic or Cr(VI) was able to activate both p53 and p21. Chronic exposure of BEAS-2B cells to these two metals caused malignant cell transformation and tumorigenesis. In arsenic-transformed BEAS-2B cells reductions in p53 promoter activity, mRNA expression, and phosphorylation of p53 at Ser392 were observed, while the total p53 protein level remained the same compared to those in passage-matched parent ones. p21 promoter activity and expression were decreased in arsenic-transformed cells. Cr(VI)-transformed cells exhibit elevated p53 promoter activity, mRNA expression, and phosphorylation at Ser15, but reduced phosphorylation at Ser392 and total p53 protein level compared to passage-matched parent ones. p21 promoter activity and expression were elevated in Cr(VI)-transformed cells. These results demonstrate that p53 is able to respond to exposure of arsenic or Cr(VI), suggesting that BEAS-2B cells are an appropriate in vitro model to investigate arsenic or Cr(VI) induced lung cancer. - Highlights: • Short-term exposure of BEAS-2B cells to arsenic or Cr(VI) activates p53 and p21. • Chronic exposure of BEAS-2B cells to arsenic or Cr(VI) causes cell transformation and tumorigenesis. • Arsenic-transformed cells exhibit

  9. Proteomic analysis of proteins associated with tt-DDE induced toxicity in BEAS-2B cells.

    Science.gov (United States)

    Lin, Pin-Pin; Yang, Ming-Hui; Liao, Pao-Chi; Wu, Hsin-Yi; Chang, Louis W; Tsai, Hui-Ti; Tyan, Yu-Chang

    2008-11-21

    Trans, trans-2,4-decadienal (tt-DDE), a specific type of dienaldehyde, is abundant in heated oils or cooking oil fumes. Ingestion of heated oils and exposure to cooking oil fumes has been suggested to have a great health impact in a variety of organs, including the lungs. Previous studies have demonstrated that acute exposures to high doses of tt-DDE have induced oxidative stress, genotoxicity, and cytotoxicity in human lung cells. The objective in utilizing proteomic techniques of this study was to identify protein biomarkers associated with tt-DDE-induced oxidative stress and cytotoxicity in human bronchial epithelial cells BEAS-2B. Experimental results suggested that DJ-1 and cofilin proteins were protein biomarkers for tt-DDE-induced cytotoxicity and oxidative stress in lung cells. DJ-1 was especially an early biomarker for tt-DDE exposure.

  10. The crosstalk between α-irradiated Beas-2B cells and its bystander U937 cells through MAPK and NF-κB signaling pathways

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    Fu, Jiamei; Yuan, Dexiao; Xiao, Linlin; Tu, Wenzhi; Dong, Chen; Liu, Weili; Shao, Chunlin, E-mail: clshao@shmu.edu.cn

    2016-01-15

    Highlights: • α-irradiated Beas-2B cells induced bystander effects in macrophage U937 cells. • The neighboring macrophages enhanced the damage of α-irradiated Beas-2B cells. • MAPK and NF-κB pathways were activated in U937 cells after cell co-culture. • NF-κB and MAPK pathways participated in the bilateral bystander responses. - Abstract: Although accumulated evidence suggests that α-particle irradiation induced bystander effect may relevant to lung injury and cancer risk assessment, the exact mechanisms are not yet elucidated. In the present study, a cell co-culture system was used to investigate the interaction between α-particle irradiated human bronchial epithelial cells (Beas-2B) and its bystander macrophage U937 cells. It was found that the cell co-culture amplified the detrimental effects of α-irradiation including cell viability decrease and apoptosis promotion on both irradiated cells and bystander cells in a feedback loop which was closely relevant to the activation of MAPK and NF-κB pathways in the bystander U937 cells. When these two pathways in U937 cells were disturbed by special pharmacological inhibitors before cell co-culture, it was found that a NF-κB inhibitor of BAY 11-7082 further enhanced the proliferation inhibition and apoptosis induction in bystander U937 cells, but MAPK inhibitors of SP600125 and SB203580 protected cells from viability loss and apoptosis and U0126 presented more beneficial effect on cell protection. For α-irradiated epithelial cells, the activation of NF-κB and MAPK pathways in U937 cells participated in detrimental cellular responses since the above inhibitors could largely attenuate cell viability loss and apoptosis of irradiated cells. Our results demonstrated that there are bilateral bystander responses between irradiated lung epithelial cells and macrophages through MAPK and NF-κB signaling pathways, which accounts for the enhancement of α-irradiation induced damage.

  11. Proteasome inhibitor MG-132 regulates the expression of VEGF in human bronchial epithelial cell line, BEAS-2B

    Institute of Scientific and Technical Information of China (English)

    Xuefan Cui; Kaisheng Yin; Mao Huang; Linfu Zhou

    2005-01-01

    Objective: To explore the effects of MG-132 on the expression of VEGF in bronchial epithelial cell line, BEAS2B. Methods: Semi-quantitive RT-PCR for VEGF mRNA and enzyme-linked immunosorbent assay (ELISA) for VEGF protein were performed. Results: MG-132 increased the expression of VEGF mRNA and protein BEAS-2B cells in time-and concentration-dependent manners. After 24-h stimulation, 25 μmol/L MG-132 increased the maximal levels of VEGF protein in cell-conditioned medium. When the cells were stimulated with cycloheximide(CHX) before treatment with MG-132, the MG-132-induced production of VEGF protein was inhibited compared to the unstimulated cells. Supernatant of condition-medium treatment with MG-132 enhanced the growth of HUVEC.Conclusion: MG-132 induces VEGF gene expression in human bronchial epithelial cells line, BEAS-2B, and the MG-132-induced expression of VEGF may modulate lung tissue injury due to airway inflammation.

  12. Genotoxicity of polyvinylpyrrolidone-coated silver nanoparticles in BEAS 2B cells.

    Science.gov (United States)

    Nymark, Penny; Catalán, Julia; Suhonen, Satu; Järventaus, Hilkka; Birkedal, Renie; Clausen, Per Axel; Jensen, Keld Alstrup; Vippola, Minnamari; Savolainen, Kai; Norppa, Hannu

    2013-11-08

    Silver nanoparticles (AgNPs) are widely utilized in various consumer products and medical devices, especially due to their antimicrobial properties. However, several studies have associated these particles with toxic effects, such as inflammation and oxidative stress in vivo and cytotoxic and genotoxic effects in vitro. Here, we assessed the genotoxic effects of AgNPs coated with polyvinylpyrrolidone (PVP) (average diameter 42.5±14.5 nm) on human bronchial epithelial BEAS 2B cells in vitro. AgNPs were dispersed in bronchial epithelial growth medium (BEGM) with 0.6 mg/ml bovine serum albumin (BSA). The AgNP were partially well-dispersed in the medium and only limited amounts (ca. 0.02 μg Ag(+) ion/l) could be dissolved after 24h. The zeta-potential of the AgNPs was found to be highly negative in pure water but was at least partially neutralized in BEGM with 0.6 mg BSA/ml. Cytotoxicity was measured by cell number count utilizing Trypan Blue exclusion and by an ATP-based luminescence cell viability assay. Genotoxicity was assessed by the alkaline single cell gel electrophoresis (comet) assay, the cytokinesis-block micronucleus (MN) assay, and the chromosomal aberration (CA) assay. The cells were exposed to various doses (0.5-48 μg/cm(2) corresponding to 2.5-240 μg/ml) of AgNPs for 4 and 24 h in the comet assay, for 48 h in the MN assay, and for 24 and 48 h in the CA assay. DNA damage measured by the percent of DNA in comet tail was induced in a dose-dependent manner after both the 4-h and the 24-h exposures to AgNPs, with a statistically significant increase starting at 16 μg/cm(2) (corresponding to 60.8 μg/ml) and doubling of the percentage of DNA in tail at 48 μg/cm(2). However, no induction of MN or CAs was observed at any of the doses or time points. The lack of induction of chromosome damage by the PVP-coated AgNPs is possibly due to the coating which may protect the cells from direct interaction with the AgNPs, either by reducing ion leaching from the

  13. Identification of PM10 characteristics involved in cellular responses in human bronchial epithelial cells (Beas-2B).

    Science.gov (United States)

    Van Den Heuvel, Rosette; Den Hond, Elly; Govarts, Eva; Colles, Ann; Koppen, Gudrun; Staelens, Jeroen; Mampaey, Maja; Janssen, Nicole; Schoeters, Greet

    2016-08-01

    Notwithstanding evidence is present that physicochemical characteristics of ambient particles attribute to adverse health effects, there is still some lack of understanding in this complex relationship. At this moment it is not clear which properties (such as particle size, chemical composition) or sources of the particles are most relevant for health effects. This study investigates the in vitro toxicity of PM10 in relation to PM chemical composition, black carbon (BC), endotoxin content and oxidative potential (OP). In 2013-2014 PM10 was sampled (24h sampling, 108 sampling days) in ambient air at three sites in Flanders (Belgium) with different pollution characteristics: an urban traffic site (Borgerhout), an industrial area (Zelzate) and a rural background location (Houtem). To characterize the toxic potential of PM10, airway epithelial cells (Beas-2B cells) have been exposed to particles in vitro. Different endpoints were studied including cell damage and death (cell viability) using the Neutral red Uptake assay, the production of pro-inflammatory molecules by interleukin 8 (IL-8) induction and DNA-damaging activity using the FPG-modified Comet assay. The endotoxin levels in the collected samples were analysed and the capacity of PM10 particles to produce reactive oxygen species (OP) was evaluated by electron paramagnetic resonance (EPR) spectroscopy. Chemical characteristics of PM10 (BC, As, Cd, Cr, Cu, Mn, Ni, Pb, Zn) and meteorological conditions were recorded on the sampling days. PM10 particles exhibited dose-dependent cytotoxicity in Beas-2B cells and were found to significantly induce the release of IL-8 in samples from the three locations. Oxidatively damaged DNA was observed in exposed Beas-2B cells. Endotoxin levels above the detection limit were detected in half of the samples. OP was measurable in all samples. Associations between PM10 characteristics and biological effects of PM10 were assessed by single and multiple regression analyses. The

  14. Role of reactive oxygen species in arsenic-induced transformation of human lung bronchial epithelial (BEAS-2B) cells

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    Zhang, Zhuo, E-mail: zhuo.zhang@uky.edu [Graduate Center for Toxicology, University of Kentucky, Lexington, KY 40536 (United States); Pratheeshkumar, Poyil; Budhraja, Amit; Son, Young-Ok [Center for Research on Environmental Diseases, University of Kentucky, Lexington, KY 40536 (United States); Kim, Donghern [Graduate Center for Toxicology, University of Kentucky, Lexington, KY 40536 (United States); Shi, Xianglin [Center for Research on Environmental Diseases, University of Kentucky, Lexington, KY 40536 (United States)

    2015-01-09

    Highlights: • Short term exposure of cells to arsenic causes ROS generation. • Chronical exposure of cells to arsenic causes malignant cell transformation. • Inhibition of ROS generation reduces cell transformation by arsenic. • Arsenic-transformed cells exhibit reduced capacity of generating ROS. • Arsenic-transformed cells exhibit increased levels of antioxidants. - Abstract: Arsenic is an environmental carcinogen, its mechanisms of carcinogenesis remain to be investigated. Reactive oxygen species (ROS) are considered to be important. A previous study (Carpenter et al., 2011) has measured ROS level in human lung bronchial epithelial (BEAS-2B) cells and arsenic-transformed BEAS-2B cells and found that ROS levels were higher in transformed cells than that in parent normal cells. Based on these observations, the authors concluded that cell transformation induced by arsenic is mediated by increased cellular levels of ROS. This conclusion is problematic because this study only measured the basal ROS levels in transformed and parent cells and did not investigate the role of ROS in the process of arsenic-induced cell transformation. The levels of ROS in arsenic-transformed cells represent the result and not the cause of cell transformation. Thus question concerning whether ROS are important in arsenic-induced cell transformation remains to be answered. In the present study, we used expressions of catalase (antioxidant against H{sub 2}O{sub 2}) and superoxide dismutase 2 (SOD2, antioxidant against O{sub 2}{sup ·−}) to decrease ROS level and investigated their role in the process of arsenic-induced cell transformation. Our results show that inhibition of ROS by antioxidant enzymes decreased arsenic-induced cell transformation, demonstrating that ROS are important in this process. We have also shown that in arsenic-transformed cells, ROS generation was lower and levels of antioxidants are higher than those in parent cells, in a disagreement with the previous

  15. High basal expression of interferon-stimulated genes in human bronchial epithelial (BEAS-2B cells contributes to influenza A virus resistance.

    Directory of Open Access Journals (Sweden)

    Lai-Giea Seng

    Full Text Available Respiratory epithelial cells play a key role in influenza A virus (IAV pathogenesis and host innate response. Transformed human respiratory cell lines are widely used in the study of IAV-host interactions due to their relative convenience, and inherent difficulties in working with primary cells. Transformed cells, however, may have altered susceptibility to virus infection. Proper characterization of different respiratory cell types in their responses to IAV infection is therefore needed to ensure that the cell line chosen will provide results that are of relevance in vivo. We compared replication kinetics of human H1N1 (A/USSR/77 IAVs in normal primary human bronchial epithelial (NHBE and two commonly used respiratory epithelial cell lines namely BEAS-2B and A549 cells. We found that IAV replication was distinctly poor in BEAS-2B cells in comparison with NHBE, A549 and Madin-Darby canine kidney (MDCK cells. IAV resistance in BEAS-2B cells was accompanied by an activated antiviral state with high basal expression of interferon (IFN regulatory factor-7 (IRF-7, stimulator of IFN genes (STING and IFN stimulated genes (ISGs. Treatment of BEAS-2B cells with a pan-Janus-activated-kinase (JAK inhibitor decreased IRF-7 and ISG expression and resulted in increased IAV replication. Therefore, the use of highly resistant BEAS-2B cells in IAV infection may not reflect the cytopathogenicity of IAV in human epithelial cells in vivo.

  16. Biological responses according to the shape and size of carbon nanotubes in BEAS-2B and MESO-1 cells.

    Science.gov (United States)

    Haniu, Hisao; Saito, Naoto; Matsuda, Yoshikazu; Tsukahara, Tamotsu; Usui, Yuki; Maruyama, Kayo; Takanashi, Seiji; Aoki, Kaoru; Kobayashi, Shinsuke; Nomura, Hiroki; Tanaka, Manabu; Okamoto, Masanori; Kato, Hiroyuki

    2014-01-01

    This study aimed to investigate the influence of the shape and size of multi-walled carbon nanotubes (MWCNTs) and cup-stacked carbon nanotubes (CSCNTs) on biological responses in vitro. Three types of MWCNTs - VGCF(®)-X, VGCF(®)-S, and VGCF(®) (vapor grown carbon fibers; with diameters of 15, 80, and 150 nm, respectively) - and three CSCNTs of different lengths (CS-L, 20-80 μm; CS-S, 0.5-20 μm; and CS-M, of intermediate length) were tested. Human bronchial epithelial (BEAS-2B) and malignant pleural mesothelioma cells were exposed to the CNTs (1-50 μg/mL), and cell viability, permeability, uptake, total reactive oxygen species/superoxide production, and intracellular acidity were measured. CSCNTs were less toxic than MWCNTs in both cell types over a 24-hour exposure period. The cytotoxicity of endocytosed MWCNTs varied according to cell type/size, while that of CSCNTs depended on tube length irrespective of cell type. CNT diameter and length influenced cell aggregation and injury extent. Intracellular acidity increased independently of lysosomal activity along with the number of vacuoles in BEAS-2B cells exposed for 24 hours to either CNT (concentration, 10 μg/mL). However, total reactive oxygen species/superoxide generation did not contribute to cytotoxicity. The results demonstrate that CSCNTs could be suitable for biological applications and that CNT shape and size can have differential effects depending on cell type, which can be exploited in the development of highly specialized, biocompatible CNTs.

  17. Long-term exposures to low doses of silver nanoparticles enhanced in vitro malignant cell transformation in non-tumorigenic BEAS-2B cells.

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    Choo, Wun Hak; Park, Cho Hee; Jung, Shi Eun; Moon, Byeonghak; Ahn, Huiyeon; Ryu, Jung Seok; Kim, Keun-Soo; Lee, Yong Hwa; Yu, Il Je; Oh, Seung Min

    2016-12-01

    To predict carcinogenic potential of AgNPs on the respiratory system, BEAS-2B cells (human bronchial epithelial cells) were chronically exposed to low- and non-cytotoxic dose (0.13 and 1.33μg/ml) of AgNPs for 4months (#40 passages). To assess malignant cell transformation of chronic exposure to AgNPs, several bioassays including anchorage independent agar colony formation, cell migration/invasion assay, and epithelial-mesenchymal transition (EMT) were performed in BEAS-2B cells. Chronic exposure to AgNPs showed a significant increase of anchorage independent agar colony formation and cell migration/invasion. EMT, which is the loss of epithelial markers (E-Cadherin and Keratin) and the gain of mesenchymal marker (N-cadherin and Vimentin), was induced by chronic exposure to AgNPs. These responses indicated that chronic exposure to AgNPs could acquire characteristics of tumorigenic cells from normal BEAS-2B cells. In addition, caspase-3, p-p53, p-p38, and p-JNK were significantly decreased, while p-ERK1/2 was significantly increased. MMP-9 related to cell migration/invasion was upregulated, while a MMP-9 inhibitor, TIMP-1 was down-regulated. These results indicated that BEAS-2B cells exposed to AgNPs could induce anti-apoptotic response/anoikis resistance, and cell migration/invasion by complex regulation of MAPK kinase (p38, JNK, and ERK) and p53 signaling pathways. Therefore, we suggested that long-term exposure to low-dose of AgNPs could enhance malignant cell transformation in non-tumorigenic BEAS-2B cells. Our findings provide useful information needed to assess the carcinogenic potential of AgNPs.

  18. Biological responses according to the shape and size of carbon nanotubes in BEAS-2B and MESO-1 cells

    Directory of Open Access Journals (Sweden)

    Haniu H

    2014-04-01

    Full Text Available Hisao Haniu,1,2 Naoto Saito,2,3 Yoshikazu Matsuda,4 Tamotsu Tsukahara,5 Yuki Usui,1,6,7 Kayo Maruyama,2,3 Seiji Takanashi,1 Kaoru Aoki,1 Shinsuke Kobayashi,1 Hiroki Nomura,1 Manabu Tanaka,1 Masanori Okamoto,1 Hiroyuki Kato1 1Department of Orthopaedic Surgery, Shinshu University School of Medicine, Nagano, Japan; 2Insutitute for Biomedical Sciences, Shinshu University, Nagano, Japan; 3Department of Applied Physical Therapy, Shinshu University School of Health Sciences, Nagano, Japan; 4Clinical Pharmacology Educational Center, Nihon Pharmaceutical University, Saitama, Japan; 5Department of Hematology and Immunology, Kanazawa Medical University, Ishikawa, Japan; 6Research Center for Exotic Nanocarbons, Shinshu University, Nagano, Japan; 7Aizawa Hospital, Sports Medicine Center, Nagano, Japan Abstract: This study aimed to investigate the influence of the shape and size of multi-walled carbon nanotubes (MWCNTs and cup-stacked carbon nanotubes (CSCNTs on biological responses in vitro. Three types of MWCNTs – VGCF®-X, VGCF®-S, and VGCF® (vapor grown carbon fibers; with diameters of 15, 80, and 150 nm, respectively – and three CSCNTs of different lengths (CS-L, 20–80 µm; CS-S, 0.5–20 µm; and CS-M, of intermediate length were tested. Human bronchial epithelial (BEAS-2B and malignant pleural mesothelioma cells were exposed to the CNTs (1–50 µg/mL, and cell viability, permeability, uptake, total reactive oxygen species/superoxide production, and intracellular acidity were measured. CSCNTs were less toxic than MWCNTs in both cell types over a 24-hour exposure period. The cytotoxicity of endocytosed MWCNTs varied according to cell type/size, while that of CSCNTs depended on tube length irrespective of cell type. CNT diameter and length influenced cell aggregation and injury extent. Intracellular acidity increased independently of lysosomal activity along with the number of vacuoles in BEAS-2B cells exposed for 24 hours to either CNT

  19. Differential Response of Mono Mac 6, BEAS-2B, and Jurkat Cells to Indoor Dust

    National Research Council Canada - National Science Library

    Herbert Riechelmann; Tom Deutschle; Ariane Grabow; Birger Heinzow; Werner Butte; Rudolf Reiter

    2007-01-01

    Background: Airway toxicity of indoor dust is not sufficiently understood. Objectives: Our goal in this study was to describe the effects of indoor dust on human monocyte, epithelial, and lymphocyte cell lines...

  20. Differential Response of Mono Mac 6, BEAS-2B, and Jurkat Cells to Indoor Dust

    OpenAIRE

    Riechelmann, Herbert; Deutschle, Tom; Grabow, Ariane; Heinzow, Birger; Butte, Werner; Reiter, Rudolf

    2007-01-01

    Background Airway toxicity of indoor dust is not sufficiently understood. Objectives Our goal in this study was to describe the effects of indoor dust on human monocyte, epithelial, and lymphocyte cell lines. We aimed to a) obtain a comprehensive and intelligible outline of the transcriptional response; b) correlate differential transcription with cellular protein secretion; c) identify cell line–specific features; and d) search for indoor dust–specific responses. Methods Settled dust was sam...

  1. Poly (I:C, an agonist of toll-like receptor-3, inhibits replication of the Chikungunya virus in BEAS-2B cells

    Directory of Open Access Journals (Sweden)

    Li Yong-Gang

    2012-06-01

    Full Text Available Abstract Background Double-stranded RNA (dsRNA and its mimic, polyinosinic acid: polycytidylic acid [Poly (I:C], are recognized by toll-like receptor 3 (TLR3 and induce interferon (IFN-β in many cell types. Poly (I:C is the most potent IFN inducer. In in vivo mouse studies, intraperitoneal injection of Poly (I:C elicited IFN-α/β production and natural killer (NK cells activation. The TLR3 pathway is suggested to contribute to innate immune responses against many viruses, including influenza virus, respiratory syncytial virus, herpes simplex virus 2, and murine cytomegalovirus. In Chikungunya virus (CHIKV infection, the viruses are cleared within 7–10 days postinfection before adaptive immune responses emerge. The innate immune response is important for CHIKV clearance. Results The effects of Poly (I:C on the replication of CHIKV in human bronchial epithelial cells, BEAS-2B, were studied. Poly (I:C suppressed cytopathic effects (CPE induced by CHIKV infection in BEAS-2B cells in the presence of Poly (I:C and inhibited the replication of CHIKV in the cells. The virus titers of Poly (I:C-treated cells were much lower compared with those of untreated cells. CHIKV infection and Poly (I:C treatment of BEAS-2B cells induced the production of IFN-β and increased the expression of anti-viral genes, including IFN-α, IFN-β, MxA, and OAS. Both Poly (I:C and CHIKV infection upregulate the expression of TLR3 in BEAS-2B cells. Conclusions CHIKV is sensitive to innate immune response induced by Poly (I:C. The inhibition of CHIKV replication by Poly (I:C may be through the induction of TLR3, which triggers the production of IFNs and other anti-viral genes. The innate immune response is important to clear CHIKV in infected cells.

  2. Comparative Analysis of Toxic Responses of Organic Extracts from Diesel and Selected Alternative Fuels Engine Emissions in Human Lung BEAS-2B Cells

    OpenAIRE

    Helena Libalova; Pavel Rossner; Kristyna Vrbova; Tana Brzicova; Jitka Sikorova; Michal Vojtisek-Lom; Vit Beranek; Jiri Klema; Miroslav Ciganek; Jiri Neca; Katerina Pencikova; Miroslav Machala; Jan Topinka

    2016-01-01

    This study used toxicogenomics to identify the complex biological response of human lung BEAS-2B cells treated with organic components of particulate matter in the exhaust of a diesel engine. First, we characterized particles from standard diesel (B0), biodiesel (methylesters of rapeseed oil) in its neat form (B100) and 30% by volume blend with diesel fuel (B30), and neat hydrotreated vegetable oil (NEXBTL100). The concentration of polycyclic aromatic hydrocarbons (PAHs) and their derivatives...

  3. Comparative analysis of toxic responses of organic extracts from diesel and selected alternative fuels engine emissions in human lung BEAS-2B cells

    OpenAIRE

    Líbalová, Helena; Rossner, Pavel, Jr.; Vrbová, Kristýna; Brzicová, Táňa; Sikorová, Jitka; Vojtíšek-Lom, Michal; Beranek, Vít; Kléma, Jiří; Ciganek,Miroslav; Neča, Jiří; Pěnčíková, Kateřina; Machala, Miroslav; Topinka, Jan

    2016-01-01

    This study used toxicogenomics to identify the complex biological response of human lung BEAS-2B cells treated with organic components of particulate matter in the exhaust of a diesel engine. First, we characterized particles from standard diesel (B0), biodiesel (methylesters of rapeseed oil) in its neat form (B100) and 30% by volume blend with diesel fuel (B30), and neat hydrotreated vegetable oil (NEXBTL100). The concentration of polycyclic aromatic hydrocarbons (PAHs) and their derivatives...

  4. Roles of MAPK pathway activation during cytokine induction in BEAS-2B cells exposed to fine World Trade Center (WTC) dust.

    Science.gov (United States)

    Wang, Shang; Prophete, Colette; Soukup, Joleen M; Chen, Lung-Chi; Costa, Max; Ghio, Andrew; Qu, Qingshan; Cohen, Mitchell D; Chen, Haobin

    2010-01-01

    The World Trade Center (WTC) collapse on September 11, 2001 released copious amounts of particulate matter (PM) into the atmosphere of New York City. Follow-up studies on persons exposed to the dusts have revealed a severely increased rate for asthma and other respiratory illnesses. There have only been a few studies that have sought to discern the possible mechanisms underlying these untoward pathologies. In one study, an increased cytokine release was detected in cells exposed to WTC fine dusts (PM₂.₅ fraction or WTC₂.₅). However, the mechanism(s) for these increases has yet to be fully defined. Because activation of the mitogen-activated protein kinase (MAPK) signaling pathways is known to cause cytokine induction, the current study was undertaken to analyze the possible involvement of these pathways in any increased cytokine formation by lung epithelial cells (as BEAS-2B cells) exposed to WTC₂.₅. Our results showed that exposure to WTC₂.₅ for 5 hr increased interleukin-6 (IL-6) mRNA expression in BEAS-2B cells, as well as its protein levels in the culture media, in a dose-dependent manner. Besides IL-6, cytokine multiplex analyses revealed that formation of IL-8 and -10 was also elevated by the exposure. Both extracellular signal-regulated kinase (ERK) and p38, but not c-Jun N-terminal protein kinase, signaling pathways were found to be activated in cells exposed to WTC₂.₅. Inactivation of ERK signaling pathways by PD98059 effectively blocked IL-6, -8, and -10 induction by WTC₂.₅; the p38 kinase inhibitor SB203580 significantly decreased induction of IL-8 and -10. Together, our data demonstrated activation of MAPK signaling pathway(s) likely played an important role in the WTC₂.₅-induced formation of several inflammatory (and, subsequently, anti-inflammatory) cytokines. The results are important in that they help to define one mechanism via which the WTC dusts may have acted to cause the documented increases in asthma and other

  5. PI3K-delta mediates double-stranded RNA-induced upregulation of B7-H1 in BEAS-2B airway epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Kan-o, Keiko [Research Institute for Diseases of the Chest, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Matsumoto, Koichiro, E-mail: koichi@kokyu.med.kyushu-u.ac.jp [Research Institute for Diseases of the Chest, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Asai-Tajiri, Yukari; Fukuyama, Satoru; Hamano, Saaka; Seki, Nanae; Nakanishi, Yoichi [Research Institute for Diseases of the Chest, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Inoue, Hiromasa [Research Institute for Diseases of the Chest, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Department of Pulmonary Medicine, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8520 (Japan)

    2013-05-31

    Highlights: •Double-stranded RNA upregulates B7-H1 on BEAS-2B airway epithelial cells. •The upregulation of B7-H1 is attenuated by inhibition of PI3Kδ isoform. •PI3Kδ-mediated upregulation of B7-H1 is independent of NF-κB activation. •Inhibition of PI3Kδ may prevent persistent viral infection induced by B7-H1. -- Abstract: Airway viral infection disturbs the health-related quality of life. B7-H1 (also known as PD-L1) is a coinhibitory molecule associated with the escape of viruses from the mucosal immunity, leading to persistent infection. Most respiratory viruses generate double-stranded (ds) RNA during replication. The stimulation of cultured airway epithelial cells with an analog of viral dsRNA, polyinosinic-polycytidylic acid (poly IC) upregulates the expression of B7-H1 via activation of the nuclear factor κB(NF-κB). The mechanism of upregulation was investigated in association with phosphatidylinositol 3-kinases (PI3Ks). Poly IC-induced upregulation of B7-H1 was profoundly suppressed by a pan-PI3K inhibitor and partially by an inhibitor or a small interfering (si)RNA for PI3Kδ in BEAS-2B cells. Similar results were observed in the respiratory syncytial virus-infected cells. The expression of p110δ was detected by Western blot and suppressed by pretreatment with PI3Kδ siRNA. The activation of PI3Kδ is typically induced by oxidative stress. The generation of reactive oxygen species was increased by poly IC. Poly IC-induced upregulation of B7-H1 was attenuated by N-acetyl-L-cysteine, an antioxidant, or by oxypurinol, an inhibitor of xanthine oxidase. Poly IC-induced activation of NF-κB was suppressed by a pan-PI3K inhibitor but not by a PI3Kδ inhibitor. These results suggest that PI3Kδ mediates dsRNA-induced upregulation of B7-H1 without affecting the activation of NF-κB.

  6. RNA-binding motif protein 5 inhibits the proliferation of cigarette smoke-transformed BEAS-2B cells through cell cycle arrest and apoptosis.

    Science.gov (United States)

    Lv, Xue-Jiao; Du, Yan-Wei; Hao, Yu-Qiu; Su, Zhen-Zhong; Zhang, Lin; Zhao, Li-Jing; Zhang, Jie

    2016-04-01

    Cigarette smoking has been shown to be the most significant risk factor for lung cancer. Recent studies have also indicated that RNA-binding motif protein 5 (RBM5) can modulate apoptosis and suppress tumor growth. The present study focused on the role of RBM5 in the regulation of cigarette smoke extract (CSE)-induced transformation of bronchial epithelial cells into the cancerous phenotype and its mechanism of action. Herein, we exposed normal BEAS-2B cells for 8 days to varying concentrations of CSE or dimethylsulfoxide (DMSO), followed by a recovery period of 2 weeks. Next, the RBM5 protein was overexpressed in these transformed BEAS-2B cells though lentiviral infection. Later, the morphological changes, cell proliferation, cell cycle, apoptosis, invasion and migration were assessed. In addition, we analyzed the role of RBM5 in xenograft growth. The expression of RBM5 along with the genes related to cell cycle regulation, apoptosis and invasion were also examined. Finally, our results revealed that BEAS-2B cells exposed to 100 µg/ml CSE acquired phenotypic changes and formed tumors in nude mice, indicative of their cancerous transformation and had reduced RBM5 expression. Subsequent overexpression of RBM5 in these cells significantly inhibited their proliferation, induced G1/S arrest, triggered apoptosis and inhibited their invasion and migration, including xenograft growth. Thus, we established an in vitro model of CSE-induced cancerous transformation and concluded that RBM5 overexpression inhibited the growth of these transformed cells through cell cycle arrest and induction of apoptosis. Therefore, our study suggests the importance of RBM5 in the pathogenesis of smoking-related cancer.

  7. Streptococcus pneumoniae induced c-Jun-N-terminal kinase- and AP-1 -dependent IL-8 release by lung epithelial BEAS-2B cells

    Directory of Open Access Journals (Sweden)

    Rosseau Simone

    2006-07-01

    Full Text Available Abstract Background Although pneumococcal pneumonia is one of the most common causes of death due to infectious diseases, little is known about pneumococci-lung cell interaction. Herein we tested the hypothesis that pneumococci activated pulmonary epithelial cell cytokine release by c-Jun-NH2-terminal kinase (JNK Methods Human bronchial epithelial cells (BEAS-2B or epithelial HEK293 cells were infected with S. pneumoniae R6x and cytokine induction was measured by RT-PCR, ELISA and Bioplex assay. JNK-phosphorylation was detected by Western blot and nuclear signaling was assessed by electrophoretic mobility shift assay (EMSA and chromatin immunoprecipitation (ChIP. JNK was modulated by the small molecule inhibitor SP600125 and AP1 by transfection of a dominant negative mutant. Results S. pneumoniae induced the release of distinct CC and CXC, as well as Th1 and Th2 cytokines and growth factors by human lung epithelial cell line BEAS-2B. Furthermore, pneumococci infection resulted in JNK phosphorylation in BEAS-2B cells. Inhibition of JNK by small molecule inhibitor SP600125 reduced pneumococci-induced IL-8 mRNA expression and release of IL-8 and IL-6. One regulator of the il8 promoter is JNK-phosphorylated activator protein 1 (AP-1. We showed that S. pneumoniae time-dependently induced DNA binding of AP-1 and its phosphorylated subunit c-Jun with a maximum at 3 to 5 h after infection. Recruitment of Ser63/73-phosphorylated c-Jun and RNA polymerase II to the endogenous il8 promoter was found 2 h after S. pneumoniae infection by chromatin immunoprecipitation. AP-1 repressor A-Fos reduced IL-8 release by TLR2-overexpressing HEK293 cells induced by pneumococci but not by TNFα. Antisense-constructs targeting the AP-1 subunits Fra1 and Fra2 had no inhibitory effect on pneumococci-induced IL-8 release. Conclusion S. pneumoniae-induced IL-8 expression by human epithelial BEAS-2B cells depended on activation of JNK and recruitment of phosphorylated c

  8. Use of human bronchial epithelial cells (BEAS-2B) to study immunological markers resulting from exposure to PM(2.5) organic extract from Puerto Rico.

    Science.gov (United States)

    Fuentes-Mattei, Enrique; Rivera, Evasomary; Gioda, Adriana; Sanchez-Rivera, Diana; Roman-Velazquez, Felix R; Jimenez-Velez, Braulio D

    2010-03-15

    Fine particulate air pollutants, mainly their organic fraction, have been demonstrated to be associated with cardiovascular and respiratory health problems. Puerto Rico has been reported to have the highest prevalence of pulmonary diseases (e.g., asthma) in the United States. The aim of this study was to assess, for the first time, the immunological response of human bronchial epithelial cells (BEAS-2B) to organic extracts isolated from airborne particulate matter (PM(2.5)) in Puerto Rico. Organic extracts from PM(2.5) collected throughout an 8-month period (2000-2001) were pooled (composite) in order to perform chemical analysis and biological activity testing. BEAS-2B cells were exposed to PM(2.5) organic extract to assess cytotoxicity, levels of cytokines and relative gene expression of MHC-II, hPXR and CYP3A5. Our findings show that organic PM(2.5) consist of toxic as well as bioactive components that can regulate the secretion of cytokines in BEAS-2B, which could modulate inflammatory response in the lung. Trace element analyses confirmed the presence of metals in organic extracts highlighting the relative high abundance of Cu and Zn in polar organic extracts. Polar organic extracts exhibited dose-dependant toxicity and were found to significantly induce the release of interleukin 6 (IL-6), IL-1beta and IL-7 while significantly inhibiting the secretion of IL-8, G-CSF and MCP-1. Moreover, MHC-II transcriptional activity was up-regulated after 24 h of exposure, whereas PXR and CYP3A5 were down-regulated. This research provides a new insight into the effects of PM(2.5) organic fractions on specific effectors and their possible role in the development of respiratory inflammatory diseases in Puerto Rico.

  9. Comparative Analysis of Toxic Responses of Organic Extracts from Diesel and Selected Alternative Fuels Engine Emissions in Human Lung BEAS-2B Cells

    Directory of Open Access Journals (Sweden)

    Helena Libalova

    2016-11-01

    Full Text Available This study used toxicogenomics to identify the complex biological response of human lung BEAS-2B cells treated with organic components of particulate matter in the exhaust of a diesel engine. First, we characterized particles from standard diesel (B0, biodiesel (methylesters of rapeseed oil in its neat form (B100 and 30% by volume blend with diesel fuel (B30, and neat hydrotreated vegetable oil (NEXBTL100. The concentration of polycyclic aromatic hydrocarbons (PAHs and their derivatives in organic extracts was the lowest for NEXBTL100 and higher for biodiesel. We further analyzed global gene expression changes in BEAS-2B cells following 4 h and 24 h treatment with extracts. The concentrations of 50 µg extract/mL induced a similar molecular response. The common processes induced after 4 h treatment included antioxidant defense, metabolism of xenobiotics and lipids, suppression of pro-apoptotic stimuli, or induction of plasminogen activating cascade; 24 h treatment affected fewer processes, particularly those involved in detoxification of xenobiotics, including PAHs. The majority of distinctively deregulated genes detected after both 4 h and 24 h treatment were induced by NEXBTL100; the deregulated genes included, e.g., those involved in antioxidant defense and cell cycle regulation and proliferation. B100 extract, with the highest PAH concentrations, additionally affected several cell cycle regulatory genes and p38 signaling.

  10. PLD1 activation mediates Amb a 1-induced Th2-associated cytokine expression via the JNK/ATF-2 pathway in BEAS-2B cells.

    Science.gov (United States)

    Kim, Joo-Hwa; Choi, Hye-Jin; Oh, Cheong-Hae; Oh, Jae-Won; Han, Joong-Soo

    2015-01-01

    The purpose of this study was to identify the role of phospholipase D1 (PLD1) in Amb a 1-induced IL-5 and IL-13 expression. When BEAS-2B cells were stimulated with Amb a 1, PLD activity increased, and knockdown of PLD1 decreased Amb a 1-induced IL-5 and IL-13 expression. Amb a 1 also activated the PLCγ/p70S6K/JNK pathway. Furthermore, Amb a 1-induced PLD activation was also attenuated by PLCγ inhibition, and knockdown of PLD1 decreased Amb a 1-induced activation of P70S6K and JNK. When ATF-2 activity was blocked with ATF-2 siRNA, Amb a 1-induced IL-5 and IL-13 expression was completely abolished, indicating that ATF-2 is a transcriptional factor required for the expression of IL-5 and IL-13 in response to Amb a 1. Taken together, we suggest that PLD1 acts as an important regulator in Amb a 1-induced expression of IL-5 and IL-13 via a PLCγ/p70S6K/JNK/ATF-2 pathway in BEAS-2B cells. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Cadmium induces carcinogenesis in BEAS-2B cells through ROS-dependent activation of PI3K/AKT/GSK-3β/β-catenin signaling

    Energy Technology Data Exchange (ETDEWEB)

    Son, Young-Ok; Wang, Lei; Poyil, Pratheeshkumar; Budhraja, Amit; Hitron, J. Andrew; Zhang, Zhuo [Graduate Center for Toxicology, College of Medicine, University of Kentucky, Lexington, KY (United States); Lee, Jeong-Chae [Graduate Center for Toxicology, College of Medicine, University of Kentucky, Lexington, KY (United States); School of Dentistry and Institute of Oral Biosciences (BK21 program), Research Center of Bioactive Materials, Chonbuk National University, Jeonju 561-756 (Korea, Republic of); Shi, Xianglin, E-mail: xshi5@email.uky.edu [Graduate Center for Toxicology, College of Medicine, University of Kentucky, Lexington, KY (United States)

    2012-10-15

    Cadmium has been widely used in industry and is known to be carcinogenic to humans. Although it is widely accepted that chronic exposure to cadmium increases the incidence of cancer, the mechanisms underlying cadmium-induced carcinogenesis are unclear. The main aim of this study was to investigate the role of reactive oxygen species (ROS) in cadmium-induced carcinogenesis and the signal transduction pathways involved. Chronic exposure of human bronchial epithelial BEAS-2B cells to cadmium induced cell transformation, as evidenced by anchorage-independent growth in soft agar and clonogenic assays. Chronic cadmium treatment also increased the potential of these cells to invade and migrate. Injection of cadmium-stimulated cells into nude mice resulted in the formation of tumors. In contrast, the cadmium-mediated increases in colony formation, cell invasion and migration were prevented by transfection with catalase, superoxide dismutase-1 (SOD1), or SOD2. In particular, chronic cadmium exposure led to activation of signaling cascades involving PI3K, AKT, GSK-3β, and β-catenin and transfection with each of the above antioxidant enzymes markedly inhibited cadmium-mediated activation of these signaling proteins. Inhibitors specific for AKT or β-catenin almost completely suppressed the cadmium-mediated increase in total and active β-catenin proteins and colony formation. Moreover, there was a marked induction of AKT, GSK-3β, β-catenin, and carcinogenic markers in tumor tissues formed in mice after injection with cadmium-stimulated cells. Collectively, our findings suggest a direct involvement of ROS in cadmium-induced carcinogenesis and implicate a role of AKT/GSK-3β/β-catenin signaling in this process. -- Highlights: ► Chronic exposure to cadmium induces carcinogenic properties in BEAS-2B cells. ► ROS involved in cadmium-induced tumorigenicity of BEAS-2B cells. ► Cadmium activates ROS-dependent AKT/GSK-3β/β-catenin-mediated signaling. ► ROS

  12. Effect of silymarin and harpagoside on inflammation reaction of BEAS-2B cells, on ciliary beat frequency (CBF) of trachea explants and on mucociliary clearance (MCC).

    Science.gov (United States)

    Boeckenholt, Corinna; Begrow, Frank; Verspohl, Eugen J

    2012-05-01

    Silymarin and harpagoside are derived from drugs which are used for their protective effects against hepatotoxicity and inflammatory processes. Both are now investigated with respect to the respiratory tract. They were able to reduce the release of the inflammatory cytokine RANTES (regulated on activation, normal T cells expressed and secreted) from BEAS-2B cells in a concentration-dependent manner when stimulated by a cytokine mix (10 ng/mL of TNF- α and IFN- γ). This effect was not due to a possible toxic effect (control experiments using LDH release as a marker). Silymarin but not harpagoside was able to increase ciliary beat frequency. Effects were comparable to positive controls (isoprenaline and salbutamol). Silymarin also increases mucociliary clearance. In conclusion, silymarin should be further investigated for its clinical use in distinct respiratory diseases.

  13. Downregulation of B-cell lymphoma/leukemia-2 by overexpressed microRNA 34a enhanced titanium dioxide nanoparticle-induced autophagy in BEAS-2B cells.

    Science.gov (United States)

    Bai, Wenlin; Chen, Yujiao; Sun, Pengling; Gao, Ai

    2016-01-01

    Titanium dioxide (TiO2) nanoparticles (TNPs) are manufactured worldwide for a wide range of applications and the toxic effect of TNPs on biological systems is gaining attention. Autophagy is recognized as an emerging toxicity mechanism triggered by nanomaterials. MicroRNA 34a (miR34a) acts as a tumor suppressor gene by targeting many oncogenes, but how it affects autophagy induced by TNPs is not completely understood. Here, we observed the activation of TNP-induced autophagy through monodansylcadaverine staining and LC3-I/LC3-II conversion. Meanwhile, the transmission electron microscope ultrastructural analysis showed typical morphological characteristics in autophagy process. We detected the expression of miR34a and B-cell lymphoma/leukemia-2 (Bcl-2). In addition, the underlying mechanism of TNP-induced autophagy was performed using overexpression of miR34a by lentivirus vector transfection. Results showed that TNPs induced autophagy generation evidently. Typical morphological changes in the process of autophagy were observed by the transmission electron microscope ultrastructural analysis and LC3-I/LC3-II conversion increased significantly in TNP-treated cells. Meanwhile, TNPs induced the downregulation of miR34a and increased the expression of Bcl-2. Furthermore, overexpressed miR34a decreased the expression of Bcl-2 both in messenger RNA and protein level, following which the level of autophagy and cell death rate increased after the transfected cells were incubated with TNPs for 24 hours. These findings provide the first evidence that overexpressed miR34a enhanced TNP-induced autophagy and cell death through targeted downregulation of Bcl-2 in BEAS-2B cells.

  14. Repeated PM2.5 exposure inhibits BEAS-2B cell P53 expression through ROS-Akt-DNMT3B pathway-mediated promoter hypermethylation.

    Science.gov (United States)

    Zhou, Wei; Tian, Dongdong; He, Jun; Wang, Yimei; Zhang, Lijun; Cui, Lan; Jia, Li; Zhang, Li; Li, Lizhong; Shu, Yulei; Yu, Shouzhong; Zhao, Jun; Yuan, Xiaoyan; Peng, Shuangqing

    2016-04-12

    Long-term exposure to fine particulate matter (PM2.5) has been reported to be closely associated with the increased lung cancer risk in populations, but the mechanisms underlying PM-associated carcinogenesis are not yet clear. Previous studies have indicated that aberrant epigenetic alterations, such as genome-wide DNA hypomethylation and gene-specific DNA hypermethylation contribute to lung carcinogenesis. And silence or mutation of P53 tumor suppressor gene is the most prevalent oncogenic driver in lung cancer development. To explore the effects of PM2.5 on global and P53 promoter methylation changes and the mechanisms involved, we exposed human bronchial epithelial cells (BEAS-2B) to low concentrations of PM2.5 for 10 days. Our results indicated that PM2.5-induced global DNA hypomethylation was accompanied by reduced DNMT1 expression. PM2.5 also induced hypermethylation of P53 promoter and inhibited its expression by increasing DNMT3B protein level. Furthermore, ROS-induced activation of Akt was involved in PM2.5-induced increase in DNMT3B. In conclusion, our results strongly suggest that repeated exposure to PM2.5 induces epigenetic silencing of P53 through ROS-Akt-DNMT3B pathway-mediated promoter hypermethylation, which not only provides a possible explanation for PM-induced lung cancer, but also may help to identify specific interventions to prevent PM-induced lung carcinogenesis.

  15. DNA damage and DNA damage response in human bronchial epithelial BEAS-2B cells following exposure to 2-nitrobenzanthrone and 3-nitrobenzanthrone: role in apoptosis.

    Science.gov (United States)

    Oya, Elisabeth; Ovrevik, Johan; Arlt, Volker M; Nagy, Eszter; Phillips, David H; Holme, Jørn A

    2011-11-01

    Nitro-polycyclic aromatic hydrocarbons (nitro-PAHs) are mutagenic and carcinogenic environmental pollutants found in diesel exhaust and on urban air pollution particles. In the present study, human bronchial epithelial BEAS-2B cells were exposed to 2-nitrobenzanthrone (2-NBA) and 3-nitrobenzanthrone (3-NBA). DNA damage responses were compared to those observed after exposure to 1-nitropyrene (1-NP) and benzo[a]pyrene (B[a]P). Examination by microscopy revealed that 3-NBA was the most potent toxic compound while weaker responses were observed with 1-NP and B[a]P. Most interestingly, 2-NBA did not induce cell death or any other stress-related responses. 3-NBA induced a typical apoptotic cell death judged by nuclear condensation and little plasma membrane damage as well as cleavage of caspase 3 and poly-(ADP-ribose) polymerase (PARP). Exposure to 3-NBA resulted in an accumulation of cells in S-phase, and further analysis by Western blotting, immunocytochemistry and flow cytometry revealed that 3-NBA induced a DNA damage response characterized by phosphorylation of ATM (ataxia-telangiectasia mutated), checkpoint kinase (Chk) 2/Chk1, H2AX and p53. The p53 inhibitor pifithrin-α inhibited 3-NBA-induced apoptosis while small effects were seen using pifithrin-μ, suggesting that 3-NBA-induced cell death is a result of transcriptional activation of p53. In conclusion, 3-NBA is a potent inducer of apoptosis, which seemed to be triggered by the DNA damage response. Furthermore, a change of the nitro-group to the second position (i.e. 2-NBA) dramatically changed the cellular reactivity of the compound.

  16. Perilla frutescens leaf extract inhibits mite major allergen Der p 2-induced gene expression of pro-allergic and pro-inflammatory cytokines in human bronchial epithelial cell BEAS-2B.

    Directory of Open Access Journals (Sweden)

    Jer-Yuh Liu

    Full Text Available Perilla frutescens has been used in traditional medicine for respiratory diseases due to its anti-bacterial and anti-inflammatory activity. This study aimed to investigate effects of Perilla frutescens leaf extract (PFE on expression of pro-allergic and pro-inflammatory cytokines in airway epithelial cells exposed to mite major allergen Der p 2 (DP2 and the underlying mechanisms. Our results showed that PFE up to 100 µg/mL had no cytotoxic effect on human bronchial epithelial cell BEAS-2B. Further investigations revealed that PFE dose-dependently diminished mRNA expression of pro-allergic cytokine IL-4, IL-5, IL-13 and GM-CSF, as well as pro-inflammatory cytokine IL-6, IL-8 and MCP-1 in BEAS-2B cells treated with DP2. In parallel to mRNA, the DP-2-elevated levels of the tested cytokines were decreased. Further investigation showed that DP2-indued phosphorylation of p38 MAPK (P38 and JNK, but not Erk1/2, was also suppressed by PFE. In addition, PFE elevated cytosolic IκBα level and decreased nuclear NF-κB level in DP2-stimulated BEAS-2B cells. Taken together, these findings revealed that PFE significantly diminished both mRNA expression and protein levels of pro-allergic and pro-inflammatory cytokines in response to DP2 through inhibition of P38/JNK and NK-κB activation. These findings suggest that PFE should be beneficial to alleviate both allergic and inflammatory responses on airway epithelium in response to aeroallergens.

  17. Proinflammatory effects of diesel exhaust particles from moderate blend concentrations of 1st and 2nd generation biodiesel in BEAS-2B bronchial epithelial cells-The FuelHealth project.

    Science.gov (United States)

    Skuland, Tonje S; Refsnes, Magne; Magnusson, Pål; Oczkowski, Michał; Gromadzka-Ostrowska, Joanna; Kruszewski, Marcin; Mruk, Remigiusz; Myhre, Oddvar; Lankoff, Anna; Øvrevik, Johan

    2017-06-01

    Biodiesel fuel fuels are introduced at an increasing extent as a more carbon-neutral alternative to reduce CO2-emissions, compared to conventional diesel fuel. In the present study we have investigated the impact of increasing the use of 1st generation fatty acid methyl ester (FAME) biodiesel from current 7% blend (B7) to 20% blend (B20), or by increasing the biodiesel content by adding 2nd generation hydrotreated vegetable oil (HVO) based biodiesel (SHB; Synthetic Hydrocarbon Biofuel) on toxicity of diesel exhaust particles (DEP) in an in vitro system. Human bronchial epithelial BEAS-2B cells were exposed for 4 and 20h to DEP from B7, B20 and SHB at different concentrations, and examined for effects on gene expression of interleukin 6 (IL-6), CXCL8 (IL-8), CYP1A1 and heme oxygenase-1 (HO-1). The results show that both B20 and SHB were more potent inducers of IL-6 expression compared to B7. Only B20 induced statistically significant increases in CXCL8 expression. By comparison the rank order of potency to induce CYP1A1 was SHB>B7>B20. No statistically significant difference were observed form HO-1 expression, suggesting that the differences in cytokine responses were not due to oxidative stress. The results show that even moderate increases in biodiesel blends, from 7% to 20%, may increase the proinflammatory potential of emitted DEP in BEAS-2B cells. This effect was observed for both addition of 1st generation FAME and 2nd generation HVO biodiesel. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Chromosomal Instability of Human Tracheal Epithelia Cells BEAS-2B Induced by Extract of Coal Tar Pitch Fume%煤焦沥青烟提取物致人支气管上皮细胞BEAS-2B染色体不稳定性研究

    Institute of Scientific and Technical Information of China (English)

    李智涛; 冯艳铭; 王威; 王丽霞; 赵勇; 祝寒松; 吴卫东; 吴逸明

    2011-01-01

    .When morphological changes were shown, cell cycle was detected by flow cytometry. Results Half lethal concentration(LC50)of the fume extract of coal tar pitch was 8. 64 mg/L with BEAS-2B cells. BEAS-2B cells were induced at dosage of 2. 0 mg/L. After the cells were cultured for 30 passages, the malignant transformation was shown in the induced cells. At the 30th generation, the cell clone formation rate was 21. 50‰ in the coal tar pitch group, which was significantly higher than those of the normal control group and DMSO group. The Gl phase cell ratio significantly reduced and G2/M phase cell ratio increased significantly in the coal tar pitch group measured by flow cytometry. Beginning from the 10 th generation, the proportion of diploid cells decreased and aneuploid cells increased distinctively in the coal tar pitch induced group.Chromosomal instability increased as the cell passage increased. Cells in soft agar colony formation rate and the percentage of chromosome aberrations showed a positive correlation. Conclusions The fume extract of coal tar pitch could injury the cell DNA, and could induce BEAS-2B cells to produce chromosomal instability and malignant transformation in vitro.

  19. 卷烟烟气暴露下A549和BEAS-2B细胞促炎性因子的释放变化%Release of proinflammatory cytokines from A549 cells and BEAS-2B cells exposed to mainstream cigarette smoke

    Institute of Scientific and Technical Information of China (English)

    李翔; 张世敏; 赵俊伟; 尚平平; 彭斌; 谢复炜; 刘惠民; 谢剑平

    2016-01-01

    为比较不同类型细胞对卷烟烟气诱导的促炎症反应的应答差异,采用中性红细胞毒性试验测试了卷烟烟气总粒相物(TPM)和全烟气(WS)对人肺腺癌细胞A549和人支气管上皮细胞BEAS-2B的细胞毒性效应;采用酶联免疫法(ELISA)分析了TPM和WS暴露后,A549和BEAS-2B促炎性细胞因子白细胞介素-6(IL-6)和白细胞介素-8(IL-8)的释放水平。结果表明:BEAS-2B细胞较A549细胞对卷烟烟气的细胞毒性效应敏感。卷烟烟气暴露下A549细胞没有增加促炎性细胞因子IL-6和IL-8的释放;而BEAS-2B细胞在TPM和WS暴露下其IL-6和IL-8的释放显著增加。卷烟烟气诱导的促炎症反应存在细胞类型的差异。%In order to compare the differences of response to proinflammatory reaction induced by mainstream cigarette smoke among cells of different types, neutral red uptake (NRU) assay was employed to assess the cytotoxicity of cigarette smoke total particulate matter (TPM) or whole smoke (WS) to A549 cells (human adenocarcinoma cells) and BEAS-2B cells (human bronchial epithelial cells). The levels of proinflammatory cytokines interleukin-6 and interleukin-8 (IL-6 and IL-8) released from A549 and BEAS-2B cells exposed to TPM and WS were analyzed by ELISA. The results showed that BEAS-2B cells were more sensitive to smoke-induced cytotoxicity than A549 cells. The induction of IL-6 and IL-8 released from A549 cells was not impacted by cigarette smoke exposure, while TPM and WS all caused dose-dependently increase of IL-6 and IL-8 released from BEAS-2B cells. The proinflammatory response to cigarette smoke thus might be different depending on cell types.

  20. H5N1禽流感病毒NS1蛋白与干扰素诱导蛋白10表达的相关性研究%Influence of avian influenza virus NS1 protein on the expression of IP-10 in BEAS-2B cells

    Institute of Scientific and Technical Information of China (English)

    贾晓俊; 周剑芳; 王晶钰; 董婕; 薄洪; 李梓; 李魁彪; 蓝雨; 舒跃龙

    2008-01-01

    目的 研究高致病性禽流感(HPAI)H5N1病毒NS1蛋白对干扰素诱导蛋白10(IP-10)的影响.方法 分别将禽流感病毒A/Anhui/1/2005(H5N1)的NS1基因、插入80-84位缺失氨基酸的NS1突变基因及流感病毒A/Puerto Rico/8/1934(H1N1)的NS1基因克隆至真核表达载体pEGFP-N1,转染人支气管上皮细胞BEAS-2B,流式细胞仪检测转染细胞内IP-10的表达情况.结果 与pEGFP-N1对照组相比,三种NS1蛋白均能下调BEAS-2B细胞IP-10的表达(P0.01).结论 A/Anhui/1/2005(H5N1)禽流感病毒单一NS1蛋白能够抑制BEAS-2B细胞IP-10表达,但这并不能完全阐明其与病毒致病性之间的关系.%Objective To investigate the influence of avian influenza virus (AIV) NS1 protein on the expression of interferon-inducible protein 10 (IP-10). Methods NS1 gene from virus A/Anhui/1/2005 (H5N1),NS1 gene inserted with 80-84 amino acids from virus A/Anhui/1/2005(H5N1)and NS1 gene from virus A/Puerto Rico/8/1934 (H1N1) were cloned into the eukaryotic expression vector pEGFP-N1, and transected into BEAS-2Bcells, IP-10 expression level in transected cells was detected by flow cytometry. Results Compared with the control group pEGFP-N1, Expression of these three different NS1 genes can down-regulate the expression of IP-10in BEAS-2B cells, but there is no significant difference as to the lower level among them. Conclusion NS1protein of A/Anhui/1/2005(H5N1) can down-regulate the expression level of IP-10, but this may not clarify its relationship with the virulence of AIV.

  1. Proliferative activity of a blend of Echinacea angustifolia and Echinacea purpurea root extracts in human vein epithelial, HeLa, and QBC-939 cell lines, but not in Beas-2b cell lines.

    Science.gov (United States)

    Cichello, Simon Angelo; Yao, Qian; He, Xiao Qiong

    2016-04-01

    Echinacea is used for its immunostimulating properties and may have a role in modulating adverse immune effects of chemotherapy (i.e., use of 5-fluorouracil (5-FU); fluorouracil and its immunosuppressive effect). Patients may seek herbal remedies such as Echinacea (Echinacea angustifolia and Echinacea purpurea) for immune stimulation. Echinacea extracts have been prescribed to supplement cancer chemotherapy for their immune-supportive effects; however, the extracts may also influence tumourgenesis. Our study aimed to determine the proliferative effect of the ethanolic blend of E. angustifolia and E. purpurea on various cancer cervical and bile duct cell lines, including HELA and QBC-939. Various cancer cells (HeLa and QBC-939) and human vein epithelial cells (HUVEC) were treated with the Echinacea blend sample that was evaporated and reconstituted in Dimethyl sulfoxide (DMSO). As the extract concentration of Echinacea was increased from 12.5 μg/mL to 25 μg/mL, there was an increase in cell inhibition up to 100%, which then reduced to 90% over the next three concentrations, 50 μg/mL, 100 μg/mL, and 200 μg/mL, in HeLa cells; further inhibitory effects were observed in QBC-939 cells, from 9% inhibition at a concentration of 25 μg/mL up to 37.96% inhibition at 100 μg/mL concentration. Moreover, this is the first study to report the growth-promoting effects of this Echinacea blend in HUVEC, up to 800% at a dose concentration of 200 μg/mL. Previous studies have suggested that chicoric acid of Echinacea spp. is responsible for the increased cell growth. The results of this study show that the hydroethanolic extract of Echinacea herbal medicine promotes the growth of HeLa cells and QBC-939 cancer cell proliferation, and may interfere with cancer treatment (i.e., chemotherapy drugs such as 5-fluorouracil and Cisplatin (DDP)). However, the Echinacea blend shows potential in neurodegenerative diseases with growth-promoting effects in HUVEC. Further animal

  2. Proliferative activity of a blend of Echinacea angustifolia and Echinacea purpurea root extracts in human vein epithelial, HeLa, and QBC-939 cell lines, but not in Beas-2b cell lines

    Directory of Open Access Journals (Sweden)

    Simon Angelo Cichello

    2016-04-01

    Full Text Available Echinacea is used for its immunostimulating properties and may have a role in modulating adverse immune effects of chemotherapy (i.e., use of 5-fluorouracil (5-FU; fluorouracil and its immunosuppressive effect. Patients may seek herbal remedies such as Echinacea (Echinacea angustifolia and Echinacea purpurea for immune stimulation. Echinacea extracts have been prescribed to supplement cancer chemotherapy for their immune-supportive effects; however, the extracts may also influence tumourgenesis. Our study aimed to determine the proliferative effect of the ethanolic blend of E. angustifolia and E. purpurea on various cancer cervical and bile duct cell lines, including HELA and QBC-939. Various cancer cells (HeLa and QBC-939 and human vein epithelial cells (HUVEC were treated with the Echinacea blend sample that was evaporated and reconstituted in Dimethyl sulfoxide (DMSO. As the extract concentration of Echinacea was increased from 12.5 μg/mL to 25 μg/mL, there was an increase in cell inhibition up to 100%, which then reduced to 90% over the next three concentrations, 50 μg/mL, 100 μg/mL, and 200 μg/mL, in HeLa cells; further inhibitory effects were observed in QBC-939 cells, from 9% inhibition at a concentration of 25 μg/mL up to 37.96% inhibition at 100 μg/mL concentration. Moreover, this is the first study to report the growth-promoting effects of this Echinacea blend in HUVEC, up to 800% at a dose concentration of 200 μg/mL. Previous studies have suggested that chicoric acid of Echinacea spp. is responsible for the increased cell growth. The results of this study show that the hydroethanolic extract of Echinacea herbal medicine promotes the growth of HeLa cells and QBC-939 cancer cell proliferation, and may interfere with cancer treatment (i.e., chemotherapy drugs such as 5-fluorouracil and Cisplatin (DDP. However, the Echinacea blend shows potential in neurodegenerative diseases with growth-promoting effects in HUVEC

  3. Protective effects of tea polyphenols on oxidative damage induced by uranium-mineral dust in BEAS-2B cells%茶多酚对铀矿尘致支气管上皮细胞损伤保护作用

    Institute of Scientific and Technical Information of China (English)

    黄波; 龙颖; 罗振华; 黄锐; 贺性鹏

    2011-01-01

    目的 探讨铀矿尘诱发细胞氧化损伤及茶多酚(tea polyphenols,TPs)的保护作用.方法 以人支气管上皮细胞(BEAS-2B)为靶细胞,分别用辣根过氧化物酶介导的酚红氧化法、细胞色素C还原法和番红花红褪色法测定细胞外液过氧化氢、超氧阴离子和羟自由基含量;细胞内H2O2和O2·-分别用2,7-二氯荧光黄双乙酸盐和氢化乙锭标记,荧光法测定荧光产物2,7'-二氯荧光黄和溴乙锭荧光强度,多核细胞法体外研究细胞次黄嘌呤磷核糖基转移酶基因(HPRT)突变,自动生化仪测量乳酸脱氢酶.结果 BEAS-2B细胞经铀矿尘染毒后,细胞内和细胞外ROS含量明显增高,高剂量组细胞上清中超氧阴离子为(10.48±0.75)μmol/L,茶多酚保护组为(9.3l±0.71)μmol/L;细胞乳酸脱氢酶溢出,HPRT突变率升高,高剂量组细胞突变率为1.092%,茶多酚保护组为0.925‰,TPS对铀矿尘诱发的损伤有明显的抑制作用.结论 铀矿尘可造成细胞的氧化损伤,TPS通过清除活性氧而达到保护作用,可为铀矿尘损伤的防护提供有效措施.%Objective To observe oxidative damage of human bronchial epithelial cells (BEAS-2B) induced by uranium-mineral dust(UD) and protective effect of tea polyphenols(TPs). Methods The measurement of extracellular superoxide anions(O2 · - ) was based on the reduction of ferricytochrome C. The quantitation of extracellular hydrogen peroxides( H2O2 ) was based on horseradish peroxidase-dependent oxidation of phenol red. The determination of extracellular hydroxyl radicals(·OH) was based on the decoloration of safranine T. Ethidium bromide, 2,7'-dichlorofluorescein, and fluorescent products of membrane-permeable dyes-hydroethine and 2,7' -dichloroflurescein diacetate were used to monitor intracellular production of O2· and H2 O2 , respectively. The content of lactate dehydrogenase was measured by AUTOLAB. Cytokinesis-block( CB ) assay was used to detect the mutation frequency of

  4. Macrophages facilitate coal tar pitch extract-induced tumorigenic transformation of human bronchial epithelial cells mediated by NF-κB.

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    Feifei Feng

    Full Text Available OBJECTIVE: Chronic respiratory inflammation has been associated with lung cancer. Tumor-associated macrophages (TAMs play a critical role in the formation of inflammation microenvironment. We sought to characterize the role of TAMs in coal tar pitch extract (CTPE-induced tumorigenic transformation of human bronchial epithelial cells and the underlying mechanisms. METHODS: The expression of TAMs-specific CD68 in lung cancer tissues and paired adjacent tissues from cancer patients was determined using immunostaining. Co-culture of human bronchial epithelial cells (BEAS-2B and macrophage-like THP-1 cells were conducted to evaluate the promotive effect of macrophages on CTPE-induced tumorigenic transformation of BEAS-2B cells. BEAS-2B cells were first treated with 2.4 µg/mL CTPE for 72 hours. After removal of CTPE, the cells were continuously cultured either with or without THP-1 cells and passaged using trypsin-EDTA. Alterations of cell cycle, karyotype, colony formation in soft agar and tumor xenograft growth in nude mice of BEAS-2B cells at passages 10, 20 and 30, indicative of tumorigenecity, were determined, respectively. In addition, mRNA and protein levels of NF-κB in BEAS-2B cells were measured with RT-PCR and western blot, respectively. B(aP was used as the positive control. RESULTS: The over-expression of TAMs-specific CD68 around lung tumor tissues was detected and associated with lung cancer progression. The tumorigenic alterations of BEAS-2B cells including increase in cell growth rate, number of cells with aneuploidy, clonogenicity in soft agar, and tumor size in nude mice in vivo occurred at passage 10, becoming significant at passages 20 and 30 of the co-culture following CTPE removal in compared to BEAS-2B cells alone. In addition, the expression levels of NF-κB in BEAS-2B cells were positively correlated to the malignancy of BEAS-2B cells under different conditions of treatment. CONCLUSION: The presence of macrophages

  5. Puerarin protects human bronchial epithelial cells from apoptosis induced by gunpowder smog

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    Yun-xia CHEN

    2016-03-01

    Full Text Available Objective  To investigate protective effects of puerarin on the human bronchial epithelial (BEAS-2B cell line against apoptosis caused by gunpowder smog and its mechanisms. Methods  BEAS-2B cells cultured in vitro were randomly divided into control group, smog group (the group treated with 4g gunpowder smog for 10min, and smog + puerarin group [puerarin group, the cells were pre-incubated with various concentrations of puerarin (12.5, 25.0, 50.0, 100.0µg/ml and then exposed to smoke]. Puerarin was added into the cells after innoculation for 12h and then the cells were sequentially cultured for 24h and followed by exposure to smoke for 10min. After being cultured again for 2h, the smoked cells were examined for cell viability using Cell Counting Kit-8(CCK-8, cell apoptosis was observed using Hoechst33258 nucleus staining, and positive rates of Annexin V-PI staining cells and caspase-3 were determined with flow cytometer. Resu lts  Compared with control, treatment of BEAS-2B cells with 4g gunpowder smog induced a characteristic apoptotic cell death (P<0.01. Pretreatment with various concentrations of puerarin antagonized the action of gunpowder smog in different degrees. The 25µg/ml was determined as the optimal effective concentration of puerarin. Compared with smog group, the apoptosis rate of BEAS-2B cells and positive rates of Annexin V-PI staining cells and caspase-3 were decreased significantly in smog + puerarin group (P<0.05, P<0.01. Conclusion  Gunpowder smog can induce apoptosis of BEAS-2B cells in vitro, while pretreatment with puerarin could protect BEAS-2B cells against apoptosis induced by gunpowder smog. DOI: 10.11855/j.issn.0577-7402.2016.01.16

  6. Evaluation of Differentiated Human Bronchial Epithelial Cell Culture Systems for Asthma Research

    Directory of Open Access Journals (Sweden)

    Ceri E. Stewart

    2012-01-01

    Full Text Available The aim of the current study was to evaluate primary (human bronchial epithelial cells, HBEC and non-primary (Calu-3, BEAS-2B, BEAS-2B R1 bronchial epithelial cell culture systems as air-liquid interface- (ALI- differentiated models for asthma research. Ability to differentiate into goblet (MUC5AC+ and ciliated (β-Tubulin IV+ cells was evaluated by confocal imaging and qPCR. Expression of tight junction/adhesion proteins (ZO-1, E-Cadherin and development of transepithelial electrical resistance (TEER were assessed. Primary cells showed localised MUC5AC, β-Tubulin IV, ZO-1, and E-Cadherin and developed TEER with, however, a large degree of inter- and intradonor variation. Calu-3 cells developed a more reproducible TEER and a phenotype similar to primary cells although with diffuse β-Tubulin IV staining. BEAS-2B cells did not differentiate or develop tight junctions. These data highlight the challenges in working with primary cell models and the need for careful characterisation and selection of systems to answer specific research questions.

  7. House dust mite allergen Der p 1 elevates the release of inflammatory cytokines and expression of adhesion molecules in co-culture of human eosinophils and bronchial epithelial cells.

    Science.gov (United States)

    Wong, Chun K; Li, Mandy L Y; Wang, Cheng B; Ip, Wai K; Tian, Ya P; Lam, Christopher W K

    2006-08-01

    House dust mite (HDM) is a common allergen of allergic asthma. Eosinophils are principal effector cells of allergic inflammation and their adhesion onto human bronchial epithelial cells is mediated by a CD18-intracellular adhesion molecule-1 (ICAM-1)-dependent interaction. We studied the effects of HDM Dermatophagoides pteronyssinus (Der p) 1 on the activation of eosinophils and bronchial epithelial BEAS-2B cells. Cytokines and adhesion molecules were measured using flow cytometry. Transcription factor nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) and signaling molecule p38 mitogen-activated protein kinase (MAPK) were analyzed using electromobility shift assay and western blot, respectively. Der p 1 protein was found to potently induce the release of IL-1beta, IL-6, IL-10, tumor necrosis factor (TNF)-alpha and granulocyte macrophage colony-stimulating factor from eosinophils. Such induction was further up-regulated for IL-6 and IL-10, and down-regulated for TNF-alpha and IL-1beta in eosinophil-BEAS-2B cells co-culture. Surface expression of CD18 and ICAM-1 on eosinophils was greatly increased by Der p 1; such inductive effect on ICAM-1 was also found to be more prominent on BEAS-2B cells from the co-culture than BEAS-2B cells alone. Der p 1 was found to activate NF-kappaB and AP-1 activity in eosinophils alone and in co-culture and BEAS-2B cells in co-culture. Moreover, Der p 1 could activate p38 MAPK in BEAS-2B cells and eosinophils alone and in co-culture. Selective inhibition of NF-kappaB, AP-1 and p38 MAPK resulted in differential suppression of the Der p 1-induced cytokine release and adhesion molecule expression. As an allergen, HDM could therefore induce the release of inflammatory cytokines and expression of adhesion molecules from the interaction of human eosinophils and bronchial epithelial cells.

  8. Effects of cell type and culture media on Interleukin-6 secretion in response to environmental particles.

    Science.gov (United States)

    Veranth, John M; Cutler, N Shane; Kaser, Erin G; Reilly, Christopher A; Yost, Garold S

    2008-03-01

    Cultured lung cells provide an alternative to animal exposures for comparing the effects of different types of air pollution particles. Studies of particulate matter in vitro have reported proinflammatory cytokine signaling in response to many types of environmental particles, but there have been few studies comparing identical treatments in multiple cell types or identical cells with alternative cell culture protocols. We compared soil-derived, diesel, coal fly ash, titanium dioxide, and kaolin particles along with soluble vanadium and lipopolysaccharide, applied to airway-derived cells grown in submerged culture. Cell types included A549, BEAS-2B, RAW 264.7, and primary macrophages. The cell culture models (specific combinations of cell types and culture conditions) were reproducibly different in the cytokine signaling responses to the suite of treatments. Further, Interleukin-6 (IL-6) response to the treatments changed when the same cells, BEAS-2B, were grown in KGM versus LHC-9 media or in media containing bovine serum. The effect of changing media composition was reversible over multiple changes of media type. Other variables tested included culture well size and degree of confluence. The observation that sensitivity of a cell type to environmental agonists can be manipulated by modifying culture conditions suggests a novel approach for studying biochemical mechanisms of particle toxicity.

  9. Effects of cell type and culture media on Interleukin-6 secretion in response to environmental particles

    Energy Technology Data Exchange (ETDEWEB)

    Veranth, J.M.; Cutler, N.S.; Kaser, E.G.; Reilly, C.A.; Yost, G.S. [University of Utah, Salt Lake City, UT (United States)

    2008-03-15

    Cultured lung cells provide an alternative to animal exposures for comparing the effects of different types of air pollution particles. Studies of particulate matter in vitro have reported proinflammatory cytokine signaling in response to many types of environmental particles, but there have been few studies comparing identical treatments in multiple cell types or identical cells with alternative cell culture protocols. We compared soil-derived, diesel, coal fly ash, titanium dioxide, and kaolin particles along with soluble vanadium and lipopolysaccharide, applied to airway-derived cells grown in submerged culture. Cell types included A549, BEAS-2B, RAW 264.7, and primary macrophages. The cell culture models (specific combinations of cell types and culture conditions) were reproducibly different in the cytokine signaling responses to the suite of treatments. Further, Interleukin-6 (IL-6) response to the treatments changed when the same cells, BEAS-2B, were grown in KGM versus LHC-9 media or in media containing bovine serum. The effect of changing media composition was reversible over multiple changes of media type. Other variables tested included culture well size and degree of confluence. The observation that sensitivity of a cell type to environmental agonists can be manipulated by modifying culture conditions suggests a novel approach for studying biochemical mechanisms of particle toxicity.

  10. Effects of bile acids on human airway epithelial cells: implications for aerodigestive diseases

    Directory of Open Access Journals (Sweden)

    Adil Aldhahrani

    2017-03-01

    Full Text Available Gastro-oesophageal reflux and aspiration have been associated with chronic and end-stage lung disease and with allograft injury following lung transplantation. This raises the possibility that bile acids may cause lung injury by damaging airway epithelium. The aim of this study was to investigate the effect of bile acid challenge using the immortalised human bronchial epithelial cell line (BEAS-2B. The immortalised human bronchial epithelial cell line (BEAS-2B was cultured. A 48-h challenge evaluated the effect of individual primary and secondary bile acids. Post-challenge concentrations of interleukin (IL-8, IL-6 and granulocyte−macrophage colony-stimulating factor were measured using commercial ELISA kits. The viability of the BEAS-2B cells was measured using CellTiter-Blue and MTT assays. Lithocholic acid, deoxycholic acid, chenodeoxycholic acid and cholic acid were successfully used to stimulate cultured BEAS-2B cells at different concentrations. A concentration of lithocholic acid above 10 μmol·L−1 causes cell death, whereas deoxycholic acid, chenodeoxycholic acid and cholic acid above 30 μmol·L−1 was required for cell death. Challenge with bile acids at physiological levels also led to a significant increase in the release of IL-8 and IL6 from BEAS-2B. Aspiration of bile acids could potentially cause cell damage, cell death and inflammation in vivo. This is relevant to an integrated gastrointestinal and lung physiological paradigm of chronic lung disease, where reflux and aspiration are described in both chronic lung diseases and allograft injury.

  11. SILAC-based quantitative proteomic analysis of human lung cell response to copper oxide nanoparticles.

    Science.gov (United States)

    Edelmann, Mariola J; Shack, Leslie A; Naske, Caitlin D; Walters, Keisha B; Nanduri, Bindu

    2014-01-01

    Copper (II) oxide (CuO) nanoparticles (NP) are widely used in industry and medicine. In our study we evaluated the response of BEAS-2B human lung cells to CuO NP, using Stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics and phosphoproteomics. Pathway modeling of the protein differential expression showed that CuO NP affect proteins relevant in cellular function and maintenance, protein synthesis, cell death and survival, cell cycle and cell morphology. Some of the signaling pathways represented by BEAS-2B proteins responsive to the NP included mTOR signaling, protein ubiquitination pathway, actin cytoskeleton signaling and epithelial adherens junction signaling. Follow-up experiments showed that CuO NP altered actin cytoskeleton, protein phosphorylation and protein ubiquitination level.

  12. SILAC-based quantitative proteomic analysis of human lung cell response to copper oxide nanoparticles.

    Directory of Open Access Journals (Sweden)

    Mariola J Edelmann

    Full Text Available Copper (II oxide (CuO nanoparticles (NP are widely used in industry and medicine. In our study we evaluated the response of BEAS-2B human lung cells to CuO NP, using Stable isotope labeling by amino acids in cell culture (SILAC-based proteomics and phosphoproteomics. Pathway modeling of the protein differential expression showed that CuO NP affect proteins relevant in cellular function and maintenance, protein synthesis, cell death and survival, cell cycle and cell morphology. Some of the signaling pathways represented by BEAS-2B proteins responsive to the NP included mTOR signaling, protein ubiquitination pathway, actin cytoskeleton signaling and epithelial adherens junction signaling. Follow-up experiments showed that CuO NP altered actin cytoskeleton, protein phosphorylation and protein ubiquitination level.

  13. Loss of fructose-1,6-bisphosphatase induces glycolysis and promotes apoptosis resistance of cancer stem-like cells: an important role in hexavalent chromium-induced carcinogenesis.

    Science.gov (United States)

    Dai, Jin; Ji, Yanli; Wang, Wei; Kim, Donghern; Fai, Leonard Yenwong; Wang, Lei; Luo, Jia; Zhang, Zhuo

    2017-09-15

    Hexavalent chromium (Cr(VI)) compounds are confirmed human carcinogens for lung cancer. Our previous studies has demonstrated that chronic exposure of human bronchial epithelial BEAS-2B cells to low dose of Cr(VI) causes malignant cell transformation. The acquisition of cancer stem cell-like properties is involved in the initiation of cancers. The present study has observed that a small population of cancer stem-like cells (BEAS-2B-Cr-CSC) exists in the Cr(VI)-transformed cells (BEAS-2B-Cr). Those BEAS-2B-Cr-CSC exhibit extremely reduced capability of generating reactive oxygen species (ROS) and apoptosis resistance. BEAS-2B-Cr-CSC are metabolic inactive as evidenced by reductions in oxygen consumption, glucose uptake, ATP production, and lactate production. Most importantly, BEAS-2B-Cr-CSC are more tumorigenic with high levels of cell self-renewal genes, Notch1 and p21. Further study has found that fructose-1,6-bisphosphatase (FBP1), an rate-limiting enzyme driving glyconeogenesis, was lost in BEAS-2B-Cr-CSC. Forced expression of FBP1 in BEAS-2B-Cr-CSC restored ROS generation, resulting in increased apoptosis, leading to inhibition of tumorigenesis. In summary, the present study suggests that loss of FBP1 is a critical event in tumorigenesis of Cr(VI)-transformed cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Exposure to Cigarette Smoke Disrupts CCL20-Mediated Antimicrobial Activity in Respiratory Epithelial Cells.

    Science.gov (United States)

    Crane-Godreau, Mardi A; Maccani, Matthew A; Eszterhas, Susan K; Warner, Sandra L; Jukosky, James A; Fiering, Steven

    2009-01-01

    Cigarette smoke (CS) exposure is known to increase infection rates, but the mechanisms are not well understood. These studies tested the hypothesis that CS exposure would impair antimicrobial activity of apical conditioned media from human airway (BEAS-2B) cultures by reducing induction and release of the antimicrobial peptide CCL20. BEAS-2B cultures were exposed to CS extract and assayed for temporal and physical characteristics of release as well as for antimicrobial activity. E. coli were exposed to Beas-2B-conditioned media (BCM) and subsequent bacterial colonies were enumerated. In time course studies TLR-agonist-induced CCL20 transcription and release were rapid, of short duration and release was consistently targeted to the apical/luminal compartment. Cells treated with CS extract had diminished release of CCL20 under both constitutive and toll-like receptor (TLR) agonist stimulating conditions. Exposure of the cells to CS significantly reduced the antimicrobial activity in BCM and neutralizing antibodies to CCL20 brought antibacterial activity back to baseline levels demonstrating that antimicrobial activity in this culture system was primarily attributable to CCL20. These studies add to the understanding of CCL20 as a mucosal antimicrobial and improve insight into a likely mechanism linking infection to CS exposure.

  15. Alteration of Cell Cycle Mediated by Zinc in Human Bronchial ...

    Science.gov (United States)

    Zinc (Zn2+), a ubiquitous ambient air contaminant, presents an oxidant challenge to the human lung and is linked to adverse human health effects. To further elucidate the adaptive and apoptotic cellular responses of human airway cells to Zn2+, we performed pilot studies to examine cell cycle perturbation upon exposure using a normal human bronchial epithelial cell culture (BEAS-2B). BEAS-2B cells were treated with low (0, 1, 2 µM) and apoptotic (3 µM) doses of Zn2+ plus 1 µM pyrithione, a Zn2+-specific ionophore facilitating cellular uptake, for up to 24 h. Fixed cells were then stained with propidium iodine (PI) and cell cycle phase was determined by fluorescent image cytometry. Initial results report the percentage of cells in the S phase after 18 h exposure to 1, 2, and 3 µM Zn2+ were similar (8%, 7%, and 12%, respectively) compared with 7% in controls. Cells exposed to 3 µM Zn2+ increased cell populations in G2/M phase (76% versus 68% in controls). Interestingly, exposure to 1 µM Zn2+ resulted in decreased (59%) cells in G2/M. While preliminary, these pilot studies suggest Zn2+ alters cell cycle in BEAS-2B cells, particularly in the G2/M phase. The G2/M checkpoint maintains DNA integrity by enabling initiation of DNA repair or apoptosis. Our findings suggest that the adaptive and apoptotic responses to Zn2+ exposure may be mediated via perturbation of the cell cycle at the G2/M checkpoint. This work was a collaborative summer student project. The st

  16. Particulate metal bioaccessibility in physiological fluids and cell culture media: Toxicological perspectives.

    Science.gov (United States)

    Leclercq, Bérénice; Alleman, Laurent Yves; Perdrix, Esperanza; Riffault, Véronique; Happillon, Mélanie; Strecker, Alain; Lo-Guidice, Jean-Marc; Garçon, Guillaume; Coddeville, Patrice

    2017-07-01

    According to the literature, tiny amounts of transition metals in airborne fine particles (PM2.5) may induce proinflammatory cell response through reactive oxygen species production. The solubility of particle-bound metals in physiological fluids, i.e. the metal bioaccessibility is driven by factors such as the solution chemical composition, the contact time with the particles, and the solid-to-liquid phase ratio (S/L). In this work, PM2.5-bound metal bioaccessibility was assessed in various physiological-like solutions including cell culture media in order to evidence the potential impact on normal human bronchial epithelial cells (NHBE) when studying the cytotoxicity and inflammatory responses of PM2.5 towards the target bronchial compartment. Different fluids (H2O, PBS, LHC-9 culture medium, Gamble and human respiratory mucus collected from COPD patients), various S/L conditions (from 1/6000 to 1/100,000) and exposure times (6, 24 and 72h) were tested on urban PM2.5 samples. In addition, metals' total, soluble and insoluble fractions from PM2.5 in LHC-9 were deposited on NHBE cells (BEAS-2B) to measure their cytotoxicity and inflammatory potential (i.e., G6PDH activity, secretion of IL-6 and IL-8). The bioaccessibility is solution-dependent. A higher salinity or organic content may increase or inhibit the bioaccessibiliy according to the element, as observed in the complex mucus matrix. Decreasing the S/L ratio also affect the bioaccessibility depending on the solution tested while the exposure time appears less critical. The LHC-9 culture medium appears to be a good physiological proxy as it induces metal bioaccessibilities close to the mucus values and is little affected by S/L ratios or exposure time. Only the insoluble fraction can be linked to the PM2.5-induced cytotoxicity. By contrast, both soluble and insoluble fractions can be related to the secretion of cytokines. The metal bioaccessibility in LHC-9 of the total, soluble, and insoluble fractions of the

  17. Trans, trans-2,4-decadienal, a product found in cooking oil fumes, induces cell proliferation and cytokine production due to reactive oxygen species in human bronchial epithelial cells.

    Science.gov (United States)

    Chang, Louis W; Lo, Wai-Sze; Lin, Pinpin

    2005-10-01

    Dienaldehydes are by-products of peroxidation of polyunsaturated lipids and commonly found in many foods or food-products. Both National Cancer Institute (NCI) and NTP have expressed great concern on the potential genotoxicity and carcinogenicity of dienaldehydes. Trans, trans-2,4-decadienal (tt-DDE or 2,4-De), a specific type of dienaldehyde, is abundant in heated oils and has been associated with lung adenocarcinoma development in women due to their exposure to oil fumes during cooking. Cultured human bronchial epithelial cells (BEAS-2B cells) were exposed to 0.1 or 1.0 microM tt-DDE for 45 days, and oxidative stress, reactive oxygen species (ROS) production, GSH/GSSG ratio, cell proliferation, and expression of TNFalpha and IL-1beta were measured. The results show that tt-DDE induced oxidative stress, an increase in ROS production, and a decrease in GSH/GSSG ratio (glutathione status) in a dose-dependent manner. Treatment of BEAS-2B cells with 1.0 microM tt-DDE for 45 days increased cell proliferation and the expression and release of pro-inflammatory cytokines TNFalpha and IL-1beta. Cotreatment of BEAS-2B cells with antioxidant N-acetylcysteine prevented tt-DDE-induced cell proliferation and release of cytokines. Therefore, these results suggest that tt-DDE-induced changes may be due to increased ROS production and enhanced oxidative stress. Since increased cell proliferation and the release of TNF-alpha and IL-1beta are believed to be involved in tumor promotion, our results suggest that tt-DDE may play a role in cancer promotion. Previous studies on dienaldehydes have focused on their genotoxic or carcinogenic effects in the gastrointestinal tract; the present study suggests a potential new role of tt-DDE as a tumor promoter in human lung epithelial cells.

  18. Particle and ozone-induced inflammation reactions of the lungs: Interactions and reactions of bronchial epithelial and endothelial cells in vitro; Partikel- und ozoninduzierte Entzuendungsreaktionen in der Lunge: Wechselwirkungen und Reaktionen von Bronchialepithelzellen und Endothelzellen in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Moegel, M.

    1998-07-01

    In the present work the influence of ozone, quartz dust (DQ12) and global extracts of atmospheric dust (GEX) on the release of the inflammatory mediators interleukin-6 (IL-6) and interleukin-8 (IL-8) as well as the expression of the intercellular adhesion molecule-1 (ICAM-1) was investigated by use of a human bronchial epithelial cell line (BEAS-2B). Exposure of the cells with 0.15 ppm ozone for 90 minutes resulted in an elevated IL-6- and IL-8-release and an increased expression of ICAM-1. On the other hand, incubations with extracts of atmospheric dust (GEX) only affected the ICAM-1-expression of the cells. The treatment with quartz dust, however, induced a time- and dose-dependent formation of the investigated markers of inflammation. Further investigations dealth with possible immunmodulating effects of ozone determined after a preceding or a subsequent stimulation of the cells with the standard stimuli lipopolysaccharide (LPS) and zymosan or the cytokines tumor-necrosis-factor-{alpha} and interleukin-1{beta}. For all those stimuli an immunsuppressive effect of ozone, manifested in decreased amounts of IL-6, IL-8 and ICAM-1, could be demonstrated. Additionally, the influence of a combined exposure of cells with ozone and quartz dust or ozone and GEX has been investigated. Ozone-treated BEAS-2B cells showed a diminished formation of all investigated markers of inflammation, when treated subsequently with quartz dust, compared with the air-treated control cells. Concerning successive incubations with GEX these effects could not be observed. In the second part of this work a coculture-system of bronchial epithelial cells (BEAS-2B) and endothelial cells (ECV304) has been established. The new culture-system offers the possibility by gas exposure of the epithelial cells to answer questions within the field of inhalation toxicology with respect to possible interactions between different cell types, e.g. epithelial and endothelial cells. (orig.) [Deutsch] In der

  19. Human bronchial epithelial cells exposed in vitro to diesel exhaust particles exhibit alterations in cell rheology and cytotoxicity associated with decrease in antioxidant defenses and imbalance in pro- and anti-apoptotic gene expression.

    Science.gov (United States)

    Seriani, Robson; de Souza, Claudia Emanuele Carvalho; Krempel, Paloma Gava; Frias, Daniela Perroni; Matsuda, Monique; Correia, Aristides Tadeu; Ferreira, Márcia Zotti Justo; Alencar, Adriano Mesquita; Negri, Elnara Marcia; Saldiva, Paulo Hilário Nascimento; Mauad, Thais; Macchione, Mariangela

    2016-05-01

    Diesel exhaust particles (DEPs) from diesel engines produce adverse alterations in cells of the airways by activating intracellular signaling pathways and apoptotic gene overexpression, and also by influencing metabolism and cytoskeleton changes. This study used human bronchial epithelium cells (BEAS-2B) in culture and evaluates their exposure to DEPs (15ug/mL for 1 and 2 h) in order to determine changes to cell rheology (viscoelasticity) and gene expression of the enzymes involved in oxidative stress, apoptosis, and cytotoxicity. BEAS-2B cells exposed to DEPs were found to have a significant loss in stiffness, membrane stability, and mitochondrial activity. The genes involved in apoptosis [B cell lymphoma 2 (BCL-2 and caspase-3)] presented inversely proportional expressions (p = 0.05, p = 0.01, respectively), low expression of the genes involved in antioxidant responses [SOD1 (superoxide dismutase 1); SOD2 (superoxide dismutase 2), and GPx (glutathione peroxidase) (p = 0.01)], along with an increase in cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A1) (p = 0.01). These results suggest that alterations in cell rheology and cytotoxicity could be associated with oxidative stress and imbalance between pro- and anti-apoptotic genes.

  20. Malignant human cell transformation of Marcellus shale gas drilling flow back water

    Science.gov (United States)

    Yao, Yixin; Chen, Tingting; Shen, Steven S.; Niu, Yingmei; DesMarais, Thomas L; Linn, Reka; Saunders, Eric; Fan, Zhihua; Lioy, Paul; Kluz, Thomas; Chen, Lung-Chi; Wu, Zhuangchun; Costa, Max

    2015-01-01

    The rapid development of high-volume horizontal hydraulic fracturing for mining natural gas from shale has posed potential impacts on human health and biodiversity. The produced flow back waters after hydraulic stimulation is known to carry high levels of saline and total dissolved solids. To understand the toxicity and potential carcinogenic effects of these waste waters, flow back water from five Marcellus hydraulic fracturing oil and gas wells were analyzed. The physicochemical nature of these samples was analyzed by inductively coupled plasma mass spectrometry and scanning electron microscopy / energy dispersive X-ray spectroscopy. A cytotoxicity study using colony formation as the endpoint was carried out to define the LC50 values of test samples using human bronchial epithelial cells (BEAS-2B). The BEAS-2B cell transformation assay was employed to assess the carcinogenic potential of the samples. Barium and strontium were among the most abundant metals in these samples and the same metals were found elevated in BEAS-2B cells after long-term treatment. BEAS-2B cells treated for 6 weeks with flow back waters produced colony formation in soft agar that was concentration dependant. In addition, flow back water-transformed BEAS-2B cells show a better migration capability when compared to control cells. This study provides information needed to assess the potential health impact of post-hydraulic fracturing flow back waters from Marcellus Shale natural gas mining. PMID:26210350

  1. Stability of mineral fibres in contact with human cell cultures. An in situ μXANES, μXRD and XRF iron mapping study.

    Science.gov (United States)

    Pollastri, Simone; Gualtieri, Alessandro F; Vigliaturo, Ruggero; Ignatyev, Konstantin; Strafella, Elisabetta; Pugnaloni, Armanda; Croce, Alessandro

    2016-12-01

    Relevant mineral fibres of social and economic importance (chrysotile UICC, crocidolite UICC and a fibrous erionite from Jersey, Nevada, USA) were put in contact with cultured diploid human non-tumorigenic bronchial epithelial (Beas2B) and pleural transformed mesothelial (MeT5A) cells to test their cytotoxicity. Slides of each sample at different contact times up to 96 h were studied in situ using synchrotron XRF, μ-XRD and μ-XAS (I18 beamline, Diamond Light Source, UK) and TEM investigations. XRF maps of samples treated for 96 h evidenced that iron is still present within the chrysotile and crocidolite fibres and retained at the surface of the erionite fibres, indicating its null to minor mobilization in contact with cell media; this picture was confirmed by the results of XANES pre-edge analyses. μ-XRD and TEM data indicate greater morphological and crystallinity modifications occurring in chrysotile, whereas crocidolite and erionite show to be resistant in the biological environment. The contact of chrysotile with the cell cultures seems to lead to earlier amorphization, interpreted as the first dissolution step of these fibres. The formation of such silica-rich fibre skeleton may prompt the production of HO in synergy with surface iron species and could indicate that chrysotile may be much more reactive and cytotoxic in vitro in the (very) short term whereas the activity of crocidolite and erionite would be much more sluggish but persistent in the long term.

  2. The IL-5 receptor expression on bronchial epithelial cell line-BEAS-2B%白细胞介素5受体α链在支气管上皮细胞BEAS-2B的表达

    Institute of Scientific and Technical Information of China (English)

    汤葳; Albert Chan; Stanley Chik; Adrain Wu; 邓伟吾

    2007-01-01

    目的 白细胞介素5(IL-5)作为参与嗜酸粒细胞成熟、分化过程的最主要因子,其作用通过特异性受体IL-5Rα介导.本文以支气管上皮细胞株为研究对象,证实该上皮细胞株在炎症因子的刺激下可以表达IL-5Rα.方法 通过促炎症因子TNF-α和TH2型细胞因子IL-4、IL-5、IL-13以及白三烯成分之一的LTCA(白三烯C4)对细胞进行单独或协同刺激,以RT-PCR方法、流式细胞检测技术评价上皮细胞对IL-5Rα的基因和蛋白质的表达,同时评价在IL-5和TNF-α的协同刺激下IL-5Rα、ICAM-1和VCAM-1的协同表达.结果 正常支气管上皮细胞在TH2型细胞因子、LTCA和TNF-α的刺激下可以表达IL-5Rα基因,在TNF-α单独或与其他因子的协同刺激下表达显著增强,更可以表达IL-5Rα蛋白.流式细胞检测分析显示TNF-α单独或协同刺激后16 h IL-5Rα蛋白表达最高,并随时间的延长表达下降.当TNF-α与IL-5协同刺激后上皮细胞可以同时表达VCAM-1和IL-5Rα,而ICAM-1呈持续表达.结论 通过对支气管上皮细胞的研究证实了支气管上皮细胞可以在炎症因子TNF-α和TH2型细胞因子的协同刺激下进一步表达IL-5特异性受体IL-5Rα,并在IL-5和TNF-α的刺激下进一步表达黏附分子VCAM-1,吸引嗜酸粒细胞及其前体细胞浸润至气道参与炎症.说明支气管上皮细胞不仅在哮喘炎症反应中是最主要的受累器官,也是导致炎症的参与者之一.

  3. Flagellin of Pseudomonas aeruginosa induces transforming growth factor beta 1 expression in normal bronchial epithelial cells through mitogen activated protein kinase cascades

    Institute of Scientific and Technical Information of China (English)

    YANG Jing-jing; WANG Dan-dan; SUN Tie-ying

    2011-01-01

    Background Acute lung infection due to Pseudomonas aeruginosa (P. Aeruginosa) is a serious problem, especially in patients with structural lung conditions or immune compromised hosts, leading to an overwhelming threat with a high risk of morbidity and mortality. As an outcome of infection, fibrosis can be linked with chronic lung diseases. But some fibrotic manifestations, such as an irreversible decrease of lung function and fibrous bands seen on chest imaging, have been found after an acute infection with P. Aeruginosa. Fibrogenesis/remodeling resulting from acute lung infection by P.aeruginosa is rarely reported. This study was designed to explore the relation between fibrogenesis/remodeling and acute infection by P. Aeruginosa in vitro. We used flagellin protein from P. Aeruginosa, a key initiator of acute P.aeruginosa lung infection, to elucidate mechanisms by which acute lung infection with P. Aeruginosa can cause fibrogenesis/remodeling.Methods We studied the effect of flagellin from P. Aeruginosa (flagellin for short) on the transforming growth factor beta 1 (TGF-β1) and interleukin-8 (IL-8) expression, and the possible involvement of the signaling pathway, tumor necrosis factor receptor-associated factor 6 (TRAF6)/mitogen activated protein kinase (MAPK) pathway. Flagellin was purified from the P. Aeruginosa standard strain, PAO1. Normal bronchial epithelial cells BEAS-2B were challenged with different concentrations of flagellin, and cell viability assessment was performed by cell counting kit-8. BEAS-2B cells were incubated with flagellin with the specific MAPK inhibitors or TRAF6 siRNA. Cell lysates and the cultured supernatant were collected. The level of TGF-β1 and IL-8 were detected by enzyme-linked immunosorbant assay (ELISA). Western blotting was used to detect the protein levels of MAPK signal proteins p38, c-Jun NH2-terminal kinase (JNK) and extracellular regulated kinase (ERK).Results Expression of TGF-β1 in BEAS-2B cells was elevated by

  4. Multiplexed quantitative high content screening reveals that cigarette smoke condensate induces changes in cell structure and function through alterations in cell signaling pathways in human bronchial cells.

    Science.gov (United States)

    Carter, Charleata A; Hamm, Jonathan T

    2009-07-10

    Human bronchial cells are one of the first cell types exposed to environmental toxins. Toxins often activate nuclear factor-kappaB (NF-kappaB) and protein kinase C (PKC). We evaluated the hypothesis that cigarette smoke condensate (CSC), the particulate fraction of cigarette smoke, activates PKC-alpha and NF-kappaB, and concomitantly disrupts the F-actin cytoskeleton, induces apoptosis and alters cell function in BEAS-2B human bronchial epithelial cells. Compared to controls, exposure of BEAS-2B cells to doses of 30mug/ml CSC significantly activated PKC-alpha, while CSC doses above 20mug/ml CSC significantly activated NF-kappaB. As NF-kappaB was activated, cell number decreased. CSC treatment of BEAS-2B cells induced a decrease in cell size and an increase in cell surface extensions including filopodia and lamellipodia. CSC treatment of BEAS-2B cells induced F-actin rearrangement such that stress fibers were no longer prominent at the cell periphery and throughout the cells, but relocalized to perinuclear regions. Concurrently, CSC induced an increase in the focal adhesion protein vinculin at the cell periphery. CSC doses above 30mug/ml induced a significant increase in apoptosis in BEAS-2B cells evidenced by an increase in activated caspase 3, an increase in mitochondrial mass and a decrease in mitochondrial membrane potential. As caspase 3 increased, cell number decreased. CSC doses above 30mug/ml also induced significant concurrent changes in cell function including decreased cell spreading and motility. CSC initiates a signaling cascade in human bronchial epithelial cells involving PKC-alpha, NF-kappaB and caspase 3, and consequently decreases cell spreading and motility. These CSC-induced alterations in cell structure likely prevent cells from performing their normal function thereby contributing to smoke-induced diseases.

  5. Luteolin inhibits Cr(VI)-induced malignant cell transformation of human lung epithelial cells by targeting ROS mediated multiple cell signaling pathways

    Energy Technology Data Exchange (ETDEWEB)

    Pratheeshkumar, Poyil; Son, Young-Ok; Divya, Sasidharan Padmaja; Roy, Ram Vinod; Hitron, John Andrew; Wang, Lei [Center for Research on Environmental Disease, University of Kentucky, 1095 VA Drive, Lexington, KY 40536 (United States); Graduate Center for Toxicology, University of Kentucky, 1095 VA Drive, Lexington, KY 40536 (United States); Kim, Donghern; Dai, Jin [Graduate Center for Toxicology, University of Kentucky, 1095 VA Drive, Lexington, KY 40536 (United States); Asha, Padmaja [National Centre for Aquatic Animal Health, Cochin University of Science and Technology, Cochin (India); Zhang, Zhuo [Graduate Center for Toxicology, University of Kentucky, 1095 VA Drive, Lexington, KY 40536 (United States); Wang, Yitao [State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macau (China); Shi, Xianglin, E-mail: xshi5@email.uky.edu [Center for Research on Environmental Disease, University of Kentucky, 1095 VA Drive, Lexington, KY 40536 (United States); Graduate Center for Toxicology, University of Kentucky, 1095 VA Drive, Lexington, KY 40536 (United States)

    2014-12-01

    Hexavalent chromium [Cr(VI)] is a well-known human carcinogen associated with the incidence of lung cancer. Inhibition of metal induced carcinogenesis by a dietary antioxidant is a novel approach. Luteolin, a natural dietary flavonoid found in fruits and vegetables, possesses potent antioxidant and anti-inflammatory activity. We found that short term exposure of human bronchial epithelial cells (BEAS-2B) to Cr(VI) (5 μM) showed a drastic increase in ROS generation, NADPH oxidase (NOX) activation, lipid peroxidation, and glutathione depletion, which were significantly inhibited by the treatment with luteolin in a dose dependent manner. Treatment with luteolin decreased AP-1, HIF-1α, COX-2, and iNOS promoter activity induced by Cr(VI) in BEAS-2B cells. In addition, luteolin protected BEAS-2B cells from malignant transformation induced by chronic Cr(VI) exposure. Moreover, luteolin also inhibited the production of pro-inflammatory cytokines (IL-1β, IL-6, IL-8, TNF-α) and VEGF in chronic Cr(VI) exposed BEAS-2B cells. Western blot analysis showed that luteolin inhibited multiple gene products linked to survival (Akt, Fak, Bcl-2, Bcl-xL), inflammation (MAPK, NF-κB, COX-2, STAT-3, iNOS, TNF-α) and angiogenesis (HIF-1α, VEGF, MMP-9) in chronic Cr(VI) exposed BEAS-2B cells. Nude mice injected with BEAS-2B cells chronically exposed to Cr(VI) in the presence of luteolin showed reduced tumor incidence compared to Cr(VI) alone treated group. Overexpression of catalase (CAT) or SOD2, eliminated Cr(VI)-induced malignant transformation. Overall, our results indicate that luteolin protects BEAS-2B cells from Cr(VI)-induced carcinogenesis by scavenging ROS and modulating multiple cell signaling mechanisms that are linked to ROS. Luteolin, therefore, serves as a potential chemopreventive agent against Cr(VI)-induced carcinogenesis. - Highlights: • Luteolin inhibited Cr(VI)-induced oxidative stress. • Luteolin inhibited chronic Cr(VI)-induced malignant transformation.

  6. Particle complexation of mitochondrial iron produces superoxide generation and activates MAP kinases, NF-kappa B, nrf-2 in human respiratory epithelial cell

    Science.gov (United States)

    The biological effect of particles is associated with a disruption in cell iron homeostasis. We tested the postulate that complexation of cell iron by silica (Si02) results in both an oxidative stress and biological effect. BEAS-2B cells were exposed to either media or 100 ug/ml....

  7. Comparative DNA damage and transcriptomic effects of engineered nanoparticles in human lung cells in vitro

    Science.gov (United States)

    A series of six titanium dioxide and two cerium oxide engineered nanomaterials were assessed for their ability to induce cytotoxicity, reactive oxygen species (ROS), various types of DNA damage, and transcriptional changes in human respiratory BEAS-2B cells exposed in vitro at se...

  8. Effect of epithelial cell type on in vitro invasion of non-typeable Haemophilus influenzae.

    Science.gov (United States)

    Singh, Neeraj Kumar; Kunde, Dale A; Tristram, Stephen G

    2016-10-01

    Non-typeable Haemophilus influenzae (NTHi) have been shown to have variable ability for in vitro invasion with a range of epithelial cells, and increased invasion of BEAS-2B cells has been associated with altered penicillin binding protein3 (PBP3), which is concerning as these strains are increasing worldwide. The aim of the study was to investigate the effect of respiratory cell type and the presence of altered PBP3 on the in vitro invasion of NTHi. A collection of 16 clinical NTHi isolates was established, 7 had normal PBP3, and 9 had altered PBP3 as defined by an N526K substitution. The isolates were tested for invasion of BEAS-2B, NHBE, A549 and NCI-H292 respiratory epithelial cells in vitro using a gentamicin survival assay, with invasion measured as the percentage of intracellular organisms relative to the initial inoculum. The overall median invasion for the 16 NTHi isolates for cell types BEAS-2B, NHBE, A549 and NCI-H292 cells were 3.17, 2.31, 0.11 and 1.52 respectively. The differences were statistically significant for BEAS-2B compared to A549 (P=0.015) and A549 compared to NCI-H292 (P=0.015), and there were also very marked differences in invasion for some individual isolates depending on the cell type used. There was a consistent bias for invasion of isolates with normal versus abnormal PBP3: and this was statistically significant for BEAS-2B (0.07 to 9.90, P=0.031) and A549 cells (0.02 to 1.68, P=0.037). These results show that NTHi invasion of respiratory epithelial cells in vitro is both strain dependant and influenced significantly by the cell line used, and that the association between altered PBP3 and increased invasion is conserved across multiple cell lines.

  9. Differences in cytotoxic, genotoxic, and inflammatory response of bronchial and alveolar human lung epithelial cells to pristine and COOH-functionalized multiwalled carbon nanotubes.

    Science.gov (United States)

    Ursini, Cinzia Lucia; Cavallo, Delia; Fresegna, Anna Maria; Ciervo, Aureliano; Maiello, Raffaele; Buresti, Giuliana; Casciardi, Stefano; Bellucci, Stefano; Iavicoli, Sergio

    2014-01-01

    Functionalized MWCNTs are used in many commercial and biomedical applications, but their potential health effects are not well defined. We investigated and compared cytotoxic, genotoxic/oxidative, and inflammatory effects of pristine and carboxyl MWCNTs exposing human respiratory (A549 and BEAS-2B) cells to 1-40 μg/mL of CNTs for 24 h. Both MWCNTs induced low viability reduction (by WST1 assay) in A549 cells and only MWCNTs-COOH caused high viability reduction in BEAS-2B cells reaching 28.5% viability at 40 μg/mL. Both CNTs induced membrane damage (by LDH assay) with higher effects in BEAS-2B cells at the highest concentrations reaching 20% cytotoxicity at 40 μg/mL. DNA damage (by Fpg-comet assay) was induced by pristine MWCNTs in A549 cells and by both MWCNTs in BEAS-2B cells reaching for MWCNTs-COOH a tail moment of 22.2 at 40 μg/mL versus 10.2 of unexposed cells. Increases of IL-6 and IL-8 release (by ELISA) were detected in A549 cells exposed to MWCNTs-COOH from 10 μg/mL while IL-8 increased in BEAS-2B cells exposed to pristine MWCNTs at 20 and 40 μg/mL. The results show higher cytogenotoxicity of MWCNTs-COOH in bronchial and of pristine MWCNTs in alveolar cells. Different inflammatory response was also found. The findings suggest the use of in vitro models with different end points and cells to study CNT toxicity.

  10. Differences in Cytotoxic, Genotoxic, and Inflammatory Response of Bronchial and Alveolar Human Lung Epithelial Cells to Pristine and COOH-Functionalized Multiwalled Carbon Nanotubes

    Science.gov (United States)

    Fresegna, Anna Maria; Ciervo, Aureliano; Buresti, Giuliana

    2014-01-01

    Functionalized MWCNTs are used in many commercial and biomedical applications, but their potential health effects are not well defined. We investigated and compared cytotoxic, genotoxic/oxidative, and inflammatory effects of pristine and carboxyl MWCNTs exposing human respiratory (A549 and BEAS-2B) cells to 1–40 μg/mL of CNTs for 24 h. Both MWCNTs induced low viability reduction (by WST1 assay) in A549 cells and only MWCNTs-COOH caused high viability reduction in BEAS-2B cells reaching 28.5% viability at 40 μg/mL. Both CNTs induced membrane damage (by LDH assay) with higher effects in BEAS-2B cells at the highest concentrations reaching 20% cytotoxicity at 40 μg/mL. DNA damage (by Fpg-comet assay) was induced by pristine MWCNTs in A549 cells and by both MWCNTs in BEAS-2B cells reaching for MWCNTs-COOH a tail moment of 22.2 at 40 μg/mL versus 10.2 of unexposed cells. Increases of IL-6 and IL-8 release (by ELISA) were detected in A549 cells exposed to MWCNTs-COOH from 10 μg/mL while IL-8 increased in BEAS-2B cells exposed to pristine MWCNTs at 20 and 40 μg/mL. The results show higher cytogenotoxicity of MWCNTs-COOH in bronchial and of pristine MWCNTs in alveolar cells. Different inflammatory response was also found. The findings suggest the use of in vitro models with different end points and cells to study CNT toxicity. PMID:25147797

  11. Insect Cell Culture

    NARCIS (Netherlands)

    Oers, van M.M.; Lynn, D.E.

    2010-01-01

    Insect cell cultures are widely used in studies on insect cell physiology, developmental biology and microbial pathology. In particular, insect cell culture is an indispensable tool for the study of insect viruses. The first continuously growing insect cell cultures were established from lepidoptera

  12. Insect Cell Culture

    NARCIS (Netherlands)

    Oers, van M.M.; Lynn, D.E.

    2010-01-01

    Insect cell cultures are widely used in studies on insect cell physiology, developmental biology and microbial pathology. In particular, insect cell culture is an indispensable tool for the study of insect viruses. The first continuously growing insect cell cultures were established from lepidoptera

  13. Phosphodiesterase 4 inhibitors augment the ability of formoterol to enhance glucocorticoid-dependent gene transcription in human airway epithelial cells: a novel mechanism for the clinical efficacy of roflumilast in severe chronic obstructive pulmonary disease.

    Science.gov (United States)

    Moodley, Thunicia; Wilson, Sylvia M; Joshi, Taruna; Rider, Christopher F; Sharma, Pawan; Yan, Dong; Newton, Robert; Giembycz, Mark A

    2013-04-01

    Post-hoc analysis of two phase III clinical studies found that the phosphodiesterase 4 (PDE4) inhibitor, roflumilast, reduced exacerbation frequency in patients with severe chronic obstructive pulmonary disease (COPD) who were taking inhaled corticosteroids (ICS) concomitantly, whereas patients not taking ICS derived no such benefit. In contrast, in two different trials also performed in patients with severe COPD, roflumilast reduced exacerbation rates in the absence of ICS, indicating that PDE4 inhibition alone is sufficient for therapeutic activity to be realized. Given that roflumilast is recommended as an "add-on" medication to patients with severe disease who will inevitably be taking a long-acting β2-adrenoceptor agonist (LABA)/ICS combination therapy, we tested the hypothesis that roflumilast augments the ability of glucocorticoids to induce genes with anti-inflammatory activity. Using a glucocorticoid response element (GRE) luciferase reporter transfected into human airway epithelial cells [both bronchial epithelium + adenovirus 12 - SV40 hybrid (BEAS-2B) cells and primary cultures], roflumilast enhanced fluticasone propionate-induced GRE-dependent transcription. Roflumilast also produced a sinistral displacement of the concentration-response curves that described the augmentation of GRE-dependent transcription by the LABA formoterol. In BEAS-2B cells and primary airway epithelia, roflumilast interacted with formoterol in a positive cooperative manner to enhance the expression of several glucocorticoid-inducible genes that have anti-inflammatory potential. We suggest that the ability of roflumilast and formoterol to interact in this way supports the concept that these drugs together may impart clinical benefit beyond that achievable by an ICS alone, a PDE4 inhibitor alone, or an ICS/LABA combination therapy. Roflumilast may, therefore, be especially effective in patients with severe COPD.

  14. Discovery of novel xylosides in co-culture of basidiomycetes Trametes versicolor and Ganoderma applanatum by integrated metabolomics and bioinformatics

    Science.gov (United States)

    Yao, Lu; Zhu, Li-Ping; Xu, Xiao-Yan; Tan, Ling-Ling; Sadilek, Martin; Fan, Huan; Hu, Bo; Shen, Xiao-Ting; Yang, Jie; Qiao, Bin; Yang, Song

    2016-01-01

    Transcriptomic analysis of cultured fungi suggests that many genes for secondary metabolite synthesis are presumably silent under standard laboratory condition. In order to investigate the expression of silent genes in symbiotic systems, 136 fungi-fungi symbiotic systems were built up by co-culturing seventeen basidiomycetes, among which the co-culture of Trametes versicolor and Ganoderma applanatum demonstrated the strongest coloration of confrontation zones. Metabolomics study of this co-culture discovered that sixty-two features were either newly synthesized or highly produced in the co-culture compared with individual cultures. Molecular network analysis highlighted a subnetwork including two novel xylosides (compounds 2 and 3). Compound 2 was further identified as N-(4-methoxyphenyl)formamide 2-O-β-D-xyloside and was revealed to have the potential to enhance the cell viability of human immortalized bronchial epithelial cell line of Beas-2B. Moreover, bioinformatics and transcriptional analysis of T. versicolor revealed a potential candidate gene (GI: 636605689) encoding xylosyltransferases for xylosylation. Additionally, 3-phenyllactic acid and orsellinic acid were detected for the first time in G. applanatum, which may be ascribed to response against T.versicolor stress. In general, the described co-culture platform provides a powerful tool to discover novel metabolites and help gain insights into the mechanism of silent gene activation in fungal defense. PMID:27616058

  15. Discovery of novel xylosides in co-culture of basidiomycetes Trametes versicolor and Ganoderma applanatum by integrated metabolomics and bioinformatics

    Science.gov (United States)

    Yao, Lu; Zhu, Li-Ping; Xu, Xiao-Yan; Tan, Ling-Ling; Sadilek, Martin; Fan, Huan; Hu, Bo; Shen, Xiao-Ting; Yang, Jie; Qiao, Bin; Yang, Song

    2016-09-01

    Transcriptomic analysis of cultured fungi suggests that many genes for secondary metabolite synthesis are presumably silent under standard laboratory condition. In order to investigate the expression of silent genes in symbiotic systems, 136 fungi-fungi symbiotic systems were built up by co-culturing seventeen basidiomycetes, among which the co-culture of Trametes versicolor and Ganoderma applanatum demonstrated the strongest coloration of confrontation zones. Metabolomics study of this co-culture discovered that sixty-two features were either newly synthesized or highly produced in the co-culture compared with individual cultures. Molecular network analysis highlighted a subnetwork including two novel xylosides (compounds 2 and 3). Compound 2 was further identified as N-(4-methoxyphenyl)formamide 2-O-β-D-xyloside and was revealed to have the potential to enhance the cell viability of human immortalized bronchial epithelial cell line of Beas-2B. Moreover, bioinformatics and transcriptional analysis of T. versicolor revealed a potential candidate gene (GI: 636605689) encoding xylosyltransferases for xylosylation. Additionally, 3-phenyllactic acid and orsellinic acid were detected for the first time in G. applanatum, which may be ascribed to response against T.versicolor stress. In general, the described co-culture platform provides a powerful tool to discover novel metabolites and help gain insights into the mechanism of silent gene activation in fungal defense.

  16. Impairment of cell cycle progression by sterigmatocystin in human pulmonary cells in vitro.

    Science.gov (United States)

    Huang, Shujuan; Wang, Juan; Xing, Lingxiao; Shen, Haitao; Yan, Xia; Wang, Junling; Zhang, Xianghong

    2014-04-01

    Sterigmatocystin (ST) is a carcinogenic mycotoxin that is commonly found in human food, animal feed and in the indoor environment. Although the correlation between ST exposure and lung cancer has been widely reported in many studies, the cytotoxicity of ST on human pulmonary cells is not yet fully understood. In the current study, we found that ST could induce DNA double-strand breaks in a human immortalized bronchial epithelial cell line (BEAS-2B cells) and a human lung cancer cell line (A549 cells). In addition, the effects of ST on cell cycle arrest were complex and dependent on the tested ST concentration and cell type. Low concentrations of ST arrested cells in the G2/M phase in BEAS-2B cells and in the S phase in A549 cells, while at high concentration both cells lines were arrested in S and G2/M phases. Furthermore, we observed that the modulation of cyclins and CDK expression showed concomitant changes with cell cycle arrest upon ST exposure in BEAS-2B and A549 cells. In conclusion, ST induced DNA damage and affected key proteins involved in cell cycle regulation to trigger genomic instability, which may be a potential mechanism underlying the developmental basis of lung carcinogenesis.

  17. Cell Culture Made Easy.

    Science.gov (United States)

    Dye, Frank J.

    1985-01-01

    Outlines steps to generate cell samples for observation and experimentation. The procedures (which use ordinary laboratory equipment) will establish a short-term primary culture of normal mammalian cells. Information on culture vessels and cell division and a list of questions to generate student interest and involvement in the topics are…

  18. Molluscan cells in culture: primary cell cultures and cell lines

    OpenAIRE

    2013-01-01

    In vitro cell culture systems from molluscs have significantly contributed to our basic understanding of complex physiological processes occurring within or between tissue-specific cells, yielding information unattainable using intact animal models. In vitro cultures of neuronal cells from gastropods show how simplified cell models can inform our understanding of complex networks in intact organisms. Primary cell cultures from marine and freshwater bivalve and gastropod species are used as bi...

  19. Subcellular Distribution and Genotoxicity of Silica Nanoparticles 
in Human Bronchial Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Guangqiang ZHAO

    2013-03-01

    Full Text Available Background and objective Silicon nanoparticles are widely used in daily life. Therefore, they attract increased attention because of their potential biotoxicity to the lungs when inhaled. The aims of this study are to explore the organism distribution and genotoxicity of silica nanoparticles in human bronchial epithelial cells (BEAS-2B. Methods The biodistribution of silica with different particle sizes in human bronchial epithelial cells was observed by transmission electron microscopy (TEM. DNA damage was detected by single-cell gel electrophoresis (comet assay. Results TEM revealed that SiO2 nanoparticles with different sizes can be uptaken by cells and be localized in the cytoplasm and the nucleus. Compared with micro-silica, nano-silica in BEAS-2B cells can inflict more severe DNA damage (P<0.05. Conclusion The particle size of silica nanoparticles can be used to determine their distribution in biological cells. Compared with micro-silica, nano-silica has higher genotoxicity.

  20. Gene expression analysis uncovers novel hedgehog interacting protein (HHIP) effects in human bronchial epithelial cells.

    Science.gov (United States)

    Zhou, Xiaobo; Qiu, Weiliang; Sathirapongsasuti, J Fah; Cho, Michael H; Mancini, John D; Lao, Taotao; Thibault, Derek M; Litonjua, Augusto A; Bakke, Per S; Gulsvik, Amund; Lomas, David A; Beaty, Terri H; Hersh, Craig P; Anderson, Christopher; Geigenmuller, Ute; Raby, Benjamin A; Rennard, Stephen I; Perrella, Mark A; Choi, Augustine M K; Quackenbush, John; Silverman, Edwin K

    2013-05-01

    Hedgehog interacting protein (HHIP) was implicated in chronic obstructive pulmonary disease (COPD) by genome-wide association studies (GWAS). However, it remains unclear how HHIP contributes to COPD pathogenesis. To identify genes regulated by HHIP, we performed gene expression microarray analysis in a human bronchial epithelial cell line (Beas-2B) stably infected with HHIP shRNAs. HHIP silencing led to differential expression of 296 genes; enrichment for variants nominally associated with COPD was found. Eighteen of the differentially expressed genes were validated by real-time PCR in Beas-2B cells. Seven of 11 validated genes tested in human COPD and control lung tissues demonstrated significant gene expression differences. Functional annotation indicated enrichment for extracellular matrix and cell growth genes. Network modeling demonstrated that the extracellular matrix and cell proliferation genes influenced by HHIP tended to be interconnected. Thus, we identified potential HHIP targets in human bronchial epithelial cells that may contribute to COPD pathogenesis.

  1. Mammalian Cell Culture Simplified.

    Science.gov (United States)

    Moss, Robert; Solomon, Sondra

    1991-01-01

    A tissue culture experiment that does not require elaborate equipment and that can be used to teach sterile technique, the principles of animal cell line maintenance, and the concept of cell growth curves is described. The differences between cancerous and normal cells can be highlighted. The procedure is included. (KR)

  2. Fish stem cell cultures.

    Science.gov (United States)

    Hong, Ni; Li, Zhendong; Hong, Yunhan

    2011-04-13

    Stem cells have the potential for self-renewal and differentiation. First stem cell cultures were derived 30 years ago from early developing mouse embryos. These are pluripotent embryonic stem (ES) cells. Efforts towards ES cell derivation have been attempted in other mammalian and non-mammalian species. Work with stem cell culture in fish started 20 years ago. Laboratory fish species, in particular zebrafish and medaka, have been the focus of research towards stem cell cultures. Medaka is the second organism that generated ES cells and the first that gave rise to a spermatogonial stem cell line capable of test-tube sperm production. Most recently, the first haploid stem cells capable of producing whole animals have also been generated from medaka. ES-like cells have been reported also in zebrafish and several marine species. Attempts for germline transmission of ES cell cultures and gene targeting have been reported in zebrafish. Recent years have witnessed the progress in markers and procedures for ES cell characterization. These include the identification of fish homologs/paralogs of mammalian pluripotency genes and parameters for optimal chimera formation. In addition, fish germ cell cultures and transplantation have attracted considerable interest for germline transmission and surrogate production. Haploid ES cell nuclear transfer has proven in medaka the feasibility of semi-cloning as a novel assisted reproductive technology. In this special issue on "Fish Stem Cells and Nuclear Transfer", we will focus our review on medaka to illustrate the current status and perspective of fish stem cells in research and application. We will also mention semi-cloning as a new development to conventional nuclear transfer.

  3. Fish Stem Cell Cultures

    Directory of Open Access Journals (Sweden)

    Ni Hong, Zhendong Li, Yunhan Hong

    2011-01-01

    Full Text Available Stem cells have the potential for self-renewal and differentiation. First stem cell cultures were derived 30 years ago from early developing mouse embryos. These are pluripotent embryonic stem (ES cells. Efforts towards ES cell derivation have been attempted in other mammalian and non-mammalian species. Work with stem cell culture in fish started 20 years ago. Laboratory fish species, in particular zebrafish and medaka, have been the focus of research towards stem cell cultures. Medaka is the second organism that generated ES cells and the first that gave rise to a spermatogonial stem cell line capable of test-tube sperm production. Most recently, the first haploid stem cells capable of producing whole animals have also been generated from medaka. ES-like cells have been reported also in zebrafish and several marine species. Attempts for germline transmission of ES cell cultures and gene targeting have been reported in zebrafish. Recent years have witnessed the progress in markers and procedures for ES cell characterization. These include the identification of fish homologs/paralogs of mammalian pluripotency genes and parameters for optimal chimera formation. In addition, fish germ cell cultures and transplantation have attracted considerable interest for germline transmission and surrogate production. Haploid ES cell nuclear transfer has proven in medaka the feasibility of semi-cloning as a novel assisted reproductive technology. In this special issue on “Fish Stem Cells and Nuclear Transfer”, we will focus our review on medaka to illustrate the current status and perspective of fish stem cells in research and application. We will also mention semi-cloning as a new development to conventional nuclear transfer.

  4. [Grape seed proanthocyanidins inhibits the invasion and migration of A549 lung cancer cells].

    Science.gov (United States)

    Zhou, Yehan; Ye, Xiufeng; Shi, Yao; Wang, Ke; Wan, Dan

    2016-02-01

    To explore the effect of grape seed proanthocyanidins (GSPs) on the invasion and migration of A549 lung cancer cells and the underlying mechanism. Trypan blue dye exclusion assay was used to determine the cytotoxic effect of varying doses of GSPs on the BEAS-2B normal human pulmonary epithelial cells. After treated with 0, 10, 20, 40, 80 μg/mL GSP, the proliferation of A549 cells was detected by MTT assay; the invasion and migration of A549 cells were determined by Transwell(TM) assay and scratch wound assay, respectively. The levels of epithelial growth factor receptor (EGFR), E-cadherin, N-cadherin in A549 cells treated with GSPs were detected by Western blotting. (0-40) μg/mL GSPs had no significant toxic effect on BEAS-2B cells, while 80 μg/mL GSPs had significant cytotoxicity to BEAS-2B cells. The proliferation of A549 cells was significantly inhibited within limited dosage in a dose-dependent manner, and the abilities of invasion and migration of A549 cells were also inhibited. Western blotting showed that the expression of EGFR and N-cadherin decreased, while E-cadherin increased after GSPs treatment. GSPs could inhibit the abilities of proliferation, invasion and migration of A549 cells, which might be related to the dow-regulation of EGFR and N-cadherin and the up-regulation of E-cadherin.

  5. Highly purified, multi-wall carbon nanotubes induce light-chain 3B expression in human lung cells

    Energy Technology Data Exchange (ETDEWEB)

    Tsukahara, Tamotsu, E-mail: ttamotsu@kanazawa-med.ac.jp [Department of Hematology and Immunology, Kanazawa Medical University, 1-1 Daigaku, Uchinada, Ishikawa 920-0293 (Japan); Matsuda, Yoshikazu [Clinical Pharmacology Educational Center, Nihon Pharmaceutical University, Ina-machi, Saitama 362-0806 (Japan); Usui, Yuki [Research Center for Exotic Nanocarbons, Shinshu University, 4-17-1 Wakasato, Nagano-shi, Nagano 380-8553 (Japan); Haniu, Hisao [Department of Orthopaedic Surgery, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621 (Japan)

    2013-10-18

    Highlights: •HTT2800-treated BEAS-2B cells induced LC3B in a time-dependent manner. •HTT2800-treated BEAS-2B cells showed decreased cell proliferation that was both time- and dose-dependent. •Addition of 3-MA, LC3B-II protein and mRNA levels were significantly decreased. •3-MA and E64-d + pepstatin A, but not brefeldin A, provided protection against HTT2800-induced cell death. •These results suggest that HTT2800 predominantly causes autophagy rather than apoptotic cell death in BEAS-2B cells. -- Abstract: Bronchial epithelial cells are targets of inhalation and play a critical role in the maintenance of mucosal integrity as mechanical barriers against various particles. Our previous result suggest that vapor-grown carbon fiber, HTT2800, which is one of the most highly purified multi-wall carbon nanotubes (MWCNT) showed cellular uptake of the carbon nanotube, increased cell death, enhanced DNA damage, and induced cytokine release. Increasing evidence suggests that autophagy may critically influence vital cellular processes such as apoptosis, cell proliferation and inflammation and thereby may play a critical role in pulmonary diseases. Autophagy was recently recognized as a critical cell death pathway, and autophagosome accumulation has been found to be associated with the exposure of various nanoparticles. In this study, the authors focus on the autophagic responses of HTT2800 exposure. The HTT2800-exposed cells induced LC3B expression and induced cell growth inhibition.

  6. Phosphorylation of p65 Is Required for Zinc Oxide Nanoparticle–Induced Interleukin 8 Expression in Human Bronchial Epithelial Cells

    OpenAIRE

    Wu, Weidong; Samet, James M.; Peden, David B.; Bromberg, Philip A.

    2010-01-01

    Background Exposure to zinc oxide (ZnO) in environmental and occupational settings causes acute pulmonary responses through the induction of proinflammatory mediators such as interleukin-8 (IL-8). Objective We investigated the effect of ZnO nanoparticles on IL-8 expression and the underlying mechanisms in human bronchial epithelial cells. Methods We determined IL-8 mRNA and protein expression in primary human bronchial epithelial cells and the BEAS-2B human bronchial epithelial cell line usin...

  7. 单核巨噬细胞在煤焦沥青烟提取物诱导永生化人支气管上皮细胞恶变中的作用%The Effects of monocyte-macrophages on malignant transformation of human bronchial epithelial cells induced by extracts from coal tar pitch

    Institute of Scientific and Technical Information of China (English)

    周凡静; 张少峰; 冯斐斐; 燕贞; 王威; 吴逸明

    2012-01-01

    目的 探讨单核巨噬细胞(THP-1)在煤焦沥青烟提取物(coal tar pitch,CTP)致永生化人支气管上皮细胞(human bronchial epithelial cells,BEAS-2B)恶变过程中的作用及肿瘤坏死因子α(tumor necrosis factor alpha,TNF-α)在该过程中的表达.方法 以THP-1和BEAS-2B为研究对象,设立CTP组、苯并(a)芘[B(a)P]组(阳性对照组)、二甲亚砜对照组(溶剂对照组)、BEAS-2B与THP-1共培养组,建立细胞恶性变模型.应用软琼脂集落形成实验、染色体数目畸变分析、流式细胞仪测定细胞周期中不同培养时期(10、20、30代)细胞的恶变情况,利用ELISA方法测定CTP组及共培养组细胞培养上清中TNF-α的含量.结果 染色体数目异常在实验的早期(10代)已经显现,表现为非整倍体和多倍体比例增加,二倍体数目减少.在第20代:共培养组克隆形成率(17.63‰±0.97‰)明显高于CTP组(13.94‰±0.84‰)和阳性对照组组(12.96‰± 1.62‰),差异有统计学意义(P<0.05);共培养组S期细胞比例(44.49%±0.68%)明显高于CTP组(38.19%±1.26%)和阳性对照组(36.41%±1.19%),差异有统计学意义(P<0.05).共培养组细胞培养上清中TNF-α含量明显高于CTP组,差异有统计学意义(P<0.01).结论 THP-1能够加速CTP诱导的BEAS-2B恶变,增加TNF-α的表达水平.%Objective To study the effects of monocyte-macrophages (THP-1) in malignant transformation of human bronchial epithelial cells (BEAS-2B) cells induced by coal tar pitch (CTP) and the expression of TNF-α in the process of the cell malignant transformation.Methods BEAS-2B cells and THP-1 Cells were divided into four groups:coal tar pitch (CTP) group,benzo(a)pyrene [B(a)P] group,dimethyl sulfoxide (DMSO) group,BEAS-2B and THP-1 co-culture (co-culture group) group.Carcinogenesis model was established.The soft agar colony formation,chromosome aberrations and cell cycle tests were used to detect the cellular malignant transformation.The ELISA

  8. Perfusion based cell culture chips

    DEFF Research Database (Denmark)

    Heiskanen, Arto; Emnéus, Jenny; Dufva, Martin

    2010-01-01

    Performing cell culture in miniaturized perfusion chambers gives possibilities to experiment with cells under near in vivo like conditions. In contrast to traditional batch cultures, miniaturized perfusion systems provide precise control of medium composition, long term unattended cultures and ti...

  9. Dectin-1-Mediated Pathway Contributes to Fusarium proliferatum-Induced CXCL-8 Release from Human Respiratory Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Chang-Ching Yeh

    2017-03-01

    Full Text Available Fusarium species are causative agents of human respiratory disorders and are distributed widely in our environment. Little is known of their interaction with human respiratory epithelial cells, which may contribute to allergic airway responses. In this study, we report on the release of C–X–C motif chemokine ligand 8 (CXCL-8 from human bronchial epithelial BEAS-2B cells upon stimulation with Fusarium proliferatum extracts. F. proliferatum-induced cytokine release from BEAS-2B cells was determined by cytokine array and CXCL-8 enzyme-linked immunosorbent assay (ELISA kits. Blocking antibodies and signaling pathway inhibitors were employed to delineate cell surface receptors and signaling pathways participating in CXCL-8 release. F. proliferatum extracts induced the release of CXCL-8 in a time-dependent manner. The dectin-1 receptor ligands, curdlan and laminarin, reduced CXCL-8 release. Cells pre-treated with anti-Dectin-1 antibodies (2 µg/mL decreased CXCL-8 release by 24%. Furthermore, F. proliferatum-stimulated CXCL-8 release was reduced by 32%, 53%–81%, 40% and 26% after BEAS-2B cells were pretreated with activation inhibitors of spleen tyrosine kinase (Syk—piceatannol—, mitogen-activated protein kinases (MAPKs—PD98059, U0126, SB202190, SP600125—, phosphatidylinositol-3-kinase (PI3K—LY294002—and nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB—BAY117082—, respectively. These results suggest that Dectin-1-mediated activation of the Syk, MAPKs, PI3K and NF-κB signaling pathways contributes to F. proliferatum-stimulated CXCL-8 release from BEAS-2B cells and provides an important basis for developing novel therapeutic strategies in clinical allergy.

  10. Molluscan cells in culture: primary cell cultures and cell lines.

    Science.gov (United States)

    Yoshino, T P; Bickham, U; Bayne, C J

    2013-06-01

    In vitro cell culture systems from molluscs have significantly contributed to our basic understanding of complex physiological processes occurring within or between tissue-specific cells, yielding information unattainable using intact animal models. In vitro cultures of neuronal cells from gastropods show how simplified cell models can inform our understanding of complex networks in intact organisms. Primary cell cultures from marine and freshwater bivalve and gastropod species are used as biomonitors for environmental contaminants, as models for gene transfer technologies, and for studies of innate immunity and neoplastic disease. Despite efforts to isolate proliferative cell lines from molluscs, the snail Biomphalaria glabrata Say, 1818 embryonic (Bge) cell line is the only existing cell line originating from any molluscan species. Taking an organ systems approach, this review summarizes efforts to establish molluscan cell cultures and describes the varied applications of primary cell cultures in research. Because of the unique status of the Bge cell line, an account is presented of the establishment of this cell line, and of how these cells have contributed to our understanding of snail host-parasite interactions. Finally, we detail the difficulties commonly encountered in efforts to establish cell lines from molluscs and discuss how these difficulties might be overcome.

  11. Cell Culturing of Cytoskeleton

    Science.gov (United States)

    2004-01-01

    Biomedical research offers hope for a variety of medical problems, from diabetes to the replacement of damaged bone and tissues. Bioreactors, which are used to grow cells and tissue cultures, play a major role in such research and production efforts. Cell culturing, such as this bone cell culture, is an important part of biomedical research. The BioDyn payload includes a tissue engineering investigation. The commercial affiliate, Millenium Biologix, Inc., has been conducting bone implant experiments to better understand how synthetic bone can be used to treat bone-related illnesses and bone damaged in accidents. On STS-95, the BioDyn payload will include a bone cell culture aimed to help develop this commercial synthetic bone product. Millenium Biologix, Inc., is exploring the potential for making human bone implantable materials by seeding its proprietary artificial scaffold material with human bone cells. The product of this tissue engineering experiment using the Bioprocessing Modules (BPMs) on STS-95 is space-grown bone implants, which could have potential for dental implants, long bone grafts, and coating for orthopedic implants such as hip replacements.

  12. Oscillating Cell Culture Bioreactor

    Science.gov (United States)

    Freed, Lisa E.; Cheng, Mingyu; Moretti, Matteo G.

    2010-01-01

    To better exploit the principles of gas transport and mass transport during the processes of cell seeding of 3D scaffolds and in vitro culture of 3D tissue engineered constructs, the oscillatory cell culture bioreactor provides a flow of cell suspensions and culture media directly through a porous 3D scaffold (during cell seeding) and a 3D construct (during subsequent cultivation) within a highly gas-permeable closed-loop tube. This design is simple, modular, and flexible, and its component parts are easy to assemble and operate, and are inexpensive. Chamber volume can be very low, but can be easily scaled up. This innovation is well suited to work with different biological specimens, particularly with cells having high oxygen requirements and/or shear sensitivity, and different scaffold structures and dimensions. The closed-loop changer is highly gas permeable to allow efficient gas exchange during the cell seeding/culturing process. A porous scaffold, which may be seeded with cells, is fixed by means of a scaffold holder to the chamber wall with scaffold/construct orientation with respect to the chamber determined by the geometry of the scaffold holder. A fluid, with/without biological specimens, is added to the chamber such that all, or most, of the air is displaced (i.e., with or without an enclosed air bubble). Motion is applied to the chamber within a controlled environment (e.g., oscillatory motion within a humidified 37 C incubator). Movement of the chamber induces relative motion of the scaffold/construct with respect to the fluid. In case the fluid is a cell suspension, cells will come into contact with the scaffold and eventually adhere to it. Alternatively, cells can be seeded on scaffolds by gel entrapment prior to bioreactor cultivation. Subsequently, the oscillatory cell culture bioreactor will provide efficient gas exchange (i.e., of oxygen and carbon dioxide, as required for viability of metabolically active cells) and controlled levels of fluid

  13. House dust mite allergen Der f enhanced bronchial epithelial cell cytokine expression

    Institute of Scientific and Technical Information of China (English)

    BAO QING SUN; WEI TANG; ALBERT CHAN; ADRIAN WU; NAN SHAN ZHONG

    2006-01-01

    The house dust mites ( Dermatophagoides farinae, Der f) are the major source of aeroallergens implicated in the expression of atopic disorders, including asthma, allergic rhinitis and atopic dermatitis. In particular, strong circumstantial evidence suggests that house dust mite antigens are important precipitating factors of asthma. Many house dust mite allergens are proteases that can elicit airway inflammation by stimulating the release of cytokines from bronchial epithelial cells. To investigate whether Derf allergen proteases induced cytokine production from the epithelial cell line BEAS-2B,BEAS-2B cells were cultured with 4 different concentrations of Derf (0.02, 0.2, 2, 20 μg/ml) for 24-96 h, after which supernatants were assayed for interleukin (IL)-6 and IL-8 with ELISA. Reverse transcription-PCR was also performed. The cell sheets were intact throughout the observation in control group without any exposure to Derf antigen. In the experimental groups cells treated with Der f allergen showed changes in the anchorage status of the monolayer. There was a significant increase in the level of cytokine production compared with the untreated sample. The release of IL-6 and IL-8 increased in a concentration-dependent manner ( P < 0.05, respectively) with the addition of increasing dosage of Der f to the cell sheets. Levels of IL-6 and IL-8 began to rise at 24 h and 48 h after allergen exposure, and they increased significantly in the supernatants at 72 h and 96 h. At the same time the concentration dependence of induction of IL-6 and IL-8 expression as well as an increase in the expression of IL-6 and IL-8 mRNA manifested evidently. HDM-induced airway inflammation may include Der f-mediated release of inflammatory mediators, and the proteolytic activity of an allergen may stimulate the release of proinflammatory cytokines from human bronchial epithelium. It is suggested that IL-6 and IL-8 production by bronchial epithelial cells may play a role in the

  14. Blockade of Airway Inflammation by Kaempferol via Disturbing Tyk-STAT Signaling in Airway Epithelial Cells and in Asthmatic Mice

    OpenAIRE

    Ju-Hyun Gong; Daekeun Shin; Seon-Young Han; Sin-Hye Park; Min-Kyung Kang; Jung-Lye Kim; Young-Hee Kang

    2013-01-01

    Asthma is characterized by bronchial inflammation causing increased airway hyperresponsiveness and eosinophilia. The interaction between airway epithelium and inflammatory mediators plays a key role in the asthmatic pathogenesis. The in vitro study elucidated inhibitory effects of kaempferol, a flavonoid found in apples and many berries, on inflammation in human airway epithelial BEAS-2B cells. Nontoxic kaempferol at ≤20  μ M suppressed the LPS-induced IL-8 production through the TLR4 activat...

  15. Perfusion Based Cell Culture Chips

    Science.gov (United States)

    Heiskanen, A.; Emnéus, J.; Dufva, M.

    Performing cell culture in miniaturized perfusion chambers gives possibilities to experiment with cells under near in vivo like conditions. In contrast to traditional batch cultures, miniaturized perfusion systems provide precise control of medium composition, long term unattended cultures and tissue like structuring of the cultures. However, as this chapter illustrates, many issues remain to be identified regarding perfusion cell culture such as design, material choice and how to use these systems before they will be widespread amongst biomedical researchers.

  16. Perfusion based cell culture chips

    DEFF Research Database (Denmark)

    Heiskanen, Arto; Emnéus, Jenny; Dufva, Martin

    2010-01-01

    and tissue like structuring of the cultures. However, as this chapter illustrates, many issues remain to be identified regarding perfusion cell culture such as design, material choice and how to use these systems before they will be widespread amongst biomedical researchers.......Performing cell culture in miniaturized perfusion chambers gives possibilities to experiment with cells under near in vivo like conditions. In contrast to traditional batch cultures, miniaturized perfusion systems provide precise control of medium composition, long term unattended cultures...

  17. Analysis of coal tar pitch and smoke extract components and their cytotoxicity on human bronchial epithelial cells.

    Science.gov (United States)

    Li, Zhitao; Wu, Yongjun; Zhao, Yong; Wang, Lixia; Zhu, Hansong; Qin, Lijuan; Feng, Feifei; Wang, Wei; Wu, Yiming

    2011-02-28

    Coal tar pitch and its smoke are considered hazardous by-products and common pollutant generated from coal industry processing. In this study, coal tar pitch and its smoke extracts were characterized by gas chromatography/mass spectrometry (GC/MS) with dimethylsulfoxide. We identified only 0.3025% of components in the total coal tar pitch using GC/MS. Among 18 identified compounds, polycyclic aromatic hydrocarbons (PAHs) has the highest relative abundance (0.19%). The remaining components were composed of monocyclic aromatic hydrocarbons, heterocyclic compounds and alkenes. In contrast, among 38 coal tar pitch smoke extract constituents that have been profiled, 87.91% were PAHs, and the remaining 12.09% were composed of monocyclic aromatic hydrocarbons, heterocyclic compounds and alkenes. The cytotoxic effect of coal tar pitch and its smoke extracts on BEAS-2B cells were also evaluated by MTT assay. BEAS-2B cells exposed to coal tar pitch showed a non dose-dependent U-shaped cytotoxicity with a dosage for maximal inhibitory of 3.75 mg/L. In contrast, BEAS-2B cells exposed to coal tar pitch smoke extracts showed a dose dependent cytotoxicity with a LC(50) of 8.64 mg/L. Our study demonstrated the significant different composition and cytotoxicity of coal tar pitch and its extracts, suggesting two different underlying mechanisms that are pending future investigation.

  18. Microfluidic Cell Culture Device

    Science.gov (United States)

    Takayama, Shuichi (Inventor); Cabrera, Lourdes Marcella (Inventor); Heo, Yun Seok (Inventor); Smith, Gary Daniel (Inventor)

    2014-01-01

    Microfluidic devices for cell culturing and methods for using the same are disclosed. One device includes a substrate and membrane. The substrate includes a reservoir in fluid communication with a passage. A bio-compatible fluid may be added to the reservoir and passage. The reservoir is configured to receive and retain at least a portion of a cell mass. The membrane acts as a barrier to evaporation of the bio-compatible fluid from the passage. A cover fluid may be added to cover the bio-compatible fluid to prevent evaporation of the bio-compatible fluid.

  19. The response of a human bronchial epithelial cell line to histamine: Intracellular calcium changes and extracellular release of inflammatory mediators

    Energy Technology Data Exchange (ETDEWEB)

    Noah, T.L.; Paradiso, A.M.; Madden, M.C.; McKinnon, K.P.; Devlin, R.B. (Department of Pediatrics, University of North Carolina School of Medicine, Chapel Hill (United States))

    1991-11-01

    Epithelial cells are likely to modulate inflammation and tissue repair in the airways, but the factors responsible for these processes remain unclear. Because human airway epithelia are infrequently available for in vitro studies, transformed epithelial cell lines are of interest as models. The authors therefore investigated the response of an SV-40/adenovirus-transformed human bronchial epithelial cell line (BEAS-2B) to histamine, a mediator with relevance for airway diseases. The intracellular calcium response to histamine (10(-4) M) was measured, using Fura-2 and microspectrofluorimetry. Histamine induced a transient increase in intracellular calcium that originated from intracellular sources; this effect was inhibited by the H1 receptor antagonist diphenhydramine, suggesting that BEAS cells retain functioning histamine receptors. BEAS cells were grown to confluence on microporous, collagen-coated filters, allowing measurement of vectorial release of soluble mediators. Monolayers exposed to histamine for 30 min released interleukin-6 and fibronectin in the apical direction, in a dose-dependent manner. Little eicosanoid production was induced by histamine, either in the apical or the basolateral direction, although BEAS cells constitutively produced small amounts of prostaglandin E2 and 15-HETE. However, these cells formed large amounts of eicosanoids in response to ozone exposure as a positive control. Comparison of their data with published reports for human airway epithelia in primary culture suggests that the BEAS cell line is, in a number of respects, a relevant model for the study of airway epithelial responses to a variety of stimuli.

  20. Rhinovirus infection induces cytotoxicity and delays wound healing in bronchial epithelial cells

    Directory of Open Access Journals (Sweden)

    Constantopoulos Andreas G

    2005-10-01

    Full Text Available Abstract Background Human rhinoviruses (RV, the most common triggers of acute asthma exacerbations, are considered not cytotoxic to the bronchial epithelium. Recent observations, however, have questioned this knowledge. The aim of this study was to evaluate the ability of RV to induce epithelial cytotoxicity and affect epithelial repair in-vitro. Methods Monolayers of BEAS-2B bronchial epithelial cells, seeded at different densities were exposed to RV serotypes 1b, 5, 7, 9, 14, 16. Cytotoxicity was assessed chromatometrically. Epithelial monolayers were mechanically wounded, exposed or not to RV and the repopulation of the damaged area was assessed by image analysis. Finally epithelial cell proliferation was assessed by quantitation of proliferating cell nuclear antigen (PCNA by flow cytometry. Results RV1b, RV5, RV7, RV14 and RV16 were able to induce considerable epithelial cytotoxicity, more pronounced in less dense cultures, in a cell-density and dose-dependent manner. RV9 was not cytotoxic. Furthermore, RV infection diminished the self-repair capacity of bronchial epithelial cells and reduced cell proliferation. Conclusion RV-induced epithelial cytotoxicity may become considerable in already compromised epithelium, such as in the case of asthma. The RV-induced impairment on epithelial proliferation and self-repair capacity may contribute to the development of airway remodeling.

  1. Nitric oxide- and cisplatin-releasing silica nanoparticles for use against non-small cell lung cancer.

    Science.gov (United States)

    Munaweera, Imalka; Shi, Yi; Koneru, Bhuvaneswari; Patel, Amit; Dang, Mai H; Di Pasqua, Anthony J; Balkus, Kenneth J

    2015-12-01

    Nitric oxide (NO) and cisplatin releasing wrinkle-structured amine-modified mesoporous silica (AMS) nanoparticles have been developed for the treatment of non-small cell lung cancer (NSCLC). The AMS and NO- and cisplatin-loaded AMS materials were characterized using TEM, BET surface area, FTIR and ICP-MS, and tested in cell culture. The results show that for NSCLC cell lines (i.e., H596 and A549), the toxicity of NO- and cisplatin-loaded silica nanoparticles (NO-Si-DETA-cisplatin-AMS) is significantly higher than that of silica nanoparticles loaded with only cisplatin (Si-DETA-cisplatin-AMS). In contrast, the toxicity of NO-Si-DETA-cisplatin-AMS toward normal lung cell lines is not significantly different from that of Si-DETA-cisplatin-AMS (normal lung fibroblast cells WI-38) or is even lower than that of Si-DETA-cisplatin-AMS (normal lung epithelial cells BEAS-2B). The NO-induced sensitization of tumor cell death demonstrates that NO is a promising enhancer of platinum-based lung cancer therapy.

  2. Inhibition of Cigarettes Smoke-induced Epithelial to Mesenchymal Transition by the SMO Inhibitor PF-5274857 in Beas-2b Epithelial Cells%PF-5274857阻断香烟烟雾诱导的上皮-间质转化的作用

    Institute of Scientific and Technical Information of China (English)

    周闻捷; 陈娇; 冯云; 樊亚平; 李倩; 傅健; 张平

    2016-01-01

    目的 探讨Smoothened (Smo)抑制剂PF-5274857在香烟烟雾(CS)诱导所致的上皮-间质转化(EMT)转变中的作用,为口腔鳞状细胞癌的临床治疗和预防提供依据.方法 以支气管上皮细胞株Beas-2b建立体外CS诱导EMT模型,实验分为预防实验和治疗实验两部分进行.预防实验:用3μtmol/L PF-5274857预刺激Beas-2b细胞2h后用CS溶液培养8d;治疗实验:CS培养8d后用3μmol/L PF-52748573处理Beas-2b细胞4d.通过Western blot、免疫荧光检测细胞蛋白中EMT标志物E-钙黏蛋白(E-cadherin)、波形蛋白(vimentin)、a平滑肌肌动蛋白(α-SMA)的含量及位置变化,小室迁移实验测定治疗实验中细胞迁移能力改变.结果 PF-5274857预刺激2h,间质标志物vimentin和α-SMA蛋白表达降低,上皮标志物E-cadherin表达升高.而已被CS诱导产生EMT改变的Beas-2b细胞经PF-5274857治疗4d后部分恢复E-cadherin表达,并且vimentin和α-SMA蛋白表达降低,其升高的迁移能力也降低.结论 PF-5274857可以预防和治疗由CS引起的Beas-2b细胞EMT.

  3. Evaluation of uptake, cytotoxicity and inflammatory effects in respiratory cells exposed to pristine and -OH and -COOH functionalized multi-wall carbon nanotubes.

    Science.gov (United States)

    Ursini, Cinzia Lucia; Maiello, Raffaele; Ciervo, Aureliano; Fresegna, Anna Maria; Buresti, Giuliana; Superti, Fabiana; Marchetti, Magda; Iavicoli, Sergio; Cavallo, Delia

    2016-03-01

    Toxic effects were reported for pristine-multi-wall carbon nanotubes (p-MWCNTs) while the role of the functionalization on MWCNT-induced toxicity is not yet well defined. We evaluated on human alveolar (A549) epithelial cells and normal bronchial (BEAS-2B) cells exposed to p-MWCNTs, MWCNTs-OH and MWCNTs-COOH: uptake by TEM, cell viability by different assays, membrane damage by the LDH assay and cytokine release by ELISA. The aims of the present study were to: (i) confirm MWCNT cytotoxicity mechanisms hypothesized in our previous studies; (ii) identify the most reliable viability assay to screen MWCNT toxicity; and (iii) to test our model to clarify the role of functionalization on MWCNT-induced toxicity. In A549 cells, p-MWCNTs and MWCNTs-OH were localized free in the cytoplasm and inside vacuoles whereas MWCNTs-COOH were confined inside filled cytoplasmic vesicles. WST-1 and Trypan blue assays showed in A549 cells a similar slight viability reduction for all MWCNTs whereas in BEAS-2B cells WST1 showed a high viability reduction at the highest concentrations, particularly for MWCNTs-COOH. The MTT assay showed a false cytotoxicity as a result of MWCNTs-interference. Pristine and MWCNTs-COOH induced membrane damage, particularly in BEAS-2B cells. MWCNTs-COOH induced interleukin-6 (IL-6) and IL-8 release in A549 cells whereas p-MWCNTs induced IL-8 release in BEAS-2B cells. MWCNTs intracellular localization in A549 cells confirms the toxicity mechanisms previously hypothesized, with p-MWCNTs disrupting the membrane and vesicle-confined MWCNTs-COOH inducing inflammation. WST-1 was more reliable than MTT to test MWCNT-toxicity. BEAS-2B cells were more susceptible then A549 cells, particularly to MWCNT-COOH cytotoxicity. Our results confirm the toxicity of p-MWCNTs and demonstrate, also for the two kinds of tested functionalized MWCNTs toxic effects with a different mechanism of action.

  4. Early detection of ozone-induced hydroperoxides in epithelial cells by a novel infrared spectroscopic method.

    Science.gov (United States)

    Hemmingsen, A; Allen, J T; Zhang, S; Mortensen, J; Spiteri, M A

    1999-11-01

    In the lower atmosphere ozone is a toxic and an unwanted oxidising pollutant causing injury to the airway epithelial cells by lipid peroxidation to yield products such as phospholipid hydroperoxides (PLHP). Measurements of PLHP, which are primary oxidation products, may reflect an early susceptibility of the target cell to oxidative stress. Biphasic cultures of bronchial epithelial cells (BEAS-2B) were exposed to ozone at environmentally relevant concentrations (0.1-1.0 ppm) for 4 and 12 h. Detection of PLHP was made using a novel technique based on fourier transform infrared spectroscopy (FTIR) in combination with high performance thin-layer chromatography (HPTLC). Six phospholipids were identified on the HPTLC plate; lysophosphatidylcholine (LPC), sphingomyelin (SM), phosphatidylcholine (PC), lysophosphatidylethanolamine (LPE), phosphatidylinositol (PI), and phosphatidylethanolamine (PE). From the FTIR spectra, O-O stretching of hydroperoxides was identified in the range 890-820cm(-1). Multivariate data analysis revealed a positive correlation (r = 0.99 for 4 h exposure and r = 0.98 for 12h exposure) between ozone exposure levels and the region of the FTIR-spectrum comprising the main wavelengths for hydroperoxides. These data support this alternative, versatile and novel spectroscopic approach for the early detection of ozone-mediated damage in human airway epithelial cells.

  5. Differences in gene expression and cytokine production by crystalline vs. amorphous silica in human lung epithelial cells

    Directory of Open Access Journals (Sweden)

    Perkins Timothy N

    2012-02-01

    Full Text Available Abstract Background Exposure to respirable crystalline silica particles, as opposed to amorphous silica, is associated with lung inflammation, pulmonary fibrosis (silicosis, and potentially with lung cancer. We used Affymetrix/GeneSifter microarray analysis to determine whether gene expression profiles differed in a human bronchial epithelial cell line (BEAS 2B exposed to cristobalite vs. amorphous silica particles at non-toxic and equal surface areas (75 and 150 × 106μm2/cm2. Bio-Plex analysis was also used to determine profiles of secreted cytokines and chemokines in response to both particles. Finally, primary human bronchial epithelial cells (NHBE were used to comparatively assess silica particle-induced alterations in gene expression. Results Microarray analysis at 24 hours in BEAS 2B revealed 333 and 631 significant alterations in gene expression induced by cristobalite at low (75 and high (150 × 106μm2/cm2 amounts, respectively (p 6μm2/cm2 induced 108 significant gene changes. Bio-Plex analysis of 27 human cytokines and chemokines revealed 9 secreted mediators (p FOS, ATF3, IL6 and IL8 early and over time (2, 4, 8, and 24 h. Patterns of gene expression in NHBE cells were similar overall to BEAS 2B cells. At 75 × 106μm2/cm2, there were 339 significant alterations in gene expression induced by cristobalite and 42 by amorphous silica. Comparison of genes in response to cristobalite (75 × 106μm2/cm2 revealed 60 common, significant gene alterations in NHBE and BEAS 2B cells. Conclusions Cristobalite silica, as compared to synthetic amorphous silica particles at equal surface area concentrations, had comparable effects on the viability of human bronchial epithelial cells. However, effects on gene expression, as well as secretion of cytokines and chemokines, drastically differed, as the crystalline silica induced more intense responses. Our studies indicate that toxicological testing of particulates by surveying viability and

  6. Neutrophil and asbestos fiber-induced cytotoxicity in cultured human mesothelial and bronchial epithelial cells.

    Science.gov (United States)

    Kinnula, V L; Raivio, K O; Linnainmaa, K; Ekman, A; Klockars, M

    1995-03-01

    This study investigates reactive oxygen species generation and oxidant-related cytotoxicity induced by amosite asbestos fibers and polymorphonuclear leucocytes (PMNs) in human mesothelial cells and human bronchial epithelial cells in vitro. Transformed human pleural mesothelial cells (MET 5A) and bronchial epithelial cells (BEAS 2B) were treated with amosite (2 micrograms/cm2) for 48 h. After 24 h of incubation, the cells were exposed for 1 h to nonactivated or amosite (50 micrograms) activated PMNs, washed, and incubated for another 23 h. Reactive oxygen species generation by the PMNs and the target cells was measured by chemiluminescence. Cell injury was assessed by cellular adenine nucleotide depletion, extracellular release of nucleotides, and lactate dehydrogenase (LDH). Amosite-activated (but also to a lesser degree nonactivated) PMNs released substantial amounts of reactive oxygen metabolites, whereas the chemiluminescence of amosite-exposed mesothelial cells and epithelial cells did not differ from the background. Amosite treatment (48 h) of the target cells did not change intracellular adenine nucleotides (ATP, ADP, AMP) or nucleotide catabolite products (xanthine, hypoxanthine, and uric acid). When the target cells were exposed to nonactivated PMNs, significant adenine nucleotide depletion and nucleotide catabolite accumulation was observed in mesothelial cells only. In separate experiments, when the target cells were exposed to amosite-activated PMNs, the target cell injury was further potentiated compared with the amosite treatment alone or exposure to nonactivated PMNs. In conclusion, this study suggests the importance of inflammatory cell-derived free radicals in the development of amosite-induced mesothelial cell injury.

  7. Cell culture's spider silk road.

    Science.gov (United States)

    Perkel, Jeffrey

    2014-06-01

    A number of synthetic and natural materials have been tried in cell culture and tissue engineering applications in recent years. Now Jeffrey Perkel takes a look at one new culture component that might surprise you-spider silk.

  8. Telomere damage effect of human tracheal epithelial cells induced by POT1 RNA interference and coal tar pitch extract%POT1 RNA干扰与煤焦沥青烟提取物对BEAS-2B细胞端粒的损伤效应

    Institute of Scientific and Technical Information of China (English)

    张建功; 王思华; 李小龙; 姚武; 燕贞; 吴拥军; 吴逸明; 王威

    2013-01-01

    Objective To explore the effect of down regulation of protection of telomere 1 (POT1) and Coal Tar Pitch on the telomere damage of human bronchial epithelial cells(BEAS-2B) caused by CTP.Methods BEAS-2B cells were cultured respectively in CTP group,dimethylsulfoxide (DMSO) solvent control group and blank control group,and then,they were RNA interferenced in POT1 gene in the 1st,the 10th,the 20th and the 30th generations,the telomere length,the mRNAs expression of POT1 and human telomerase reverse transcriptase(hTERT),and proteins expression of POT1 and hTERT were detected.Results Compared with control group,suppression of POT1 gene could reduce the mRNA expression of hTERT(F =25.008,P <0.001),and the relative length of telomere DNA was shorterned(F =14.617,P =0.001).With the increase of cell culture generation,the relative length of telomere DNA was shortern (F =15.988,P <0.001).Conclusions Inhibiting the expression of POT1 gene can further the telomeredamage of BASE-2B cells by%目的 探讨永生化人支气管上皮细胞(human bronchial epithelial cells,BEAS-2B)端粒保卫蛋白1(protection of telomere 1,POTl)的表达下调与煤焦沥青(coal tar pitch,CTP)致端粒损伤的作用.方法 BEAS-2B细胞以CTP组,二甲基亚砜(dimethylsulfoxide,DMSO)溶剂对照组和空白对照组分别进行培养.培养至1代、10代、20代和30代时分别对细胞进行POT1基因RNA干扰,然后进行端粒长度、POT1和人端粒酶逆转录酶(human telomerase reverse transcriptase,hTERT)的mRNA及蛋白表达情况的检测.结果 与对照组相比,抑制POT1基因的表达可导致hTERT的mRNA表达水平下调(F=25.008,P<0.001),端粒DNA相对长度缩短(F=14.617,P=0.001),随着培养代数的增加,端粒DNA相对长度缩短(F=15.988,P<0.001).结论 抑制POT1基因表达可间接促进CTP对BASE-2B细胞的端粒损伤作用.

  9. Nanodiamond internalization in cells and the cell uptake mechanism

    Energy Technology Data Exchange (ETDEWEB)

    Perevedentseva, E. [National Dong Hwa University, Department of Physics (China); Hong, S.-F.; Huang, K.-J. [National Dong Hwa University, Department of Life Sciences (China); Chiang, I.-T.; Lee, C.-Y. [National Dong Hwa University, Department of Physics (China); Tseng, Y.-T. [National Dong Hwa University, Department of Life Sciences (China); Cheng, C.-L., E-mail: clcheng@mail.ndhu.edu.tw [National Dong Hwa University, Department of Physics (China)

    2013-08-15

    Cell type-dependent penetration of nanodiamond in living cells is one of the important factors for using nanodiamond as cellular markers/labels, for drug delivery as well as for other biomedical applications. In this work, internalization of 100 nm nanodiamonds by A549 lung human adenocarcinoma cell, Beas-2b non-tumorigenic human bronchial epithelial cell, and HFL-1 fibroblast-like human fetal lung cell is studied and compared. The penetration of nanodiamond into the cells was observed using confocal fluorescence imaging and Raman imaging methods. Visualization of the nanodiamond in cells allows comparison of the internalization for diamond nanoparticles in cancer A549 cell, non-cancer HFL-1, and Beas-2b cells. The dose-dependent and time-dependent behavior of nanodiamond uptake is observed in both cancer as well as non-cancer cells. The mechanism of nanodiamond uptake by cancer and non-cancer cells is analyzed by blocking different pathways. The uptake of nanodiamond in both cancer and non-cancer cells was found predominantly via clathrin-dependent endocytosis. In spite of observed similarity in the uptake mechanism for cancer and non-cancer cells, the nanodiamond uptake for cancer cell quantitatively exceeds the uptake for non-cancer cells, for the studied cell lines. The observed difference in internalization of nanodiamond by cancer and non-cancer cells is discussed.

  10. Nanodiamond internalization in cells and the cell uptake mechanism

    Science.gov (United States)

    Perevedentseva, E.; Hong, S.-F.; Huang, K.-J.; Chiang, I.-T.; Lee, C.-Y.; Tseng, Y.-T.; Cheng, C.-L.

    2013-08-01

    Cell type-dependent penetration of nanodiamond in living cells is one of the important factors for using nanodiamond as cellular markers/labels, for drug delivery as well as for other biomedical applications. In this work, internalization of 100 nm nanodiamonds by A549 lung human adenocarcinoma cell, Beas-2b non-tumorigenic human bronchial epithelial cell, and HFL-1 fibroblast-like human fetal lung cell is studied and compared. The penetration of nanodiamond into the cells was observed using confocal fluorescence imaging and Raman imaging methods. Visualization of the nanodiamond in cells allows comparison of the internalization for diamond nanoparticles in cancer A549 cell, non-cancer HFL-1, and Beas-2b cells. The dose-dependent and time-dependent behavior of nanodiamond uptake is observed in both cancer as well as non-cancer cells. The mechanism of nanodiamond uptake by cancer and non-cancer cells is analyzed by blocking different pathways. The uptake of nanodiamond in both cancer and non-cancer cells was found predominantly via clathrin-dependent endocytosis. In spite of observed similarity in the uptake mechanism for cancer and non-cancer cells, the nanodiamond uptake for cancer cell quantitatively exceeds the uptake for non-cancer cells, for the studied cell lines. The observed difference in internalization of nanodiamond by cancer and non-cancer cells is discussed.

  11. Low-solubility particles and a Trojan-horse type mechanism of toxicity: the case of cobalt oxide on human lung cells

    Science.gov (United States)

    2014-01-01

    Background The mechanisms of toxicity of metal oxide particles towards lung cells are far from being understood. In particular, the relative contribution of intracellular particulate versus solubilized fractions is rarely considered as it is very challenging to assess, especially for low-solubility particles such as cobalt oxide (Co3O4). Methods This study was possible owing to two highly sensitive, independent, analytical techniques, based on single-cell analysis, using ion beam microanalysis, and on bulk analysis of cell lysates, using mass spectrometry. Results Our study shows that cobalt oxide particles, of very low solubility in the culture medium, are readily incorporated by BEAS-2B human lung cells through endocytosis via the clathrin-dependent pathway. They are partially solubilized at low pH within lysosomes, leading to cobalt ions release. Solubilized cobalt was detected within the cytoplasm and the nucleus. As expected from these low-solubility particles, the intracellular solubilized cobalt content is small compared with the intracellular particulate cobalt content, in the parts-per-thousand range or below. However, we were able to demonstrate that this minute fraction of intracellular solubilized cobalt is responsible for the overall toxicity. Conclusions Cobalt oxide particles are readily internalized by pulmonary cells via the endo-lysosomal pathway and can lead, through a Trojan-horse mechanism, to intracellular release of toxic metal ions over long periods of time, involving specific toxicity. PMID:24669904

  12. Interpretation of cell culture phenomena.

    Science.gov (United States)

    Vierck, J L; Dodson, M V

    2000-03-01

    This paper discusses the dilemma of interpreting unusual or abnormal phenomena seen in cell cultures and is not intended to address the statistical design of experiments. Problems that can be encountered when growing cells in experimental situations include low or decreasing cell numbers, abnormal cell morphology, microbial contamination, and detachment of the cell monolayer. If any of these situations occur, it is not realistic to proceed with data analysis until the problem is corrected. The best policy is to attempt to standardize all types of cultures used for analysis and to avoid using any cultures that display atypical characteristics.

  13. Role of size and surface area for pro-inflammatory responses to silica nanoparticles in epithelial lung cells: importance of exposure conditions.

    Science.gov (United States)

    Skuland, T; Ovrevik, J; Låg, M; Refsnes, M

    2014-03-01

    The present study compared non-crystalline silica particles of nano (50nm)- and submicro (500nm)-size (Si50 and Si500) for the potential to induce cytokine responses in bronchial epithelial lung cells (BEAS-2B). The cell cultures were exposed to equal mass and surface area concentrations of the two particles in different exposure media; LHC-9 and DMEM:F12. The state of agglomeration was different in the two media; with marked agglomeration in LHC-9 and nearly no agglomeration in DMEM:F12. On a mass basis, Si50 was more potent than Si500 in inducing cytokine responses in both exposure media. In contrast, upon exposure to similar surface area concentrations, Si500 was more potent than Si50 in DMEM:F12. This might be due to different agglomeration/sedimentation properties of Si50 versus Si500 in the two media. However, influence of differences in particle reactivity or particle uptake cannot be excluded. The data indicated no qualitative changes in the cytokine gene-expression patterns induced by the two particles, suggesting effects through similar mechanisms. These aspects might be of importance for interpretation of in vitro studies of nanomaterials. Copyright © 2013 Elsevier Ltd. All rights reserved.

  14. Importance of agglomeration state and exposure conditions for uptake and pro-inflammatory responses to amorphous silica nanoparticles in bronchial epithelial cells.

    Science.gov (United States)

    Gualtieri, Maurizio; Skuland, Tonje; Iversen, Tore-Geir; Låg, Marit; Schwarze, Per; Bilaničová, Dagmar; Pojana, Giulio; Refsnes, Magne

    2012-11-01

    Amorphous silica nanoparticles (SiNPs, 30 and 50 nm) and rhodamine-coated SiNPs (50 nm) were examined for their ability to induce pro-inflammatory responses and cytotoxicity in BEAS-2B cells under different experimental conditions. The SiNPs formed micrometre-sized agglomerates in the absence of bovine serum albumin (BSA) in the culture medium, whereas with BSA (0.1%) they were much less agglomerated. All the SiNPs induced IL-6 and IL-8 responses, as measured by ELISA and real-time PCR. The responses were more marked without BSA and higher for the rhodamine SiNPs than the plain ones. Rhodamine SiNPs were not taken up by cells during a 3-h exposure, even though cytokine mRNAs were up-regulated. In conclusion, agglomerated SiNPs induced more potent cytokine responses than the non-agglomerated ones; either due to the agglomeration state per se or more conceivably to a change in surface reactivity against cellular targets due to BSA. Furthermore, cytokine expression was up-regulated independently of SiNP uptake.

  15. Cell culture purity issues and DFAT cells

    Energy Technology Data Exchange (ETDEWEB)

    Wei, Shengjuan [College of Animal Science and Technology, Northwest A and F University, Yangling, Shaanxi Province 712100 (China); Department of Animal Sciences, Washington State University, Pullman, WA 99164 (United States); Bergen, Werner G. [Program in Cellular and Molecular Biosciences/Department of Animal Sciences, Auburn University, Auburn, AL 36849 (United States); Hausman, Gary J. [Animal Science Department, University of Georgia, Athens, GA 30602-2771 (United States); Zan, Linsen, E-mail: zanls@yahoo.com.cn [College of Animal Science and Technology, Northwest A and F University, Yangling, Shaanxi Province 712100 (China); Dodson, Michael V., E-mail: dodson@wsu.edu [Department of Animal Sciences, Washington State University, Pullman, WA 99164 (United States)

    2013-04-12

    Highlights: •DFAT cells are progeny cells derived from dedifferentiated mature adipocytes. •Common problems in this research is potential cell contamination of initial cultures. •The initial cell culture purity is crucial in DFAT cell research field. -- Abstract: Dedifferentiation of mature adipocytes, in vitro, has been pursued/documented for over forty years. The subsequent progeny cells are named dedifferentiated adipocyte-derived progeny cells (DFAT cells). DFAT cells are proliferative and likely to possess mutilineage potential. As a consequence, DFAT cells and their progeny/daughter cells may be useful as a potential tool for various aspects of tissue engineering and as potential vectors for the alleviation of several disease states. Publications in this area have been increasing annually, but the purity of the initial culture of mature adipocytes has seldom been documented. Consequently, it is not always clear whether DFAT cells are derived from dedifferentiated mature (lipid filled) adipocytes or from contaminating cells that reside in an impure culture.

  16. Cell culture purity issues and DFAT cells.

    Science.gov (United States)

    Wei, Shengjuan; Bergen, Werner G; Hausman, Gary J; Zan, Linsen; Dodson, Michael V

    2013-04-12

    Dedifferentiation of mature adipocytes, in vitro, has been pursued/documented for over forty years. The subsequent progeny cells are named dedifferentiated adipocyte-derived progeny cells (DFAT cells). DFAT cells are proliferative and likely to possess mutilineage potential. As a consequence, DFAT cells and their progeny/daughter cells may be useful as a potential tool for various aspects of tissue engineering and as potential vectors for the alleviation of several disease states. Publications in this area have been increasing annually, but the purity of the initial culture of mature adipocytes has seldom been documented. Consequently, it is not always clear whether DFAT cells are derived from dedifferentiated mature (lipid filled) adipocytes or from contaminating cells that reside in an impure culture.

  17. ADAM28 is expressed by epithelial cells in human normal tissues and protects from C1q-induced cell death.

    Science.gov (United States)

    Miyamae, Yuka; Mochizuki, Satsuki; Shimoda, Masayuki; Ohara, Kentaro; Abe, Hitoshi; Yamashita, Shuji; Kazuno, Saiko; Ohtsuka, Takashi; Ochiai, Hiroki; Kitagawa, Yuko; Okada, Yasunori

    2016-05-01

    ADAM28 (disintegrin and metalloproteinase 28), which was originally reported to be lymphocyte-specific, is over-expressed by carcinoma cells and plays a key role in cell proliferation and progression in human lung and breast carcinomas. We studied ADAM28 expression in human normal tissues and examined its biological function. By using antibodies specific to ADAM28, ADAM28 was immunolocalized mainly to epithelial cells in several tissues, including epididymis, bronchus and stomach, whereas lymphocytes in lymph nodes and spleen were negligibly immunostained. RT-PCR, immunoblotting and ELISA analyses confirmed the expression in these tissues, and low or negligible expression by lymphocytes was found in the lymph node and spleen. C1q was identified as a candidate ADAM28-binding protein from a human lung cDNA library by yeast two-hybrid system, and specific binding was demonstrated by binding assays, immunoprecipitation and surface plasmon resonance. C1q treatment of normal bronchial epithelial BEAS-2B and NHBE cells, both of which showed low-level expression of ADAM28, caused apoptosis through activation of p38 and caspase-3, and cell death with autophagy through accumulation of LC3-II and autophagosomes, respectively. C1q-induced cell death was attenuated by treatment of the cells with antibodies against the C1q receptor gC1qR/p33 or cC1qR/calreticulin. Treatment of C1q with recombinant ADAM28 prior to addition to culture media reduced C1q-induced cell death, and knockdown of ADAM28 using siRNAs increased cell death. These data demonstrate that ADAM28 is expressed by epithelial cells of several normal organs, and suggest that ADAM28 plays a role in cell survival by suppression of C1q-induced cytotoxicity in bronchial epithelial cells.

  18. Cell culture compositions

    Science.gov (United States)

    Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yiao, Jian

    2014-03-18

    The present invention provides a novel endoglucanase nucleic acid sequence, designated egl6 (SEQ ID NO:1 encodes the full length endoglucanase; SEQ ID NO:4 encodes the mature form), and the corresponding endoglucanase VI amino acid sequence ("EGVI"; SEQ ID NO:3 is the signal sequence; SEQ ID NO:2 is the mature sequence). The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVI, recombinant EGVI proteins and methods for producing the same.

  19. Coffee diterpenes prevent benzo[a]pyrene genotoxicity in rat and human culture systems.

    Science.gov (United States)

    Cavin, C; Bezencon, C; Guignard, G; Schilter, B

    2003-06-27

    The coffee-specific diterpenes cafestol and kahweol (C+K) have been identified as two important chemoprotective agents in coffee. In the present study, the potential preventive effects of C+K against the genotoxicity of B[a]P were investigated in rat primary hepatocytes and in human bronchial Beas-2B cells. Several independent mechanisms were identified and their respective contribution to the overall protective effects was determined. A marked dose-dependent inhibition by C+K of B[a]P DNA-binding was found in cells of both origins. However, data showed that the significant induction by C+K of the detoxifying enzyme GST-Yp subunit is the key mechanism of protection against B[a]P DNA-binding in rat liver. In contrast, the phase I-mediated mechanism where C+K produce an inhibition of CYP 1A1 induction by B[a]P is of key significance for the C+K protection in human Beas-2B cells. Moreover, this effect suggests a novel mechanism of chemoprotection by the coffee diterpenes cafestol and kahweol.

  20. Acute toxicity of silver and carbon nanoaerosols to normal and cystic fibrosis human bronchial epithelial cells.

    Science.gov (United States)

    Jeannet, Natalie; Fierz, Martin; Schneider, Sarah; Künzi, Lisa; Baumlin, Nathalie; Salathe, Matthias; Burtscher, Heinz; Geiser, Marianne

    2016-01-01

    Inhalation of engineered nanoparticles (NP) poses a still unknown risk. Individuals with chronic lung diseases are expected to be more vulnerable to adverse effects of NP than normal subjects, due to altered respiratory structures and functions. Realistic and dose-controlled aerosol exposures were performed using the deposition chamber NACIVT. Well-differentiated normal and cystic fibrosis (CF) human bronchial epithelia (HBE) with established air-liquid interface and the human bronchial epithelial cell line BEAS-2B were exposed to spark-generated silver and carbon nanoaerosols (20 nm diameter) at three different doses. Necrotic and apoptotic cell death, pro-inflammatory response, epithelial function and morphology were assessed within 24 h after aerosol exposure. NP exposure resulted in significantly higher necrosis in CF than normal HBE and BEAS-2B cells. Before and after NP treatment, CF HBE had higher caspase-3 activity and secreted more IL-6 and MCP-1 than normal HBE. Differentiated HBE had higher baseline secretion of IL-8 and less caspase-3 activity and MCP-1 secretion compared to BEAS-2B cells. These biomarkers increased moderately in response to NP exposure, except for MCP-1, which was reduced in HBE after AgNP treatment. No functional and structural alterations of the epithelia were observed in response to NP exposure. Significant differences between cell models suggest that more than one and fully differentiated HBE should be used in future toxicity studies of NP in vitro. Our findings support epidemiologic evidence that subjects with chronic airway diseases are more vulnerable to adverse effects of particulate air pollution. Thus, this sub-population needs to be included in nano-toxicity studies.

  1. [Cellular distribution and behavior of metallothionein in mammalian cells following exposure to silver nanoparticles and silver ions].

    Science.gov (United States)

    Miyayama, Takamitsu; Arai, Yuta; Suzuki, Noriyuki; Hirano, Seishiro

    2014-01-01

    Silver nanoparticles (AgNPs) are commercially used mainly as antibacterial reagents in wound dressing and deodorant powders. However, the mechanisms underlying Ag toxicity in mammals are not fully understood. In the present study, we assessed cellular distribution and toxicity of AgNPs and AgNO3 in mouse macrophage cell line (J774.1) and those of AgNO3 in human bronchial epithelial cell line (BEAS-2B) focusing on behavior of metallothionein (MT). J774.1 cells were exposed to 0-100 μg Ag/mL AgNPs or AgNO3 and BEAS-2B cells were exposed to 0-100 μM AgNO3 for 24 h. The cytotoxicity was assayed by a modified MTT method. The cellular concentration and distribution of Ag were evaluated by inductively coupled plasma-mass spectorometry (ICP-MS) and laser scanning microscopy. Distribution of Ag to MT and other proteins was determined using HPLC-ICP-MS. Most AgNPs were found in lysosomes in J774.1 at 3 h after post exposure. Ag was distributed to high molecular weight proteins in AgNPs-exposed cells, while most Ag was bound to MT in AgNO3-exposed cells. In AgNO3-exposed BEAS-2B cells cellular Ag concentration and Ag-bound MT (Ag-MT) were sharply increased up to 3 h and then decreased. ROS production appeared to cause relocation of MT-bound Ag to mitochondria, which evoked inhibition of electron transport chain. AgNPs were sequestered by high-molecular weight proteins rather than MT, probably because they were taken up by lysosomes before induction of MT.

  2. Asthmatic bronchial epithelium activated by the proteolytic allergen Der p 1 increases selective dendritic cell recruitment.

    Science.gov (United States)

    Pichavant, Muriel; Charbonnier, Anne-Sophie; Taront, Solenne; Brichet, Anne; Wallaert, Benoît; Pestel, Joel; Tonnel, André-Bernard; Gosset, Philippe

    2005-04-01

    Airway dendritic cells (DCs) are crucial for allergen-induced sensitization and inflammation in allergic asthma. After allergen challenge, an increased number of DCs is observed in airway epithelium from patients with allergy. Because Der p 1, a cysteine protease allergen from Dermatophagoides pteronyssinus , induces chemokine production by bronchial epithelial cells (BECs), the purpose of this investigation was to evaluate the capacity of BEC exposed to Der p 1 to recruit DCs. Chemotactic activity of BEAS-2B, a bronchial epithelial cell line, and BECs from nonatopic controls and patients with allergic asthma was evaluated on the migration of precursors, immature and mature monocyte-derived DCs (MDDCs), and CD34 + -derived Langerhans cells (LCs). C-C chemokine ligand (CCL)-2, CCL5, and C-X-C chemokine ligand 10 production by BEAS-2B and BEC was increased after Der p 1 exposure, whereas the proenzyme proDer p 1 devoid of enzymatic activity had no effect. Der p 1 stimulation of BEAS-2B and BEC from both groups increased significantly the recruitment of MDDC precursors, depending on CCL2, CCL5, and C-X-C chemokine ligand 10 production. In a reconstituted polarized epithelium, apical application of Der p 1 enhanced MDDC precursor migration into the epithelial layer. Moreover, Der p 1 stimulation of BEC from patients with asthma but not from controls increased the migration of LC precursors, mainly dependent on CCL20 secretion. No migration of immature and mature DCs was observed. These data confirmed that BECs participate in the homeostasis of the DC network present within the bronchial epithelium through the secretion of chemokines. In allergic asthma, upregulation of CCL20 production induced LC recruitment, the role of which remains to be determined.

  3. Insect Cell Culture and Biotechnology

    Institute of Scientific and Technical Information of China (English)

    Robert R.Granados; Guoxun Li; G.W.Blissard

    2007-01-01

    The continued development of new cell culture technology is essential for the future growth and application of insect cell and baculovirus biotechnology. The use of cell lines for academic research and for commercial applications is currently dominated by two cell lines; the Spodoptera frugiperda line, SF21 (and its clonal isolate, SF9), and the Trichoplusia ni line, BTI 5B1-4, commercially known as High Five cells. The long perceived prediction that the immense potential application of the baculovirus-insect cell system, as a tool in cell and molecular biology, agriculture, and animal health, has been achieved. The versatility and recent applications of this popular expression system has been demonstrated by both academia and industry and it is clear that this cell-based system has been widely accepted for biotechnological applications. Numerous small to midsize startup biotechnology companies in North America and the Europe are currently using the baculovirus-insect cell technology to produce custom recombinant proteins for research and commercial applications. The recent breakthroughs using the baculovirus-insect cell-based system for the development of several commercial products that will impact animal and human health will further enhance interest in this technology by pharma. Clearly, future progress in novel cell and engineering advances will lead to fundamental scientific discoveries and serve to enhance the utility and applications of this baculovirus-insect cell system.

  4. s-Ethyl Cysteine and s-Methyl Cysteine Protect Human Bronchial Epithelial Cells Against Hydrogen Peroxide Induced Injury.

    Science.gov (United States)

    Hsia, Te-chun; Yin, Mei-chin

    2015-09-01

    Protective effects and actions from s-ethyl cysteine (SEC) and s-methyl cysteine (SMC) for BEAS-2B cells were examined. BEAS-2B cells were pretreated with SEC or SMC at 4, 8, or 16 μmol/L, and followed by hydrogen peroxide (H2 O2 ) treatment. Data showed that H2 O2 enhanced Bax, caspase-3 and caspase-8 expression, and declined Bcl-2 expression. However, SEC or SMC dose-dependently decreased caspase-3 expression and reserved Bcl-2 expression. H2 O2 increased reactive oxygen species (ROS) production, and lowered glutathione level, glutathione peroxide, and glutathione reductase activities in BEAS-2B cells. SEC or SMC pretreatments reduced ROS generation, and maintained glutathione redox cycle in those cells. H2 O2 upregulated the expression of both p47(phox) and gp91(phox) . SEC and SMC downregulated p47(phox) expression. SEC or SMC at 8 and 16 μmol/L decreased H2 O2 -induced release of inflammatory cytokines. H2 O2 stimulated the activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinase. SEC and SMC pretreatments dose-dependently downregulated NF-κB p65 and p-p38 expression. Pyrrolidine dithiocarbamate or SB203580 inhibited NF-κB activation and p38 phosphorylation; thus, SEC or SMC pretreatments failed to affect protein expression of these factors. These novel findings suggest that SEC or SMC could protect bronchial cells and benefit respiratory epithelia stability and functions.

  5. 36. Study on p16INK4a and p15INK4b genes of human bronchial epithelial cells malignantly transformed by cyclophosphamide and thiotepa

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    @@Transformed human bronchial epithelial cells BEAS-2B induced by CP and TEPA were used to study abnormity of the tumor suppressor genes p15INK4b and p16INK4a, through which we can provide clues for explanations of the molecular mechanism in carcinogenesis of human bronchial epithelial cells induced by CP and TEPA. Analysis of the genomic DNA from the transformed BEAS-CP, and BEAS-T cells using PCR amplification, singe strand conformation polymorphism(SSCP) and DNA sequencing

  6. NEIL2 protects against oxidative DNA damage induced by sidestream smoke in human cells.

    Directory of Open Access Journals (Sweden)

    Altaf H Sarker

    Full Text Available Secondhand smoke (SHS is a confirmed lung carcinogen that introduces thousands of toxic chemicals into the lungs. SHS contains chemicals that have been implicated in causing oxidative DNA damage in the airway epithelium. Although DNA repair is considered a key defensive mechanism against various environmental attacks, such as cigarette smoking, the associations of individual repair enzymes with susceptibility to lung cancer are largely unknown. This study investigated the role of NEIL2, a DNA glycosylase excising oxidative base lesions, in human lung cells treated with sidestream smoke (SSS, the main component of SHS. To do so, we generated NEIL2 knockdown cells using siRNA-technology and exposed them to SSS-laden medium. Representative SSS chemical compounds in the medium were analyzed by mass spectrometry. An increased production of reactive oxygen species (ROS in SSS-exposed cells was detected through the fluorescent detection and the induction of HIF-1α. The long amplicon-quantitative PCR (LA-QPCR assay detected significant dose-dependent increases of oxidative DNA damage in the HPRT gene of cultured human pulmonary fibroblasts (hPF and BEAS-2B epithelial cells exposed to SSS for 24 h. These data suggest that SSS exposure increased oxidative stress, which could contribute to SSS-mediated toxicity. siRNA knockdown of NEIL2 in hPF and HEK 293 cells exposed to SSS for 24 h resulted in significantly more oxidative DNA damage in HPRT and POLB than in cells with control siRNA. Taken together, our data strongly suggest that decreased repair of oxidative DNA base lesions due to an impaired NEIL2 expression in non-smokers exposed to SSS would lead to accumulation of mutations in genomic DNA of lung cells over time, thus contributing to the onset of SSS-induced lung cancer.

  7. Inflammatory Response and Barrier Dysfunction by Different e-Cigarette Flavoring Chemicals Identified by Gas Chromatography–Mass Spectrometry in e-Liquids and e-Vapors on Human Lung Epithelial Cells and Fibroblasts

    Science.gov (United States)

    Gerloff, Janice; Sundar, Isaac K.; Freter, Robert; Sekera, Emily R.; Friedman, Alan E.; Robinson, Risa; Pagano, Todd

    2017-01-01

    Abstract Recent studies suggest that electronic cigarette (e-cig) flavors can be harmful to lung tissue by imposing oxidative stress and inflammatory responses. The potential inflammatory response by lung epithelial cells and fibroblasts exposed to e-cig flavoring chemicals in addition to other risk-anticipated flavor enhancers inhaled by e-cig users is not known. The goal of this study was to evaluate the release of the proinflammatory cytokine (interleukin-8 [IL-8]) and epithelial barrier function in response to different e-cig flavoring chemicals identified in various e-cig e-liquid flavorings and vapors by chemical characterization using gas chromatography–mass spectrometry analysis. Flavorings, such as acetoin (butter), diacetyl, pentanedione, maltol (malt), ortho-vanillin (vanilla), coumarin, and cinnamaldehyde in comparison with tumor necrosis factor alpha (TNFα), were used in this study. Human bronchial epithelial cells (Beas2B), human mucoepidermoid carcinoma epithelial cells (H292), and human lung fibroblasts (HFL-1) were treated with each flavoring chemical for 24 hours. The cells and conditioned media were then collected and analyzed for toxicity (viability %), lung epithelial barrier function, and proinflammatory cytokine IL-8 release. Cell viability was not significantly affected by any of the flavoring chemicals tested at a concentration of 10 μM to 1 mM. Acetoin and diacetyl treatment induced IL-8 release in Beas2B cells. Acetoin- and pentanedione-treated HFL-1 cells produced a differential, but significant response for IL-8 release compared to controls and TNFα. Flavorings, such as ortho-vanillin and maltol, induced IL-8 release in Beas2B cells, but not in H292 cells. Of all the flavoring chemicals tested, acetoin and maltol were more potent inducers of IL-8 release than TNFα in Beas2B and HFL-1 cells. Flavoring chemicals rapidly impaired epithelial barrier function in human bronchial epithelial cells (16-HBE) as measured by electric cell

  8. Inflammatory Response and Barrier Dysfunction by Different e-Cigarette Flavoring Chemicals Identified by Gas Chromatography-Mass Spectrometry in e-Liquids and e-Vapors on Human Lung Epithelial Cells and Fibroblasts.

    Science.gov (United States)

    Gerloff, Janice; Sundar, Isaac K; Freter, Robert; Sekera, Emily R; Friedman, Alan E; Robinson, Risa; Pagano, Todd; Rahman, Irfan

    2017-03-01

    Recent studies suggest that electronic cigarette (e-cig) flavors can be harmful to lung tissue by imposing oxidative stress and inflammatory responses. The potential inflammatory response by lung epithelial cells and fibroblasts exposed to e-cig flavoring chemicals in addition to other risk-anticipated flavor enhancers inhaled by e-cig users is not known. The goal of this study was to evaluate the release of the proinflammatory cytokine (interleukin-8 [IL-8]) and epithelial barrier function in response to different e-cig flavoring chemicals identified in various e-cig e-liquid flavorings and vapors by chemical characterization using gas chromatography-mass spectrometry analysis. Flavorings, such as acetoin (butter), diacetyl, pentanedione, maltol (malt), ortho-vanillin (vanilla), coumarin, and cinnamaldehyde in comparison with tumor necrosis factor alpha (TNFα), were used in this study. Human bronchial epithelial cells (Beas2B), human mucoepidermoid carcinoma epithelial cells (H292), and human lung fibroblasts (HFL-1) were treated with each flavoring chemical for 24 hours. The cells and conditioned media were then collected and analyzed for toxicity (viability %), lung epithelial barrier function, and proinflammatory cytokine IL-8 release. Cell viability was not significantly affected by any of the flavoring chemicals tested at a concentration of 10 μM to 1 mM. Acetoin and diacetyl treatment induced IL-8 release in Beas2B cells. Acetoin- and pentanedione-treated HFL-1 cells produced a differential, but significant response for IL-8 release compared to controls and TNFα. Flavorings, such as ortho-vanillin and maltol, induced IL-8 release in Beas2B cells, but not in H292 cells. Of all the flavoring chemicals tested, acetoin and maltol were more potent inducers of IL-8 release than TNFα in Beas2B and HFL-1 cells. Flavoring chemicals rapidly impaired epithelial barrier function in human bronchial epithelial cells (16-HBE) as measured by electric cell surface

  9. 9 CFR 101.6 - Cell cultures.

    Science.gov (United States)

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Cell cultures. 101.6 Section 101.6..., SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS DEFINITIONS § 101.6 Cell cultures. When used in conjunction with or in reference to cell cultures, which may be referred to as tissue...

  10. Cadmium induces cytotoxicity in human bronchial epithelial cells through upregulation of eIF5A1 and NF-kappaB

    Energy Technology Data Exchange (ETDEWEB)

    Chen, De-Ju; Xu, Yan-Ming; Du, Ji-Ying [Laboratory of Cancer Biology and Epigenetics, Shantou University Medical College, Shantou, Guangdong 515041 (China); Department of Cell Biology and Genetics, Shantou University Medical College, Shantou, Guangdong 515041 (China); Huang, Dong-Yang [Department of Cell Biology and Genetics, Shantou University Medical College, Shantou, Guangdong 515041 (China); Lau, Andy T.Y., E-mail: andytylau@stu.edu.cn [Laboratory of Cancer Biology and Epigenetics, Shantou University Medical College, Shantou, Guangdong 515041 (China); Department of Cell Biology and Genetics, Shantou University Medical College, Shantou, Guangdong 515041 (China)

    2014-02-28

    Highlights: • Normal human bronchial epithelial cells (BEAS-2B) were dosed with cadmium (Cd). • A low level (2 μM) of Cd treatment for 36 h elicited negligible cytotoxicity. • High levels (20 or 30 μM) of Cd treatment for 36 h induced cell death. • High levels of Cd can upregulate the protein levels of eIF5A1 and NF-κB p65. • We suggest that eIF5A1 level is possibly modulated by NF-κB. - Abstract: Cadmium (Cd) and Cd compounds are widely-distributed in the environment and well-known carcinogens. Here, we report that in CdCl{sub 2}-exposed human bronchial epithelial cells (BEAS-2B), the level of p53 is dramatically decreased in a time- and dose-dependent manner, suggesting that the observed Cd-induced cytotoxicity is not likely due to the pro-apoptotic function of p53. Therefore, this prompted us to further study the responsive pro-apoptotic factors by proteomic approaches. Interestingly, we identified that high levels (20 or 30 μM) of Cd can significantly upregulate the protein levels of eukaryotic translation initiation factor 5A1 (eIF5A1) and redox-sensitive transcription factor NF-κB p65. Moreover, there is an enhanced NF-κB nuclear translocation as well as chromatin-binding in Cd-treated BEAS-2B cells. We also show that small interfering RNA-specific knockdown of eIF5A1 in Cd-exposed cells attenuated the Cd cytotoxicity, indicating the potential role of eIF5A1 in Cd cytotoxicity. As eIF5A1 is reported to be related with cell apoptosis but little is known about its transcriptional control, we hypothesize that NF-κB might likely modulate eIF5A1 gene expression. Notably, by bioinformatic analysis, several potential NF-κB binding sites on the upstream promoter region of eIF5A1 gene can be found. Subsequent chromatin immunoprecipitation assay revealed that indeed there is enhanced NF-κB binding on eIF5A1 promoter region of Cd-treated BEAS-2B cells. Taken together, our findings suggest for the first time a regulatory mechanism for the pro

  11. Defining viability in mammalian cell cultures

    OpenAIRE

    Browne, Susan M.; Al-Rubeai, Mohamed

    2011-01-01

    Abstract A large number of assays are available to monitor viability in mammalian cell cultures with most defining loss of viability as a loss of plasma membrane integrity, a characteristic of necrotic cell death. However, the majority of cultured cells die by apoptosis and early apoptotic cells, although non-viable, maintain an intact plasma membrane and are thus ignored. Here we measure the viability of cultures of a number of common mammalian cell lines by assays that measure me...

  12. Dynamized Preparations in Cell Culture

    Directory of Open Access Journals (Sweden)

    Ellanzhiyil Surendran Sunila

    2009-01-01

    Full Text Available Although reports on the efficacy of homeopathic medicines in animal models are limited, there are even fewer reports on the in vitro action of these dynamized preparations. We have evaluated the cytotoxic activity of 30C and 200C potencies of ten dynamized medicines against Dalton's Lymphoma Ascites, Ehrlich's Ascites Carcinoma, lung fibroblast (L929 and Chinese Hamster Ovary (CHO cell lines and compared activity with their mother tinctures during short-term and long-term cell culture. The effect of dynamized medicines to induce apoptosis was also evaluated and we studied how dynamized medicines affected genes expressed during apoptosis. Mother tinctures as well as some dynamized medicines showed significant cytotoxicity to cells during short and long-term incubation. Potentiated alcohol control did not produce any cytotoxicity at concentrations studied. The dynamized medicines were found to inhibit CHO cell colony formation and thymidine uptake in L929 cells and those of Thuja, Hydrastis and Carcinosinum were found to induce apoptosis in DLA cells. Moreover, dynamized Carcinosinum was found to induce the expression of p53 while dynamized Thuja produced characteristic laddering pattern in agarose gel electrophoresis of DNA. These results indicate that dynamized medicines possess cytotoxic as well as apoptosis-inducing properties.

  13. Expanding intestinal stem cells in culture

    NARCIS (Netherlands)

    Heo, Inha; Clevers, Hans

    2015-01-01

    Culturing intestinal stem cells into 3D organoids results in heterogeneous cell populations, reflecting the in vivo cell type diversity. In a recent paper published in Nature, Wang et al. established a culture condition for a highly homogeneous population of intestinal stem cells.

  14. Expanding intestinal stem cells in culture

    NARCIS (Netherlands)

    Heo, Inha; Clevers, Hans

    Culturing intestinal stem cells into 3D organoids results in heterogeneous cell populations, reflecting the in vivo cell type diversity. In a recent paper published in Nature, Wang et al. established a culture condition for a highly homogeneous population of intestinal stem cells.

  15. Quantitative evaluation of berberine subcellular distribution and cellular accumulation in non-small cell lung cancer cells by UPLC-MS/MS.

    Science.gov (United States)

    Yuan, Zhong-Wen; Leung, Elaine Lai-Han; Fan, Xing-Xing; Zhou, Hua; Ma, Wen-Zhe; Liu, Liang; Xie, Ying

    2015-11-01

    Berberine, an isoquinoline alkaloid, has been demonstrated to be a safe anti-cancer agent with multiple effects on mitochondria. Intracellular concentration and distribution around the targeting sites are determinants of efficacy, but subcellular distribution of berberine has not been fully elucidated yet, which relies on the sensitive and robustness assay. In this study, a sensitive and robust UPLC-MS/MS method has been developed and validated with optimized extraction solvents and detection conditions. Key factors such as the purity and integrity of isolated organelle fractions, and the effects of isolation procedures on the subcellular concentration of berberine were systemically evaluated. With the developed assay, we found that the intracellular accumulations of berberine in two gefitinib resistant NSCLC cell lines H1650 and H1975 were 2-3 folds higher than that of normal epithelial cells BEAS-2B. Moreover, significantly different subcellular distribution profiles in NSCLC cancer cells from that of BEAS-2B cells with a striking increase in content in most organelles may contribute to its selective cytotoxicity to cancer cells. Furthermore, a predominant accumulation of berberine was observed for the first time in microsomal fraction for all three cell lines. Therefore, this method could be used for quantitative evaluation of subcellular distribution and cellular accumulation of berberine and for further evaluation of the concentration-effects relationship.

  16. Nickel oxide nanoparticles induce inflammation and genotoxic effect in lung epithelial cells.

    Science.gov (United States)

    Capasso, Laura; Camatini, Marina; Gualtieri, Maurizio

    2014-04-07

    Nickel oxide nanoparticles (NiONPs) toxicity has been evaluated in the human pulmonary epithelial cell lines: BEAS-2B and A549. The nanoparticles, used at the doses of 20, 40, 60, 80, 100 μg/ml, induced a significant reduction of cell viability and an increase of apoptotic and necrotic cells at 24h. A significant release of interleukin-6 and -8 was assessed after 24h of treatment, even intracellular ROS increased already at 45 min after exposure. The results obtained evidenced that the cytokines release was dependent on mitogen activated protein kinases (MAPK) cascade through the induction of NF-kB pathway. NiONPs induced cell cycle alteration in both the cell lines even in different phases and these modifications may be induced by the NPs genotoxic effect, suggested by the nuclear translocation of phospho-ATM and phospho-ATR. Our results confirm the cytotoxic and pro-inflammatory potential of NiONPs. Moreover their ability in inducing DNA damage responses has been demonstrated. Such effects were present in A549 cells which internalize the NPs and BEAS-2B cells in which endocytosis has not been observed.

  17. An antagonist of the platelet-activating factor receptor inhibits adherence of both nontypeable Haemophilus influenzae and Streptococcus pneumoniae to cultured human bronchial epithelial cells exposed to cigarette smoke

    Directory of Open Access Journals (Sweden)

    Shukla SD

    2016-07-01

    Full Text Available Shakti D Shukla,1,* Rory L Fairbairn,1,* David A Gell,1 Roger D Latham,1 Sukhwinder S Sohal,1,2 Eugene H Walters,1 Ronan F O’Toole11Breathe Well Centre, School of Medicine, Faculty of Health, University of Tasmania, Hobart, TAS, Australia; 2School of Health Sciences, Faculty of Health, University of Tasmania, Launceston, TAS, Australia*These authors contributed equally to this workBackground: COPD is emerging as the third largest cause of human mortality worldwide after heart disease and stroke. Tobacco smoking, the primary risk factor for the development of COPD, induces increased expression of platelet-activating factor receptor (PAFr in the lung epithelium. Nontypeable Haemophilus influenzae (NTHi and Streptococcus pneumoniae adhere to PAFr on the luminal surface of human respiratory tract epithelial cells.Objective: To investigate PAFr as a potential drug target for the prevention of infections caused by the main bacterial drivers of acute exacerbations in COPD patients, NTHi and S. pneumoniae.Methods: Human bronchial epithelial BEAS-2B cells were exposed to cigarette smoke extract (CSE. PAFr expression levels were determined using immunocytochemistry and quantitative polymerase chain reaction. The epithelial cells were challenged with either NTHi or S. pneumoniae labeled with fluorescein isothiocyanate, and bacterial adhesion was measured using immunofluorescence. The effect of a well-evaluated antagonist of PAFr, WEB-2086, on binding of the bacterial pathogens to BEAS-2B cells was then assessed. In silico studies of the tertiary structure of PAFr and the binding pocket for PAF and its antagonist WEB-2086 were undertaken.Results: PAFr expression by bronchial epithelial cells was upregulated by CSE, and significantly associated with increased bacterial adhesion. WEB-2086 reduced the epithelial adhesion by both NTHi and S. pneumoniae to levels observed for non-CSE-exposed cells. Furthermore, it was nontoxic toward the bronchial epithelial

  18. Best practices in cell culture: an overview.

    Science.gov (United States)

    Baust, John M; Buehring, Gertrude Case; Campbell, Lia; Elmore, Eugene; Harbell, John W; Nims, Raymond W; Price, Paul; Reid, Yvonne A; Simione, Frank

    2017-08-14

    This overview describes a series of articles to provide an unmet need for information on best practices in animal cell culture. The target audience primarily consists of entry-level scientists with minimal experience in cell culture. It also include scientists, journalists, and educators with some experience in cell culture, but in need of a refresher in best practices. The articles will be published in this journal over a six-month period and will emphasize best practices in: (1) media selection; (2) use and evaluation of animal serum as a component of cell culture medium; (3) receipt of new cells into the laboratory; (4) naming cell lines; (5) authenticating cell line identity; (6) detecting and mitigating risk of cell culture contamination; (7) cryopreservation and thawing of cells; and (8) storing and shipping viable cells.

  19. Cell density monitoring and control of microencapsulated CHO cell cultures

    OpenAIRE

    Cole, Harriet Emma

    2015-01-01

    Though mammalian cells play a key role in the manufacturing of recombinant glycosylated proteins, cell cultures and productivity are limited by the lack of suitable systems to enable stable perfusion culture. Microencapsulation, or entrapping cells within a semi-permeable membrane, offers the potential to generate high cell density cultures and improve the productivity by mimicking the cells natural environment. However, the cells being secluded by the microcapsules membrane are difficult to ...

  20. Respiratory virus infection up-regulates TRPV1, TRPA1 and ASICS3 receptors on airway cells

    Science.gov (United States)

    Omar, Shadia; Clarke, Rebecca; Abdullah, Haniah; Brady, Clare; Corry, John; Winter, Hanagh; Touzelet, Olivier; Power, Ultan F.; Lundy, Fionnuala; McGarvey, Lorcan P. A.

    2017-01-01

    Receptors implicated in cough hypersensitivity are transient receptor potential vanilloid 1 (TRPV1), transient receptor potential cation channel, Subfamily A, Member 1 (TRPA1) and acid sensing ion channel receptor 3 (ASIC3). Respiratory viruses, such as respiratory syncytial virus (RSV) and measles virus (MV) may interact directly and/or indirectly with these receptors on sensory nerves and epithelial cells in the airways. We used in vitro models of sensory neurones (SHSY5Y or differentiated IMR-32 cells) and human bronchial epithelium (BEAS-2B cells) as well as primary human bronchial epithelial cells (PBEC) to study the effect of MV and RSV infection on receptor expression. Receptor mRNA and protein levels were examined by qPCR and flow cytometry, respectively, following infection or treatment with UV inactivated virus, virus-induced soluble factors or pelleted virus. Concentrations of a range of cytokines in resultant BEAS-2B and PBEC supernatants were determined by ELISA. Up-regulation of TRPV1, TRPA1 and ASICS3 expression occurred by 12 hours post-infection in each cell type. This was independent of replicating virus, within the same cell, as virus-induced soluble factors alone were sufficient to increase channel expression. IL-8 and IL-6 increased in infected cell supernatants. Antibodies against these factors inhibited TRP receptor up-regulation. Capsazepine treatment inhibited virus induced up-regulation of TRPV1 indicating that these receptors are targets for treating virus-induced cough. PMID:28187208

  1. Cell culture techniques in honey bee research

    Science.gov (United States)

    Cell culture techniques are indispensable in most if not all life science disciplines to date. Wherever cell culture models are lacking scientific development is hampered. Unfortunately this has been and still is the case in honey bee research because permanent honey bee cell lines have not yet been...

  2. Cell Culture as an Alternative in Education.

    Science.gov (United States)

    Nardone, Roland M.

    1990-01-01

    Programs that are intended to inform and provide "hands-on" experience for students and to facilitate the introduction of cell culture-based laboratory exercises into the high school and college laboratory are examined. The components of the CellServ Program and the Cell Culture Toxicology Training Programs are described. (KR)

  3. The surface charge of visible particulate matter predicts biological activation in human bronchial epithelial cells.

    Science.gov (United States)

    Veronesi, Bellina; de Haar, Colin; Lee, Lseng; Oortgiesen, Marga

    2002-02-01

    The physicochemical complexity of airborne particulate matter (PM) has hampered identifying a specific mechanism(s) for its toxicity. In this study, selected physicochemical characteristics (i.e., size, particle number, acidity, and surface charge) were measured on various field PM, derived from urban ambient (St. Louis, Ottawa, Canada), residential (Woodstove), volcanic dust from Mt. St. Helen (MSH), and industrial [oil fly ash (OFA) coal fly ash (CFA)] sources. Morphometric analysis of visible (10 microm) field particles indicated that the industrial PM (OFA, CFA) had the smallest diameter and lowest total number of particles per weight while Woodstove and Ottawa had the largest diameter and highest number of particles. All PM lowered the pH of an unbuffered 10 mM NaCl solution from pH 7.4 to pH 4.7-6.8 but did not change the neutral pH of the cell culture medium, keratinocyte growth media (KGM). The surface charge (i.e., zeta potential) of microscopically visible (> or = 2.0 microm) field particles, suspended in either a Hepes-buffered KCl solution or in KGM, was measured by microelectrophoresis. In KCl solution, the mean zeta potential of all tested PM ranged from -36 +/- 2 (Woodstove) to -27 +/- 4.3 mV (MSH). When measured in KGM medium, the mean zeta potential value of each PM was significantly less (p > 0.001) than those measured in KCl solution, with values ranging from -17 +/- 0.3 mV (St. Louis) to -9 +/- 0.6 mV (MSH). Suspensions of field PM, its soluble and washed particulate fractions, were next prepared from each PM. The biological effects (i.e., increases in intracellular calcium ([Ca2+]i), cytokine release) of their exposure were measured in human, immortalized, tracheal-bronchial epithelial cells (BEAS-2B). Exposure of BEAS-2B cells to each fraction produced an immediate, but differential increase in [Ca2+]i and the subsequent release of the inflammatory cytokine IL-6, 4 and 16 h later. Increases in [Ca2+]i by field PM significantly correlated with

  4. Primary Culture of Porcine Pancreatic Acinar Cells

    OpenAIRE

    2001-01-01

    OBJECTIVE: To develop a method for the primary culture of porcine pancreatic acinar cells. INTERVENTIONS: Dispersed pancreatic acinar cells available utilizing RPMI-1640 medium containing collagenase III. After purification, the isolated acinar cells were cultured in RPMI-1640 medium with the addition of 2.5% fetal bovine serum. MAIN OUTCOME MEASURES: The morphological characteristics of acinar cells were described. (3)H-thymidine incorporation of acinar cells and the activity of amylase or l...

  5. Differences in cytotoxicity versus pro-inflammatory potency of different PM fractions in human epithelial lung cells.

    Science.gov (United States)

    Gualtieri, Maurizio; Øvrevik, Johan; Holme, Jørn A; Perrone, M Grazia; Bolzacchini, Ezio; Schwarze, Per E; Camatini, Marina

    2010-02-01

    Air pollution in Milan causes health concern due to the high concentrations of particulate matter (PM10 and PM2.5). The aim of this study was to investigate possible seasonal differences in PM10 and PM2.5 chemical composition and their biological effects on pro-inflammatory cytokine release and cytotoxicity. The PM was sampled during winter and summer seasons. The winter PMs had higher levels of PAHs than the summer samples which contained a greater amount of mineral dust elements. The PM toxicity was tested in the human pulmonary epithelial cell lines BEAS-2B and A549. The winter PMs were more cytotoxic than summer samples, whereas the summer PM10 exhibited a higher pro-inflammatory potential, as measured by ELISA. This inflammatory potential seemed partly due to biological components such as bacterial lipopolysaccharides (LPS), as evaluated by the use of Polymixin B. Interestingly, in the BEAS-2B cells the winter PM2.5 reduced proliferation due to a mitotic delay/arrest, while no such effects were observed in the A549 cells. These results underline that the in vitro responsiveness to PM may be cell line dependent and suggest that the PM different properties may trigger different endpoints such as inflammation, perturbation of cell cycle and cell death.

  6. Culture of Cells from Amphibian Embryos.

    Science.gov (United States)

    Stanisstreet, Martin

    1983-01-01

    Describes a method for in vitro culturing of cells from amphibian early embryos. Such cells can be used to demonstrate such properties of eukaryote cells as cell motility, adhesion, differentiation, and cell sorting into tissues. The technique may be extended to investigate other factors. (Author/JN)

  7. Physico-chemical properties based differential toxicity of graphene oxide/reduced graphene oxide in human lung cells mediated through oxidative stress

    Science.gov (United States)

    Mittal, Sandeep; Kumar, Veeresh; Dhiman, Nitesh; Chauhan, Lalit Kumar Singh; Pasricha, Renu; Pandey, Alok Kumar

    2016-12-01

    Goraphene derivatives (GD) are currently being evaluated for technological and biomedical applications owing to their unique physico-chemical properties over other carbon allotrope such as carbon nanotubes (CNTs). But, the possible association of their properties with underlying in vitro effects have not fully examined. Here, we assessed the comparative interaction of three GD - graphene oxide (GO), thermally reduced GO (TRGO) and chemically reduced GO (CRGO), which significantly differ in their lateral size and functional groups density, with phenotypically different human lung cells; bronchial epithelial cells (BEAS-2B) and alveolar epithelial cells (A549). The cellular studies demonstrate that GD significantly ineternalize and induce oxidative stress mediated cytotoxicity in both cells. The toxicity intensity was in line with the reduced lateral size and increased functional groups revealed more toxicity potential of TRGO and GO respectively. Further, A549 cells showed more susceptibility than BEAS-2B which reflected cell type dependent differential cellular response. Molecular studies revealed that GD induced differential cell death mechanism which was efficiently prevented by their respective inhibitors. This is prior study to the best of our knowledge involving TRGO for its safety evaluation which provided invaluable information and new opportunities for GD based biomedical applications.

  8. Primary Culture of Porcine Pancreatic Acinar Cells

    Directory of Open Access Journals (Sweden)

    Zhao X

    2001-03-01

    Full Text Available OBJECTIVE: To develop a method for the primary culture of porcine pancreatic acinar cells. INTERVENTIONS: Dispersed pancreatic acinar cells available utilizing RPMI-1640 medium containing collagenase III. After purification, the isolated acinar cells were cultured in RPMI-1640 medium with the addition of 2.5% fetal bovine serum. MAIN OUTCOME MEASURES: The morphological characteristics of acinar cells were described. (3H-thymidine incorporation of acinar cells and the activity of amylase or lipase were determined during the culture process. RESULTS: There were no remarkable morphological changes in the pancreatic acinar cells during the 20 days culture. The acini showed a tendency to gather but did not attach to the walls of the culture disks. A good (3H-thymidine incorporation of acinar cells in the primary culture was maintained. The secretion of amylase or lipase from the acini decreased with the length of time of the culture. DISCUSSION: The primary culture of acinar cells from a porcine pancreas which was carried out in this study maintained the normal morphology of the acinar cells and their ability to grow but not their secretion of amylase or lipase. The method would benefit by the further experiments on acini of porcine pancreas.

  9. Embryonic Stem Cells: Isolation, Characterization and Culture

    Science.gov (United States)

    Amit, Michal; Itskovitz-Eldor, Joseph

    Embryonic stem cells are pluripotent cells isolated from the mammalian blastocyst. Traditionally, these cells have been derived and cultured with mouse embryonic fibroblast (MEF) supportive layers, which allow their continuous growth in an undifferentiated state. However, for any future industrial or clinical application hESCs should be cultured in reproducible, defined, and xeno-free culture system, where exposure to animal pathogens is prevented. From their derivation in 1998 the methods for culturing hESCs were significantly improved. This chapter wills discuss hESC characterization and the basic methods for their derivation and maintenance.

  10. Cell Suspension Culture of Neem Tree

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    The establishment of suspension culture system for neem (Azadirachta indica A. Juss) cells and the suspension culture condition was studied. It shows that the neem cell suspension culture system was best in B5 liquid medium, 2.0~4.0mg/L NAA with direct spill method. Based on the integrated analysis of cell biomass, Azadirachtin content and productivity, the optimum culture conditions were B5 liquid medium, 2.0-4.0 mg/L NAA, 3% sucrose at 25 ℃. The optimum rotating speed of the shaker and broth content d...

  11. Analysis of miRNA profiles identified miR-196a as a crucial mediator of aberrant PI3K/AKT signaling in lung cancer cells.

    Science.gov (United States)

    Guerriero, Ilaria; D'Angelo, Daniela; Pallante, Pierlorenzo; Santos, Mafalda; Scrima, Marianna; Malanga, Donatella; De Marco, Carmela; Ravo, Maria; Weisz, Alessandro; Laudanna, Carmelo; Ceccarelli, Michele; Falco, Geppino; Rizzuto, Antonia; Viglietto, Giuseppe

    2016-11-17

    Hyperactivation of the PI3K/AKT pathway is observed in most human cancer including lung carcinomas. Here we have investigated the role of miRNAs as downstream targets of activated PI3K/AKT signaling in Non Small Cell Lung Cancer (NSCLC). To this aim, miRNA profiling was performed in human lung epithelial cells (BEAS-2B) expressing active AKT1 (BEAS-AKT1-E17K), active PI3KCA (BEAS-PIK3CA-E545K) or with silenced PTEN (BEAS-shPTEN).Twenty-four differentially expressed miRNAs common to BEAS-AKT1-E17K, BEAS-PIK3CA-E545K and BEAS-shPTEN cells were identified through this analysis, with miR-196a being the most consistently up-regulated miRNA. Interestingly, miR-196a was significantly overexpressed also in human NSCLC-derived cell lines (n=11) and primary lung cancer samples (n=28).By manipulating the expression of miR-196a in BEAS-2B and NCI-H460 cells, we obtained compelling evidence that this miRNA acts downstream the PI3K/AKT pathway, mediating some of the proliferative, pro-migratory and tumorigenic activity that this pathway exerts in lung epithelial cells, possibly through the regulation of FoxO1, CDKN1B (hereafter p27) and HOXA9.

  12. Emissions from commercial-grade charbroiling meat operations induce oxidative stress and inflammatory responses in human bronchial epithelial cells

    Directory of Open Access Journals (Sweden)

    Ning Li

    2014-01-01

    Full Text Available Commercial charbroiling emissions are a significant source of ambient particulate matter (PM in urban settings. The objective of this study was to determine whether organic extract of PM emissions from commercial charbroiling meat operations could induce an inflammatory response in human bronchial epithelial cells and whether this effect was mediated by oxidative stress. PM samples were collected during cooking hamburgers on a commercial-grade under-fired charbroiler and sequentially extracted with water and methanol to obtain the aqueous PM suspension (AqPM and organic extract (OE. The pro-oxidative and pro-inflammatory effects of OE were assessed using human bronchial epithelial cell line BEAS-2B. While AqPM did not have any effect, OE effectively induced the expression of heme oxygennase-1 and cyclooxygenase-2 in BEAS-2B cells. OE also up-regulated the levels of IL-6, IL-8, and prostaglandin E2. OE-induced cellular inflammatory response could be effectively suppressed by the antioxidant N-acetyl cysteine, nuclear factor (erythroid-derived 2-like 2 activator sulforaphane and p38 MAPK inhibitor SB203580. In conclusion, organic chemicals emitted from commercial charbroiling meat operations could induce an inflammatory response in human bronchial epithelial cells, which was mediated by oxidative stress and p38 MAPK.

  13. [Effects of beryllium chloride on cultured cells].

    Science.gov (United States)

    Sakaguchi, T; Sakaguchi, S; Nakamura, I; Kagami, M

    1984-05-01

    The effects of beryllium on cultured cells were investigated. Three cell-lines (HeLa-S3, Vero, HEL-R66) were used in these experiments and they were cultured in Eagle's MEM plus 5 or 10% FBS (Fetal Bovine Serum) containing beryllium in various concentrations. HeLa cells or Vero cells were able to grow in the medium with 10 micrograms Be/ml (1.1 mM). On the other hand, the growth of HEL cells were strongly inhibited, even when cultured in the medium with 1 microgram Be/ml (1.1 X 10(-1) mM) and the number of living cells showed markedly low level as compared to that of the control samples cultured in the medium without beryllium. The cytotoxic effects of beryllium on these cells, which were cultured for three days in the medium with beryllium, were observed. None of cytotoxic effects were found on HeLa cells cultured with 0.5 micrograms/ml (5.5 X 10(-2) mM) and on Vero cells cultured with 0.05 micrograms Be/ml (5.5 X 10(-3) mM), while HEL cells received cytotoxic effects even when cultured in the medium containing 0.05 micrograms Be/ml (5.5 X 10(-3) mM), and these effects on the cells appeared strong when cultured in the medium without FBS. It was revealed from these experiments that HEL cells are very sensitive in terms of toxic effects of beryllium. Therefore, there cells can be used for the toxicological study on low level concentrations of the metal.

  14. Dynamic culture improves cell reprogramming efficiency.

    Science.gov (United States)

    Sia, Junren; Sun, Raymond; Chu, Julia; Li, Song

    2016-06-01

    Cell reprogramming to pluripotency is an inefficient process and various approaches have been devised to improve the yield of induced pluripotent stem cells. However, the effect of biophysical factors on cell reprogramming is not well understood. Here we showed that, for the first time, dynamic culture with orbital shaking significantly improved the reprogramming efficiency in adherent cells. Manipulating the viscosity of the culture medium suggested that the improved efficiency is mainly attributed to convective mixing rather than hydrodynamic shear stress. Temporal studies demonstrated that the enhancement of reprogramming efficiency required the dynamic culture in the middle but not early phase. In the early phase, fibroblasts had a high proliferation rate, but as the culture became over-confluent in the middle phase, expression of p57 was upregulated to inhibit cell proliferation and consequently, cell reprogramming. Subjecting the over confluent culture to orbital shaking prevented the upregulation of p57, thus improving reprogramming efficiency. Seeding cells at low densities to avoid over-confluency resulted in a lower efficiency, and optimal reprogramming efficiency was attained at a high seeding density with dynamic culture. Our findings provide insight into the underlying mechanisms of how dynamic culture condition regulate cell reprogramming, and will have broad impact on cell engineering for regenerative medicine and disease modeling.

  15. New culture medium concepts for cell transplantation.

    Science.gov (United States)

    Lee, S; Kim, B Y; Yeo, J E; Nemeno, J G; Jo, Y H; Yang, W; Nam, B M; Namoto, S; Tanaka, S; Sato, M; Lee, K M; Hwang, H S; Lee, J I

    2013-10-01

    Before cell or tissue transplantation, cells or tissues have to be maintained for a certain period in vitro using culture medium and methods. Most culture media contain substances such as pH indicators and buffers. It is not known whether some of these substances are safe for subsequent application in the transplantation of cells or tissues into the human body. We investigated culture media and methods with respect to the safety of the components in future transplantation applications. A modified culture medium--medical fluid-based culture medium (FCM)--was designed by using various fluids and injectable drugs that are already currently permitted for use in clinical medicine. Medium components necessary for optimal cell growth were obtained from approved drugs. FCM was manufactured with adjusted final concentrations of the medium components similar to those in commercial Dulbecco's modified Eagle's medium (DMEM). In particular, 1029.40 mg/L amino acids, approximately 88.85 mg/L vitamins, 13,525.77 mg/L inorganic salts, and 4500 mg/L D-glucose comprise the high-glucose FCM. Next, human fat synovium-derived mesenchymal stem cells and rat H9c2 (2-1) cells were cultured under 2 conditions: (1) DMEM-high glucose (HG), an original commercial medium, and (2) optimized FCM-HG. We assessed the morphologies and proliferation rates of these cells. We observed that FCM-HG was able to induce the growth of FS-MSC and commercially available H9c2 cell. The morphologies and proliferation patterns of these cells cultured under FCM-HG showed no differences compared with cells grown in DMEM-HG. Our data suggest that FCM, which we developed for the first time according to the concept of drug repositioning, was a useful culture medium, especially in cultured cells intended for human cell transplantation. Crown Copyright © 2013. Published by Elsevier Inc. All rights reserved.

  16. Autofluorescence of viable cultured mammalian cells.

    Science.gov (United States)

    Aubin, J E

    1979-01-01

    The autofluorescence other than intrinsic protein emission of viable cultured mammalian cells has been investigated. The fluorescence was found to originate in discrete cytoplasmic vesicle-like regions and to be absent from the nucleus. Excitation and emission spectra of viable cells revealed at least two distinct fluorescent species. Comparison of cell spectra with spectra of known cellular metabolites suggested that most, if not all, of the fluorescence arises from intracellular nicotinamide adenine dinucleotide (NADH) and riboflavin and flavin coenzymes. Various changes in culture conditions did not affect the observed autofluorescence intensity. A multiparameter flow system (MACCS) was used to compare the fluorescence intensities of numerous cultured mammalian cells.

  17. Emulsions Containing Perfluorocarbon Support Cell Cultures

    Science.gov (United States)

    Ju, Lu-Kwang; Lee, Jaw Fang; Armiger, William B.

    1990-01-01

    Addition of emulsion containing perfluorocarbon liquid to aqueous cell-culture medium increases capacity of medium to support mammalian cells. FC-40 Fluorinert (or equivalent) - increases average density of medium so approximately equal to that of cells. Cells stay suspended in medium without mechanical stirring, which damages them. Increases density enough to prevent cells from setting, and increases viscosity of medium so oxygen bubbled through it and nutrients stirred in with less damage to delicate cells.

  18. Cell culture processes for monoclonal antibody production

    OpenAIRE

    LI Feng; Vijayasankaran, Natarajan; Shen, Amy (Yijuan); Kiss, Robert; Amanullah, Ashraf

    2010-01-01

    Animal cell culture technology has advanced significantly over the last few decades and is now generally considered a reliable, robust and relatively mature technology. A range of biotherapeutics are currently synthesized using cell culture methods in large scale manufacturing facilities that produce products for both commercial use and clinical studies. The robust implementation of this technology requires optimization of a number of variables, including (1) cell lines capable of synthesizin...

  19. Toluene diisocyanate (TDI) induces production of inflammatory cytokines and chemokines by bronchial epithelial cells via the epidermal growth factor receptor and p38 mitogen-activated protein kinase pathways.

    Science.gov (United States)

    Ogawa, Hirohisa; Inoue, Shizuka; Ogushi, Fumitaka; Ogura, Hideo; Nakamura, Yoichi

    2006-01-01

    Toluene diisocyanate (TDI) is known as one of causes of occupational asthma and hypersensitivity pneumonitis. To investigate the stimulatory effect on bronchial epithelial cells in response to TDI, the authors examined production of cytokines by the bronchial epithelial cell line BEAS-2B and intercellular signal transduction stimulated by TDI-human serum albumin (HSA) conjugate. The production of interleukin (IL)-8, granulocyte-macrophage colony-stimulating factor (GM-CSF), and regulated on activation normal T cell expressed and secreted (RANTES) from the bronchial epithelial cells were augmented by the TDI-HSA conjugate. Extracellular signal-regulated kinase (Erk) 1/2 and p38 mitogen-activated protein kinase (MAPK) were phosphorylated by the TDI-HSA conjugate. AG1478, SB203580, and dexamethasone prevented augmentation of these cytokine production. TDI-HSA conjugate did not augment release of epidermal growth factor (EGF) ligands from BEAS-2B. These results suggest that TDI directly induces production of proinflammatory cytokines and chemokines through p38 MAPK and EGF receptor (EGFR)-Erk pathway without an autocrine mechanism. Thus, TDI was shown to have a stimulatory effect on bronchial epithelial cells, suggesting the potent role of bronchial epithelial cells in TDI-induced asthma.

  20. 3D Cell Culture in Alginate Hydrogels

    Directory of Open Access Journals (Sweden)

    Therese Andersen

    2015-03-01

    Full Text Available This review compiles information regarding the use of alginate, and in particular alginate hydrogels, in culturing cells in 3D. Knowledge of alginate chemical structure and functionality are shown to be important parameters in design of alginate-based matrices for cell culture. Gel elasticity as well as hydrogel stability can be impacted by the type of alginate used, its concentration, the choice of gelation technique (ionic or covalent, and divalent cation chosen as the gel inducing ion. The use of peptide-coupled alginate can control cell–matrix interactions. Gelation of alginate with concomitant immobilization of cells can take various forms. Droplets or beads have been utilized since the 1980s for immobilizing cells. Newer matrices such as macroporous scaffolds are now entering the 3D cell culture product market. Finally, delayed gelling, injectable, alginate systems show utility in the translation of in vitro cell culture to in vivo tissue engineering applications. Alginate has a history and a future in 3D cell culture. Historically, cells were encapsulated in alginate droplets cross-linked with calcium for the development of artificial organs. Now, several commercial products based on alginate are being used as 3D cell culture systems that also demonstrate the possibility of replacing or regenerating tissue.

  1. Insect cell culture in reagent bottles.

    Science.gov (United States)

    Rieffel, S; Roest, S; Klopp, J; Carnal, S; Marti, S; Gerhartz, B; Shrestha, B

    2014-01-01

    Growing insect cells with high air space in culture vessel is common from the early development of suspension cell culture. We believed and followed it with the hope that it allows sufficient air for optimal cell growth. However, we missed to identify how much air exactly cells need for its growth and multiplication. Here we present the innovative method that changed the way we run insect cell culture. The method is easy to adapt, cost-effective and useful for both academic and industrial research labs. We believe this method will revolutionize the way we run insect cell culture by increasing throughput in a cost-effective way. In our study we identified:•Insect cells need to be in suspension; air space in culture vessel and type of culture vessel is of less importance. Shaking condition that introduces small air bubbles and maintains it in suspension for longer time provides better oxygen transfer in liquid. For this, high-fill volume in combination with speed and shaking diameter are important.•Commercially available insect cells are not fragile as original isolates. These cells can easily withstand higher shaking speed.•Growth condition in particular lab set-up needs to be optimized. The condition used in one lab may not be optimum for another lab due to different incubators from different vendors.

  2. Cell culture from sponges: pluripotency and immortality.

    Science.gov (United States)

    de Caralt, Sònia; Uriz, María J; Wijffels, René H

    2007-10-01

    Sponges are a source of compounds with potential pharmaceutical applications. In this article, methods of sponge cell culture for production of these bioactive compounds are reviewed, and new approaches for overcoming the problem of metabolite supply are examined. The use of embryos is proposed as a new source of sponge material for cell culture. Stem cells are present in high amounts in embryos and are more versatile and resistant to infections than adult cells. Additionally, genetic engineering and cellular research on apoptotic mechanisms are promising new fields that might help to improve cell survival in sponge-cell lines. We propose that one topic for future research should be how to reduce apoptosis, which appears to be very high in sponge cell cultures.

  3. Safe-by-Design CuO Nanoparticles via Fe-Doping, Cu-O Bond Length Variation, and Biological Assessment in Cells and Zebrafish Embryos.

    Science.gov (United States)

    Naatz, Hendrik; Lin, Sijie; Li, Ruibin; Jiang, Wen; Ji, Zhaoxia; Chang, Chong Hyun; Köser, Jan; Thöming, Jorg; Xia, Tian; Nel, Andre E; Mädler, Lutz; Pokhrel, Suman

    2017-01-24

    The safe implementation of nanotechnology requires nanomaterial hazard assessment in accordance with the material physicochemical properties that trigger the injury response at the nano/bio interface. Since CuO nanoparticles (NPs) are widely used industrially and their dissolution properties play a major role in hazard potential, we hypothesized that tighter bonding of Cu to Fe by particle doping could constitute a safer-by-design approach through decreased dissolution. Accordingly, we designed a combinatorial library in which CuO was doped with 1-10% Fe in a flame spray pyrolysis reactor. The morphology and structural properties were determined by XRD, BET, Raman spectroscopy, HRTEM, EFTEM, and EELS, which demonstrated a significant reduction in the apical Cu-O bond length while simultaneously increasing the planar bond length (Jahn-Teller distortion). Hazard screening was performed in tissue culture cell lines and zebrafish embryos to discern the change in the hazardous effects of doped vs nondoped particles. This demonstrated that with increased levels of doping there was a progressive decrease in cytotoxicity in BEAS-2B and THP-1 cells, as well as an incremental decrease in the rate of hatching interference in zebrafish embryos. The dissolution profiles were determined and the surface reactions taking place in Holtfreter's solution were validated using cyclic voltammetry measurements to demonstrate that the Cu(+)/Cu(2+) and Fe(2+)/Fe(3+) redox species play a major role in the dissolution process of pure and Fe-doped CuO. Altogether, a safe-by-design strategy was implemented for the toxic CuO particles via Fe doping and has been demonstrated for their safe use in the environment.

  4. Rotating cell culture systems for human cell culture: human trophoblast cells as a model.

    Science.gov (United States)

    Zwezdaryk, Kevin J; Warner, Jessica A; Machado, Heather L; Morris, Cindy A; Höner zu Bentrup, Kerstin

    2012-01-18

    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 lines support our understanding of invasion of the uterine wall and remodeling of uterine spiral arteries 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 counterparts. 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 differentiation. 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 assemblies. 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

  5. Porcine mitral valve interstitial cells in culture.

    Science.gov (United States)

    Lester, W; Rosenthal, A; Granton, B; Gotlieb, A I

    1988-11-01

    There are connective tissue cells present within the interstitium of the heart valves. This study was designed to isolate and characterize mitral valve interstitial cells from the anterior leaflet of the mitral valve. Explants obtained from the distal part of the leaflet, having been scraped free of surface endocardial cells, were incubated in medium 199 supplemented with 10% fetal bovine serum. Cells grew out of the explant after 3 to 5 days and by 3 weeks these cells were harvested and passaged. Passages 1 to 22 were characterized in several explant sets. The cells showed a growth pattern reminiscent of fibroblasts. Growth was dependent on serum concentration. Cytoskeletal localization of actin and myosin showed prominent stress fibers. Ultrastructural studies showed many elongated cells with prominent stress fibers and some gap junctions and few adherens junctions. There were as well cells with fewer stress fibers containing prominent Golgi complex and dilated endoplasmic reticulum. In the multilayered superconfluent cultures, the former cells tended to be on the substratum of the dish or surface of the multilayered culture, whereas the latter was generally located within the layer of cells. Extracellular matrix was prominent in superconfluent cultures, often within the layers as well. Labeling of the cells with antibody HHF 35 (Tsukada T, Tippens D, Gordon D, Ross R, Gown AM: Am J Pathol 126:51, 1987), which recognizes smooth muscle cell actin, showed prominent staining of the elongated stress fiber-containing cells and much less in the secretory type cells. These studies show that interstitial mitral valve cells can be grown in culture and that either two different cell types or one cell type with two phenotypic expressions is present in culture.

  6. Cell culture processes for monoclonal antibody production.

    Science.gov (United States)

    Li, Feng; Vijayasankaran, Natarajan; Shen, Amy Yijuan; Kiss, Robert; Amanullah, Ashraf

    2010-01-01

    Animal cell culture technology has advanced significantly over the last few decades and is now generally considered a reliable, robust and relatively mature technology. A range of biotherapeutics are currently synthesized using cell culture methods in large scale manufacturing facilities that produce products for both commercial use and clinical studies. The robust implementation of this technology requires optimization of a number of variables, including 1) cell lines capable of synthesizing the required molecules at high productivities that ensure low operating cost; 2) culture media and bioreactor culture conditions that achieve both the requisite productivity and meet product quality specifications; 3) appropriate on-line and off-line sensors capable of providing information that enhances process knowledge; and 4) good understanding of culture performance at different scales to ensure smooth scale-up. Successful implementation also requires appropriate strategies for process development, scale-up and process characterization and validation that enable robust operation that is compliant with current regulations. This review provides an overview of the state-of-the art technology in key aspects of cell culture, e.g., engineering of highly productive cell lines and optimization of cell culture process conditions. We also summarize the current thinking on appropriate process development strategies and process advances that might affect process development.

  7. Cell culture processes for monoclonal antibody production

    Science.gov (United States)

    Li, Feng; Vijayasankaran, Natarajan; Shen, Amy (Yijuan); Kiss, Robert

    2010-01-01

    Animal cell culture technology has advanced significantly over the last few decades and is now generally considered a reliable, robust and relatively mature technology. A range of biotherapeutics are currently synthesized using cell culture methods in large scale manufacturing facilities that produce products for both commercial use and clinical studies. The robust implementation of this technology requires optimization of a number of variables, including (1) cell lines capable of synthesizing the required molecules at high productivities that ensure low operating cost; (2) culture media and bioreactor culture conditions that achieve both the requisite productivity and meet product quality specifications; (3) appropriate on-line and off-line sensors capable of providing information that enhances process control; and (4) good understanding of culture performance at different scales to ensure smooth scale-up. Successful implementation also requires appropriate strategies for process development, scale-up and process characterization and validation that enable robust operation and ensure compliance with current regulations. This review provides an overview of the state-of-the art technology in key aspects of cell culture, e.g., generation of highly productive cell lines and optimization of cell culture process conditions. We also summarize the current thinking on appropriate process development strategies and process advances that might affect process development. PMID:20622510

  8. Hyperoside induces both autophagy and apoptosis in non-small cell lung cancer cells in vitro.

    Science.gov (United States)

    Fu, Ting; Wang, Ling; Jin, Xiang-nan; Sui, Hai-juan; Liu, Zhou; Jin, Ying

    2016-04-01

    Hyperoside (quercetin-3-O-β-D-galactopyranoside) is a flavonol glycoside found in plants of the genera Hypericum and Crataegus, which exhibits anticancer, anti-oxidant, and anti-inflammatory activities. In this study we investigated whether autophagy was involved in the anticancer mechanisms of hyperoside in human non-small cell lung cancer cells in vitro. Human non-small cell lung cancer cell line A549 was tested, and human bronchial epithelial cell line BEAS-2B was used for comparison. The expression of LC3-II, apoptotic and signaling proteins was measured using Western blotting. Autophagosomes were observed with MDC staining, LC3 immunocytochemistry, and GFP-LC3 fusion protein techniques. Cell viability was assessed using MTT assay. Hyperoside (0.5, 1, 2 mmol/L) dose-dependently increased the expression of LC3-II and autophagosome numbers in A549 cells, but had no such effects in BEAS-2B cells. Moreover, hyperoside dose-dependently inhibited the phosphorylation of Akt, mTOR, p70S6K and 4E-BP1, but increased the phosphorylation of ERK1/2 in A549 cells. Insulin (200 nmol/L) markedly enhanced the phosphorylation of Akt and decreased LC3-II expression in A549 cells, which were reversed by pretreatment with hyperoside, whereas the MEK1/2 inhibitor U0126 (20 μmol/L) did not blocked hyperoside-induced LC3-II expression. Finally, hyperoside dose-dependently suppressed the cell viability and induced apoptosis in A549 cells, which were significantly attenuated by pretreatment with the autophagy inhibitor 3-methyladenine (2.5 mmol/L). Hyperoside induces both autophagy and apoptosis in human non-small cell lung cancer cells in vitro. The autophagy is induced through inhibiting the Akt/mTOR/p70S6K signal pathways, which contributes to anticancer actions of hyperoside.

  9. Isolation of mitochondria from tissue culture cells.

    Science.gov (United States)

    Clayton, David A; Shadel, Gerald S

    2014-10-01

    The number of mitochondria per cell varies substantially from cell line to cell line. For example, human HeLa cells contain at least twice as many mitochondria as smaller mouse L cells. This protocol starts with a washed cell pellet of 1-2 mL derived from ∼10⁹ cells grown in culture. The cells are swollen in a hypotonic buffer and ruptured with a Dounce or Potter-Elvehjem homogenizer using a tight-fitting pestle, and mitochondria are isolated by differential centrifugation. © 2014 Cold Spring Harbor Laboratory Press.

  10. Cross-contamination of a UROtsa stock with T24 cells--molecular comparison of different cell lines and stocks.

    Directory of Open Access Journals (Sweden)

    Georg Johnen

    Full Text Available BACKGROUND: UROtsa is an authentic, immortalized human urothelial cell line that is used to study the effects of metals and other toxic substances, mostly in the context of bladder cancer carcinogenesis. Unusual properties on the molecular level of a provided UROtsa cell line stock prompted us to verify its identity. METHODS: UROtsa cell line stocks from different sources were tested on several molecular levels and compared with other cell lines. MicroRNA and mRNA expression was determined by Real-Time PCR. Chromosome numbers were checked and PCR of different regions of the large T-antigen was performed. DNA methylation of RARB, PGR, RASSF1, CDH1, FHIT, ESR1, C1QTNF6, PTGS2, SOCS3, MGMT, and LINE1 was analyzed by pyrosequencing and compared with results from the cell lines RT4, T24, HeLa, BEAS-2B, and HepG2. Finally, short tandem repeat (STR profiling was applied. RESULTS: All tested UROtsa cell line stocks lacked large T-antigen. STR analysis unequivocally identified our main UROtsa stock as the bladder cancer cell line T24, which was different from two authentic UROtsa stocks that served as controls. Analysis of DNA methylation patterns and RNA expression confirmed their differences. Methylation pattern and mRNA expression of the contaminating T24 cell line showed moderate changes even after long-term culture of up to 56 weeks, whereas miRNAs and chromosome numbers varied markedly. CONCLUSIONS: It is important to check the identity of cell lines, especially those that are not distributed by major cell banks. However, for some cell lines STR profiles are not available. Therefore, new cell lines should either be submitted to cell banks or at least their STR profile determined and published as part of their initial characterization. Our results should help to improve the identification of UROtsa and other cells on different molecular levels and provide information on the use of urothelial cells for long-term experiments.

  11. Diatom-derived polyunsaturated aldehydes activate cell death in human cancer cell lines but not normal cells.

    Directory of Open Access Journals (Sweden)

    Clementina Sansone

    Full Text Available Diatoms are an important class of unicellular algae that produce bioactive polyunsaturated aldehydes (PUAs that induce abortions or malformations in the offspring of invertebrates exposed to them during gestation. Here we compare the effects of the PUAs 2-trans,4-trans-decadienal (DD, 2-trans,4-trans-octadienal (OD and 2-trans,4-trans-heptadienal (HD on the adenocarcinoma cell lines lung A549 and colon COLO 205, and the normal lung/brunch epithelial BEAS-2B cell line. Using the viability MTT/Trypan blue assays, we show that PUAs have a toxic effect on both A549 and COLO 205 tumor cells but not BEAS-2B normal cells. DD was the strongest of the three PUAs tested, at all time-intervals considered, but HD was as strong as DD after 48 h. OD was the least active of the three PUAs. The effect of the three PUAs was somewhat stronger for A549 cells. We therefore studied the death signaling pathway activated in A549 showing that cells treated with DD activated Tumor Necrosis Factor Receptor 1 (TNFR1 and Fas Associated Death Domain (FADD leading to necroptosis via caspase-3 without activating the survival pathway Receptor-Interacting Protein (RIP. The TNFR1/FADD/caspase pathway was also observed with OD, but only after 48 h. This was the only PUA that activated RIP, consistent with the finding that OD causes less damage to the cell compared to DD and HD. In contrast, cells treated with HD activated the Fas/FADD/caspase pathway. This is the first report that PUAs activate an extrinsic apoptotic machinery in contrast to other anticancer drugs that promote an intrinsic death pathway, without affecting the viability of normal cells from the same tissue type. These findings have interesting implications also from the ecological viewpoint considering that HD is one of the most common PUAs produced by diatoms.

  12. Diatom-derived polyunsaturated aldehydes activate cell death in human cancer cell lines but not normal cells.

    Science.gov (United States)

    Sansone, Clementina; Braca, Alessandra; Ercolesi, Elena; Romano, Giovanna; Palumbo, Anna; Casotti, Raffaella; Francone, Maria; Ianora, Adrianna

    2014-01-01

    Diatoms are an important class of unicellular algae that produce bioactive polyunsaturated aldehydes (PUAs) that induce abortions or malformations in the offspring of invertebrates exposed to them during gestation. Here we compare the effects of the PUAs 2-trans,4-trans-decadienal (DD), 2-trans,4-trans-octadienal (OD) and 2-trans,4-trans-heptadienal (HD) on the adenocarcinoma cell lines lung A549 and colon COLO 205, and the normal lung/brunch epithelial BEAS-2B cell line. Using the viability MTT/Trypan blue assays, we show that PUAs have a toxic effect on both A549 and COLO 205 tumor cells but not BEAS-2B normal cells. DD was the strongest of the three PUAs tested, at all time-intervals considered, but HD was as strong as DD after 48 h. OD was the least active of the three PUAs. The effect of the three PUAs was somewhat stronger for A549 cells. We therefore studied the death signaling pathway activated in A549 showing that cells treated with DD activated Tumor Necrosis Factor Receptor 1 (TNFR1) and Fas Associated Death Domain (FADD) leading to necroptosis via caspase-3 without activating the survival pathway Receptor-Interacting Protein (RIP). The TNFR1/FADD/caspase pathway was also observed with OD, but only after 48 h. This was the only PUA that activated RIP, consistent with the finding that OD causes less damage to the cell compared to DD and HD. In contrast, cells treated with HD activated the Fas/FADD/caspase pathway. This is the first report that PUAs activate an extrinsic apoptotic machinery in contrast to other anticancer drugs that promote an intrinsic death pathway, without affecting the viability of normal cells from the same tissue type. These findings have interesting implications also from the ecological viewpoint considering that HD is one of the most common PUAs produced by diatoms.

  13. Culture and transfection of axolotl cells.

    Science.gov (United States)

    Denis, Jean-François; Sader, Fadi; Ferretti, Patrizia; Roy, Stéphane

    2015-01-01

    The use of cells grown in vitro has been instrumental for multiple aspects of biomedical research and especially molecular and cellular biology. The ability to grow cells from multicellular organisms like humans, squids, or salamanders is important to simplify the analyses and experimental designs to help understand the biology of these organisms. The advent of the first cell culture has allowed scientists to tease apart the cellular functions, and in many situations these experiments help understand what is happening in the whole organism. In this chapter, we describe techniques for the culture and genetic manipulation of an established cell line from axolotl, a species widely used for studying epimorphic regeneration.

  14. Culture of Mouse Neural Stem Cell Precursors

    OpenAIRE

    Currle, D. Spencer; Hu, Jia Sheng; Kolski-Andreaco, Aaron; Monuki, Edwin S

    2007-01-01

    Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase our understanding of the nervous system and may allow us to develop treatments for currently incurable brain diseases and injuries. In addition, stem cells should be used for stem cell research aimed at the detailed study of mechanisms of neural differentiation and transdifferentiation and the genetic and environmental signals that direct the...

  15. Wound Coverage by Cultured Skin Cells

    Science.gov (United States)

    1988-11-01

    and spread. 6 We later coated collagen sponges with human or porcine plasma. Although this coating improved the plating of epidermal cells, it did not...healing by cultured epidermal grafts, we have found that: - We were able to grow epidermal cells on collapsed collagen sponges . As a result, we can create...plastic. Epidermal cells grown on collagen sponges formed four to five layers of nucleated cells, compared to only one layer on plastic surfaces. The use of

  16. Sponge cell culture? A molecular identification method for sponge cells

    NARCIS (Netherlands)

    Sipkema, D.; Heilig, G.H.J.; Akkermans, A.D.L.; Osinga, R.; Tramper, J.; Wijffels, R.H.

    2003-01-01

    Dissociated sponge cells are easily confused with unicellular organisms. This has been an obstacle in the development of sponge-cell lines. We developed a molecular detection method to identify cells of the sponge Dysidea avara in dissociated cell cultures. The 18S ribosomal RNA gene from a Dysidea

  17. Primary cell cultures of bovine colon epithelium: isolation and cell culture of colonocytes.

    Science.gov (United States)

    Föllmann, W; Weber, S; Birkner, S

    2000-10-01

    Epithelial cells from bovine colon were isolated by mechanical preparation combined with an enzymatic digestion from colon specimens derived from freshly slaughtered animals. After digestion with collagenase I, the isolated tissue was centrifuged on a 2% D-sorbitol gradient to separate epithelial crypts which were seeded in collagen I-coated culture flasks. By using colon crypts and omitting the seeding of single cells a contamination by fibroblasts was prevented. The cells proliferated under the chosen culture conditions and formed monolayer cultures which were maintained for several weeks, including subcultivation steps. A population doubling time of about 21 hr was estimated in the log phase of the corresponding growth curve. During the culture period the cells were characterized morphologically and enzymatically. By using antibodies against cytokeratine 7 and 13 the isolated cells were identified as cells of epithelial origin. Antibodies against vimentin served as negative control. Morphological features such as microvilli, desmosomes and tight junctions, which demonstrated the ability of the cultured cells to restore an epithelial like monolayer, were shown by ultrastructural investigations. The preservation of the secretory function of the cultured cells was demonstrated by mucine cytochemistry with alcian blue staining. A stable expression of enzyme activities over a period of 6 days in culture occurred for gamma-glutamyltranspeptidase, acid phosphatase and NADH-dehydrogenase activity under the chosen culture conditions. Activity of alkaline phosphatase decreased to about 50% of basal value after 6 days in culture. Preliminary estimations of the metabolic competence of these cells revealed cytochrome P450 1A1-associated EROD activity in freshly isolated cells which was stable over 5 days in cultured cells. Then activity decreased completely. This culture system with primary epithelial cells from the colon will be used further as a model for the colon

  18. Human cell culture in a space bioreactor

    Science.gov (United States)

    Morrison, Dennis R.

    1988-01-01

    Microgravity offers new ways of handling fluids, gases, and growing mammalian cells in efficient suspension cultures. In 1976 bioreactor engineers designed a system using a cylindrical reactor vessel in which the cells and medium are slowly mixed. The reaction chamber is interchangeable and can be used for several types of cell cultures. NASA has methodically developed unique suspension type cell and recovery apparatus culture systems for bioprocess technology experiments and production of biological products in microgravity. The first Space Bioreactor was designed for microprocessor control, no gaseous headspace, circulation and resupply of culture medium, and slow mixing in very low shear regimes. Various ground based bioreactors are being used to test reactor vessel design, on-line sensors, effects of shear, nutrient supply, and waste removal from continuous culture of human cells attached to microcarriers. The small Bioreactor is being constructed for flight experiments in the Shuttle Middeck to verify systems operation under microgravity conditions and to measure the efficiencies of mass transport, gas transfer, oxygen consumption and control of low shear stress on cells.

  19. Flux analysis of mammalian cell culture

    NARCIS (Netherlands)

    Martens, D.E.; Tramper, J.

    2010-01-01

    Animal cells are used for the production of vaccines and pharmaceutical proteins. The increase in demand for these products requires an increase in volumetric productivity of animal cell culture processes, which can be attained through an increase in biomass concentration and/or specific productivit

  20. Xylogenesis in zinnia (Zinnia elegans) cell cultures

    NARCIS (Netherlands)

    Iakimova, Elena T.; Woltering, Ernst J.

    2017-01-01

    Main conclusion: Physiological and molecular studies support the view that xylogenesis can largely be determined as a specific form of vacuolar programmed cell death (PCD). The studies in xylogenic zinnia cell culture have led to many breakthroughs in xylogenesis research and provided a background

  1. Flux analysis of mammalian cell culture

    NARCIS (Netherlands)

    Martens, D.E.; Tramper, J.

    2010-01-01

    Animal cells are used for the production of vaccines and pharmaceutical proteins. The increase in demand for these products requires an increase in volumetric productivity of animal cell culture processes, which can be attained through an increase in biomass concentration and/or specific

  2. Pitfalls in cell culture work with xanthohumol.

    Science.gov (United States)

    Motyl, M; Kraus, B; Heilmann, J

    2012-01-01

    Xanthohumol, the most abundant prenylated chalcone in hop (Humulus lupulus L.) cones, is well known to exert several promising pharmacological activities in vitro and in vivo. Among these, the chemopreventive, anti-inflammatory and anti-cancer effects are probably the most interesting. As xanthohumol is hardly soluble in water and able to undergo conversion to isoxanthohumol we determined several handling characteristics for cell culture work with this compound. Recovery experiments revealed that working with xanthohumol under cell culture conditions requires a minimal amount of 10% FCS to increase its solubility to reasonable concentrations (-50-75 micromol/l) for pharmacological in vitro tests. Additionally, more than 50% of xanthohumol can be absorbed to various plastic materials routinely used in the cell culture using FCS concentrations below 10%. In contrast, experiments using fluorescence microscopy in living cells revealed that detection of cellular intake of xanthohumol is hampered by concentrations above 1% FCS.

  3. Increasing cell culture population doublings for long-term growth of finite life span human cell cultures

    Science.gov (United States)

    Stampfer, Martha R; Garbe, James C

    2015-02-24

    Cell culture media formulations for culturing human epithelial cells are herein described. Also described are methods of increasing population doublings in a cell culture of finite life span human epithelial cells and prolonging the life span of human cell cultures. Using the cell culture media disclosed alone and in combination with addition to the cell culture of a compound associated with anti-stress activity achieves extended growth of pre-stasis cells and increased population doublings and life span in human epithelial cell cultures.

  4. Increasing cell culture population doublings for long-term growth of finite life span human cell cultures

    Energy Technology Data Exchange (ETDEWEB)

    Stampfer, Martha R.; Garbe, James C.

    2016-06-28

    Cell culture media formulations for culturing human epithelial cells are herein described. Also described are methods of increasing population doublings in a cell culture of finite life span human epithelial cells and prolonging the life span of human cell cultures. Using the cell culture media disclosed alone and in combination with addition to the cell culture of a compound associated with anti-stress activity achieves extended growth of pre-stasis cells and increased population doublings and life span in human epithelial cell cultures.

  5. Pinoresinol from Ipomoea cairica cell cultures.

    Science.gov (United States)

    Páska, Csilla; Innocenti, Gabbriella; Ferlin, Mariagrazia; Kunvári, Mónika; László, Miklós

    2002-10-01

    Ipomoea cairica cell cultures produced a tetrahydrofuran lignan, (+)-pinoresinol, identified by UV, IR, MS and NMR methods, not yet found in the intact plant, and new in the Convolvulaceae family. Pinoresinol was found to have antioxidant and Ca2+ antagonist properties. As it could be requested for its biological activity, we examined the possibility to raise the pinoresinol yield of I. cairica cultures, as well as we continued investigations on lignans' response to optimization.

  6. Cell culture experiments planned for the space bioreactor

    Science.gov (United States)

    Morrison, Dennis R.; Cross, John H.

    1987-01-01

    Culturing of cells in a pilot-scale bioreactor remains to be done in microgravity. An approach is presented based on several studies of cell culture systems. Previous and current cell culture research in microgravity which is specifically directed towards development of a space bioprocess is described. Cell culture experiments planned for a microgravity sciences mission are described in abstract form.

  7. 21 CFR 864.2280 - Cultured animal and human cells.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Cultured animal and human cells. 864.2280 Section... (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture Products § 864.2280 Cultured animal and human cells. (a) Identification. Cultured animal and human cells are in vitro...

  8. General overview of neuronal cell culture.

    Science.gov (United States)

    Gordon, Jennifer; Amini, Shohreh; White, Martyn K

    2013-01-01

    In this introductory chapter, we provide a general overview of neuronal cell culture. This is a rapidly evolving area of research and we provide an outline and contextual framework for the different chapters of this book. These chapters were all contributed by scientists actively working in the field who are currently using state-of-the-art techniques to advance our understanding of the molecular and cellular biology of the central nervous system. Each chapter provides detailed descriptions and experimental protocols for a variety of techniques ranging in scope from basic neuronal cell line culturing to advanced and specialized methods.

  9. A Novel Natural Product, KL-21, Inhibits Proliferation and Induces Apoptosis in Chronic Lymphocytic Leukemia Cells

    Directory of Open Access Journals (Sweden)

    Aysun Adan Gökbulut

    2015-06-01

    Full Text Available INTRODUCTION: The aims of this study were to examine the cytotoxic and apoptotic effects of KL-21, a novel plant product (produced by Naturin Natural Products, İzmir, Turkey, on 232B4 chronic lymphocytic leukemia (CLL cells and to determine the cytotoxic effects on healthy BEAS-2B human bronchial epithelial cells. METHODS: The cytotoxic effect of KL-21 was determined by MTT cell proliferation assay. Changes in caspase-3 enzyme activity were measured using the caspase-3 colorimetric assay. Changes in mitochondrial membrane potential were determined using the JC-1 dye-based method. Annexin V-FITC/PI double staining was performed to measure the apoptotic cell population. Effects of KL-21 on cell cycle profiles of CLL cells were investigated by flow cytometry. RESULTS: We detected time- and concentration-dependent increases in the cytotoxic effect of KL-21 on 232B4 CLL cells. However, we also showed that, especially at higher concentrations, KL-21 was less cytotoxic towards BEAS-2B healthy cells than towards CLL cells. Annexin-V/PI double staining results showed that the apoptotic cell population increased in 232B4 cells. Increasing concentrations of KL-21 increased caspase-3 enzyme activity and induced loss of mitochondrial membrane potential. KL-21 administration resulted in small increases in the percentage of the cells in the G0/G1 phase while it decreased the S phase cell population up to 1 mg/mL. At the highest concentration, most of the cells accumulated in the G0/G1 phase. DISCUSSION AND CONCLUSION: KL-21 has a growth-inhibitory effect on 232B4 CLL cells. KL-21 causes apoptosis and cell cycle arrest at G0/G1.

  10. Wnt-Dependent Control of Cell Polarity in Cultured Cells.

    Science.gov (United States)

    Runkle, Kristin B; Witze, Eric S

    2016-01-01

    The secreted ligand Wnt5a regulates cell polarity and polarized cell movement during development by signaling through the poorly defined noncanonical Wnt pathway. Cell polarity regulates most aspects of cell behavior including the organization of apical/basolateral membrane domains of epithelial cells, polarized cell divisions along a directional plane, and front rear polarity during cell migration. These characteristics of cell polarity allow coordinated cell movements required for tissue formation and organogenesis during embryonic development. Genetic model organisms have been used to identify multiple signaling pathways including Wnt5a that are required to establish cell polarity and regulate polarized cell behavior. However, the downstream signaling events that regulate these complex cellular processes are still poorly understood. The methods below describe assays to study Wnt5a-induced cell polarity in cultured cells, which may facilitate our understanding of these complex signaling pathways.

  11. Cytochrome P450 2A13 enhances the sensitivity of human bronchial epithelial cells to aflatoxin B1-induced DNA damage

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Xuejiao [Key Lab of Modern Toxicology of Ministry of Education, School of Public Health, Nanjing Medical University, 818 East Tiangyuan Rd., Nanjing 211166 (China); Jiaojiang District Center for Disease Control and Prevention, 518 Jingdong Rd., Taizhou 318000 (China); Zhang, Zhan; Wang, Xichen; Wang, Yun; Zhang, Xiaoming; Lu, Huiyuan [Key Lab of Modern Toxicology of Ministry of Education, School of Public Health, Nanjing Medical University, 818 East Tiangyuan Rd., Nanjing 211166 (China); Wang, Shou-Lin, E-mail: wangshl@njmu.edu.cn [Key Lab of Modern Toxicology of Ministry of Education, School of Public Health, Nanjing Medical University, 818 East Tiangyuan Rd., Nanjing 211166 (China)

    2013-07-15

    Cytochrome P450 2A13 (CYP2A13) mainly expresses in human respiratory system and mediates the metabolic activation of aflatoxin B1 (AFB1). Our previous study suggested that CYP2A13 could increase the cytotoxic and apoptotic effects of AFB1 in immortalized human bronchial epithelial cells (BEAS-2B). However, the role of CYP2A13 in AFB1-induced DNA damage is unclear. Using BEAS-2B cells that stably express CYP2A13 (B-2A13), CYP1A2 (B-1A2), and CYP2A6 (B-2A6), we compared their effects in AFB1-induced DNA adducts, DNA damage, and cell cycle changes. BEAS-2B cells that were transfected with vector (B-vector) were used as a control. The results showed that AFB1 (5–80 nM) dose- and time-dependently induced DNA damage in B-2A13 cells. AFB1 at 10 and 80 nM significantly augmented this effect in B-2A13 and B-1A2 cells, respectively. B-2A6 cells showed no obvious DNA damage, similar to B-vector cells and the vehicle control. Similarly, compared with B-vector, B-1A2 or B-2A6 cells, B-2A13 cells showed more sensitivity in AFB1-induced γH2AX expression, DNA adduct 8-hydroxy-deoxyguanosine formation, and S-phase cell-cycle arrest. Furthermore, AFB1 activated the proteins related to DNA damage responses, such as ATM, ATR, Chk2, p53, BRCA1, and H2AX, rather than the proteins related to DNA repair. These effects could be almost completely inhibited by 100 μM nicotine (a substrate of CYP2A13) or 1 μM 8-methoxypsoralen (8-MOP; an inhibitor of CYP enzyme). Collectively, these findings suggest that CYP2A13 plays an important role in low-concentration AFB1-induced DNA damage, possibly linking environmental airborne AFB1 to genetic injury in human respiratory system. - Highlights: • CYP2A13 plays a critical role in low concentration of AFB1-induced DNA damage. • B-2A13 cells were more sensitive to AFB1 than B-1A2 cells and B-2A6 cells. • AFB1 dose- and time-dependently induced DNA damage in B-2A13 cells • AFB1-induced DNA adducts and damage can be inhibited by nicotine and 8

  12. Cell Cycle Progression of Human Cells Cultured in Rotating Bioreactor

    Science.gov (United States)

    Parks, Kelsey

    2009-01-01

    Space flight has been shown to alter the astronauts immune systems. Because immune performance is complex and reflects the influence of multiple organ systems within the host, scientists sought to understand the potential impact of microgravity alone on the cellular mechanisms critical to immunity. Lymphocytes and their differentiated immature form, lymphoblasts, play an important and integral role in the body's defense system. T cells, one of the three major types of lymphocytes, play a central role in cell-mediated immunity. They can be distinguished from other lymphocyte types, such as B cells and natural killer cells by the presence of a special receptor on their cell surface called T cell receptors. Reported studies have shown that spaceflight can affect the expression of cell surface markers. Cell surface markers play an important role in the ability of cells to interact and to pass signals between different cells of the same phenotype and cells of different phenotypes. Recent evidence suggests that cell-cycle regulators are essential for T-cell function. To trigger an effective immune response, lymphocytes must proliferate. The objective of this project is to investigate the changes in growth of human cells cultured in rotating bioreactors and to measure the growth rate and the cell cycle distribution for different human cell types. Human lymphocytes and lymphoblasts will be cultured in a bioreactor to simulate aspects of microgravity. The bioreactor is a cylindrical culture vessel that incorporates the aspects of clinostatic rotation of a solid fluid body around a horizontal axis at a constant speed, and compensates gravity by rotation and places cells within the fluid body into a sustained free-fall. Cell cycle progression and cell proliferation of the lymphocytes will be measured for a number of days. In addition, RNA from the cells will be isolated for expression of genes related in cell cycle regulations.

  13. Cell Cycle Progression of Human Cells Cultured in Rotating Bioreactor

    Science.gov (United States)

    Parks, Kelsey

    2009-01-01

    Space flight has been shown to alter the astronauts immune systems. Because immune performance is complex and reflects the influence of multiple organ systems within the host, scientists sought to understand the potential impact of microgravity alone on the cellular mechanisms critical to immunity. Lymphocytes and their differentiated immature form, lymphoblasts, play an important and integral role in the body's defense system. T cells, one of the three major types of lymphocytes, play a central role in cell-mediated immunity. They can be distinguished from other lymphocyte types, such as B cells and natural killer cells by the presence of a special receptor on their cell surface called T cell receptors. Reported studies have shown that spaceflight can affect the expression of cell surface markers. Cell surface markers play an important role in the ability of cells to interact and to pass signals between different cells of the same phenotype and cells of different phenotypes. Recent evidence suggests that cell-cycle regulators are essential for T-cell function. To trigger an effective immune response, lymphocytes must proliferate. The objective of this project is to investigate the changes in growth of human cells cultured in rotating bioreactors and to measure the growth rate and the cell cycle distribution for different human cell types. Human lymphocytes and lymphoblasts will be cultured in a bioreactor to simulate aspects of microgravity. The bioreactor is a cylindrical culture vessel that incorporates the aspects of clinostatic rotation of a solid fluid body around a horizontal axis at a constant speed, and compensates gravity by rotation and places cells within the fluid body into a sustained free-fall. Cell cycle progression and cell proliferation of the lymphocytes will be measured for a number of days. In addition, RNA from the cells will be isolated for expression of genes related in cell cycle regulations.

  14. Shape memory polymers for active cell culture.

    Science.gov (United States)

    Davis, Kevin A; Luo, Xiaofan; Mather, Patrick T; Henderson, James H

    2011-07-04

    Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent shape upon the application of a stimulus such as heat(1-5). In a typical shape memory cycle, the SMP is first deformed at an elevated temperature that is higher than its transition temperature, T(trans;) [either the melting temperature (T(m;)) or the glass transition temperature (T(g;))]. The deformation is elastic in nature and mainly leads to a reduction in conformational entropy of the constituent network chains (following the rubber elasticity theory). The deformed SMP is then cooled to a temperature below its T(trans;) while maintaining the external strain or stress constant. During cooling, the material transitions to a more rigid state (semi-crystalline or glassy), which kinetically traps or "freezes" the material in this low-entropy state leading to macroscopic shape fixing. Shape recovery is triggered by continuously heating the material through T(trans;) under a stress-free (unconstrained) condition. By allowing the network chains (with regained mobility) to relax to their thermodynamically favored, maximal-entropy state, the material changes from the temporary shape to the permanent shape. Cells are capable of surveying the mechanical properties of their surrounding environment(6). The mechanisms through which mechanical interactions between cells and their physical environment control cell behavior are areas of active research. Substrates of defined topography have emerged as powerful tools in the investigation of these mechanisms. Mesoscale, microscale, and nanoscale patterns of substrate topography have been shown to direct cell alignment, cell adhesion, and cell traction forces(7-14). These findings have underscored the potential for substrate topography to control and assay the mechanical interactions between cells and their physical environment during cell culture, but the substrates used to date

  15. Microanalysis of gene expression in cultured cells

    NARCIS (Netherlands)

    E. van der Veer (Eveliene)

    1982-01-01

    textabstractIn this thesis two aspects of gene expression in cultured cells have been studied: the heterogeneity in gene expression in relation with the development and application of microchemical techniques for the prenatal diagnosis of inborn errors of metabolism and the possibility of inducing g

  16. 3D culture for cardiac cells.

    Science.gov (United States)

    Zuppinger, Christian

    2016-07-01

    This review discusses historical milestones, recent developments and challenges in the area of 3D culture models with cardiovascular cell types. Expectations in this area have been raised in recent years, but more relevant in vitro research, more accurate drug testing results, reliable disease models and insights leading to bioartificial organs are expected from the transition to 3D cell culture. However, the construction of organ-like cardiac 3D models currently remains a difficult challenge. The heart consists of highly differentiated cells in an intricate arrangement.Furthermore, electrical “wiring”, a vascular system and multiple cell types act in concert to respond to the rapidly changing demands of the body. Although cardiovascular 3D culture models have been predominantly developed for regenerative medicine in the past, their use in drug screening and for disease models has become more popular recently. Many sophisticated 3D culture models are currently being developed in this dynamic area of life science. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel.

  17. Cell Culture Microfluidic Biochips: Experimental Throughput Maximization

    DEFF Research Database (Denmark)

    Minhass, Wajid Hassan; Pop, Paul; Madsen, Jan

    2011-01-01

    Microfluidic biochips offer a promising alternative to a conventional biochemical laboratory, integrating all necessary functionalities on-chip in order to perform biochemical applications. Researchers have started to propose computer-aided design tools for the synthesis of such biochips. Our focus...... metaheuristic for experimental design generation for the cell culture microfluidic biochips, and we have evaluated our approach using multiple experimental setups....

  18. Nanotechnology, Cell Culture and Tissue Engineering

    Directory of Open Access Journals (Sweden)

    Kazutoshi Haraguchi

    2011-01-01

    Full Text Available We have fabricated new types of polymer hydrogels and polymer nanocomposites, i.e., nanocomposite gels (NC gels and soft, polymer nanocomposites (M-NCs: solid, with novel organic/inorganic network structures. Both NC gels and M-NCs were synthesized by in-situ free-radical polymerization in the presence of exfoliated clay platelets in aqueous systems and were obtained in various forms such as film, sheet, tube, coating, etc. and sizes with a wide range of clay contents. Here, disk-like inorganic clay nanoparticles act as multi-functional crosslinkers to form new types of network systems. Both NC gels and M-NCs have extraordinary optical and mechanical properties including ultra-high reversible extensibility, as well as a number of new characteristics relating to optical anisotropy, polymer/clay morphology, biocompatibility, stimuli-sensitive surfaces, micro-patterning, etc. For examples, the biological testing of medical devices, comprised of a sensitization test, an irritation test, an intracutaneous test and an in vitro cytotoxicity test,was carried out for NC gels and M-NCs. The safety of NC gels and M-NCs was confirmed in all tests. Also, the interaction of living tissue with NC gel was investigated in vivo by implantation in live goats; neither inflammation nor concrescence occurred around the NC gels. Furthermore, it was found that both N-NC gels consisting of poly(N-isopropylacrylamide(PNIPA/clay network and M-NCs consisting of poly(2-methoxyethyacrylate(PMEA/clay network show characteristic cell culture and subsequent cell detachment on their surfaces, although it was almost impossible to culture cells on conventional, chemically-crosslinked PNIPA hydrogels and chemically crossslinked PMEA, regardless of their crosslinker concentration. Various kinds of cells, such ashumanhepatoma cells (HepG2, normal human dermal fibroblast (NHDF, and human umbilical vein endothelial cells (HUVEC, could be cultured to be confluent on the surfaces of N

  19. Trans, trans-2,4-decadienal induced cell proliferation via p27 pathway in human bronchial epithelial cells.

    Science.gov (United States)

    Chang, Yun-Ching; Lin, Pinpin

    2008-04-01

    Lung cancer is the leading cause of cancer deaths worldwide. Epidemiological studies have shown that exposure to cooking oil fumes (COF) is a risk factor for lung cancer. Trans, trans-2,4-decadienal (tt-DDE), a dienaldehyde, is abundant in heated oils and COF. Previously, we found that long-term exposure (45 days) to a sub-lethal dose (1 microM) of tt-DDE significantly increased growth of human bronchial epithelial cells (BEAS-2B). Aims of this study are to understand the mechanism of tt-DDE-induced cell proliferation and possible protective effects of antioxidant, vitamin C and N-acetylcysteine (NAC) in BEAS-2B cells. Utilizing the real-time RT-PCR and Western immunoblotting, we found that p27 mRNA and protein levels were significantly increased by 1 microM tt-DDE treatment. Co-treatment with vitamin C or NAC partially prevented tt-DDE-induced cell proliferation. In addition, the downstream targets of p27, including CDK4, cyclin D1 and phosphorylated-Rb proteins, increased in 1 microM tt-DDE-treated cells and these changes were prevented by NAC co-treatment. Therefore, these results suggest that tt-DDE increased cell proliferation via inhibition of p27 expression, increase in CDK4/cyclin D1 protein accumulation and enhancement of Rb phosphorylation. Increased cell proliferation is considered as the early stages of lung carcinogenesis. Administration of antioxidants may prevent COF-associated lung carcinogenesis.

  20. Cell Culture Assay for Human Noroviruses [response

    Energy Technology Data Exchange (ETDEWEB)

    Straub, Tim M.; Honer Zu Bentrup, Kerstin; Orosz Coghlan, Patricia; Dohnalkova, Alice; Mayer, Brooke K.; Bartholomew, Rachel A.; Valdez, Catherine O.; Bruckner-Lea, Cindy J.; Gerba, Charles P.; Abbaszadegan, Morteza A.; Nickerson, Cheryl A.

    2007-07-01

    We appreciate the comments provided by Leung et al., in response to our recently published article “In Vitro Cell Culture Infectivity Assay for Human Noroviruses” by Straub et al. (1). The specific aim of our project was to develop an in vitro cell culture infectivity assay for human noroviruses (hNoV) to enhance risk assessments when they are detected in water supplies. Reverse transcription (RT) qualitative or quantitative PCR are the primary assays for waterborne NoV monitoring. However, these assays cannot distinguish between infectious vs. non-infectious virions. When hNoV is detected in water supplies, information provided by our infectivity assay will significantly improve risk assessment models and protect human health, regardless of whether we are propagating NoV. Indeed, in vitro cell culture infectivity assays for the waterborne pathogen Cryptosporidium parvum that supplement approved fluorescent microscopy assays, do not result in amplification of the environmentally resistant hard-walled oocysts (2). However, identification of life cycle stages in cell culture provides evidence of infectious oocysts in a water supply. Nonetheless, Leung et al.’s assertion regarding the suitability of our method for the in vitro propagation of high titers of NoV is valid for the medical research community. In this case, well-characterized challenge pools of virus would be useful for developing and testing diagnostics, therapeutics, and vaccines. As further validation of our published findings, we have now optimized RT quantitative PCR to assess the level of viral production in cell culture, where we are indeed finding significant increases in viral titer. The magnitude and time course of these increases is dependent on both virus strain and multiplicity of infection. We are currently preparing a manuscript that will discuss these findings in greater detail, and the implications this may have for creating viral challenge pools

  1. EOTAXIN AND EOTAXIN-2 EXPRESSION IN HUMAN BRONCHIAL EPITHELIAL CELL

    Institute of Scientific and Technical Information of China (English)

    TANG Wei; DENG Wei-wu; Albert CHAN; Stanley CHIK; Adrain WU

    2005-01-01

    Objective To study the role of eotaxin and eotaxin-2 expression by Th2 cytokine and analyze their relationship in normal human bronchial epithelial cell line-BEAS-2B cell. Methods Levels of eotaxin mRNA and protein expression in the bronchial epithelial cell line BEAS-2B cell were determined with RT-PCR and ELISA. We also used RT-PCR to evaluate eotaxin-2 expression under the regulation of Th2 cytokine IL-4 and IL-13 as well as proinflammatory agent-TNFα. Results Eotaxin mRNA expression was the highest at the time point of 12h under the stimulation of TNF-α. While Th2 cytokine IL-4 and IL-13 had the amplification effect on the expression. Eotaxin protein was also elevated with the combination stimulation of proinflammatory agent TNF-α and IL-4 in dose and time dependent manner(P<0.01). These results were also seen when the cells were stimulated by TNF-α and IL-13. Eotaxin-2 mRNA expression was the highest at the time point of 8h. The expression evaluated by semi-quantitative RT-PCR also elevated under the co-stimulation of TNF-α and IL-4 or TNF-α and IL-13 and it should significantly correlate with Eotaxin(P<0.05). Conclusion This study demonstrated that Th2 cytokine like IL-4 and IL-13 enhances eotaxin and eotaxin-2 expression when co-stimulated with proinflammatory agent TNF-α. These results showed that Th2 cytokines existence is the strong evidence for bronchial epithelial cells taking part in the allergic inflammation especially in eosinophils recruitment.

  2. Cell Culture Microfluidic Biochips: Experimental Throughput Maximization

    DEFF Research Database (Denmark)

    Minhass, Wajid Hassan; Pop, Paul; Madsen, Jan

    2011-01-01

    Microfluidic biochips offer a promising alternative to a conventional biochemical laboratory, integrating all necessary functionalities on-chip in order to perform biochemical applications. Researchers have started to propose computer-aided design tools for the synthesis of such biochips. Our foc...... metaheuristic for experimental design generation for the cell culture microfluidic biochips, and we have evaluated our approach using multiple experimental setups....... in this paper is on the optimization of how a biochemical application is performed on a biochip. In this paper, we consider cell culture biochips, where several cell colonies are exposed to soluble compounds and monitored in real-time to determine the right combination of factors that leads to the desired...

  3. Use of an adaptable cell culture kit for performing lymphocyte and monocyte cell cultures in microgravity

    Science.gov (United States)

    Hatton, J. P.; Lewis, M. L.; Roquefeuil, S. B.; Chaput, D.; Cazenave, J. P.; Schmitt, D. A.

    1998-01-01

    The results of experiments performed in recent years on board facilities such as the Space Shuttle/Spacelab have demonstrated that many cell systems, ranging from simple bacteria to mammalian cells, are sensitive to the microgravity environment, suggesting gravity affects fundamental cellular processes. However, performing well-controlled experiments aboard spacecraft offers unique challenges to the cell biologist. Although systems such as the European 'Biorack' provide generic experiment facilities including an incubator, on-board 1-g reference centrifuge, and contained area for manipulations, the experimenter must still establish a system for performing cell culture experiments that is compatible with the constraints of spaceflight. Two different cell culture kits developed by the French Space Agency, CNES, were recently used to perform a series of experiments during four flights of the 'Biorack' facility aboard the Space Shuttle. The first unit, Generic Cell Activation Kit 1 (GCAK-1), contains six separate culture units per cassette, each consisting of a culture chamber, activator chamber, filtration system (permitting separation of cells from supernatant in-flight), injection port, and supernatant collection chamber. The second unit (GCAK-2) also contains six separate culture units, including a culture, activator, and fixation chambers. Both hardware units permit relatively complex cell culture manipulations without extensive use of spacecraft resources (crew time, volume, mass, power), or the need for excessive safety measures. Possible operations include stimulation of cultures with activators, separation of cells from supernatant, fixation/lysis, manipulation of radiolabelled reagents, and medium exchange. Investigations performed aboard the Space Shuttle in six different experiments used Jurkat, purified T-cells or U937 cells, the results of which are reported separately. We report here the behaviour of Jurkat and U937 cells in the GCAK hardware in ground

  4. Dynamic cell culture system (7-IML-1)

    Science.gov (United States)

    Cogoli, Augusto

    1992-01-01

    This experiment is one of the Biorack experiments being flown on the International Microgravity Laboratory 1 (MIL-1) mission as part of an investigation studying cell proliferation and performance in space. One of the objectives of this investigation is to assess the potential benefits of bioprocessing in space with the ultimate goal of developing a bioreactor for continuous cell cultures in space. This experiment will test the operation of an automated culture chamber that was designed for use in a Bioreactor in space. The device to be tested is called the Dynamic Cell Culture System (DCCS). It is a simple device in which media are renewed or chemicals are injected automatically, by means of osmotic pumps. This experiment uses four Type I/O experiment containers. One DCCS unit, which contains a culture chamber with renewal of medium and a second chamber without a medium supply fits in each container. Two DCCS units are maintained under zero gravity conditions during the on-orbit period. The other two units are maintained under 1 gh conditions in a 1 g centrifuge. The schedule for incubator transfer is given.

  5. Culturing of retinal pigment epithelium cells.

    Science.gov (United States)

    Valtink, Monika; Engelmann, Katrin

    2009-01-01

    The retinal pigment epithelium (RPE) is a monolayer of cells adjacent to the photoreceptors of the retina. It plays a crucial role in maintaining photoreceptor health and survival. Degeneration or dysfunction of the RPE can lead to photoreceptor degeneration and as a consequence to visual impairment. The most common diseased state of the RPE becomes manifest in age-related macular degeneration, an increasing cause of blindness in the elderly. RPE cells are therefore of great interest to researchers working in the field of tissue engineering and cell transplantation. In fact, studies in animal models have proven that the transplantation of RPE cells can delay the course of photoreceptor degenerative diseases. Although first attempts to transplant RPE cells into the subretinal space in human individuals suffering from age-related macular degeneration were less successful, RPE cell transplantation is still favored as a future therapeutic option, and much work is done to develop and design cell transplants. Cell banking is a prerequisite to have well-differentiated and characterized cells at hand when needed for research purposes, but also for therapeutic approaches. In this chapter the authors will describe methods to isolate, culture and preserve adult human RPE cells for the purpose of RPE cell banking. Copyright 2009 S. Karger AG, Basel.

  6. Prevention and Detection of Mycoplasma Contamination in Cell Culture

    Directory of Open Access Journals (Sweden)

    Parvaneh Farzaneh

    2012-01-01

    Full Text Available One of the main problems in cell culture is mycoplasma infection. It can extensively affectcell physiology and metabolism. As the applications of cell culture increase in research,industrial production and cell therapy, more concerns about mycoplasma contaminationand detection will arise. This review will provide valuable information about: 1. the waysin which cells are contaminated and the frequency and source of mycoplasma species incell culture; 2. the ways to prevent mycoplasma contamination in cell culture; 3. the importanceof mycoplasma tests in cell culture; 4. different methods to identify mycoplasmacontamination; 5. the consequences of mycoplasma contamination in cell culture and 6.available methods to eliminate mycoplasma contamination. Awareness about the sourcesof mycoplasma and pursuing aseptic techniques in cell culture along with reliable detectionmethods of mycoplasma contamination can provide an appropriate situation to preventmycoplasma contamination in cell culture.

  7. Human ES cells: starting culture from frozen cells.

    Science.gov (United States)

    Trish, Erin; Dimos, John; Eggan, Kevin

    2006-11-09

    Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock. First, a one to two day old ten cm plate of approximately one (to two) million irradiated mouse embryonic fibroblast feeder cells is rinsed with HuES media to remove residual serum and cell debris, and then HuES media added and left to equilibrate in the cell culture incubator. A frozen vial of cells from long term liquid nitrogen storage or a -80 C freezer is sourced and quickly submerged in a 37 C water bath for quick thawing. Cells in freezing media are then removed from the vial and placed in a large volume of HuES media. The large volume of HuES media facilitates removal of excess serum and DMSO, which can cause HuES human embryonic stem cells to differentiate. Cells are gently spun out of suspension, and then re-suspended in a small volume of fresh HuES media that is then used to seed the MEF plate. It is considered important to seed the MEF plate by gently adding the HuES cells in a drop wise fashion to evenly disperse them throughout the plate. The newly established HuES culture plate is returned to the incubator for 48 hrs before media is replaced, then is fed every 24 hours thereafter.

  8. Mouse cell culture - Methods and protocols

    Directory of Open Access Journals (Sweden)

    CarloAlberto Redi

    2010-12-01

    Full Text Available The mouse is, out of any doubt, the experimental animal par excellence for many many colleagues within the scientific community, notably for those working in mammalian biology (in a broad sense, from basic genetic to modeling human diseases, starting at least from 1664 Robert Hooke experiments on air’s propertyn. Not surprising then that mouse cell cultures is a well established field of research itself and that there are several handbooks devoted to this discipline. Here, Andrew Ward and David Tosh provide a necessary update of the protocols currently needed. In fact, nearly half of the book is devoted to stem cells culture protocols, mainly embryonic, from a list of several organs (kidney, lung, oesophagus and intestine, pancreas and liver to mention some........

  9. Sequencing technologies for animal cell culture research.

    Science.gov (United States)

    Kremkow, Benjamin G; Lee, Kelvin H

    2015-01-01

    Over the last 10 years, 2nd and 3rd generation sequencing technologies have made the use of genomic sequencing within the animal cell culture community increasingly commonplace. Each technology's defining characteristics are unique, including the cost, time, sequence read length, daily throughput, and occurrence of sequence errors. Given each sequencing technology's intrinsic advantages and disadvantages, the optimal technology for a given experiment depends on the particular experiment's objective. This review discusses the current characteristics of six next-generation sequencing technologies, compares the differences between them, and characterizes their relevance to the animal cell culture community. These technologies are continually improving, as evidenced by the recent achievement of the field's benchmark goal: sequencing a human genome for less than $1,000.

  10. Mouse cell culture: methods and protocols

    OpenAIRE

    Elvira M. Guerra Shinohara

    2010-01-01

    The mouse is, out of any doubt, the experimental animal par excellence for many many colleagues within the scientific community, notably for those working in mammalian biology (in a broad sense, from basic genetic to modeling human diseases), starting at least from 1664 Robert Hooke experiments on air’s propertyn. Not surprising then that mouse cell cultures is a well established field of research itself and that there are several handbooks devoted to this discipline. Here, Andrew Ward ...

  11. Embryo forming cells in carrot suspension cultures.

    OpenAIRE

    Toonen, M.A.J.

    1997-01-01

    Somatic cells of many plant species can be cultured in vitro and induced to form embryos that are able to develop into mature plants. This process, termed somatic embryogenesis, was originally described in carrot (Daucus carota L.). Somatic embryos develop through the same characteristic morphological stages, i.e. the globular-, heartand torpedo-stage respectively, as their zygotic counterparts. Due to the different cellular origin of somatic embryos, it is less clear to what extent the earli...

  12. PHYTOCHEMICAL STUDY OF CELL CULTURE JATROPHA CURCAS

    OpenAIRE

    KOMAR RUSLAN; ARTRI; ELFAHMI

    2011-01-01

    Jatropha curcas belongs to the Euphorbiaceae family which has potential economically. This plant has been reported to contain toxic compounds such as curcin and phorbol ester and its derivatives. These compounds may become a problem if J. curcas will be explored as a source of biofuel. In order to provide safety plants, the research on the study of phytochemical and initiation of cell and organ culture have been carried out. J curcas which has been collected from different regions in Indonesi...

  13. A Versatile Bioreactor for Dynamic Suspension Cell Culture. Application to the Culture of Cancer Cell Spheroids.

    Science.gov (United States)

    Massai, Diana; Isu, Giuseppe; Madeddu, Denise; Cerino, Giulia; Falco, Angela; Frati, Caterina; Gallo, Diego; Deriu, Marco A; Falvo D'Urso Labate, Giuseppe; Quaini, Federico; Audenino, Alberto; Morbiducci, Umberto

    2016-01-01

    A versatile bioreactor suitable for dynamic suspension cell culture under tunable shear stress conditions has been developed and preliminarily tested culturing cancer cell spheroids. By adopting simple technological solutions and avoiding rotating components, the bioreactor exploits the laminar hydrodynamics establishing within the culture chamber enabling dynamic cell suspension in an environment favourable to mass transport, under a wide range of tunable shear stress conditions. The design phase of the device has been supported by multiphysics modelling and has provided a comprehensive analysis of the operating principles of the bioreactor. Moreover, an explanatory example is herein presented with multiphysics simulations used to set the proper bioreactor operating conditions for preliminary in vitro biological tests on a human lung carcinoma cell line. The biological results demonstrate that the ultralow shear dynamic suspension provided by the device is beneficial for culturing cancer cell spheroids. In comparison to the static suspension control, dynamic cell suspension preserves morphological features, promotes intercellular connection, increases spheroid size (2.4-fold increase) and number of cycling cells (1.58-fold increase), and reduces double strand DNA damage (1.5-fold reduction). It is envisioned that the versatility of this bioreactor could allow investigation and expansion of different cell types in the future.

  14. Conversion of primordial germ cells to pluripotent stem cells: methods for cell tracking and culture conditions.

    Science.gov (United States)

    Nagamatsu, Go; Suda, Toshio

    2013-01-01

    Primordial germ cells (PGCs) are unipotent cells committed to germ lineage: PGCs can only differentiate into gametes in vivo. However, upon fertilization, germ cells acquire the capacity to differentiate into all cell types in the body, including germ cells. Therefore, germ cells are thought to have the potential for pluripotency. PGCs can convert to pluripotent stem cells in vitro when cultured under specific conditions that include bFGF, LIF, and the membrane-bound form of SCF (mSCF). Here, the culture conditions which efficiently convert PGCs to pluripotent embryonic germ (EG) cells are described, as well as methods used for identifying pluripotent candidate cells during culture.

  15. An Introductory Undergraduate Course Covering Animal Cell Culture Techniques

    Science.gov (United States)

    Mozdziak, Paul E.; Petitte, James N.; Carson, Susan D.

    2004-01-01

    Animal cell culture is a core laboratory technique in many molecular biology, developmental biology, and biotechnology laboratories. Cell culture is a relatively old technique that has been sparingly taught at the undergraduate level. The traditional methodology for acquiring cell culture training has been through trial and error, instruction when…

  16. An Introductory Undergraduate Course Covering Animal Cell Culture Techniques

    Science.gov (United States)

    Mozdziak, Paul E.; Petitte, James N.; Carson, Susan D.

    2004-01-01

    Animal cell culture is a core laboratory technique in many molecular biology, developmental biology, and biotechnology laboratories. Cell culture is a relatively old technique that has been sparingly taught at the undergraduate level. The traditional methodology for acquiring cell culture training has been through trial and error, instruction when…

  17. Inhibition of lung cancer cells A549 and H460 by curcuminoid extracts and nanoemulsions prepared from Curcuma longa Linnaeus

    Science.gov (United States)

    Chang, Hong-Bin; Chen, Bing-Huei

    2015-01-01

    The objectives of this study were to explore the inhibition mechanism of lung cancer cells A549 and H460 by curcuminoid extracts and nanoemulsions prepared from Curcuma longa Linnaeus. In addition, human bronchus epithelial cell line BEAS-2B (normal cell) was selected for comparison. A high-performance liquid chromatography (HPLC) method was developed to separate and quantify the various curcuminoids in C. longa extract, including curcumin (1,714.5 μg/mL), demethoxycurcumin (1,147.4 μg/mL), and bisdemethoxycurcumin (190.2 μg/mL). A high-stability nanoemulsion composed of Tween 80, water, and curcuminoid extract was prepared, with mean particle size being 12.6 nm. The cell cycle was retarded at G2/M for both the curcuminoid extract and nanoemulsion treatments; however, the inhibition pathway may be different. H460 cells were more susceptible to apoptosis than A549 cells for both curcuminoid extract and nanoemulsion treatments. Growth of BEAS-2B remained unaffected for both the curcuminoid extract and nanoemulsion treatments, with a concentration range from 1 to 4 μg/mL. Also, the activities of caspase-3, caspase-8, and caspase-9 followed a dose-dependent increase for both A549 and H460 cells for both the treatments, accompanied by a dose-dependent increase in cytochrome C expression and a dose-dependent decrease in CDK1 expression. Interestingly, a dose-dependent increase in cyclin B expression was shown for A549 cells for both the treatments, while a reversed trend was found for H460 cells. Both mitochondria and death receptor pathways may be responsible for apoptosis of both A549 and H460 cells. PMID:26345201

  18. Inhibition of lung cancer cells A549 and H460 by curcuminoid extracts and nanoemulsions prepared from Curcuma longa Linnaeus.

    Science.gov (United States)

    Chang, Hong-Bin; Chen, Bing-Huei

    2015-01-01

    The objectives of this study were to explore the inhibition mechanism of lung cancer cells A549 and H460 by curcuminoid extracts and nanoemulsions prepared from Curcuma longa Linnaeus. In addition, human bronchus epithelial cell line BEAS-2B (normal cell) was selected for comparison. A high-performance liquid chromatography (HPLC) method was developed to separate and quantify the various curcuminoids in C. longa extract, including curcumin (1,714.5 μg/mL), demethoxycurcumin (1,147.4 μg/mL), and bisdemethoxycurcumin (190.2 μg/mL). A high-stability nanoemulsion composed of Tween 80, water, and curcuminoid extract was prepared, with mean particle size being 12.6 nm. The cell cycle was retarded at G2/M for both the curcuminoid extract and nanoemulsion treatments; however, the inhibition pathway may be different. H460 cells were more susceptible to apoptosis than A549 cells for both curcuminoid extract and nanoemulsion treatments. Growth of BEAS-2B remained unaffected for both the curcuminoid extract and nanoemulsion treatments, with a concentration range from 1 to 4 μg/mL. Also, the activities of caspase-3, caspase-8, and caspase-9 followed a dose-dependent increase for both A549 and H460 cells for both the treatments, accompanied by a dose-dependent increase in cytochrome C expression and a dose-dependent decrease in CDK1 expression. Interestingly, a dose-dependent increase in cyclin B expression was shown for A549 cells for both the treatments, while a reversed trend was found for H460 cells. Both mitochondria and death receptor pathways may be responsible for apoptosis of both A549 and H460 cells.

  19. Cell response of Chlamydomonas actinochloris culture to repeated microwave irradiation

    Directory of Open Access Journals (Sweden)

    OLESIA O. GRYGORIEVA

    2015-05-01

    Full Text Available Abstract. Grygorieva OO, Berezovsjka MA, Dacenko OI. 2015. Cell response of Chlamydomonas actinochloris culture to repeated microwave irradiation. Nusantara Bioscience 7: 38-42. Two cultures of Chlamydomonas actinochloris Deason et Bold in the lag-phase were exposed to the microwave irradiation. One of them (culture 1 was not treated beforehand, whereas the other (culture 2 was irradiated by microwaves 2 years earlier. The measurement of cell quantity as well as measurement of change of intensities and spectra of cultures photoluminescence (PL in the range of chlorophyll a emission was regularly conducted during the cell cultures development. Cell concentration of culture 1 exposed to the microwave irradiation for the first time has quickly restored while cell concentration of culture 2 which was irradiated repeatedly has fallen significantly. The following increasing of cell concentration of culture 2 is negligible. Cell concentration reaches the steady-state level that is about a half of the cell concentration of control culture. Initially the PL efficiency of cells of both cultures decreases noticeable as a result of irradiation. Then there is the monotonic increase to the values which are significantly higher than the corresponding values in the control cultures. The ratio of the intensities at the maxima of the main emission bands of chlorophyll for control samples of both cultures remained approximately at the same level. At the same time effect of irradiation on the cell PL spectrum appears as a temporary reduction of this magnitude.

  20. Inhibitory effects of Piper betle on production of allergic mediators by bone marrow-derived mast cells and lung epithelial cells.

    Science.gov (United States)

    Wirotesangthong, Mali; Inagaki, Naoki; Tanaka, Hiroyuki; Thanakijcharoenpath, Witchuda; Nagai, Hiroichi

    2008-03-01

    The leaves of the Piper betle Linn. (Piperaceae) are used in traditional medicine and possess anti-oxidant, anti-bacterial, anti-fungal, anti-diabetic and radioprotective activities. However, little is known about their anti-allergic activity. Therefore, the effects of P. betle ethanolic extract (PE) on the production of histamine and granulocyte macrophage-colony-stimulating factor (GM-CSF) by murine bone marrow mast cells (BMMCs) and on the secretion of eotaxin and IL-8 by the human lung epithelial cell line, BEAS-2B, were investigated in vitro. PE significantly decreased histamine and GM-CSF produced by an IgE-mediated hypersensitive reaction, and inhibited eotaxin and IL-8 secretion in a TNF-alpha and IL-4-induced allergic reaction. The results suggest that P. betle may offer a new therapeutic approach for the control of allergic diseases through inhibition of production of allergic mediators.

  1. Insect cell culture in research: Indian scenario.

    Science.gov (United States)

    Sudeep, A B; Mourya, D T; Mishra, A C

    2005-06-01

    Insect cell cultures are widely used in viral diagnosis and biotechnology, for the production of recombinant proteins, viral pesticides and vaccines as well as in basic research in genetics, molecular biology, biochemistry, endocrinology and virology. Following KRP Singh's pioneering research in 1967, a large number of cell lines from diptera, hemiptera, and lepidopteran insects were established and characterized in India. With the availability of the modern tools in molecular biology and the advancements made in biotechnology, the indigenous cell lines may prove useful in creating a future without biohazardous chemical pesticides as well as producing life saving pharmaceuticals and vaccines for many diseases. This review summarizes information gathered regarding the insect cell lines established so far in India. It also covers the familiarization of the well characterized continuous cell lines and their potential applications. Special attention is given to virus susceptibility of the cell lines, the yield of virus with a comparative analysis with other conventional systems. The potential applications of dipteran and lepidopteran cell lines in agriculture and biotechnology are also briefly discussed for prospective studies.

  2. Cell culture device using spatial light modulator

    Science.gov (United States)

    Ou, Chung-Jen; Shen, Ching-I.; Ou, Chung-Ming

    2009-07-01

    Spatial light modulator is introduced for cell culturing and related illumination experiment. Two kinds of designs were used. The first type put the cell along with the bio-medium directly on top of the analyzer of the microdisplay and set a cover glass on it to retain the medium environment, which turned the microdisplay into a bio-container. The second type introduced an optical lens system placed below the spatial light modulator to focus the light spots on specific position. Details of the advantages and drawbacks for the two different approaches are discussed, and the human melanocyte cell (HMC) is introduced to prove the feasibility of the concept. Results indicate that the second type is much more suitable than the first for precision required application.

  3. Airborne urban particles (Milan winter-PM2.5) cause mitotic arrest and cell death: Effects on DNA, mitochondria, AhR binding and spindle organization

    Energy Technology Data Exchange (ETDEWEB)

    Gualtieri, Maurizio [Applied Cell Biology and Particles Effects, Department of Environmental Science, University Milano-Bicocca, Piazza della Scienza 1, 20126 Milano (Italy); Division of Environmental Medicine, Norwegian Institute of Public Health, P.O. Box 4404 Nydalen, N-0403 Oslo (Norway); Ovrevik, Johan [Division of Environmental Medicine, Norwegian Institute of Public Health, P.O. Box 4404 Nydalen, N-0403 Oslo (Norway); Mollerup, Steen [Section for Toxicology, National Institute of Occupational Health, N-0033 Oslo (Norway); Asare, Nana [Division of Environmental Medicine, Norwegian Institute of Public Health, P.O. Box 4404 Nydalen, N-0403 Oslo (Norway); Longhin, Eleonora [Applied Cell Biology and Particles Effects, Department of Environmental Science, University Milano-Bicocca, Piazza della Scienza 1, 20126 Milano (Italy); Dahlman, Hans-Jorgen [Division of Environmental Medicine, Norwegian Institute of Public Health, P.O. Box 4404 Nydalen, N-0403 Oslo (Norway); Camatini, Marina [Applied Cell Biology and Particles Effects, Department of Environmental Science, University Milano-Bicocca, Piazza della Scienza 1, 20126 Milano (Italy); Centre Research POLARIS, Department of Environmental Science, University Milano-Bicocca, Piazza della Scienza 1, 20126 Milano (Italy); Holme, Jorn A., E-mail: jorn.holme@fhi.no [Division of Environmental Medicine, Norwegian Institute of Public Health, P.O. Box 4404 Nydalen, N-0403 Oslo (Norway)

    2011-08-01

    Highlights: {yields} PM2.5 induces mitotic arrest in BEAS-2B cells. {yields} PM2.5 induces DNA damage and activates DNA damage response. {yields} AhR regulated genes (Cyp1A1, Cyp1B1 and AhRR) are upregulated after PM exposure. {yields} Mitotic spindle assembly is perturbed in PM exposed cells. - Abstract: Airborne particulate matter (PM) is considered to be an important contributor to lung diseases. In the present study we report that Milan winter-PM2.5 inhibited proliferation in human bronchial epithelial cells (BEAS-2B) by inducing mitotic arrest. The cell cycle arrest was followed by an increase in mitotic-apoptotic cells, mitotic slippage and finally an increase in 'classical' apoptotic cells. Exposure to winter-PM10 induced only a slight effect which may be due to the presence of PM2.5 in this fraction while pure combustion particles failed to disturb mitosis. Fewer cells expressing the mitosis marker phospho-histone H3 compared to cells with condensed chromosomes, suggest that PM2.5 induced premature mitosis. PM2.5 was internalized into the cells and often localized in laminar organelles, although particles without apparent plasma membrane covering were also seen. In PM-containing cells mitochondria and lysosomes were often damaged, and in mitotic cells fragmented chromosomes often appeared. PM2.5 induced DNA strands breaks and triggered a DNA-damage response characterized by increased phosphorylation of ATM, Chk2 and H2AX; as well as induced a marked increase in expression of the aryl hydrocarbon receptor (AhR)-regulated genes, CYP1A1, CYP1B1 and AhRR. Furthermore, some disturbance of the organization of microtubules was indicated. It is hypothesized that the induced mitotic arrest and following cell death was due to a premature chromosome condensation caused by a combination of DNA, mitochondrial and spindle damage.

  4. Rapid method for culturing embryonic neuron-glial cell cocultures

    DEFF Research Database (Denmark)

    Svenningsen, Åsa Fex; Shan, Wei-Song; Colman, David R;

    2003-01-01

    A streamlined, simple technique for primary cell culture from E17 rat tissue is presented. In an attempt to standardize culturing methods for all neuronal cell types in the embryo, we evaluated a commercial medium without serum and used similar times for trypsinization and tested different surfaces...... for plating. In 1 day, using one method and a single medium, it is possible to produce robust E17 cultures of dorsal root ganglia (DRG), cerebellum, and enteric plexi. Allowing the endogenous glial cells to repopulate the cultures saves time compared with existing techniques, in which glial cells are added...... to cultures first treated with antimitotic agents. It also ensures that all the cells present in vivo will be present in the culture. Myelination commences after approximately 2 weeks in culture for dissociated DRG and 3-4 weeks in cerebellar cultures. In enteric cultures, glial wrapping of the enteric...

  5. PHYTOCHEMICAL STUDY OF CELL CULTURE JATROPHA CURCAS

    Directory of Open Access Journals (Sweden)

    KOMAR RUSLAN

    2011-01-01

    Full Text Available Jatropha curcas belongs to the Euphorbiaceae family which has potential economically. This plant has been reported to contain toxic compounds such as curcin and phorbol ester and its derivatives. These compounds may become a problem if J. curcas will be explored as a source of biofuel. In order to provide safety plants, the research on the study of phytochemical and initiation of cell and organ culture have been carried out. J curcas which has been collected from different regions in Indonesia showed to contain relatively the same profile of chemical contents. Dominant compounds that were detected by GCMS are hidrocarbon such as 2-heptenal, decadienal, hexsadecane, pentadecane, cyclooctane etc, fatty acid such as oktadecanoate acid, etthyl linoleate, ethyl stearate, heksadecanoate acid and steroid such as stigmasterol, fucosterol, sitosterol. No phorbol ester and its derivatives have been detected yet by the GCMS method. Callus and suspension cultures of J. curcas have been established to be used for further investigation.

  6. How do culture media influence in vitro perivascular cell behavior?

    Science.gov (United States)

    Huber, Birgit; Volz, Ann-Cathrin; Kluger, Petra Juliane

    2015-12-01

    Perivascular cells are multilineage cells located around the vessel wall and important for wall stabilization. In this study, we evaluated a stem cell media and a perivascular cell-specific media for the culture of primary perivascular cells regarding their cell morphology, doubling time, stem cell properties, and expression of cell type-specific markers. When the two cell culture media were compared to each other, perivascular cells cultured in the stem cell medium had a more elongated morphology and a faster doubling rate and cells cultured in the pericyte medium had a more typical morphology, with several filopodia, and a slower doubling rate. To evaluate stem cell properties, perivascular cells, CD146(-) cells, and mesenchymal stem cells (MSCs) were differentiated into the adipogenic, osteogenic, and chondrogenic lineages. It was seen that perivascular cells, as well as CD146(-) cells and MSCs, cultured in stem cell medium showed greater differentiation than cells cultured in pericyte-specific medium. The expression of pericyte-specific markers CD146, neural/glial antigen 2 (NG2), platelet-derived growth factor receptor-β (PDGFR-β), myosin, and α-smooth muscle actin (α-SMA) could be found in both pericyte cultures, as well as to varying amounts in CD146(-) cells, MSCs, and endothelial cells. The here presented work shows that perivascular cells can adapt to their in vitro environment and cell culture conditions influence cell functionality, such as doubling rate or differentiation behavior. Pericyte-specific markers were shown to be expressed also from cells other than perivascular cells. We can further conclude that CD146(+) perivascular cells are inhomogeneous cell population probably containing stem cell subpopulations, which are located perivascular around capillaries.

  7. Good cell culture practices &in vitro toxicology.

    Science.gov (United States)

    Eskes, Chantra; Boström, Ann-Charlotte; Bowe, Gerhard; Coecke, Sandra; Hartung, Thomas; Hendriks, Giel; Pamies, David; Piton, Alain; Rovida, Costanza

    2017-04-25

    Good Cell Culture Practices (GCCP) is of high relevance to in vitro toxicology. The European Society of Toxicology In Vitro (ESTIV), the Center for Alternatives for Animal Testing (CAAT) and the In Vitro Toxicology Industrial Platform (IVTIP) joined forces to address by means of an ESTIV 2016 pre-congress session the different aspects and applications of GCCP. The covered aspects comprised the current status of the OECD guidance document on Good In Vitro Method Practices, the importance of quality assurance for new technological advances in in vitro toxicology including stem cells, and the optimized implementation of Good Manufacturing Practices and Good Laboratory Practices for regulatory testing purposes. General discussions raised the duality related to the difficulties in implementing GCCP in an academic innovative research framework on one hand, and on the other hand, the need for such GCCP principles in order to ensure reproducibility and robustness of in vitro test methods for toxicity testing. Indeed, if good cell culture principles are critical to take into consideration for all uses of in vitro test methods for toxicity testing, the level of application of such principles may depend on the stage of development of the test method as well as on the applications of the test methods, i.e., academic innovative research vs. regulatory standardized test method. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Cryptosporidium cell culture infectivity assay design.

    Science.gov (United States)

    King, B J; Keegan, A R; Robinson, B S; Monis, P T

    2011-05-01

    Members of the genus Cryptosporidium, which cause the gastrointestinal disease cryptosporidiosis, still represent a significant cause of water-borne disease worldwide. While intensive efforts have been invested in the development of techniques for parasite culture, in vitro growth has been hampered by a number of factors including low levels of infectivity as well as delayed life-cycle development and poor synchronicity. In this study we examined factors affecting the timing of contact between excysted sporozoites and target host cells and the subsequent impact of this upon the establishment of infection. We demonstrate that excystation rate impacts upon establishment of infection and that in our standard assay format the majority of sporozoites are not close enough to the cell monolayer when they are released from the oocyst to successfully establish infection. However, this can be easily overcome by centrifugation of oocysts onto the cell monolayer, resulting in approximately 4-fold increases in sporozoite attachment and subsequent infection. We further demonstrate that excystation procedures can be tailored to control excystation rate to match the assay end purpose and that excystation rate can influence data interpretation. Finally, the addition of both a centrifugation and washing step post-sporozoite attachment may be appropriate when considering the design of in vitro culture experiments for developmental analysis and stage-specific gene expression as this appears to increase the synchronicity of early developmental stages.

  9. Sarcoma derived from cultured mesenchymal stem cells.

    Science.gov (United States)

    Tolar, Jakub; Nauta, Alma J; Osborn, Mark J; Panoskaltsis Mortari, Angela; McElmurry, Ron T; Bell, Scott; Xia, Lily; Zhou, Ning; Riddle, Megan; Schroeder, Tania M; Westendorf, Jennifer J; McIvor, R Scott; Hogendoorn, Pancras C W; Szuhai, Karoly; Oseth, Leann; Hirsch, Betsy; Yant, Stephen R; Kay, Mark A; Peister, Alexandra; Prockop, Darwin J; Fibbe, Willem E; Blazar, Bruce R

    2007-02-01

    To study the biodistribution of MSCs, we labeled adult murine C57BL/6 MSCs with firefly luciferase and DsRed2 fluorescent protein using nonviral Sleeping Beauty transposons and coinfused labeled MSCs with bone marrow into irradiated allogeneic recipients. Using in vivo whole-body imaging, luciferase signals were shown to be increased between weeks 3 and 12. Unexpectedly, some mice with the highest luciferase signals died and all surviving mice developed foci of sarcoma in their lungs. Two mice also developed sarcomas in their extremities. Common cytogenetic abnormalities were identified in tumor cells isolated from different animals. Original MSC cultures not labeled with transposons, as well as independently isolated cultured MSCs, were found to be cytogenetically abnormal. Moreover, primary MSCs derived from the bone marrow of both BALB/c and C57BL/6 mice showed cytogenetic aberrations after several passages in vitro, showing that transformation was not a strain-specific nor rare event. Clonal evolution was observed in vivo, suggesting that the critical transformation event(s) occurred before infusion. Mapping of the transposition insertion sites did not identify an obvious transposon-related genetic abnormality, and p53 was not overexpressed. Infusion of MSC-derived sarcoma cells resulted in malignant lesions in secondary recipients. This new sarcoma cell line, S1, is unique in having a cytogenetic profile similar to human sarcoma and contains bioluminescent and fluorescent genes, making it useful for investigations of cellular biodistribution and tumor response to therapy in vivo. More importantly, our study indicates that sarcoma can evolve from MSC cultures.

  10. DNA MUTAGENESIS IN PANAX GINSENG CELL CULTURES

    Directory of Open Access Journals (Sweden)

    Kiselev K.V.

    2012-08-01

    Full Text Available At the present time, it is well documented that plant tissue culture induces a number of mutations and chromosome rearrangements termed “somaclonal variations”. However, little is known about the nature and the molecular mechanisms of the tissue culture-induced mutagenesis and the effects of long-term subculturing on the rate and specific features of the mutagenesis. The aim of the present study was to investigate and compare DNA mutagenesis in different genes of Panax ginseng callus cultures of different age. It has previously been shown that the nucleotide sequences of the Agrobacterium rhizogenes rolC locus and the selective marker nptII developed mutations during long-term cultivation of transgenic cell cultures of P. ginseng. In the present work, we analyzed nucleotide sequences of selected plant gene families in a 2-year-old and 20-year-old P. ginseng 1c cell culture and in leaves of cultivated P. ginseng plants. We analysed sequence variability between the Actin genes, which are a family of house-keeping genes; the phenylalanine ammonia-lyase (PAL and dammarenediol synthase (DDS genes, which actively participate in the biosynthesis of ginsenosides; and the somatic embryogenesis receptor kinase (SERK genes, which control plant development. The frequency of point mutations in the Actin, PAL, DDS, and SERK genes in the 2-year-old callus culture was markedly higher than that in cultivated plants but lower than that in the 20-year-old callus culture of P. ginseng. Most of the mutations in the 2- and 20-year-old P. ginseng calli were A↔G and T↔C transitions. The number of nonsynonymous mutations was higher in the 2- and 20-year-old callus cultures than the number of nonsynonymous mutations in the cultivated plants of P. ginseng. Interestingly, the total number of N→G or N→C substitutions in the analyzed genes was 1.6 times higher than the total number of N→A or N→T substitutions. Using methylation-sensitive DNA fragmentation

  11. Growth of cultured porcine retinal pigment epithelial cells

    DEFF Research Database (Denmark)

    Wiencke, A.K.; Kiilgaard, Jens Folke; Nicolini, Jair;

    2003-01-01

    To establish and characterize cultures of porcine retinal pigment epithelial (pRPE) cells in order to produce confluent monolayers of cells for transplantation.......To establish and characterize cultures of porcine retinal pigment epithelial (pRPE) cells in order to produce confluent monolayers of cells for transplantation....

  12. Equipment for large-scale mammalian cell culture.

    Science.gov (United States)

    Ozturk, Sadettin S

    2014-01-01

    This chapter provides information on commonly used equipment in industrial mammalian cell culture, with an emphasis on bioreactors. The actual equipment used in the cell culture process can vary from one company to another, but the main steps remain the same. The process involves expansion of cells in seed train and inoculation train processes followed by cultivation of cells in a production bioreactor. Process and equipment options for each stage of the cell culture process are introduced and examples are provided. Finally, the use of disposables during seed train and cell culture production is discussed.

  13. Wood dusts induce the production of reactive oxygen species and caspase-3 activity in human bronchial epithelial cells.

    Science.gov (United States)

    Pylkkänen, Lea; Stockmann-Juvala, Helene; Alenius, Harri; Husgafvel-Pursiainen, Kirsti; Savolainen, Kai

    2009-08-21

    Wood dusts are associated with several respiratory symptoms, e.g. impaired lung function and asthma, in exposed workers. However, despite the evidence from epidemiological studies, the underlying mechanisms are not well understood. In the present study, we investigated different wood dusts for their capacity to induce cytotoxicity and production of radical oxygen species (ROS) as well as activation of the apoptotic caspase-3 enzyme in human bronchial epithelial cells (BEAS-2B). Dusts from three different tree species widely used in wood industry were studied; birch and oak represented hardwood species, and pine a common softwood species. All the experiments were carried out in three different concentrations (10, 50, and 500 microg/ml) and the analysis was performed after 0.5, 2, 6, and 24h exposure. All wood dusts studied were cytotoxic to human bronchial epithelial cells in a dose-dependent manner after 2 and 6h treatment. Exposure to pine, birch, or oak dust had a significant stimulating effect on the production of ROS. Also an induction in caspase-3 protease activity, one of the central components of the apoptotic cascade, was seen in BEAS-2B cells after 2 and 6h exposure to each of the wood dusts studied. In summary, we demonstrate that dusts from pine, birch and oak are cytotoxic, able to increase the production of ROS and the apoptotic response in human broncho-epithelial cells in vitro. Thus, our current data suggest oxidative stress by ROS as an important mechanism likely to function in wood dust related pulmonary toxicity although details of the cellular targets and cell-particle interactions remain to be solved. It is though tempting to speculate that redox-regulated transcription factors such as NFkappaB or AP-1 may play a role in this wood dust-evoked process leading to apparently induced apoptosis of target cells.

  14. The differential role of human macrophage in triggering secondary bystander effects after either gamma-ray or carbon beam irradiation.

    Science.gov (United States)

    Dong, Chen; He, Mingyuan; Tu, Wenzhi; Konishi, Teruaki; Liu, Weili; Xie, Yuexia; Dang, Bingrong; Li, Wenjian; Uchihori, Yukio; Hei, Tom K; Shao, Chunlin

    2015-07-10

    The abscopal effect could be an underlying factor in evaluating prognosis of radiotherapy. This study established an in vitro system to examine whether tumor-generated bystander signals could be transmitted by macrophages to further trigger secondary cellular responses after different irradiations, where human lung cancer NCI-H446 cells were irradiated with either γ-rays or carbon ions and co-cultured with human macrophage U937 cells, then these U937 cells were used as a bystander signal transmitter and co-cultured with human bronchial epithelial cells BEAS-2B. Results showed that U937 cells were only activated by γ-irradiated NCI-H446 cells so that the secondary injuries in BEAS-2B cells under carbon ion irradiation were weaker than γ-rays. Both TNF-α and IL-1α were involved in the γ-irradiation induced secondary bystander effect but only TNF-α contributed to the carbon ion induced response. Further assay disclosed that IL-1α but not TNF-α was largely responsible for the activation of macrophages and the formation of micronucleus in BEAS-2B cells. These data suggest that macrophages could transfer secondary bystander signals and play a key role in the secondary bystander effect of photon irradiation, while carbon ion irradiation has conspicuous advantage due to its reduced secondary injury. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  15. Modelling of Mammalian cells and cell culture processes.

    Science.gov (United States)

    Sidoli, F R; Mantalaris, A; Asprey, S P

    2004-01-01

    Mammalian cell cultures represent the major source for a number of very high-value biopharmaceutical products, including monoclonal antibodies (MAbs), viral vaccines, and hormones. These products are produced in relatively small quantities due to the highly specialised culture conditions and their susceptibility to either reduced productivity or cell death as a result of slight deviations in the culture conditions. The use of mathematical relationships to characterise distinct parts of the physiological behaviour of mammalian cells and the systematic integration of this information into a coherent, predictive model, which can be used for simulation, optimisation, and control purposes would contribute to efforts to increase productivity and control product quality. Models can also aid in the understanding and elucidation of underlying mechanisms and highlight the lack of accuracy or descriptive ability in parts of the model where experimental and simulated data cannot be reconciled. This paper reviews developments in the modelling of mammalian cell cultures in the last decade and proposes a future direction - the incorporation of genomic, proteomic, and metabolomic data, taking advantage of recent developments in these disciplines and thus improving model fidelity. Furthermore, with mammalian cell technology dependent on experiments for information, model-based experiment design is formally introduced, which when applied can result in the acquisition of more informative data from fewer experiments. This represents only part of a broader framework for model building and validation, which consists of three distinct stages: theoretical model assessment, model discrimination, and model precision, which provides a systematic strategy from assessing the identifiability and distinguishability of a set of competing models to improving the parameter precision of a final validated model.

  16. Recombinant Protein Production and Insect Cell Culture and Process

    Science.gov (United States)

    Spaulding, Glenn F. (Inventor); Goodwin, Thomas J. (Inventor); OConnor, Kim C. (Inventor); Francis, Karen M. (Inventor); Andrews, Angela D. (Inventor); Prewett, Tracey L. (Inventor)

    1997-01-01

    A process has been developed for recombinant production of selected polypeptides using transformed insect cells cultured in a horizontally rotating culture vessel modulated to create low shear conditions. A metabolically transformed insect cell line is produced using the culture procedure regardless of genetic transformation. The recombinant polypeptide can be produced by an alternative process using virtually infected or stably transformed insect cells containing a gene encoding the described polypeptide. The insect cells can also be a host for viral production.

  17. Protection of cultured mammalian cells by rebamipide

    Energy Technology Data Exchange (ETDEWEB)

    Antoku, Shigetoshi; Aramaki, Ryoji [Kyushu Univ., Fukuoka (Japan). Faculty of Medicine; Tanaka, Hisashi; Kusumoto, Naotoshi

    1997-06-01

    Rebamipide which is used as a drug for gastritis and stomach ulcer has large capability for OH radical scavenging. It is expected that rebamipide has protective effect against ionizing radiations. The present paper deals with protective effect of rebamipide for cultured mammalian cells exposed to ionizing radiations. As rebamipide is insoluble in water, three solvents were used to dissolve. Rebamipide dissolved in dimethyl sulfoxide (DMSO), dimethyl formamide (DMFA) and 0.02 N NaOH was added to the cells in Eagle`s minimum essential medium (MEM) supplemented with 10% fetal calf serum and the cells were irradiated with X-rays. After irradiation, the cells were trypsinized, plated in MEM with 10% fetal calf serum and incubated for 7 days in a CO{sub 2} incubator to form colonies. Rebamipide dissolved in 0.02 N NaOH exhibited the protective effect expected its OH radical scavenging capability. However, the protective effect of rebamipide dissolved in DMSO was about half of that expected by its radical scavenging capability and that of rebamipide dissolved in DMFA was not observed. Uptake of rebamipide labeled with {sup 14}C increased with increasing contact time with rebamipide. These rebamipide mainly distributed in nucleus rather than cytoplasm. (author)

  18. Microfluidics and cancer analysis: cell separation, cell/tissue culture, cell mechanics, and integrated analysis systems.

    Science.gov (United States)

    Pappas, Dimitri

    2016-01-21

    Among the growing number of tools available for cancer studies, microfluidic systems have emerged as a promising analytical tool to elucidate cancer cell and tumor function. Microfluidic methods to culture cells have created approaches to provide a range of environments from single-cell analysis to complex three-dimensional devices. In this review we discuss recent advances in tumor cell culture, cancer cell analysis, and advanced studies enabled by microfluidic systems.

  19. Silica nanoparticles induce cytokine responses in lung epithelial cells through activation of a p38/TACE/TGF-α/EGFR-pathway and NF-κΒ signalling

    Energy Technology Data Exchange (ETDEWEB)

    Skuland, Tonje, E-mail: tonje.skuland@fhi.no; Øvrevik, Johan; Låg, Marit; Schwarze, Per; Refsnes, Magne

    2014-08-15

    Amorphous silica nanoparticles (SiNPs) have previously been shown to induce marked cytokine (interleukin-6; IL-6 and interleukin-8; CXCL8/IL-8) responses independently of particle uptake in human bronchial epithelial BEAS-2B cells. In this study the involvement of the mitogen-activated protein kinases (MAP-kinases), nuclear factor-kappa Β (NF-κΒ) and in particular tumour necrosis factor-α converting enzyme (TACE) and—epidermal growth factor receptor (EGFR) signalling pathways were examined in triggering of IL-6 and CXCL8 release after exposure to a 50 nm silica nanoparticle (Si50). Exposure to Si50 increased phosphorylation of NF-κΒ p65 and MAP-kinases p38 and JUN-N-terminal protein kinase pathways (JNK), but not extracellular signal regulated kinases (ERK). Inhibition of NF-κΒ and p38 reduced the cytokine responses to Si50, whereas neither JNK- nor ERK-inhibition exerted any significant effect on the responses to Si50. Increases in membrane-bound transforming growth factor-α (TGF-α) release and EGFR phosphorylation were also observed after Si50 exposure, and pre-treatment with inhibitors of these pathways reduced the release of IL-6 and CXCL8, but did not affect the Si50-induced phosphorylation of p38 and p65. In contrast, p38-inhibition partially reduced Si50-induced TGF-α release, while the p65-inhibition was without effect. Overall, our results indicate that Si50-induced IL-6 and CXCL8 responses in BEAS-2B cells were regulated through combined activation of several pathways, including NF-κΒ and p38/TACE/TGF-α/EGFR signalling. The study identifies critical, initial events in the triggering of pro-inflammatory responses by nanoparticles. - Highlights: • Silica nanoparticles induce IL-6 and CXCL8 via NFκB and MAPKinase p38 in BEAS-2B • Silica nanoparticles induce release of the EGF-receptor ligand TGF-α • TGF-α release contributes to the IL-6 and CXCL8 release • Phosphorylation of p38 is involved in release of TGF-α.

  20. System-level modeling and simulation of the cell culture microfluidic biochip ProCell

    DEFF Research Database (Denmark)

    Minhass, Wajid Hassan; Pop, Paul; Madsen, Jan

    2010-01-01

    -defined micro-channels using valves and pumps. We present an approach to the system-level modeling and simulation of a cell culture microfluidic biochip called ProCell, Programmable Cell Culture Chip. ProCell contains a cell culture chamber, which is envisioned to run 256 simultaneous experiments (viewed...

  1. Three-dimensional cell culturing by magnetic levitation.

    Science.gov (United States)

    Haisler, William L; Timm, David M; Gage, Jacob A; Tseng, Hubert; Killian, T C; Souza, Glauco R

    2013-10-01

    Recently, biomedical research has moved toward cell culture in three dimensions to better recapitulate native cellular environments. This protocol describes one method for 3D culture, the magnetic levitation method (MLM), in which cells bind with a magnetic nanoparticle assembly overnight to render them magnetic. When resuspended in medium, an external magnetic field levitates and concentrates cells at the air-liquid interface, where they aggregate to form larger 3D cultures. The resulting cultures are dense, can synthesize extracellular matrix (ECM) and can be analyzed similarly to the other culture systems using techniques such as immunohistochemical analysis (IHC), western blotting and other biochemical assays. This protocol details the MLM and other associated techniques (cell culture, imaging and IHC) adapted for the MLM. The MLM requires 45 min of working time over 2 d to create 3D cultures that can be cultured in the long term (>7 d).

  2. Primary culture of Rhodnius prolixus (Hemiptera: Reduviidae salivary gland cells

    Directory of Open Access Journals (Sweden)

    Fernanda F Rocha

    2010-03-01

    Full Text Available In the present paper, we developed a primary culture of Rhodnius prolixus salivary gland and main salivary canal cells. Cells remained viable in culture for 30 days. Three types of cells were indentified in the salivary gland cultures, with binuclear cells being the most abundant. The supernatants of salivary cultures contained mainly 16-24 kDa proteins and presented anticoagulant and apyrase activities. Secretion vesicles were observed budding from the cellular monolayer of the main salivary canal cells. These results indicate that R. prolixus salivary proteins may be produced in vitro and suggest that the main salivary canal may have a possible secretory role.

  3. TGF-β1 induced epithelial to mesenchymal transition (EMT in human bronchial epithelial cells is enhanced by IL-1β but not abrogated by corticosteroids

    Directory of Open Access Journals (Sweden)

    Zuraw Bruce L

    2009-10-01

    Full Text Available Abstract Background Chronic persistent asthma is characterized by ongoing airway inflammation and airway remodeling. The processes leading to airway remodeling are poorly understood, and there is increasing evidence that even aggressive anti-inflammatory therapy does not completely prevent this process. We sought to investigate whether TGFβ1 stimulates bronchial epithelial cells to undergo transition to a mesenchymal phenotype, and whether this transition can be abrogated by corticosteroid treatment or enhanced by the pro-inflammatory cytokine IL-1β. Methods BEAS-2B and primary normal human bronchial epithelial cells were stimulated with TGFβ1 and expression of epithelial and mesenchymal markers assessed by quantitative real-time PCR, immunoblotting, immunofluorescence microscopy and zymography. In some cases the epithelial cells were also incubated with corticosteroids or IL-1β. Results were analyzed using non-parametric statistical tests. Results Treatment of BEAS-2B or primary human bronchial epithelial cells with TGFβ1 significantly reduced the expression level of the epithelial adherence junction protein E-cadherin. TGFβ1 then markedly induced mesenchymal marker proteins such as collagen I, tenascin C, fibronectin and α-smooth muscle actin mRNA in a dose dependant manner. The process of mesenchymal transition was accompanied by a morphological change towards a more spindle shaped fibroblast cell type with a more motile and invasive phenotype. Corticosteroid pre-treatment did not significantly alter the TGFβ1 induced transition but IL-1β enhanced the transition. Conclusion Our results indicate, that TGFβ1 can induce mesenchymal transition in the bronchial epithelial cell line and primary cells. Since asthma has been strongly associated with increased expression of TGFβ1 in the airway, epithelial to mesenchymal transition may contribute to the contractile and fibrotic remodeling process that accompanies chronic asthma.

  4. Anti-Inflammatory Activity of a Novel Family of Aryl Ureas Compounds in an Endotoxin-Induced Airway Epithelial Cell Injury Model

    Science.gov (United States)

    Cabrera-Benitez, Nuria E.; Pérez-Roth, Eduardo; Casula, Milena; Ramos-Nuez, Ángela; Ríos-Luci, Carla; Rodríguez-Gallego, Carlos; Sologuren, Ithaisa; Jakubkiene, Virginija; Slutsky, Arthur S.; Padrón, José M.; Villar, Jesús

    2012-01-01

    Background Despite our increased understanding of the mechanisms involved in acute lung injury (ALI) and the acute respiratory distress syndrome (ARDS), there is no specific pharmacological treatment of proven benefit. We used a novel screening methodology to examine potential anti-inflammatory effects of a small structure-focused library of synthetic carbamate and urea derivatives in a well established cell model of lipopolysaccharide (LPS)-induced ALI/ARDS. Methodology/Principal Findings After a pilot study to develop an in vitro LPS-induced airway epithelial cell injury model, a library of synthetic carbamate and urea derivates was screened against representative panels of human solid tumor cell lines and bacterial and fungal strains. Molecules that were non-cytotoxic and were inactive in terms of antiproliferative and antimicrobial activities were selected to study the effects on LPS-induced inflammatory response in an in vitro cell culture model using A549 human alveolar and BEAS-2B human bronchial cells. These cells were exposed for 18 h to LPS obtained from Escherichia coli, either alone or in combination with the test compounds. The LPS antagonists rhein and emodin were used as reference compounds. The most active compound (CKT0103) was selected as the lead compound and the impact of CKT0103 on pro-inflammatory IL-6 and IL-8 cytokine levels, expression of toll-like receptor-4 (TLR4) and nuclear factor kappa B inhibitor alpha (IκBα) was measured. CKT0103 significantly inhibited the synthesis and release of IL-6 and IL-8 induced by LPS. This suppression was associated with inhibition of TLR4 up-regulation and IκBα down-regulation. Immunocytochemical staining for TLR4 and IκBα supported these findings. Conclusions/Significance Using a novel screening methodology, we identified a compound – CKT0103 – with potent anti-inflammatory effects. These findings suggest that CKT0103 is a potential target for the treatment of the acute phase of sepsis and

  5. Anti-inflammatory activity of a novel family of aryl ureas compounds in an endotoxin-induced airway epithelial cell injury model.

    Directory of Open Access Journals (Sweden)

    Nuria E Cabrera-Benitez

    Full Text Available BACKGROUND: Despite our increased understanding of the mechanisms involved in acute lung injury (ALI and the acute respiratory distress syndrome (ARDS, there is no specific pharmacological treatment of proven benefit. We used a novel screening methodology to examine potential anti-inflammatory effects of a small structure-focused library of synthetic carbamate and urea derivatives in a well established cell model of lipopolysaccharide (LPS-induced ALI/ARDS. METHODOLOGY/PRINCIPAL FINDINGS: After a pilot study to develop an in vitro LPS-induced airway epithelial cell injury model, a library of synthetic carbamate and urea derivates was screened against representative panels of human solid tumor cell lines and bacterial and fungal strains. Molecules that were non-cytotoxic and were inactive in terms of antiproliferative and antimicrobial activities were selected to study the effects on LPS-induced inflammatory response in an in vitro cell culture model using A549 human alveolar and BEAS-2B human bronchial cells. These cells were exposed for 18 h to LPS obtained from Escherichia coli, either alone or in combination with the test compounds. The LPS antagonists rhein and emodin were used as reference compounds. The most active compound (CKT0103 was selected as the lead compound and the impact of CKT0103 on pro-inflammatory IL-6 and IL-8 cytokine levels, expression of toll-like receptor-4 (TLR4 and nuclear factor kappa B inhibitor alpha (IκBα was measured. CKT0103 significantly inhibited the synthesis and release of IL-6 and IL-8 induced by LPS. This suppression was associated with inhibition of TLR4 up-regulation and IκBα down-regulation. Immunocytochemical staining for TLR4 and IκBα supported these findings. CONCLUSIONS/SIGNIFICANCE: Using a novel screening methodology, we identified a compound - CKT0103 - with potent anti-inflammatory effects. These findings suggest that CKT0103 is a potential target for the treatment of the acute phase of

  6. Polydimethylsiloxane SlipChip for mammalian cell culture applications.

    Science.gov (United States)

    Chang, Chia-Wen; Peng, Chien-Chung; Liao, Wei-Hao; Tung, Yi-Chung

    2015-11-07

    This paper reports a polydimethylsiloxane (PDMS) SlipChip for in vitro cell culture applications, multiple-treatment assays, cell co-cultures, and cytokine detection assays. The PDMS SlipChip is composed of two PDMS layers with microfluidic channels on each surface that are separated by a thin silicone fluid (Si-fluid) layer. The integration of Si-fluid enables the two PDMS layers to be slid to different positions; therefore, the channel patterns can be re-arranged for various applications. The SlipChip design significantly reduces the complexity of sample handling, transportation, and treatment processes. To apply the developed SlipChip for cell culture applications, human lung adenocarcinoma epithelial cells (A549) and lung fibroblasts (MRC-5) were cultured to examine the biocompatibility of the developed PDMS SlipChip. Moreover, embryonic pluripotent stem cells (ES-D3) were also cultured in the device to evaluate the retention of their stemness in the device. The experimental results show that cell morphology, viability and proliferation are not affected when the cells are cultured in the SlipChip, indicating that the device is highly compatible with mammalian cell culture. In addition, the stemness of the ES-D3 cells was highly retained after they were cultured in the device, suggesting the feasibility of using the SlipChip for stem cell research. Various cell experiments, such as simultaneous triple staining of cells and co-culture of MRC-5 with A549 cells, were also performed to demonstrate the functionalities of the PDMS SlipChip. Furthermore, we used a cytokine detection assay to evaluate the effect of endotoxin (lipopolysaccharides, LPS) treatment on the cytokine secretion of A549 cells using the SlipChip. The developed PDMS SlipChip provides a straightforward and effective platform for various on-chip in vitro cell cultures and consequent analysis, which is promising for a number of cell biology studies and biomedical applications.

  7. Quantitative-PCR Assessment of Cryptosporidium parvum Cell Culture Infection

    OpenAIRE

    Di Giovanni, George D.; LeChevallier, Mark W.

    2005-01-01

    A quantitative TaqMan PCR method was developed for assessing the Cryptosporidium parvum infection of in vitro cultivated human ileocecal adenocarcinoma (HCT-8) cell cultures. This method, termed cell culture quantitative sequence detection (CC-QSD), has numerous applications, several of which are presented. CC-QSD was used to investigate parasite infection in cell culture over time, the effects of oocyst treatment on infectivity and infectivity assessment of different C. parvum isolates. CC-Q...

  8. Organ culture-cell culture system for studying multistage carcinogenesis in respiratory epithelium. [Mice

    Energy Technology Data Exchange (ETDEWEB)

    Steele, Vernon E.; Marchok, Ann C.; Nettesheim, Paul

    1977-01-01

    An organ culture-cell culture system was used to demonstrate carcinogen dose-dependent transformation of tracheal epithelial cells in vitro. Tracheal explants were exposed to MNNG (N-methyl-N/sup 1/-nitro-N-nitrosoguanidine) in organ culture. Outgrowths from these explants provided epithelial cell cultures. The numbers of long term epithelial cell cultures and cell lines that were established per explant increased as MNNG exposure concentration increased. At the present time, more cell lines derived from explants exposed to the highest MNNG concentration have produced palpable tumors than cell lines derived from explants exposed to lower MNNG concentrations. No cell lines were established from primaries derived from control explants. TPA (12-0-tetradecanoyl-phorbol-13-acetate), stimulates DNA synthesis in tracheal epithelium in organ culture in a manner simular to that described for mouse skin. Short exposures to TPA not only stimulated DNA synthesis earlier, but the stimulation was greater than that obtained with continuous exposure. At the present time, exposure of tracheal organ cultures to MNNG followed by TPA has resulted in an enhanced production of morphologically altered cells in primary epithelial cell cultures, than exposure to either agent alone.

  9. Growth of the Pittsburgh Pneumonia Agent in Animal Cell Cultures

    OpenAIRE

    Rinaldo, Charles R.; Pasculle, A. William; Myerowitz, Richard L.; Gress, Francis M.; Dowling, John N.

    1981-01-01

    Pittsburgh pneumonia agent (Legionella micdadei) grew in monkey, chicken, and human cell cultures. Pittsburgh pneumonia agent grew predominantly in the cytoplasm, resulting in a nonfocal, mild cytopathic effect.

  10. Particle Trajectories in Rotating Wall Cell Culture Devices

    Science.gov (United States)

    Ramachandran N.; Downey, J. P.

    1999-01-01

    Cell cultures are extremely important to the medical community since such cultures provide an opportunity to perform research on human tissue without the concerns inherent in experiments on individual humans. Development of cells in cultures has been found to be greatly influenced by the conditions of the culture. Much work has focused on the effect of the motions of cells in the culture relative to the solution. Recently rotating wall vessels have been used with success in achieving improved cellular cultures. Speculation and limited research have focused on the low shear environment and the ability of rotating vessels to keep cells suspended in solution rather than floating or sedimenting as the primary reasons for the improved cellular cultures using these devices. It is widely believed that the cultures obtained using a rotating wall vessel simulates to some degree the effect of microgravity on cultures. It has also been speculated that the microgravity environment may provide the ideal acceleration environment for culturing of cellular tissues due to the nearly negligible levels of sedimentation and shear possible. This work predicts particle trajectories of cells in rotating wall vessels of cylindrical and annular design consistent with the estimated properties of typical cellular cultures. Estimates of the shear encountered by cells in solution and the interactions with walls are studied. Comparisons of potential experiments in ground and microgravity environments are performed.

  11. Gene expression profiling of the effects of organic dust in lung epithelial and THP-1 cells reveals inductive effects on inflammatory and immune response genes.

    Science.gov (United States)

    Boggaram, Vijay; Loose, David S; Gottipati, Koteswara R; Natarajan, Kartiga; Mitchell, Courtney T

    2016-04-01

    The intensification and concentration of animal production operations expose workers to high levels of organic dusts in the work environment. Exposure to organic dusts is a risk factor for the development of acute and chronic respiratory symptoms and diseases. Lung epithelium plays important roles in the control of immune and inflammatory responses to environmental agents to maintain lung health. To better understand the effects of organic dust on lung inflammatory responses, we characterized the gene expression profiles of A549 alveolar and Beas2B bronchial epithelial and THP-1 monocytic cells influenced by exposure to poultry dust extract by DNA microarray analysis using Illumina Human HT-12 v4 Expression BeadChip. We found that A549 alveolar and Beas2B bronchial epithelial and THP-1 cells responded with unique changes in the gene expression profiles with regulation of genes encoding inflammatory cytokines, chemokines, and other inflammatory proteins being common to all the three cells. Significantly induced genes included IL-8, IL-6, IL-1β, ICAM-1, CCL2, CCL5, TLR4, and PTGS2. Validation by real-time qRT-PCR, ELISA, Western immunoblotting, and immunohistochemical staining of lung sections from mice exposed to dust extract validated DNA microarray results. Pathway analysis indicated that dust extract induced changes in gene expression influenced functions related to cellular growth and proliferation, cell death and survival, and cellular development. These data show that a broad range of inflammatory mediators produced in response to poultry dust exposure can modulate lung immune and inflammatory responses. This is the first report on organic dust induced changes in expression profiles in lung epithelial and THP-1 monocytic cells.

  12. Cell culture chip using low-shear mass transport.

    Science.gov (United States)

    Liu, Ke; Pitchimani, Rajasekar; Dang, Dana; Bayer, Keith; Harrington, Tyler; Pappas, Dimitri

    2008-06-03

    We have developed a flow cell that allows culturing adherent cells as well as suspended cells in a stable, homogeneous, and low-shear force environment. The device features continuous medium supply and waste exchange. In this paper, a simple and fast protocol for device design, fabrication, and assembly (sealing) based on a poly(dimethylsiloxane) (PMDS)/glass slide hybrid structure is described. The cell culture system performance was monitored, and the effective shear force inside the culture well was also determined. By manipulating the device dimensions and volumetric flow rate, shear stress was controlled during experiments. Cell adhesion, growth, proliferation, and death over long-term culture periods were observed by microscopy. The growth of both endothelial and suspension cells in this device exhibited comparable characteristics to those of traditional approaches. The low-shear culture device significantly reduced shear stress encountered in microfluidic systems, allowing both adherent and suspended cells to be grown in a simple device.

  13. Three-dimensional Tissue Culture Based on Magnetic Cell Levitation

    Science.gov (United States)

    Souza, Glauco R.; Molina, Jennifer R.; Raphael, Robert M.; Ozawa, Michael G.; Stark, Daniel J.; Levin, Carly S.; Bronk, Lawrence F.; Ananta, Jeyarama S.; Mandelin, Jami; Georgescu, Maria-Magdalena; Bankson, James A.; Gelovani, Juri G.

    2015-01-01

    Cell culture is an essential tool for drug discovery, tissue engineering, and stem cell research. Conventional tissue culture produces two-dimensional (2D) cell growth with gene expression, signaling, and morphology that can differ from those in vivo and thus compromise clinical relevancy1–5. Here we report a three-dimensional (3D) culture of cells based on magnetic levitation in the presence of hydrogels containing gold and magnetic iron oxide (MIO) nanoparticles plus filamentous bacteriophage. This methodology allows for control of cell mass geometry and guided, multicellular clustering of different cell types in co-culture through spatial variance of the magnetic field. Moreover, magnetic levitation of human glioblastoma cells demonstrates similar protein expression profiles to those observed in human tumor xenografts. Taken together, these results suggest levitated 3D culture with magnetized phage-based hydrogels more closely recapitulates in vivo protein expression and allows for long-term multi-cellular studies. PMID:20228788

  14. HAIR CELL-LIKE CELL GENERATION INDUCED BY NATURE CULTURE OF ADULT RAT AUDITORY EPITHELIUM

    Institute of Scientific and Technical Information of China (English)

    Liu Hui; Zhu Hongliang; Li Shengli; Yao Xiaobao; Wang Xiaoxia

    2006-01-01

    Objective To establish adult rat auditory epithelial cell culture and try to find precursor cells of auditory hair cells in vitro. Methods With refinement of culture media and techniques, cochlear sensory epithelial cells of adult rat were cultured. Immunocytochemistry and Bromodeoxyuridine (BrdU)labeling were used to detect properties and mitotic status of cultured cells. Results The cultured auditory epithelial cells showed a large, flat epithelial morphotype and expressed F-actin and cytokeratin, a subset of cells generated from auditory epithelium were labeled by calretinin, a specific marker of early hair cell. Conclusion Adult rat auditory epithelium can be induced to generate hair cell-like cells by nature culture, this phenomenon suggests that progenitor cells may exist in rat cochlea and they may give birth to new hair cells. Whether these progenitor cells are tissue specific stem cells is still need more study.

  15. Dissociated neurons of the pupal blowfly antenna in cell culture.

    Science.gov (United States)

    Nakagawa, A; Iwama, A

    1995-12-01

    Primary cell cultures are useful for studying the function of neurons in a simplified and controlled environment. We established a primary culture of antennal cells from pupal blowflies in order to investigate olfactory receptor neurons. In cultures, neuron-like cells were identified on the basis of morphology and immunocytochemical characterization with anti-HRP staining. Neuron-like cells showed variety in the extension pattern of neurites. Many neuron-like cells extended a single prominent long process, which reached about 200 microm after four days, and several short ones. However, some neuron-like cells differentiated in other ways; some exhibited bipolar or multipolar processes, distinct from intact olfactory receptor neurons. The size of cell bodies of neuron-like cells as divisible into two groups; approx. 7 microm diameter and 10-15 microm diameter. Neuron-like cells in culture will provide a good model for electrophysiological analysis and for developmental studies of olfactory receptor neurons.

  16. Usability and Applicability of Microfluidic Cell Culture Systems

    DEFF Research Database (Denmark)

    Hemmingsen, Mette

    Microfluidic cell culture has been a research area with great attention the last decade due to its potential to mimic the in vivo cellular environment more closely compared to what is possible by conventional cell culture methods. Many exciting and complex devices have been presented providing...... possibilities for, for example, precise control of the chemical environment, 3D cultures, controlled co-culture of different cell types or automated, individual control of up to 96 cell culture chambers in one integrated system. Despite the great new opportunities to perform novel experimental designs......, these devices still lack general implementation into biological research laboratories. In this project, the usability and applicability of microfluidic cell culture systems have been investigated. The tested systems display good properties regarding optics and compatibility with standard laboratory equipment...

  17. THE ALKALOID CYTISINE IN THE CELL CULTURE

    Directory of Open Access Journals (Sweden)

    Gazaliev A.M.

    2012-08-01

    Full Text Available Alkaloids are vegetative establishments of complex and original structure with nitrous heterocycles in the basis. For a long time they drew researchers’ attention because of their unique and specific physiological effect on alive organisms. Not all the representatives of the globe’s flora contain these unique substances. Alkaloid cytisine is to be found mainly in the plants of the fabaceous family - Fabaceae. For the cytisine production the seeds of Thermopsis lanceolata R.Br (T. lanceolata R.Br and Cytisus laburnum (C. laburnum are used as a raw material. The object of the research is T. lanceolata cell culture. Sterile sprouts are used at the first stage of the experiment. Callus genesis is accompanied with dedifferentiation. It leads to the cellular organization simplification. Based on an important property of a plant cell, such as totipotency, there appears the formation of the “de novo” biosynthetic device. The cultivation algorithm consists of two basic stages: (i the cultivation conditions optimization of callus with a high level of the primary metabolites biosynthesis (Aspartat – lysine; (ii the research of cultivation chemical and physical factors influence on the secondary metabolite (cytisine biosynthesis and accumulation. During the cultivation the Murashige and Skoog classical recipe of nutrient medium will be used. Optimization of the cultivation conditions will concern the phytohormones, macro- and micronutrients content, as the purpose of optimization is the production of the determined high-level competence embriogenical callus. The main problem is genetic heterogeneity of a cellular population and instability of morpho-physiological processes. The correct management of higher plants cells population is possible at the synchronization of a cellular cycle phases. The references analysis has shown that it is almost impossible to synchronize cellular cycles in the culture of plant tissue. The application of chemical

  18. An Optically Controlled 3D Cell Culturing System

    Directory of Open Access Journals (Sweden)

    Kelly S. Ishii

    2011-01-01

    Full Text Available A novel 3D cell culture system was developed and tested. The cell culture device consists of a microfluidic chamber on an optically absorbing substrate. Cells are suspended in a thermoresponsive hydrogel solution, and optical patterns are utilized to heat the solution, producing localized hydrogel formation around cells of interest. The hydrogel traps only the desired cells in place while also serving as a biocompatible scaffold for supporting the cultivation of cells in 3D. This is demonstrated with the trapping of MDCK II and HeLa cells. The light intensity from the optically induced hydrogel formation does not significantly affect cell viability.

  19. Aeroponics for the culture of organisms, tissues and cells.

    Science.gov (United States)

    Weathers, P J; Zobel, R W

    1992-01-01

    Characteristics of aeroponics are discussed. Contrast is made, where appropriate, with hydroponics and aero-hydroponics as applies to research and commercial applications of nutrient mist technology. Topics include whole plants, plant tissue cultures, cell and microbial cultures, and animal tissue cultures with regard to operational considerations (moisture, temperature, minerals, gaseous atmosphere) and design of apparati.

  20. Systems Biology for Organotypic Cell Cultures

    Energy Technology Data Exchange (ETDEWEB)

    Grego, Sonia [RTI International, Research Triangle Park, NC (United States); Dougherty, Edward R. [Texas A & M Univ., College Station, TX (United States); Alexander, Francis J. [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Auerbach, Scott S. [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States); Berridge, Brian R. [GlaxoSmithKline, Research Triangle Park, NC (United States); Bittner, Michael L. [Translational Genomics Research Inst., Phoenix, AZ (United States); Casey, Warren [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States); Cooley, Philip C. [RTI International, Research Triangle Park, NC (United States); Dash, Ajit [HemoShear Therapeutics, Charlottesville, VA (United States); Ferguson, Stephen S. [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States); Fennell, Timothy R. [RTI International, Research Triangle Park, NC (United States); Hawkins, Brian T. [RTI International, Research Triangle Park, NC (United States); Hickey, Anthony J. [RTI International, Research Triangle Park, NC (United States); Kleensang, Andre [Johns Hopkins Univ., Baltimore, MD (United States). Center for Alternatives to Animal Testing; Liebman, Michael N. [IPQ Analytics, Kennett Square, PA (United States); Martin, Florian [Phillip Morris International, Neuchatel (Switzerland); Maull, Elizabeth A. [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States); Paragas, Jason [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Qiao, Guilin [Defense Threat Reduction Agency, Ft. Belvoir, VA (United States); Ramaiahgari, Sreenivasa [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States); Sumner, Susan J. [RTI International, Research Triangle Park, NC (United States); Yoon, Miyoung [The Hamner Inst. for Health Sciences, Research Triangle Park, NC (United States); ScitoVation, Research Triangle Park, NC (United States)

    2016-08-04

    Translating in vitro biological data into actionable information related to human health holds the potential to improve disease treatment and risk assessment of chemical exposures. While genomics has identified regulatory pathways at the cellular level, translation to the organism level requires a multiscale approach accounting for intra-cellular regulation, inter-cellular interaction, and tissue/organ-level effects. Tissue-level effects can now be probed in vitro thanks to recently developed systems of three-dimensional (3D), multicellular, “organotypic” cell cultures, which mimic functional responses of living tissue. However, there remains a knowledge gap regarding interactions across different biological scales, complicating accurate prediction of health outcomes from molecular/genomic data and tissue responses. Systems biology aims at mathematical modeling of complex, non-linear biological systems. We propose to apply a systems biology approach to achieve a computational representation of tissue-level physiological responses by integrating empirical data derived from organotypic culture systems with computational models of intracellular pathways to better predict human responses. Successful implementation of this integrated approach will provide a powerful tool for faster, more accurate and cost-effective screening of potential toxicants and therapeutics. On September 11, 2015, an interdisciplinary group of scientists, engineers, and clinicians gathered for a workshop in Research Triangle Park, North Carolina, to discuss this ambitious goal. Participants represented laboratory-based and computational modeling approaches to pharmacology and toxicology, as well as the pharmaceutical industry, government, non-profits, and academia. Discussions focused on identifying critical system perturbations to model, the computational tools required, and the experimental approaches best suited to generating key data. This consensus report summarizes the discussions held.

  1. Isolation and culture of larval cells from C. elegans.

    Directory of Open Access Journals (Sweden)

    Sihui Zhang

    Full Text Available Cell culture is an essential tool to study cell function. In C. elegans the ability to isolate and culture cells has been limited to embryonically derived cells. However, cells or blastomeres isolated from mixed stage embryos terminally differentiate within 24 hours of culture, thus precluding post-embryonic stage cell culture. We have developed an efficient and technically simple method for large-scale isolation and primary culture of larval-stage cells. We have optimized the treatment to maximize cell number and minimize cell death for each of the four larval stages. We obtained up to 7.8×10(4 cells per microliter of packed larvae, and up to 97% of adherent cells isolated by this method were viable for at least 16 hours. Cultured larval cells showed stage-specific increases in both cell size and multinuclearity and expressed lineage- and cell type-specific reporters. The majority (81% of larval cells isolated by our method were muscle cells that exhibited stage-specific phenotypes. L1 muscle cells developed 1 to 2 wide cytoplasmic processes, while L4 muscle cells developed 4 to 14 processes of various thicknesses. L4 muscle cells developed bands of myosin heavy chain A thick filaments at the cell center and spontaneously contracted ex vivo. Neurons constituted less than 10% of the isolated cells and the majority of neurons developed one or more long, microtubule-rich protrusions that terminated in actin-rich growth cones. In addition to cells such as muscle and neuron that are high abundance in vivo, we were also able to isolate M-lineage cells that constitute less than 0.2% of cells in vivo. Our novel method of cell isolation extends C. elegans cell culture to larval developmental stages, and allows use of the wealth of cell culture tools, such as cell sorting, electrophysiology, co-culture, and high-resolution imaging of subcellular dynamics, in investigation of post-embryonic development and physiology.

  2. Using Tissue Culture To Investigate Plant Cell Differentiation and Dedifferentiation.

    Science.gov (United States)

    Bozzone, Donna M.

    1997-01-01

    Describes an experimental project that uses plant tissue culture techniques to examine cell differentiation in the carrot. Allows students to gain experience in some important techniques and to explore fundamental questions about cell differentiation. (DDR)

  3. Using Tissue Culture To Investigate Plant Cell Differentiation and Dedifferentiation.

    Science.gov (United States)

    Bozzone, Donna M.

    1997-01-01

    Describes an experimental project that uses plant tissue culture techniques to examine cell differentiation in the carrot. Allows students to gain experience in some important techniques and to explore fundamental questions about cell differentiation. (DDR)

  4. Acetylsalicylic acid induces programmed cell death in Arabidopsis cell cultures.

    Science.gov (United States)

    García-Heredia, José M; Hervás, Manuel; De la Rosa, Miguel A; Navarro, José A

    2008-06-01

    Acetylsalicylic acid (ASA), a derivative from the plant hormone salicylic acid (SA), is a commonly used drug that has a dual role in animal organisms as an anti-inflammatory and anticancer agent. It acts as an inhibitor of cyclooxygenases (COXs), which catalyze prostaglandins production. It is known that ASA serves as an apoptotic agent on cancer cells through the inhibition of the COX-2 enzyme. Here, we provide evidences that ASA also behaves as an agent inducing programmed cell death (PCD) in cell cultures of the model plant Arabidopsis thaliana, in a similar way than the well-established PCD-inducing agent H(2)O(2), although the induction of PCD by ASA requires much lower inducer concentrations. Moreover, ASA is herein shown to be a more efficient PCD-inducing agent than salicylic acid. ASA treatment of Arabidopsis cells induces typical PCD-linked morphological and biochemical changes, namely cell shrinkage, nuclear DNA degradation, loss of mitochondrial membrane potential, cytochrome c release from mitochondria and induction of caspase-like activity. However, the ASA effect can be partially reverted by jasmonic acid. Taking together, these results reveal the existence of common features in ASA-induced animal apoptosis and plant PCD, and also suggest that there are similarities between the pathways of synthesis and function of prostanoid-like lipid mediators in animal and plant organisms.

  5. Dammarane-type glycosides from Gynostemma pentaphyllum and their effects on IL-4-induced eotaxin expression in human bronchial epithelial cells.

    Science.gov (United States)

    Hung, Tran Manh; Thu, Cao Van; Cuong, To Dao; Hung, Nguyen Phi; Kwack, Seung Jun; Huh, Jung-Im; Min, Byung Sun; Choi, Jae Sue; Lee, Hyeong Kyu; Bae, KiHwan

    2010-02-26

    Two new dammarane-type glycosides, 2alpha,3beta,12beta,20S-tetrahydroxydammar-24-ene-3-O-[beta-d-glucopyranosyl(1-->4)-beta-d-glucopyranosyl]-20-O-[beta-d-xylopyranosyl-(1-->6)-beta-d-glucopyranoside] (1) and 2alpha,3beta,12beta,20S-tetrahydroxydammar-24-ene-3-O-beta-d-glucopyranosyl-20-O-[beta-d-6-O-acetylglucopyranosyl-(1-->2)-beta-d-glucopyranoside] (2), were isolated from a MeOH extract of the leaves of Gynostemma pentaphyllum. Their structures were elucidated by 1D and 2D NMR spectroscopic interpretation as well as by chemical studies. The isolated compounds showed potential inhibitory effects on eotaxin expression in BEAS-2B bronchial epithelial cells.

  6. A 3D cell culture system: separation distance between INS-1 cell and endothelial cell monolayers co-cultured in fibrin influences INS-1 cells insulin secretion.

    Science.gov (United States)

    Sabra, Georges; Vermette, Patrick

    2013-02-01

    The aim of this study was to develop an in vitro cell culture system allowing studying the effect of separation distance between monolayers of rat insulinoma cells (INS-1) and human umbilical vein endothelial cells (HUVEC) co-cultured in fibrin over INS-1 cell insulin secretion. For this purpose, a three-dimensional (3D) cell culture chamber was designed, built using micro-fabrication techniques and validated. The co-culture was successfully carried out and the effect on INS-1 cell insulin secretion was investigated. After 48 and 72 h, INS-1 cells co-cultured with HUVEC separated by a distance of 100 µm revealed enhanced insulin secretion compared to INS-1 cells cultured alone or co-cultured with HUVEC monolayers separated by a distance of 200 µm. These results illustrate the importance of the separation distance between two cell niches for cell culture design and the possibility to further enhance the endocrine function of beta cells when this factor is considered.

  7. Callus production from photoautotrophic soybean cell culture protoplasts.

    Science.gov (United States)

    Chowhury, V K; Widholm, J M

    1985-10-01

    Protoplasts were prepared from a photoautotrophic (PA) cell line of Glycine max (soybean). A yield of 75 to 90% after two to three hours digestion in a mixture of 1% Cellulase R10, 0.2% Pectolyase Y23 and 2% Driselase was obtained. Cell division and colony formation occurred from approximately 18% of the plated protoplasts. The cultured protoplasts were as sensitive to the herbicide atrazine, a photosynthetic inhibitor, as the original PA cells under the same conditions. Protoplasts and cells of a heterotrophic (HT) soybean culture were not as sensitive to atrazine. The isolated protoplasts retained the PA characteristics of the parental culture in the callus and cell suspension cultures obtained from the protoplasts. The chromosome numbers in the parental cell line and in cells derived from the isolated protoplasts (both PA and HT) were found to be largely (99%) the normal diploid number of 40.

  8. HEPES inhibits the conversion of prion protein in cell culture.

    Science.gov (United States)

    Delmouly, Karine; Belondrade, Maxime; Casanova, Danielle; Milhavet, Ollivier; Lehmann, Sylvain

    2011-05-01

    HEPES is a well-known buffering reagent used in cell-culture medium. Interestingly, this compound is also responsible for significant modifications of biological parameters such as uptake of organic molecules, alteration of oxidative stress mechanisms or inhibition of ion channels. While using cell-culture medium supplemented with HEPES on prion-infected cells, it was noticed that there was a significant concentration-dependent inhibition of accumulation of the abnormal isoform of the prion protein (PrP(Sc)). This effect was present only in live cells and was thought to be related to modification of the PrP environment or biology. These results could modify the interpretation of cell-culture assays of prion therapeutic agents, as well as of previous cell biology results obtained in the field using HEPES buffers. This inhibitory effect of HEPES could also be exploited to prevent contamination or propagation of prions in cell culture.

  9. Horizontally rotated cell culture system with a coaxial tubular oxygenator

    Science.gov (United States)

    Wolf, David A. (Inventor); Schwarz, Ray P. (Inventor); Trinh, Tinh T. (Inventor)

    1991-01-01

    The present invention relates to a horizontally rotating bioreactor useful for carrying out cell and tissue culture. For processing of mammalian cells, the system is sterilized and fresh fluid medium, microcarrier beads, and cells are admitted to completely fill the cell culture vessel. An oxygen containing gas is admitted to the interior of the permeable membrane which prevents air bubbles from being introduced into the medium. The cylinder is rotated at a low speed within an incubator so that the circular motion of the fluid medium uniformly suspends the microbeads throughout the cylinder during the cell growth period. The unique design of this cell and tissue culture device was initially driven by two requirements imposed by its intended use for feasibility studies for three dimensional culture of living cells and tissues in space by JSC. They were compatible with microgravity and simulation of microgravity in one G. The vessels are designed to approximate the extremely quiescent low shear environment obtainable in space.

  10. 21 CFR 876.5885 - Tissue culture media for human ex vivo tissue and cell culture processing applications.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Tissue culture media for human ex vivo tissue and... DEVICES Therapeutic Devices § 876.5885 Tissue culture media for human ex vivo tissue and cell culture processing applications. (a) Identification. Tissue culture media for human ex vivo tissue and cell culture...

  11. PECULIARITIES OF SECONDARY METABOLITES BIOSYNTHESIS IN PLANT CELL CULTURES

    Directory of Open Access Journals (Sweden)

    A.M. NOSOV

    2014-06-01

    Full Text Available metabolites formation in plant cell cultures of Panax spp., (ginsenosides; Dioscorea deltoidea (steroid glycosides; Ajuga reptans, Serratula coronata, Rhaponticum carthamoides (ecdisteroids; Polyscias spp., (triterpene glycosides, Taxus spp. (taxoids, Stevia rebaudiana (diterpene steviol-glycosides, Stephania glabra (alkaloids. They are some regular trends of secondary metabolites synthesis in the plant cell culture:It can be noted the stable synthesis of the compound promoting cell proliferation. Indeed, cell cultures of Dioscorea deltoidea were demonstrated to accumulate only furostanol glycosides, which promoted cell division. Furostanol glycoside content of Dioscorea strain DM-0.5 was up to 6 - 12% by dry biomass.Panax ginseng and P. japonicus plant cell cultures synthesize as minimum seven triterpene glycosides (ginsenosides, the productivity of these compounds was up to 6.0 - 8.0% on dry biomass.By contrast, the detectable synthesis of diterpene steviol-glycosides in cultivated cells of Stevia rebaudiana initiated in the mixotrophic cultures during chloroplast formation only.Despite these differences, or mainly due to them, plant cell cultures have become an attractive source of phytochemicals in alternative to collecting wild plants. It provides a guideline to bioreactor-based production of isoprenoids using undifferentiated plant cell cultures

  12. Primary cell culture of human adenocarcinomas--practical considerations.

    Science.gov (United States)

    Lerescu, Lucian; Tucureanu, Cătălin; Caraş, Iuliana; Neagu, Stefan; Melinceanu, Laura; Sălăgeanu, Aurora

    2008-01-01

    Cell culture is one of the major tools for oncology research, being an excellent system in which to study the biochemistry and molecular biology associated with individual cancer types and to understand cancer cell physiology. Progress in understanding the biology of any type of carcinoma has been impeded by the inability to culture adequately malignant cells from most epithelial tissues. The ultimate in vitro tumor model would completely reflect the in vivo tumor microenvironment in function and mechanism. Unfortunately, such a model does not currently exist. Homogeneous cell lines that can be continuously propagated on plastic surfaces have been extensively used as a surrogate for tumor environment; however they are very different from the in vivo tumor cells. Model systems involving primary culture represent the situation most closely related to the original tissue although they have a number of disadvantages over cell lines, such as the limited ability to repeat studies with a well characterized culture system that can be used in multiple laboratories. The primary culture may contain many types of stromal and infiltrating cell types potentially complicating the interpretation of data. Yet, their properties better reflect the cellular interactions present in intact tissue. The present article reviews the critical steps in obtaining, routine maintenance and cryopreservation of primary tumor cell cultures, based on information from literature and personal experience on the subject. The article also includes an updated protocol for primary tumor cell isolation and culture.

  13. Optical Oxygen Sensors for Applications in Microfluidic Cell Culture

    Directory of Open Access Journals (Sweden)

    Samantha M. Grist

    2010-10-01

    Full Text Available The presence and concentration of oxygen in biological systems has a large impact on the behavior and viability of many types of cells, including the differentiation of stem cells or the growth of tumor cells. As a result, the integration of oxygen sensors within cell culture environments presents a powerful tool for quantifying the effects of oxygen concentrations on cell behavior, cell viability, and drug effectiveness. Because microfluidic cell culture environments are a promising alternative to traditional cell culture platforms, there is recent interest in integrating oxygen-sensing mechanisms with microfluidics for cell culture applications. Optical, luminescence-based oxygen sensors, in particular, show great promise in their ability to be integrated with microfluidics and cell culture systems. These sensors can be highly sensitive and do not consume oxygen or generate toxic byproducts in their sensing process. This paper presents a review of previously proposed optical oxygen sensor types, materials and formats most applicable to microfluidic cell culture, and analyzes their suitability for this and other in vitro applications.

  14. Antioxidant components of naturally-occurring oils exhibit marked anti-inflammatory activity in epithelial cells of the human upper respiratory system

    Directory of Open Access Journals (Sweden)

    Biswal Shyam

    2011-07-01

    Full Text Available Abstract Background The upper respiratory tract functions to protect lower respiratory structures from chemical and biological agents in inspired air. Cellular oxidative stress leading to acute and chronic inflammation contributes to the resultant pathology in many of these exposures and is typical of allergic disease, chronic sinusitis, pollutant exposure, and bacterial and viral infections. Little is known about the effective means by which topical treatment of the nose can strengthen its antioxidant and anti-inflammatory defenses. The present study was undertaken to determine if naturally-occurring plant oils with reported antioxidant activity can provide mechanisms through which upper respiratory protection might occur. Methods Controlled exposure of the upper respiratory system to ozone and nasal biopsy were carried out in healthy human subjects to assess mitigation of the ozone-induced inflammatory response and to assess gene expression in the nasal mucosa induced by a mixture of five naturally-occurring antioxidant oils - aloe, coconut, orange, peppermint and vitamin E. Cells of the BEAS-2B and NCI-H23 epithelial cell lines were used to investigate the source and potential intracellular mechanisms of action responsible for oil-induced anti-inflammatory activity. Results Aerosolized pretreatment with the mixed oil preparation significantly attenuated ozone-induced nasal inflammation. Although most oil components may reduce oxidant stress by undergoing reduction, orange oil was demonstrated to have the ability to induce long-lasting gene expression of several antioxidant enzymes linked to Nrf2, including HO-1, NQO1, GCLm and GCLc, and to mitigate the pro-inflammatory signaling of endotoxin in cell culture systems. Nrf2 activation was demonstrated. Treatment with the aerosolized oil preparation increased baseline levels of nasal mucosal HO-1 expression in 9 of 12 subjects. Conclusions These data indicate that selected oil-based antioxidant

  15. Coculture of osteoblasts and endothelial cells: optimization of culture medium and cell ratio

    NARCIS (Netherlands)

    Ma, J.; Beucken, J.J. van den; Yang, F.; Both, S.K.; Cui, F.Z.; Pan, J.; Jansen, J.A.

    2011-01-01

    Vascularization strategies in cell-based bone tissue engineering depend on optimal culture conditions. The present study aimed to determine optimal cell culture medium and cell ratio for cocultures of human marrow stromal cells (HMSCs) and human umbilical vein endothelial cells (HUVECs) in view of

  16. Biologic characteristics of fibroblast cells cultured from the knee ligaments

    Institute of Scientific and Technical Information of China (English)

    陈鸿辉; 唐毅; 李斯明; 沈雁; 刘向荣; 钟灿灿

    2002-01-01

    Objective: To culture fibroblast cells from the kneeligaments and to study the biological characteristics of thesecells.Methods: Cells of the anterior cruciate ligament(ACL) and the medial collateral ligament (MCL) fromNew Zealand white rabbit were cultured in vitro. Cellulargrowth and expression of the collagen were analyzed.Moreover, an in vitro wound closure model was establishedand the healing of the ACL and the MCL cells wascompared.Results: Maximal growth for all these cells wereobtained with Dulbecco's modified Eagle's mediumsupplemented with 10% fetal bovine serum, but RPMI 1640and Ham's F12 media were not suitable to maintain thesecells. Morphology of both ACL and MCL cells from NewZealand white rabbit was alike in vitro, but the MCL cellsgrew faster than the ACL cells. Both cell types producedsimilar amount of collagen in culture, but the ratio ofcollage type I to type III produced by ACL cells was higherthan that produced by MCL cells. Wound closure assayshowed that at 36 hours after injury, cell-free zones createdin the ACL cultures were occupied partially by the ACLcells; in contrast, the wounded zone in the MCL cultureswas almost completely covered by the cells.Conclusions: Although the ACL cells and the MCLcells from New Zealand white rabbit show similarappearance in morphology in culture, the cellular growthand the biochemical synthesis of collagen as well as thehealing in vitro were significantly different. Thesedifferences in intrinsic properties of the two types of cells invitro might contribute to the differential healing potentialsof these ligaments in vivo.

  17. LIF-free embryonic stem cell culture in simulated microgravity.

    Directory of Open Access Journals (Sweden)

    Yumi Kawahara

    Full Text Available BACKGROUND: Leukemia inhibitory factor (LIF is an indispensable factor for maintaining mouse embryonic stem (ES cell pluripotency. A feeder layer and serum are also needed to maintain an undifferentiated state, however, such animal derived materials need to be eliminated for clinical applications. Therefore, a more reliable ES cell culture technique is required. METHODOLOGY/PRINCIPAL FINDINGS: We cultured mouse ES cells in simulated microgravity using a 3D-clinostat. We used feeder-free and serum-free media without LIF. CONCLUSIONS/SIGNIFICANCE: Here we show that simulated microgravity allows novel LIF-free and animal derived material-free culture methods for mouse ES cells.

  18. Long-term maintenance of human induced pluripotent stem cells by automated cell culture system.

    Science.gov (United States)

    Konagaya, Shuhei; Ando, Takeshi; Yamauchi, Toshiaki; Suemori, Hirofumi; Iwata, Hiroo

    2015-11-17

    Pluripotent stem cells, such as embryonic stem cells and induced pluripotent stem (iPS) cells, are regarded as new sources for cell replacement therapy. These cells can unlimitedly expand under undifferentiated conditions and be differentiated into multiple cell types. Automated culture systems enable the large-scale production of cells. In addition to reducing the time and effort of researchers, an automated culture system improves the reproducibility of cell cultures. In the present study, we newly designed a fully automated cell culture system for human iPS maintenance. Using an automated culture system, hiPS cells maintained their undifferentiated state for 60 days. Automatically prepared hiPS cells had a potency of differentiation into three germ layer cells including dopaminergic neurons and pancreatic cells.

  19. Colorimetric pH measurement of animal cell culture media.

    Science.gov (United States)

    Jang, Juno; Moon, Soo-Jin; Hong, Sung-Hwan; Kim, Ik-Hwan

    2010-11-01

    Most animal cell culture media can be buffered using bicarbonate and high pressure CO(2) in a closed system. However, in an open system, the pH of the culture media increases continuously due to the marked difference in CO(2) pressure between the culture media and the atmosphere. Therefore, it is important to measure the exact pH of the culture media in an intact closed system. In this study, a pH measurement method was developed using visible light. The pH was calculated from light absorbance by the cells and by the culture media. This method was successfully applied to both suspension and anchorage-dependent cell cultures.

  20. Novel culturing platform for brain slices and neuronal cells

    DEFF Research Database (Denmark)

    Svendsen, Winnie Edith; Al Atraktchi, Fatima Al-Zahraa; Bakmand, Tanya

    2015-01-01

    In this paper we demonstrate a novel culturing system for brain slices and neuronal cells, which can control the concentration of nutrients and the waste removal from the culture by adjusting the fluid flow within the device. The entire system can be placed in an incubator. The system has been te...... tested successfully with brain slices and PC12 cells. The culture substrate can be modified using metal electrodes and/or nanostructures for conducting electrical measurements while culturing and for better mimicking the in vivo conditions.......In this paper we demonstrate a novel culturing system for brain slices and neuronal cells, which can control the concentration of nutrients and the waste removal from the culture by adjusting the fluid flow within the device. The entire system can be placed in an incubator. The system has been...

  1. [Application of cell co-culture techniques in medical studies].

    Science.gov (United States)

    Luo, Yun; Sun, Gui-Bo; Qin, Meng; Yao, Fan; Sun, Xiao-Bo

    2012-11-01

    As the cell co-culture techniques can better imitate an in vivo environment, it is helpful in observing the interactions among cells and between cells and the culture environment, exploring the effect mechanisms of drugs and their possible targets and filling the gaps between the mono-layer cell culture and the whole animal experiments. In recently years, they has attracted much more attention from the medical sector, and thus becoming one of research hotspots in drug research and development and bio-pharmaceutical fields. The cell co-culture techniques, including direct and indirect methods, are mainly used for studying pathological basis, new-type treatment methods and drug activity screening. Existing cell co-culture techniques are used for more pharmacological studies on single drug and less studies on interaction of combined drugs, such as collaborative compatibility and attenuation and synergistic effect among traditional Chinese medicines (TCMs). In line with the action characteristics of multi-component and multi-target, the cell co-culture techniques provide certain reference value for future studies on the effect and mechanism of combined TCMs on organisms as well as new methods for studies on TCMs and their compounds. This essay summarizes cell co-culture methods and their application and look into the future of their application in studies on TCMs and compounds.

  2. Culture materials affect ex vivo expansion of hematopoietic progenitor cells.

    Science.gov (United States)

    LaIuppa, J A; McAdams, T A; Papoutsakis, E T; Miller, W M

    1997-09-01

    Ex vivo expansion of hematopoietic cells is important for applications such as cancer treatment, gene therapy, and transfusion medicine. While cell culture systems are widely used to evaluate the biocompatibility of materials for implantation, the ability of materials to support proliferation of primary human cells in cultures for reinfusion into patients has not been addressed. We screened a variety of commercially available polymer (15 types), metal (four types), and glass substrates for their ability to support expansion of hematopoietic cells when cultured under conditions that would be encountered in a clinical setting. Cultures of peripheral blood (PB) CD34+ cells and mononuclear cells (MNC) were evaluated for expansion of total cells and colony-forming unit-granulocyte monocyte (CFU-GM; progenitors committed to the granulocyte and/or monocyte lineage). Human hematopoietic cultures in serum-free medium were found to be extremely sensitive to the substrate material. The only materials tested that supported expansion at or near the levels of polystyrene were tissue culture polystyrene, Teflon perfluoroalkoxy, Teflon fluorinated ethylene propylene, cellulose acetate, titanium, new polycarbonate, and new polymethylpentene. MNC were less sensitive to the substrate materials than the primitive CD34+ progenitors, although similar trends were seen for expansion of the two cell populations on the substrates tested. CFU-GM expansion was more sensitive to substrate materials than was total cell expansion. The detrimental effects of a number of the materials on hematopoietic cultures appear to be caused by protein adsorption and/or leaching of toxins. Factors such as cleaning, sterilization, and reuse significantly affected the performance of some materials as culture substrates. We also used PB CD34+ cell cultures to examine the biocompatibility of gas-permeable cell culture and blood storage bags and several types of tubing commonly used with biomedical equipment

  3. Culturing Schwann Cells from Neonatal Rats by Improved Enzyme Digestion Combined with Explants-culture Method.

    Science.gov (United States)

    Liu, Di; Liang, Xiao-Chun; Zhang, Hong

    2016-08-01

    Objective To develop an improved method for culturing Schwann cells(SCs) by using both enzyme digestion and explants-culture approaches and compared with traditional explants-culture method and general hemi-explants-culture method. Methods Bilaterally sciatic nerves and brachial plexus nerves were dissected from 3 to 5-day-old neonatal SD rats and explants-culture method,general hemi-explants-culture method,and improved enzyme digestion combined with explants-culture method were adopted to culture SCs,respectively. SCs were digested and passaged after 7 days in culture and counted under the microscope. The purity of SCs was identified by S-100 immunofluorescence staining. Results The SCs of improved method group grew fastest and the total number of cells obtained was(1.85±0.13)×10(6);the SCs of the hemi-explants-culture method group grew slower than the improved method group and the total number of cells obtained was (1.10±0.10)×10(6);the SCs of the explants-culture method group grew slowest and the total number of cells obtained was (0.77±0.03)×10(6).The total number of cells obtained showed significant difference among the three groups(Pculture method group,and (74.50±4.23)% in the explants-culture method group(Pculture method can obtain sufficient amount of high-purity SCs in a short time and thus may be applied in further research on peripheral nerve regeneration.

  4. Cell cycle regulation in human embryonic stem cells: links to adaptation to cell culture.

    Science.gov (United States)

    Barta, Tomas; Dolezalova, Dasa; Holubcova, Zuzana; Hampl, Ales

    2013-03-01

    Cell cycle represents not only a tightly orchestrated mechanism of cell replication and cell division but it also plays an important role in regulation of cell fate decision. Particularly in the context of pluripotent stem cells or multipotent progenitor cells, regulation of cell fate decision is of paramount importance. It has been shown that human embryonic stem cells (hESCs) show unique cell cycle characteristics, such as short doubling time due to abbreviated G1 phase; these properties change with the onset of differentiation. This review summarizes the current understanding of cell cycle regulation in hESCs. We discuss cell cycle properties as well as regulatory machinery governing cell cycle progression of undifferentiated hESCs. Additionally, we provide evidence that long-term culture of hESCs is accompanied by changes in cell cycle properties as well as configuration of several cell cycle regulatory molecules.

  5. LIF-Free Embryonic Stem Cell Culture in Simulated Microgravity

    OpenAIRE

    Yumi Kawahara; Tomotaka Manabe; Masaya Matsumoto; Teruyuki Kajiume; Masayasu Matsumoto; Louis Yuge

    2009-01-01

    BACKGROUND: Leukemia inhibitory factor (LIF) is an indispensable factor for maintaining mouse embryonic stem (ES) cell pluripotency. A feeder layer and serum are also needed to maintain an undifferentiated state, however, such animal derived materials need to be eliminated for clinical applications. Therefore, a more reliable ES cell culture technique is required. METHODOLOGY/PRINCIPAL FINDINGS: We cultured mouse ES cells in simulated microgravity using a 3D-clinostat. We used feeder-free and...

  6. Three-dimensional cell culture models for investigating human viruses.

    Science.gov (United States)

    He, Bing; Chen, Guomin; Zeng, Yi

    2016-10-01

    Three-dimensional (3D) culture models are physiologically relevant, as they provide reproducible results, experimental flexibility and can be adapted for high-throughput experiments. Moreover, these models bridge the gap between traditional two-dimensional (2D) monolayer cultures and animal models. 3D culture systems have significantly advanced basic cell science and tissue engineering, especially in the fields of cell biology and physiology, stem cell research, regenerative medicine, cancer research, drug discovery, and gene and protein expression studies. In addition, 3D models can provide unique insight into bacteriology, virology, parasitology and host-pathogen interactions. This review summarizes and analyzes recent progress in human virological research with 3D cell culture models. We discuss viral growth, replication, proliferation, infection, virus-host interactions and antiviral drugs in 3D culture models.

  7. Multizone Paper Platform for 3D Cell Cultures

    Science.gov (United States)

    Derda, Ratmir; Hong, Estrella; Mwangi, Martin; Mammoto, Akiko; Ingber, Donald E.; Whitesides, George M.

    2011-01-01

    In vitro 3D culture is an important model for tissues in vivo. Cells in different locations of 3D tissues are physiologically different, because they are exposed to different concentrations of oxygen, nutrients, and signaling molecules, and to other environmental factors (temperature, mechanical stress, etc). The majority of high-throughput assays based on 3D cultures, however, can only detect the average behavior of cells in the whole 3D construct. Isolation of cells from specific regions of 3D cultures is possible, but relies on low-throughput techniques such as tissue sectioning and micromanipulation. Based on a procedure reported previously (“cells-in-gels-in-paper” or CiGiP), this paper describes a simple method for culture of arrays of thin planar sections of tissues, either alone or stacked to create more complex 3D tissue structures. This procedure starts with sheets of paper patterned with hydrophobic regions that form 96 hydrophilic zones. Serial spotting of cells suspended in extracellular matrix (ECM) gel onto the patterned paper creates an array of 200 micron-thick slabs of ECM gel (supported mechanically by cellulose fibers) containing cells. Stacking the sheets with zones aligned on top of one another assembles 96 3D multilayer constructs. De-stacking the layers of the 3D culture, by peeling apart the sheets of paper, “sections” all 96 cultures at once. It is, thus, simple to isolate 200-micron-thick cell-containing slabs from each 3D culture in the 96-zone array. Because the 3D cultures are assembled from multiple layers, the number of cells plated initially in each layer determines the spatial distribution of cells in the stacked 3D cultures. This capability made it possible to compare the growth of 3D tumor models of different spatial composition, and to examine the migration of cells in these structures. PMID:21573103

  8. Microfluidic cardiac cell culture model (μCCCM).

    Science.gov (United States)

    Giridharan, Guruprasad A; Nguyen, Mai-Dung; Estrada, Rosendo; Parichehreh, Vahidreza; Hamid, Tariq; Ismahil, Mohamed Ameen; Prabhu, Sumanth D; Sethu, Palaniappan

    2010-09-15

    Physiological heart development and cardiac function rely on the response of cardiac cells to mechanical stress during hemodynamic loading and unloading. These stresses, especially if sustained, can induce changes in cell structure, contractile function, and gene expression. Current cell culture techniques commonly fail to adequately replicate physical loading observed in the native heart. Therefore, there is a need for physiologically relevant in vitro models that recreate mechanical loading conditions seen in both normal and pathological conditions. To fulfill this need, we have developed a microfluidic cardiac cell culture model (μCCCM) that for the first time allows in vitro hemodynamic stimulation of cardiomyocytes by directly coupling cell structure and function with fluid induced loading. Cells are cultured in a small (1 cm diameter) cell culture chamber on a thin flexible silicone membrane. Integrating the cell culture chamber with a pump, collapsible pulsatile valve and an adjustable resistance element (hemostatic valve) in series allow replication of various loading conditions experienced in the heart. This paper details the design, modeling, fabrication and characterization of fluid flow, pressure and stretch generated at various frequencies to mimic hemodynamic conditions associated with the normal and failing heart. Proof-of-concept studies demonstrate successful culture of an embryonic cardiomyoblast line (H9c2 cells) and establishment of an in vivo like phenotype within this system.

  9. Isolating highly pure rat spermatogonial stem cells in culture.

    Science.gov (United States)

    Hamra, F Kent; Chapman, Karen M; Wu, Zhuoru; Garbers, David L

    2008-01-01

    Methods are detailed for isolating highly pure populations of spermatogonial stem cells from primary cultures of testis cells prepared from 22- to 24-day-old rats. The procedure is based on the principle that testicular somatic cells bind tightly to plastic and collagen matrices when cultured in serum-containing medium, whereas spermatogonia and spermatocytes do not bind to plastic or collagen when cultured in serum-containing medium. The collagen-non-binding testis cells obtained using these procedures are thus approx. 97% pure spermatogenic cells. Stem spermatogonia are then easily isolated from the purified spermatogenic population during a short incubation step in culture on laminin matrix. The spermatogenic cells that bind to laminin are more than 90% undifferentiated, type A spermatogonia and are greatly enriched in genetically modifiable stem cells that can develop into functional spermatozoa. This method does not require flow cytometry and can also be applied to obtain enriched cultures of mouse spermatogonial stem cells. The isolated spermatogonia provide a highly potent and effective source of stem cells that have been used to initiate in vitro and in vivo culture studies on spermatogenesis.

  10. Three Dimensional Culture of Human Renal Cell Carcinoma Organoids.

    Directory of Open Access Journals (Sweden)

    Cynthia A Batchelder

    Full Text Available Renal cell carcinomas arise from the nephron but are heterogeneous in disease biology, clinical behavior, prognosis, and response to systemic therapy. Development of patient-specific in vitro models that efficiently and faithfully reproduce the in vivo phenotype may provide a means to develop personalized therapies for this diverse carcinoma. Studies to maintain and model tumor phenotypes in vitro were conducted with emerging three-dimensional culture techniques and natural scaffolding materials. Human renal cell carcinomas were individually characterized by histology, immunohistochemistry, and quantitative PCR to establish the characteristics of each tumor. Isolated cells were cultured on renal extracellular matrix and compared to a novel polysaccharide scaffold to assess cell-scaffold interactions, development of organoids, and maintenance of gene expression signatures over time in culture. Renal cell carcinomas cultured on renal extracellular matrix repopulated tubules or vessel lumens in renal pyramids and medullary rays, but cells were not observed in glomeruli or outer cortical regions of the scaffold. In the polysaccharide scaffold, renal cell carcinomas formed aggregates that were loosely attached to the scaffold or free-floating within the matrix. Molecular analysis of cell-scaffold constructs including immunohistochemistry and quantitative PCR demonstrated that individual tumor phenotypes could be sustained for up to 21 days in culture on both scaffolds, and in comparison to outcomes in two-dimensional monolayer cultures. The use of three-dimensional scaffolds to engineer a personalized in vitro renal cell carcinoma model provides opportunities to advance understanding of this disease.

  11. Development of bone marrow mesenchymal stem cell culture in vitro

    Institute of Scientific and Technical Information of China (English)

    ZHANG Li; PENG Li-pan; WU Nan; LI Le-ping

    2012-01-01

    Objective To review the in vitro development of bone marrow mesenchymal stem cells culture (BM-MSC).Data sources The data cited in this review were mainly obtained from articles listed in Medline and PubMed.The search terms were “bone marrow mesenchymal stem cell" and "cell culture".Study selection Articles regarding the in vitro development of BM-MSCs culture,as well as the challenge of optimizing cell culture environment in two-dimensional (2D) vs.3D.Results Improving the culture conditions increases the proliferation and reduces the differentiation.Optimal values for many culture parameters remain to be identified.Expansion of BM-MSCs under defined conditions remains challenging,including the development of optimal culture conditions for BMSC and large-volume production systems.Conclusions Expansion of BM-MSCs under defined conditions remains challenges,including the development of optimal culture conditions for BMSC and scale-up to large-volume production systems.Optimal values for many culture parameters remain to be identified.

  12. THE ULTRASTRUCTURE OF SEPARATED AND CULTURED CELL OF PORPHYRA YEZOENSIS

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    There are many reports that cells (protoplasts) separated from the thallus of Porphyra by enzyme can develop to normal leafy thalli in the same way as monospores. But there are few investigations on the subcellular structure of the isolated vegetative cell for comparison with the subcellular structure of monospores. To clarify whether the separated and cultured cells undergo the same or similar ultrastructure changes during culture and germination as monospores undergo in their formation and germination, we observed their ultrastructure, compared them with those of the monospore and found that the ultrastructure of separated and cultured cells did not have the characteristic feature as that of monospore formation, such as production of small and large fibrous vesicles, but was accompanied by vacuolation and starch mobilization like that in monospore germination. The paper also discusses the relations between monospores and separated and cultured cells.

  13. Quantitative volumetric Raman imaging of three dimensional cell cultures

    Science.gov (United States)

    Kallepitis, Charalambos; Bergholt, Mads S.; Mazo, Manuel M.; Leonardo, Vincent; Skaalure, Stacey C.; Maynard, Stephanie A.; Stevens, Molly M.

    2017-03-01

    The ability to simultaneously image multiple biomolecules in biologically relevant three-dimensional (3D) cell culture environments would contribute greatly to the understanding of complex cellular mechanisms and cell-material interactions. Here, we present a computational framework for label-free quantitative volumetric Raman imaging (qVRI). We apply qVRI to a selection of biological systems: human pluripotent stem cells with their cardiac derivatives, monocytes and monocyte-derived macrophages in conventional cell culture systems and mesenchymal stem cells inside biomimetic hydrogels that supplied a 3D cell culture environment. We demonstrate visualization and quantification of fine details in cell shape, cytoplasm, nucleus, lipid bodies and cytoskeletal structures in 3D with unprecedented biomolecular specificity for vibrational microspectroscopy.

  14. Surface modified alginate microcapsules for 3D cell culture

    Science.gov (United States)

    Chen, Yi-Wen; Kuo, Chiung Wen; Chueh, Di-Yen; Chen, Peilin

    2016-06-01

    Culture as three dimensional cell aggregates or spheroids can offer an ideal platform for tissue engineering applications and for pharmaceutical screening. Such 3D culture models, however, may suffer from the problems such as immune response and ineffective and cumbersome culture. This paper describes a simple method for producing microcapsules with alginate cores and a thin shell of poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) to encapsulate mouse induced pluripotent stem (miPS) cells, generating a non-fouling surface as an effective immunoisolation barrier. We demonstrated the trapping of the alginate microcapsules in a microwell array for the continuous observation and culture of a large number of encapsulated miPS cells in parallel. miPS cells cultured in the microcapsules survived well and proliferated to form a single cell aggregate. Droplet formation of monodisperse microcapsules with controlled size combined with flow cytometry provided an efficient way to quantitatively analyze the growth of encapsulated cells in a high-throughput manner. The simple and cost-effective coating technique employed to produce the core-shell microcapsules could be used in the emerging field of cell therapy. The microwell array would provide a convenient, user friendly and high-throughput platform for long-term cell culture and monitoring.

  15. Nylon-3 polymers that enable selective culture of endothelial cells.

    Science.gov (United States)

    Liu, Runhui; Chen, Xinyu; Gellman, Samuel H; Masters, Kristyn S

    2013-11-06

    Substrates that selectively encourage the growth of specific cell types are valuable for the engineering of complex tissues. Some cell-selective peptides have been identified from extracellular matrix proteins; these peptides have proven useful for biomaterials-based approaches to tissue repair or regeneration. However, there are very few examples of synthetic materials that display selectivity in supporting cell growth. We describe nylon-3 polymers that support in vitro culture of endothelial cells but do not support the culture of smooth muscle cells or fibroblasts. These materials may be promising for vascular biomaterials applications.

  16. Fundamentals of microfluidic cell culture in controlled microenvironments.

    Science.gov (United States)

    Young, Edmond W K; Beebe, David J

    2010-03-01

    Microfluidics has the potential to revolutionize the way we approach cell biology research. The dimensions of microfluidic channels are well suited to the physical scale of biological cells, and the many advantages of microfluidics make it an attractive platform for new techniques in biology. One of the key benefits of microfluidics for basic biology is the ability to control parameters of the cell microenvironment at relevant length and time scales. Considerable progress has been made in the design and use of novel microfluidic devices for culturing cells and for subsequent treatment and analysis. With the recent pace of scientific discovery, it is becoming increasingly important to evaluate existing tools and techniques, and to synthesize fundamental concepts that would further improve the efficiency of biological research at the microscale. This tutorial review integrates fundamental principles from cell biology and local microenvironments with cell culture techniques and concepts in microfluidics. Culturing cells in microscale environments requires knowledge of multiple disciplines including physics, biochemistry, and engineering. We discuss basic concepts related to the physical and biochemical microenvironments of the cell, physicochemical properties of that microenvironment, cell culture techniques, and practical knowledge of microfluidic device design and operation. We also discuss the most recent advances in microfluidic cell culture and their implications on the future of the field. The goal is to guide new and interested researchers to the important areas and challenges facing the scientific community as we strive toward full integration of microfluidics with biology.

  17. Fundamentals of microfluidic cell culture in controlled microenvironments†

    Science.gov (United States)

    Young, Edmond W. K.; Beebe, David J.

    2010-01-01

    Microfluidics has the potential to revolutionize the way we approach cell biology research. The dimensions of microfluidic channels are well suited to the physical scale of biological cells, and the many advantages of microfluidics make it an attractive platform for new techniques in biology. One of the key benefits of microfluidics for basic biology is the ability to control parameters of the cell microenvironment at relevant length and time scales. Considerable progress has been made in the design and use of novel microfluidic devices for culturing cells and for subsequent treatment and analysis. With the recent pace of scientific discovery, it is becoming increasingly important to evaluate existing tools and techniques, and to synthesize fundamental concepts that would further improve the efficiency of biological research at the microscale. This tutorial review integrates fundamental principles from cell biology and local microenvironments with cell culture techniques and concepts in microfluidics. Culturing cells in microscale environments requires knowledge of multiple disciplines including physics, biochemistry, and engineering. We discuss basic concepts related to the physical and biochemical microenvironments of the cell, physicochemical properties of that microenvironment, cell culture techniques, and practical knowledge of microfluidic device design and operation. We also discuss the most recent advances in microfluidic cell culture and their implications on the future of the field. The goal is to guide new and interested researchers to the important areas and challenges facing the scientific community as we strive toward full integration of microfluidics with biology. PMID:20179823

  18. Controlling the diversity of cell populations in a stem cell culture

    NARCIS (Netherlands)

    Heo, Inha; Clevers, Hans

    2015-01-01

    Culturing intestinal stem cells into 3D organoids results in heterogeneous cell populations, reflecting the in vivo cell type diversity. In a recent paper published in Nature, Wang et al. established a culture condition for a highly homogeneous population of intestinal stem cells.

  19. Controlling the diversity of cell populations in a stem cell culture

    NARCIS (Netherlands)

    Heo, Inha; Clevers, Hans

    2015-01-01

    Culturing intestinal stem cells into 3D organoids results in heterogeneous cell populations, reflecting the in vivo cell type diversity. In a recent paper published in Nature, Wang et al. established a culture condition for a highly homogeneous population of intestinal stem cells.

  20. Ontogeny of electrically excitable cells in cultured olfactory epithelium.

    OpenAIRE

    Schubert, D; Stallcup, W.; LaCorbiere, M; Kidokoro, Y; Orgel, L

    1985-01-01

    A primary system has been developed in which it is possible to study the production of electrically excitable neuron-like cells from a precursor population of olfactory epithelial cells. Rat nasal epithelium was dissociated and placed in culture. The initial surviving cells are flat and ciliated and contain glial fibrillary acidic protein (GFAP). After 3-5 days electrically excitable cells appear that contain neuron-specific enolase but not GFAP. These round cells originate by means of the di...

  1. Characterization of a novel miniature cell culture device

    Science.gov (United States)

    Moore, Sandra K.; Kleis, Stanley J.

    2008-05-01

    Recent advancements in the field of microfluidics have generated much interest in the advent of a miniaturized cell culture device. In this study, we developed a novel miniature culture system (cells, either prokaryotic or eukaryotic in type, for both 1 g and microgravity applications. The miniature culture system may advance the development of microanalytical remote monitoring tools such as biological sentinels, biosensors, and lab-on-a-chip. Integrating the autonomous miniature culture system with a microanalytical device makes a powerful biological tool. Cells can be cultured long-term, harvested, and released directly into an analytical tool without the need for human interaction through fluid dynamic manipulations. This work characterizes the miniature bioreactor system through numerical and experimental proof of concept studies.

  2. Enhanced casein kinase II activity in human tumour cell cultures

    DEFF Research Database (Denmark)

    Prowald, K; Fischer, H; Issinger, O G

    1984-01-01

    Casein kinase II (CKII) activity is enhanced as much as 2-3 fold in established and 4-5-fold in transformed human cell lines when compared to that of fibroblasts and primary human tumour cell cultures where CKII activity never exceeded a basic level. The high activity of CKII in transformed cells...

  3. Microfluidic bioreactors for culture of non-adherent cells

    DEFF Research Database (Denmark)

    Shah, Pranjul Jaykumar; Vedarethinam, Indumathi; Kwasny, Dorota

    2011-01-01

    Microfluidic bioreactors (μBR) are becoming increasingly popular for cell culture, sample preparation and analysis in case of routine genetic and clinical diagnostics. We present a novel μBR for non-adherent cells designed to mimic in vivo perfusion of cells based on diffusion of media through...

  4. [Cytotoxicity studies on T-3262 in cultured Chinese hamster cells].

    Science.gov (United States)

    Yoneda, T; Nakamura, S; Nojima, Y; Nishio, Y

    1989-04-01

    T-3262 is an antibacterial drug which belongs to the group of pyridonecarboxylic acids. In this study, we investigated cytotoxicity of T-3262 for inhibition of cell growth and effects on viability of, and morphological changes in cultured Chinese hamster cells (V79 cells). The following results were obtained. 1. The 50% inhibition dose of T-3262 for cell growth (ID50, cultured for 48 hours) was 12 micrograms/ml, showing that the inhibitory effect of T-3262 on the cell growth was stronger than that of enoxacin (ENX: ID50 44 micrograms/ml), norfloxacin (NFLX: ID50 105 micrograms/ml) or ofloxacin (OFLX: ID50 145 micrograms/ml). 2. The number of cells increased and dead cells were scarcely seen at the highest concentration tested in culture medium (40 micrograms/ml of T-3262 for 48 hours). At this concentration, degeneration of cytoplasm (atrophy and round shape) and decrease of mitotic cells were observed. These morphological changes were similar to those of the cells treated 400 micrograms/ml of NFLX or OFLX for 48 hours. 3. After the removal of T-3262 from culture medium, the cells began to grow actively and recovered from the morphological changes. The similar phenomenon was observed with ENX treated cells but not with fluorouracil or mitomycin C treated cells.

  5. Application of cell co-culture system to study fat and muscle cells.

    Science.gov (United States)

    Pandurangan, Muthuraman; Hwang, Inho

    2014-09-01

    Animal cell culture is a highly complex process, in which cells are grown under specific conditions. The growth and development of these cells is a highly unnatural process in vitro condition. Cells are removed from animal tissues and artificially cultured in various culture vessels. Vitamins, minerals, and serum growth factors are supplied to maintain cell viability. Obtaining result homogeneity of in vitro and in vivo experiments is rare, because their structure and function are different. Living tissues have highly ordered complex architecture and are three-dimensional (3D) in structure. The interaction between adjacent cell types is quite distinct from the in vitro cell culture, which is usually two-dimensional (2D). Co-culture systems are studied to analyze the interactions between the two different cell types. The muscle and fat co-culture system is useful in addressing several questions related to muscle modeling, muscle degeneration, apoptosis, and muscle regeneration. Co-culture of C2C12 and 3T3-L1 cells could be a useful diagnostic tool to understand the muscle and fat formation in animals. Even though, co-culture systems have certain limitations, they provide a more realistic 3D view and information than the individual cell culture system. It is suggested that co-culture systems are useful in evaluating the intercellular communication and composition of two different cell types.

  6. Establishment of sorghum cell suspension culture system for ...

    African Journals Online (AJOL)

    STORAGESEVER

    2008-03-18

    Mar 18, 2008 ... Total soluble proteins (TSP) and culture filtrate (CF) proteins were extracted from the cell culture ... Even though. Arabidopsis provides for an excellent model system for .... stained, destained and imaged using a Molecular Imager PharosFX ... system, are dynamic and heterogeneous, being com- posed of a ...

  7. Expansion of Endothelial Progenitor Cells in High Density Dot Culture of Rat Bone Marrow Cells

    Science.gov (United States)

    Wang, Ling; Kretlow, James D.; Zhou, Guangdong; Cao, Yilin; Liu, Wei; Zhang, Wen Jie

    2014-01-01

    In vitro expansion of endothelial progenitor cells (EPCs) remains a challenge in stem cell research and its application. We hypothesize that high density culture is able to expand EPCs from bone marrow by mimicking cell-cell interactions of the bone marrow niche. To test the hypothesis, rat bone marrow cells were either cultured in high density (2×105 cells/cm2) by seeding total 9×105 cells into six high density dots or cultured in regular density (1.6×104 cells/cm2) with the same total number of cells. Flow cytometric analyses of the cells cultured for 15 days showed that high density cells exhibited smaller cell size and higher levels of marker expression related to EPCs when compared to regular density cultured cells. Functionally, these cells exhibited strong angiogenic potentials with better tubal formation in vitro and potent rescue of mouse ischemic limbs in vivo with their integration into neo-capillary structure. Global gene chip and ELISA analyses revealed up-regulated gene expression of adhesion molecules and enhanced protein release of pro-angiogenic growth factors in high density cultured cells. In summary, high density cell culture promotes expansion of bone marrow contained EPCs that are able to enhance tissue angiogenesis via paracrine growth factors and direct differentiation into endothelial cells. PMID:25254487

  8. Feeding lactate for CHO cell culture processes: impact on culture metabolism and performance.

    Science.gov (United States)

    Li, Jincai; Wong, Chun Loong; Vijayasankaran, Natarajan; Hudson, Terry; Amanullah, Ashraf

    2012-05-01

    Lactate has long been regarded as one of the key metabolites of mammalian cell cultures. High levels of lactate have clear negative impacts on cell culture processes, and therefore, a great amount of efforts have been made to reduce lactate accumulation and/or to induce lactate consumption in the later stage of cultures. However, there is virtually no report on the impact of lactate depletion after initial accumulation. In this work, we observed that glucose uptake rate dropped over 50% at the onset of lactate consumption, and that catabolism of alanine due to lactate depletion led to ammonium accumulation. We explored the impact of feeding lactate as well as pyruvate to the cultures. In particular, a strategy was employed where CO(2) was replaced by lactic acid for culture pH control, which enabled automatic lactate feeding. The results demonstrated that lactate or pyruvate can serve as an alternative or even preferred carbon source during certain stage of the culture in the presence of glucose, and that by feeding lactate or pyruvate, very low levels of ammonia can be achieved throughout the culture. In addition, low levels of pCO(2) were also maintained in these cultures. This was in strong contrast to the control cultures where lactate was depleted during the culture, and ammonia and pCO(2) build-up were significant. Culture growth and productivity were similar between the control and lactate-fed cultures, as well as various product quality attributes. To our knowledge, this work represents the first comprehensive study on lactate depletion and offers a simple yet effective strategy to overcome ammonia and pCO(2) accumulation that could arise in certain cultures due to early depletion of lactate.

  9. Adherence of Moraxella bovis to cell cultures of bovine origin.

    Science.gov (United States)

    Annuar, B O; Wilcox, G E

    1985-09-01

    The adherence of five strains of Moraxella bovis to cell cultures was investigated. M bovis adhered to cultures of bovine corneal epithelial and Madin-Darby bovine kidney cells but not to cell types of non-bovine origin. Both piliated and unpiliated strains adhered but piliated strains adhered to a greater extent than unpiliated strains. Antiserum against pili of one strain inhibited adherence of piliated strains but caused only slight inhibition of adherence to the unpiliated strains. Treatment of bacteria with magnesium chloride caused detachment of pili from the bacterial cell and markedly inhibited adherence of piliated strains but caused only slight inhibition of adherence by the unpiliated strains. The results suggested that adhesion of piliated strains to cell cultures was mediated via pili but that adhesins other than pili may be involved in the attachment of unpiliated strains of M bovis to cells.

  10. Quantitative volumetric Raman imaging of three dimensional cell cultures

    KAUST Repository

    Kallepitis, Charalambos

    2017-03-22

    The ability to simultaneously image multiple biomolecules in biologically relevant three-dimensional (3D) cell culture environments would contribute greatly to the understanding of complex cellular mechanisms and cell–material interactions. Here, we present a computational framework for label-free quantitative volumetric Raman imaging (qVRI). We apply qVRI to a selection of biological systems: human pluripotent stem cells with their cardiac derivatives, monocytes and monocyte-derived macrophages in conventional cell culture systems and mesenchymal stem cells inside biomimetic hydrogels that supplied a 3D cell culture environment. We demonstrate visualization and quantification of fine details in cell shape, cytoplasm, nucleus, lipid bodies and cytoskeletal structures in 3D with unprecedented biomolecular specificity for vibrational microspectroscopy.

  11. Mixed cultures of Kimchi lactic acid bacteria show increased cell ...

    African Journals Online (AJOL)

    ufuoma

    kimchi lactobacilli on batch fermentation increased the cell density and lactic acid production with low nutrients .... first series of fermentations were carried out using pure cultures of each ... Each number represents the mean. ± SD of three ...

  12. Cell/Tissue Culture Radiation Exposure Facility Project

    Data.gov (United States)

    National Aeronautics and Space Administration — We propose to develop a Cell/Tissue Culture Radiation Exposure Facility (CTC-REF) to enable radiobiologists to investigate the real-time radiation effects on...

  13. Isolation, Culture and Identification of Porcine Skeletal Muscle Satellite Cells

    Directory of Open Access Journals (Sweden)

    Bo-jiang Li

    2015-08-01

    Full Text Available The objective of this study was to establish the optimum protocol for the isolation and culture of porcine muscle satellite cells. Mononuclear muscle satellite cells are a kind of adult stem cell, which is located between the basal lamina and sarcolemma of muscle fibers and is the primary source of myogenic precursor cells in postnatal muscle. Muscle satellite cells are a useful model to investigate the mechanisms of muscle growth and development. Although the isolation and culture protocols of muscle satellite cells in some species (e.g. mouse have been established successfully, the culture system for porcine muscle satellite cells is very limited. In this study, we optimized the isolation procedure of porcine muscle satellite cells and elaborated the isolation and culture process in detail. Furthermore, we characterized the porcine muscle satellite cells using the immunofluorecence. Our study provides a reference for the isolation of porcine muscle satellite cells and will be useful for studying the molecular mechanisms in these cells.

  14. p38丝裂原活化蛋白激酶对耐火陶瓷纤维诱导人支气管上皮细胞凋亡的作用%Effects of p38 mitogen-activated protein kinases on the apoptosis of human bronchial epithelial cells induced by refractory ceramic fibers in vitro

    Institute of Scientific and Technical Information of China (English)

    张敏; 朱丽瑾; 肖芸; 张幸

    2013-01-01

    Objective To investigate the role of p38 mitogen-activated protein kinases (MAPKs) in the apoptosis of human bronchial epithelial cells (BEAS-2B) induced by refractory ceramic fibers (RCFs).Methods BEAS-2B cells were exposed to 10,20,40,80,and 160 μg/cm2 RCF1,RCF2,and RCF3 for 24 h,and the cell viability was measured by CCK-8 assay.BEAS-2B cells were exposed to 20,40,and 100 μg/cm2 RCF1,RCF2,and RCF3 for 24 h,and the cell apoptosis rate was measured by flow cytometry.BEAS-2B cells were exposed to 40 μg/cm2 RCF1,RCF2,and RCF3,and the expression levels of phospho-p38 MAPK and caspase-3 were measured by Western blot.In each of the above treatments,the BEAS-2B cells were divided into positive control,p38 inhibitor SB203580 intervention,and normal groups.Results As the concentration of RCFs rose,the RCF exposure groups showed decreased cell viability and increased cell apoptosis rate.After SB203580 intervention,the intervention groups (all concentrations of asbestos + SB,20,40,80,and 160 μg/cm2 RCF1+SB,and 40,80,and 160 μg/cm2 RCF2 and RCF3+SB) had significantly increased cell viabilities (P<0.05),and the intervention groups (asbestos + SB and 20,40,and 100 μg/cm2 RCF1,RCF2,and RCF3 + SB) had significantly decreased cell apoptosis rates (P<0.05).Compared with the normal group,the RCF (40 μg/cm2)exposure and positive control groups had significantly increased expression of phospho-p38 MAPK (P<0.05),and the RCF (40 μg/cm2) exposure group had significantly increased expression of caspase-3 (P<0.05).The intervention groups (asbestos + SB and 40 μg/cm2 RCF1,RCF2,and RCF3 + SB) had significantly decreased expression of caspase-3 after SB203580 intervention.Conclusion p38 MAPKs play an important role in RCFinduced apoptosis of BEAS-2B cells.%目的 p38丝裂原活化蛋白激酶(p38MAPK)在RCFs诱导BEAS-2B凋亡中的作用.方法 10、20、40、80、160 μg/cm2 RCF1、RCF2和RCF3诱导BEAS-2B细胞24 h,用CCK-8法检测细胞存活率;20、40、100 μg/cm2

  15. Human hematopoietic cell culture, transduction, and analyses

    DEFF Research Database (Denmark)

    Bonde, Jesper; Wirthlin, Louisa; Kohn, Donald B;

    2008-01-01

    This unit provides methods for introducing genes into human hematopoietic progenitor cells. The Basic Protocol describes isolation of CD34(+) cells, transduction of these cells with a retroviral vector on fibronectin-coated plates, assaying the efficiency of transduction, and establishing long...

  16. Generation of a patterned co-culture system composed of adherent cells and immobilized nonadherent cells.

    Science.gov (United States)

    Yamazoe, Hironori; Ichikawa, Takashi; Hagihara, Yoshihisa; Iwasaki, Yasuhiko

    2016-02-01

    Patterned co-culture is a promising technique used for fundamental investigation of cell-cell communication and tissue engineering approaches. However, conventional methods are inapplicable to nonadherent cells. In this study, we aimed to establish a patterned co-culture system composed of adherent and nonadherent cells. Nonadherent cells were immobilized on a substrate using a cell membrane anchoring reagent conjugated to a protein, in order to incorporate them into the co-culture system. Cross-linked albumin film, which has unique surface properties capable of regulating protein adsorption, was used to control their spatial localization. The utility of our approach was demonstrated through the fabrication of a patterned co-culture consisting of micropatterned neuroblastoma cells surrounded by immobilized myeloid cells. Furthermore, we also created a co-culture system composed of cancer cells and immobilized monocytes. We observed that monocytes enhanced the drug sensitivity of cancer cells and its influence was limited to cancer cells located near the monocytes. Therefore, the incorporation of nonadherent cells into a patterned co-culture system is useful for creating culture systems containing immune cells, as well as investigating the influence of these immune cells on cancer drug sensitivity. Various methods have been proposed for creating patterned co-culture systems, in which multiple cell types are attached to a substrate with a desired pattern. However, conventional methods, including our previous report published in Acta Biomaterialia (2010, 6, 526-533), are unsuitable for nonadherent cells. Here, we developed a novel method that incorporates nonadherent cells into the co-culture system, which allows us to precisely manipulate and study microenvironments containing nonadherent and adherent cells. Using this technique, we demonstrated that monocytes (nonadherent cells) could enhance the drug sensitivity of cancer cells and that their influence had a

  17. Which form of collagen is suitable for nerve cell culture?*

    Institute of Scientific and Technical Information of China (English)

    Mohsen Fathi Najafi; Saber Zahri; Fatemeh Vahedi; Leila Esmaililian Toosi; Nazila Ariaee

    2013-01-01

    In this study, we investigated the effects of hydrolyzed and non-hydrolyzed col agen and two-dimensional and three-dimensional col agen matrices on cell survival, attachment and neurite outgrowth of primary cultured nerve cells using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay and inverted microscopy. Hydrolyzed col agen facilitated nerve cell survival and neurite outgrowth, but it had no obvious influences on cellattachment. In contrast, non-hydrolyzed two-dimensional collagen matrix had no obvious effects on neurite outgrowth. These findings suggest that hydrolyzed col agen is an ideal nerve cell culture media.

  18. Study on Cell Suspension Culture of Floribunda Rose

    Institute of Scientific and Technical Information of China (English)

    ZHANG Chun'ai; WANG Jingang; FAN Jinping; GONG Shufang; CHE Daidi

    2008-01-01

    Friable callus was induced when immature seeds of floribunda rose were inoculated on MS medium supplemented with 2,4-D 3.0 mg-L-1.When transfered onto subculture media,fi-iable callus developed into embryogenic callus,which was used to establish cell suspension lines.Cell suspensions had to be subcultured at a interval of 4-5 days at the first several culture cycles.The best subculturing cycle for the stable cell suspensions was 8-10 days.The best inoculum quantity was 1 mL PCV(Packed Cell Volume) per 40 mL culture fluid.

  19. 2D- and 3D-culture of cell

    Directory of Open Access Journals (Sweden)

    Khoruzhenko A. I.

    2011-02-01

    Full Text Available The cultivation of mammalian cells in three-dimensional conditions acquires a priority in a variety of biomedical applications. In the areas of toxicology and anticancer drug development it concerns a significant difference of responses to proapoptotic factors of the cells cultured in 2D versus 3D environment. Besides, the clear-cut differences have been found in cell polarity, cytoskeleton structure, distribution of receptors to wide range of hormones, growth factors, etc. in mammalian cells depending on culture conditions. It is resulted in different response of cultured cells to extracellular stimuli. Multicellular spheroids are regarded presently as the most convenient model of solid tumour growth in vitro. The cultivation of thyroid follicles, mammary acini and other structure units, maintaining initial tissue organization, allows studying the behavior, biochemical features and gene profile of differentiated cells. On the other hand, 3D cultures have some limitations in comparison with a well established monolayer culture. The advantages and disadvantages of each type of cultures and their application in biological and medical researches will be discussed in this review

  20. Convoluted cells as a marker for maternal cell contamination in CVS cultures

    DEFF Research Database (Denmark)

    Hertz, Jens Michael; Jensen, P K; Therkelsen, A J

    1987-01-01

    In order to identify cells of maternal origin in CVS cultures, tissue from 1st trimester abortions were cultivated and the cultures stained in situ for X-chromatin. Convoluted cells and maternal fibroblasts were found to be positive. By chromosome analysis of cultures from 105 diagnostic placenta...... biopsies, obtained by the transabdominal route, metaphases of maternal origin were found in nine cases. In eight of these cases colonies of convoluted cells were observed. We conclude that convoluted cells are of maternal origin and are a reliable marker for maternal cell contamination in CVS cultures....

  1. Xeno-free culture of human periodontal ligament stem cells.

    Science.gov (United States)

    Trubiani, Oriana; Diomede, Francesca

    2015-01-01

    The possibility of transplanting adult stem cells into damaged organs has opened a new prospective for the treatment of several human pathologies. Currently, in vitro expansion and culture of mesenchymal stem cells is founded on supplementing cell culture and differentiation medium with fetal calf serum (FCS) or fetal bovine serum (FBS) that contain numerous growth factors inducing cell attachment to plastic surfaces, proliferation, and differentiation. Mesenchymal stem cells (MSCs) cultured with medium containing FCS or FBS are unusable in the cell therapy; in fact the central issues regarding limitations in using animal sera for cell therapy is that its components are highly variable and often unknown and may trigger a xenogenic immune response, immunological reactions, and the potential transmission of prion diseases and zoonoses. Here we describe the culture system protocols for the expansion and production of human Periodontal Ligament Stem Cells (hPDLSCs) using a new xeno-free medium formulation ensuring the maintenance of the stem cells features comprising the multiple passage expansion, mesengenic lineage differentiation, cellular phenotype, and genomic stability, essential elements for conforming to translation to cell therapy.

  2. Guard cell protoplasts: isolation, culture, and regeneration of plants.

    Science.gov (United States)

    Tallman, Gary

    2006-01-01

    Guard cell protoplasts have been used extensively in short-term experiments designed to elucidate the signal transduction mechanisms that regulate stomatal movements. The utility of uard cell protoplasts for other types of longer-term signal transduction experiments is just now being realized. Because highly purified, primary isolates of guard cell protoplasts are synchronous initially, they are uniform in their responses to changes in culture conditions. Such isolates have demonstrated potential to reveal mechanisms that underlie hormonal signalling for plant cell survival, cell cycle re-entry, reprogramming of genes during dedifferentiation to an embryogenic state, and plant cell thermotolerance. Plants have been regenerated from cultured guard cell protoplasts of two species: Nicotiana glauca (Graham), tree tobacco, and Beta vulgaris, sugar beet. Plants genetically engineered for herbicide tolerance have been regenerated from cultured guard cell protoplasts of B. vulgaris. The method for isolating, culturing, and regenerating plants from guard cell protoplasts of N. glauca is described here. A recently developed procedure for large-scale isolation of these cells from as many as nine leaves per experiment is described. Using this protocol, yields of 1.5-2 x 10(7) per isolate may be obtained. Such yields are sufficient for standard methods of molecular, biochemical, and proteomic analysis.

  3. Auxin requirements of sycamore cells in suspension culture.

    Science.gov (United States)

    Moloney, M M; Hall, J F; Robinson, G M; Elliott, M C

    1983-04-01

    Sycamore (Acer pseudoplatanus L.) cell suspension cultures (strain OS) require 2,4-dichlorophenoxyacetic acid (2,4-D) in their culture medium for normal growth. If the 2,4-D is omitted, rates of cell division are dramatically reduced and cell lysis may occur. Despite this ;auxin requirement,' it has been shown by gas chromatography-mass spectrometry that the cells synthesize indol-3yl-acetic acid (IAA). Changes in free 2,4-D and IAA in the cells during a culture passage have been monitored.There is a rapid uptake of 2,4-D by the cells during the lag phase leading to a maximum concentration per cell (125 nanograms per 10(6) cells) on day 2 followed by a decline to 45 nanograms per 10(6) cells by day 9 (middle of linear phase). The initial concentration of IAA (0.08 nanograms per 10(6) cells) rises slowly to a peak of 1.4 nanograms per 10(6) cells by day 9 then decreases rapidly to 0.2 nanograms per 10(6) cells by day 15 (early declining phase) and 0.08 nanograms per 10(6) cells by day 23 (early stationary phase).

  4. Isolation and Culture of Satellite Cells from Mouse Skeletal Muscle.

    Science.gov (United States)

    Musarò, Antonio; Carosio, Silvia

    2017-01-01

    Skeletal muscle tissue is characterized by a population of quiescent mononucleated myoblasts, localized between the basal lamina and sarcolemma of myofibers, known as satellite cells. Satellite cells play a pivotal role in muscle homeostasis and are the major source of myogenic precursors in mammalian muscle regeneration.This chapter describes protocols for isolation and culturing satellite cells isolated from mouse skeletal muscles. The classical procedure, which will be discussed extensively in this chapter, involves the enzymatic dissociation of skeletal muscles, while the alternative method involves isolation of satellite cells from isolated myofibers in which the satellite cells remain in their in situ position underneath the myofiber basal lamina.In particular, we discuss the technical aspect of satellite cell isolation, the methods necessary to enrich the satellite cell fraction and the culture conditions that optimize proliferation and myotube formation of mouse satellite cells.

  5. [Effect evaluation of three cell culture models].

    Science.gov (United States)

    Wang, Aiguo; Xia, Tao; Yuan, Jing; Chen, Xuemin

    2003-11-01

    Primary rat hepatocytes were cultured using three kinds of models in vitro and the enzyme leakage, albumin secretion, and cytochrome P450 1A (CYP 1A) activity were observed. The results showed that the level of LDH in the medium decreased over time in the period of culture. However, on 5 days, LDH showed a significant increase in monolayer culture (MC) while after 8 days LDH was not detected in sandwich culture (SC). The levels of AST and ALT in the medium did not change significantly over the investigated time. The basic CYP 1A activity gradually decreased with time in MC and SC. The decline of CYP 1A in rat hepatocytes was faster in MC than that in SC. This effect was partially reversed by using cytochrome P450 (CYP450) inducers such as omeprazol and 3-methylcholanthrene (3-MC) and the CYP 1A induction was always higher in MC than that in SC. Basic CYP 1A activity in bioreactor was keeped over 2 weeks and the highest albumin production was observed in bioreactor, and next were SC and MC. In conclusion, our results clearly indicated that there have some advantages and disadvantages in each of models in which can address different questions in metabolism of toxicants and drugs.

  6. Qualitative study of three cell culture methods.

    Science.gov (United States)

    Wang, Aiguo; Xia, Tao; Ran, Peng; Chen, Xuemin; Nuessler, Andreas K

    2002-01-01

    Primary rat hepatocytes were cultured using different in vitro models and the enzyme leakage, albumin secretion, and cytochrome P450 1A (CYP 1A) activity were observed. The results showed that the level of LDH was decreased over time in culture. However, on day 5, LDH showed a significant increase in monolayer culture (MC) while after day 8 no LDH was detectable in sandwich culture (SC). The levels of AST and ALT did not change significantly over the investigated time. The CYP 1A activity was gradually decreased in a time-dependent manner in MC and SC. The decline of CYP 1A was faster in MC than in SC. This effect was partially reversed by using cytochrome P450 (CYP450) inducer such as Omeprazol and 3-methylcholanthrene (3-MC) and the CYP 1A induction was always higher in MC than in SC. In bioreactor basic CYP 1A activity was preserved over 2 weeks and the highest albumin production was observed in bioreactor followed by SC and MC. Taken together, it was indicated each investigated model had its advantages and disadvantages. It was also underlined that various in vitro models may address different questions.

  7. Morphology of primary human venous endothelial cell cultures before and after culture medium exchange.

    Science.gov (United States)

    Krüger-Genge, A; Fuhrmann, R; Jung, F; Franke, R P

    2015-01-01

    The evaluation of the interaction of human, venous endothelial cells (HUVEC) with body foreign materials on the cellular level cannot be performed in vivo, but is investigated in vitro under standard culture conditions. To maintain the vitality, proliferation and morphology of HUVEC seeded on body foreign substrates over days, the cell culture medium is usually exchanged every second day. It is well known, that alterations in the microenvironment of cells bear the risk of influencing cell morphology and function. In the current study the influence of cell culture medium exchange on HUVEC cytoskeletal microfilament structure and function was investigated. HUVEC in the third passage were seeded on extracellular matrix (ECM) - which was secreted from bovine corneal endothelial cells on glass- until functional confluence was reached. The experiment started 11 days after HUVEC seeding with an exchange of the cell culture medium followed by a staining of the actin microfilaments with phalloidin-rhodamin 1.5 and 5 minutes after medium exchange. The microfilaments were documented by use of an Olympus microscope (IMT-2) equipped with a UV lamp and online connected to a TV chain (Sony XC 50 ST/monochrome) implying an OPTIMAS - Image analysis system. Prostacyclin was analysed in the cell culture supernatant. 1.5 min after culture medium exchange in the functionally confluent cultures a slight disturbance of the actin microfilament structure with a broadening of the marginal filament band, a partial disconnection of cell-cell contacts and the appearance of intercellular fenestrations were observed. 5 minutes after medium exchange a redevelopment of the slightly disturbed microfilament structure with a condensation and narrowing of the marginal filament band was seen. 12 h later a further consolidation of the microfilament structure occurred. In addition, a perturbation of the cultured HUVEC occurred after cell culture medium exchange. The prostacyclin concentration in the

  8. Lacrimal gland primary acinar cell culture: the role of insulin

    Directory of Open Access Journals (Sweden)

    Leonardo Tannus Malki

    2016-04-01

    Full Text Available ABSTRACT Purpose: The goal of the present study was to establish a protocol for primary culture of lacrimal gland acinar cells (LGACs and to assess the effect of adding insulin to the culture media. Methods: LGACs were isolated and cultured from lacrimal glands of Wistar male rats. The study outcomes included cell number, viability, and peroxidase release over time and in response to three concentrations of insulin (0.5, 5.0, and 50.0 μg/mL. Results: In LGAC primary culture, cells started to form clusters by day 3. There was a time-response pattern of peroxidase release, which rose by day 6, in response to carbachol. Culture viability lasted for 12 days. An insulin concentration of 5.0 μg/mL in the culture medium resulted in higher viability and secretory capacity. Conclusions: The present method simplifies the isolation and culture of LGACs. The data confirmed the relevance of adding insulin to maintain LGACs in culture.

  9. Production of recombinant proteins in suspension-cultured plant cells.

    Science.gov (United States)

    Plasson, Carole; Michel, Rémy; Lienard, David; Saint-Jore-Dupas, Claude; Sourrouille, Christophe; de March, Ghislaine Grenier; Gomord, Véronique

    2009-01-01

    Plants have emerged in the past decade as a suitable alternative to the current production systems for recombinant pharmaceutical proteins and, today their potential for low-cost production of high quality, much safer and biologically active mammalian proteins is largely documented. Among various plant expression systems being explored, genetically modified suspension-cultured plant cells offer a promising system for production of biopharmaceuticals. Indeed, when compared to other plant-based production platforms that have been explored, suspension-cultured plant cells have the advantage of being totally devoid of problems associated with the vagaries of weather, pest, soil and gene flow in the environment. Because of short growth cycles, the timescale needed for the production of recombinant proteins in plant cell culture can be counted in days or weeks after transformation compared to months needed for the production in transgenic plants. Moreover, recovery and purification of recombinant proteins from plant biomass is an expensive and technically challenging business that may amount to 80-94% of the final product cost. One additional advantage of plant cell culture is that the recombinant protein fused with a signal sequence can be expressed and secreted into the culture medium, and therefore recovered and purified in the absence of large quantities of contaminating proteins. Consequently, the downstream processing of proteins extracted from plant cell culture medium is less expensive, which may/does balance the higher costs of fermentation. When needed for clinical use, recombinant proteins are easily produced in suspension-cultured plant cells under certified, controllable and sterile conditions that offer improved safety and provide advantages for good manufacturing practices and regulatory compliance. In this chapter, we present basic protocols for rapid generation of transgenic suspension-cultured cells of Nicotiana tabacum, Oriza sativa and Arabidopis

  10. Exposure to Music Alters Cell Viability and Cell Motility of Human Nonauditory Cells in Culture

    Directory of Open Access Journals (Sweden)

    Nathalia R. Lestard

    2016-01-01

    Full Text Available Although music is part of virtually all cultures in the world, little is known about how it affects us. Since the beginning of this century several studies suggested that the response to music, and to sound in general, is complex and might not be exclusively due to emotion, given that cell types other than auditory hair cells can also directly react to audible sound. The present study was designed to better understand the direct effects of acoustic vibrations, in the form of music, in human cells in culture. Our results suggest that the mechanisms of cell growth arrest and/or cell death induced by acoustic vibrations are similar for auditory and nonauditory cells.

  11. Topological defects control collective dynamics in neural progenitor cell cultures

    Science.gov (United States)

    Kawaguchi, Kyogo; Kageyama, Ryoichiro; Sano, Masaki

    2017-04-01

    Cultured stem cells have become a standard platform not only for regenerative medicine and developmental biology but also for biophysical studies. Yet, the characterization of cultured stem cells at the level of morphology and of the macroscopic patterns resulting from cell-to-cell interactions remains largely qualitative. Here we report on the collective dynamics of cultured murine neural progenitor cells (NPCs), which are multipotent stem cells that give rise to cells in the central nervous system. At low densities, NPCs moved randomly in an amoeba-like fashion. However, NPCs at high density elongated and aligned their shapes with one another, gliding at relatively high velocities. Although the direction of motion of individual cells reversed stochastically along the axes of alignment, the cells were capable of forming an aligned pattern up to length scales similar to that of the migratory stream observed in the adult brain. The two-dimensional order of alignment within the culture showed a liquid-crystalline pattern containing interspersed topological defects with winding numbers of +1/2 and -1/2 (half-integer due to the nematic feature that arises from the head-tail symmetry of cell-to-cell interaction). We identified rapid cell accumulation at +1/2 defects and the formation of three-dimensional mounds. Imaging at the single-cell level around the defects allowed us to quantify the velocity field and the evolving cell density; cells not only concentrate at +1/2 defects, but also escape from -1/2 defects. We propose a generic mechanism for the instability in cell density around the defects that arises from the interplay between the anisotropic friction and the active force field.

  12. Dynamic 3D cell culture via a chemoselective photoactuated ligand.

    Science.gov (United States)

    Westcott, Nathan P; Luo, Wei; Goldstein, Jeffrey; Yousaf, Muhammad N

    2014-09-01

    A new strategy to create a dynamic scaffold for three-dimensional (3D) cell experiments based on a photo-activated cell adhesive peptide ligand is described. After polymerization, the inert matrix becomes cell adhesive by chemoselective modification through the conjugation of oxyamine-terminated ligands. Furthermore, spatial and temporal control of cell culture within the 3D matrix was achieved by the use of a biospecific photoprotected peptide and visualized by confocal microscopy.

  13. Hydrofocusing Bioreactor for Three-Dimensional Cell Culture

    Science.gov (United States)

    Gonda, Steve R.; Spaulding, Glenn F.; Tsao, Yow-Min D.; Flechsig, Scott; Jones, Leslie; Soehnge, Holly

    2003-01-01

    The hydrodynamic focusing bioreactor (HFB) is a bioreactor system designed for three-dimensional cell culture and tissue-engineering investigations on orbiting spacecraft and in laboratories on Earth. The HFB offers a unique hydrofocusing capability that enables the creation of a low-shear culture environment simultaneously with the "herding" of suspended cells, tissue assemblies, and air bubbles. Under development for use in the Biotechnology Facility on the International Space Station, the HFB has successfully grown large three-dimensional, tissuelike assemblies from anchorage-dependent cells and grown suspension hybridoma cells to high densities. The HFB, based on the principle of hydrodynamic focusing, provides the capability to control the movement of air bubbles and removes them from the bioreactor without degrading the low-shear culture environment or the suspended three-dimensional tissue assemblies. The HFB also provides unparalleled control over the locations of cells and tissues within its bioreactor vessel during operation and sampling.

  14. Good Cell Culture Practice for stem cells and stem-cell-derived models.

    Science.gov (United States)

    Pamies, David; Bal-Price, Anna; Simeonov, Anton; Tagle, Danilo; Allen, Dave; Gerhold, David; Yin, Dezhong; Pistollato, Francesca; Inutsuka, Takashi; Sullivan, Kristie; Stacey, Glyn; Salem, Harry; Leist, Marcel; Daneshian, Mardas; Vemuri, Mohan C; McFarland, Richard; Coecke, Sandra; Fitzpatrick, Suzanne C; Lakshmipathy, Uma; Mack, Amanda; Wang, Wen Bo; Yamazaki, Daiju; Sekino, Yuko; Kanda, Yasunari; Smirnova, Lena; Hartung, Thomas

    2017-01-01

    The first guidance on Good Cell Culture Practice (GCCP) dates back to 2005. This document expands this to include aspects of quality assurance for in vitro cell culture focusing on the increasingly diverse cell types and culture formats used in research, product development, testing and manufacture of biotechnology products and cell-based medicines. It provides a set of basic principles of best practice that can be used in training new personnel, reviewing and improving local procedures, and helping to assure standard practices and conditions for the comparison of data between laboratories and experimentation performed at different times. This includes recommendations for the documentation and reporting of culture conditions. It is intended as guidance to facilitate the generation of reliable data from cell culture systems, and is not intended to conflict with local or higher level legislation or regulatory requirements. It may not be possible to meet all recommendations in this guidance for practical, legal or other reasons. However, when it is necessary to divert from the principles of GCCP, the risk of decreasing the quality of work and the safety of laboratory staff should be addressed and any conclusions or alternative approaches justified. This workshop report is considered a first step toward a revised GCCP 2.0.

  15. EXPLANTATION OF MESANGIAL CELL HILLOCKS - A METHOD FOR OBTAINING HUMAN MESANGIAL CELLS IN CULTURE

    NARCIS (Netherlands)

    MULLER, EW; KIM, Y; MICHAEL, AF; VERNIER, RL; VANDERHEM, GK; VANDERWOUDE, FJ

    1992-01-01

    A simple method is presented for selective cell culture of human mesangial cells using explanatation of mesangial cell hillocks. Glomeruli which had been incubated with collagenase were explanted on plastic tissue culture flasks. Three to 6 weeks after explantation, a rapidly growing multilayer of e

  16. EXPLANTATION OF MESANGIAL CELL HILLOCKS - A METHOD FOR OBTAINING HUMAN MESANGIAL CELLS IN CULTURE

    NARCIS (Netherlands)

    MULLER, EW; KIM, Y; MICHAEL, AF; VERNIER, RL; VANDERHEM, GK; VANDERWOUDE, FJ

    1992-01-01

    A simple method is presented for selective cell culture of human mesangial cells using explanatation of mesangial cell hillocks. Glomeruli which had been incubated with collagenase were explanted on plastic tissue culture flasks. Three to 6 weeks after explantation, a rapidly growing multilayer of e

  17. Learning about Cells as Dynamic Entities: An Inquiry-Driven Cell Culture Project

    Science.gov (United States)

    Palombi, Peggy Shadduck; Jagger, Kathleen Snell

    2008-01-01

    Using cultured fibroblast cells, undergraduate students explore cell division and the responses of cultured cells to a variety of environmental changes. The students learn new research techniques and carry out a self-designed experiment. Through this project, students enhance their creative approach to scientific inquiry, learn time-management and…

  18. Cell culture process development: advances in process engineering.

    Science.gov (United States)

    Heath, Carole; Kiss, Robert

    2007-01-01

    Representatives from the cell culture process development community met on September 11 and 12, 2006 at the ACS National Meeting in San Francisco to discuss "Cell Culture Process Development: Advances in Process Engineering". This oral session was held as part of the Division of Biochemical Technology (BIOT) program. The presentations addressed the very small scale (less than 1 mL) to the very large scale (20,000 L). The topics covered included development of high throughput cell culture screening systems, modeling and characterization of bioreactor environments from mixing and shear perspectives at both small and large scales, systematic approaches for improving scale-up and scale-down activities, development of disposable bioreactor technologies, and novel perfusion culture approaches. All told, this well-attended session resulted in a valuable exchange of technical information and demonstrated a high level of interest within the process development community.

  19. Stability of resazurin in buffers and mammalian cell culture media

    DEFF Research Database (Denmark)

    Rasmussen, Eva; Nicolaisen, G.M.

    1999-01-01

    The utility of a ferricyanide/ferrocyanide system used in the AlamarBlue(TM) (Serotec, Oxford, UK) vital. dye to inhibit the reduction of resazurin by mammalian cell culture media is questioned. Resazurin was found to be relatively stable when dissolved in phosphate-buffered saline (PBS). The use...... of HEPES resulted in a huge immediate dye reduction, which was significantly enhanced by exposure to diffuse light from fluorescent tubes in the laboratory 8 h per day. The reduction of resazurin by various cell culture media was time and temperature dependent, and it was significantly enhanced......'s nutrient mixture F-10 and F-12. Fetal calf serum (5-20%) slightly decreased resazurin reduction during the first 2 days of incubation. The reduction of resazurin by mammalian cell culture media do not appear to be problematic under normal culture conditions, and it is primarily dependent upon the presence...

  20. Modeling of cell culture damage and recovery leads to increased antibody and biomass productivity in CHO cell cultures.

    Science.gov (United States)

    Naderi, Saeideh; Nikdel, Ali; Meshram, Mukesh; McConkey, Brendan; Ingalls, Brian; Budman, Hector; Scharer, Jeno

    2014-09-01

    The development of an efficient and productive cell-culture process requires a deep understanding of intracellular mechanisms and extracellular conditions for optimal product synthesis. Mathematical modeling provides an effective strategy to predict, control, and optimize cell performance under a range of culture conditions. In this study, a mathematical model is proposed for the investigation of cell damage of a Chinese hamster ovary cell culture secreting recombinant anti-RhD monoclonal antibody (mAb). Irreversible cell damage was found to be correlated with a reduction in pH. This irreversible damage to cellular function is described mathematically by a Tessier-based model, in which the actively growing fraction of cells is dependent on an intracellular metabolic product acting as a growth inhibitor. To further verify the model, an offline model-based optimization of mAb production in the cell culture was carried out, with the goal of minimizing cell damage and thereby enhancing productivity through intermittent refreshment of the culture medium. An experimental implementation of this model-based strategy resulted in a doubling of the yield as compared to the batch operation and the resulting biomass and productivity profiles agreed with the model predictions.

  1. Introducing Mammalian Cell Culture and Cell Viability Techniques in the Undergraduate Biology Laboratory.

    Science.gov (United States)

    Bowey-Dellinger, Kristen; Dixon, Luke; Ackerman, Kristin; Vigueira, Cynthia; Suh, Yewseok K; Lyda, Todd; Sapp, Kelli; Grider, Michael; Crater, Dinene; Russell, Travis; Elias, Michael; Coffield, V McNeil; Segarra, Verónica A

    2017-01-01

    Undergraduate students learn about mammalian cell culture applications in introductory biology courses. However, laboratory modules are rarely designed to provide hands-on experience with mammalian cells or teach cell culture techniques, such as trypsinization and cell counting. Students are more likely to learn about cell culture using bacteria or yeast, as they are typically easier to grow, culture, and manipulate given the equipment, tools, and environment of most undergraduate biology laboratories. In contrast, the utilization of mammalian cells requires a dedicated biological safety cabinet and rigorous antiseptic techniques. For this reason, we have devised a laboratory module and method herein that familiarizes students with common cell culture procedures, without the use of a sterile hood or large cell culture facility. Students design and perform a time-efficient inquiry-based cell viability experiment using HeLa cells and tools that are readily available in an undergraduate biology laboratory. Students will become familiar with common techniques such as trypsinizing cells, cell counting with a hemocytometer, performing serial dilutions, and determining cell viability using trypan blue dye. Additionally, students will work with graphing software to analyze their data and think critically about the mechanism of death on a cellular level. Two different adaptations of this inquiry-based lab are presented-one for non-biology majors and one for biology majors. Overall, these laboratories aim to expose students to mammalian cell culture and basic techniques and help them to conceptualize their application in scientific research.

  2. Interactions between airway epithelial cells and dendritic cells during viral infections using an in vitro co-culture model

    Science.gov (United States)

    Rationale: Historically, single cell culture models have been limited in pathological and physiological relevance. A co-culture model of dendritic cells (DCs) and differentiated human airway epithelial cells was developed to examine potential interactions between these two cell t...

  3. Callus and cell suspension cultures of carnation

    DEFF Research Database (Denmark)

    Engvild, Kjeld Christensen

    1972-01-01

    Callus cultures of carnation, Dianthus caryophyllus L. ev. G. J. Sim, were grown on a synthetic medium of half strength Murashige and Skoog salts, 3 % sucrose, 100 mg/l of myo-inositol, 0.5 mg/l each of thiamin, HCl, pyridoxin, HCl and nicotinic acid and 10 g/l agar. Optimal concentrations of gro......, but all attempts to induce formation of shoots or em-bryoids gave negative results....

  4. In vitro methods to culture primary human breast epithelial cells.

    Science.gov (United States)

    Raouf, Afshin; Sun, Yu Jia

    2013-01-01

    Current evidence suggests that much like leukemia, breast tumors are maintained by a small subpopulation of tumor cells that have stem cell properties. These cancer stem cells are envisaged to be responsible for tumor formation and relapse. Therefore, knowledge about their nature will provide a platform to develop therapies to eliminate these breast cancer stem cells. This concept highlights the need to understand the mechanisms that regulate the normal functions of the breast stem cells and their immediate progeny as alterations to these same mechanisms can cause these primitive cells to act as cancer stem cells. The study of the primitive cell functions relies on the ability to isolate them from primary sources of breast tissue. This chapter describes processing of discarded tissue from reduction mammoplasty samples as sources of normal primary human breast epithelial cells and describes cell culture systems to grow single-cell suspensions prepared from these reduction samples in vitro.

  5. Metabolism Kinetics of Glucose in Anchorage-dependent Cell Cultures

    Institute of Scientific and Technical Information of China (English)

    孙祥明; 张元兴

    2001-01-01

    The kinetic model of glucose metabolism was established and successfully applied to batchcultures of rCHO and rBHK cells. It was found that a large amount of glucose was utilized for cellmaintenance, and the overwhelming majority of maintenance energy from glucose was by its anaerobicmetabolism in both rBHK and rCHO cell cultures. The overall maintenance coefficients from aerobicmetabolism were 1.9×10-13 mmol/(cell.h) for rCHO cells and 7×10-13 mmol/(cell.h) for rBHK cells. Inaddition, all Go/T and Eo/T gradually increased with the same trend as the cell growth in the culture ofboth rCHO and rBHK cells. The overall molecule yield coefficients of lactate to glucose were 1.61 for rCHO cells and 1.38 for rBHK cells. The yield coefficients of cell to glucose were 4.5×108 cells/mmol for rCHO cells and 1.9 × 108 cells/mmol for rBHK cells, respectively.

  6. Challenges of culturing human norovirus in three-dimensional organoid intestinal cell culture models.

    Directory of Open Access Journals (Sweden)

    Efstathia Papafragkou

    Full Text Available Human noroviruses are the most common cause of acute gastroenteritis worldwide. Recently, cell culture systems have been described using either human embryonic intestinal epithelial cells (Int-407 or human epithelial colorectal adenocarcinoma cells (Caco-2 growing on collagen-I porous micro carrier beads in a rotating bioreactor under conditions of physiological fluid shear. Here, we describe the efforts from two independent laboratories to implement this three dimensional (3D cell culture system for the replication of norovirus. Int-407 and Caco-2 were grown in a rotating bioreactor for up to 28 days. Prior to infection, cells were screened for the presence of microvilli by electron microscopy and stained for junction proteins (zonula occludens-1, claudin-1, and β-catenin. Differentiated 3D cells were transferred to 24-well plates and infected with bacteria-free filtrates of various norovirus genotypes (GI.1, GI.3, GI.8, GII.2, GII.4, GII.7, and GII.8. At 12 h, 24 h, and 48 h post inoculation, viral RNA from both cells and supernatants were collected and analyzed for norovirus RNA by real-time reverse transcription PCR. Despite observations of high expression of junction proteins and microvilli development in stained thin sections, our data suggest no significant increase in viral titer based on norovirus RNA copy number during the first 48 h after inoculation for the different samples and virus culture conditions tested. Our combined efforts demonstrate that 3D cell culture models using Int-407 or Caco-2 cells do not support norovirus replication and highlight the complexity and difficulty of developing a reproducible in vitro cell culture system for human norovirus.

  7. Seed train optimization for cell culture.

    Science.gov (United States)

    Frahm, Björn

    2014-01-01

    For the production of biopharmaceuticals a seed train is required to generate an adequate number of cells for inoculation of the production bioreactor. This seed train is time- and cost-intensive but offers potential for optimization. A method and a protocol are described for the seed train mapping, directed modeling without major effort, and its optimization regarding selected optimization criteria such as optimal points in time for cell passaging. Furthermore, the method can also be applied for the set-up of a new seed train, for example for a new cell line. Although the chapter is directed towards suspension cell lines, the method is also generally applicable, e.g. for adherent cell lines.

  8. Characterisation and germline transmission of cultured avian primordial germ cells.

    Directory of Open Access Journals (Sweden)

    Joni Macdonald

    Full Text Available BACKGROUND: Avian primordial germ cells (PGCs have significant potential to be used as a cell-based system for the study and preservation of avian germplasm, and the genetic modification of the avian genome. It was previously reported that PGCs from chicken embryos can be propagated in culture and contribute to the germ cell lineage of host birds. PRINCIPAL FINDINGS: We confirm these results by demonstrating that PGCs from a different layer breed of chickens can be propagated for extended periods in vitro. We demonstrate that intracellular signalling through PI3K and MEK is necessary for PGC growth. We carried out an initial characterisation of these cells. We find that cultured PGCs contain large lipid vacuoles, are glycogen rich, and express the stem cell marker, SSEA-1. These cells also express the germ cell-specific proteins CVH and CDH. Unexpectedly, using RT-PCR we show that cultured PGCs express the pluripotency genes c-Myc, cKlf4, cPouV, cSox2, and cNanog. Finally, we demonstrate that the cultured PGCs will migrate to and colonise the forming gonad of host embryos. Male PGCs will colonise the female gonad and enter meiosis, but are lost from the gonad during sexual development. In male hosts, cultured PGCs form functional gametes as demonstrated by the generation of viable offspring. CONCLUSIONS: The establishment of in vitro cultures of germline competent avian PGCs offers a unique system for the study of early germ cell differentiation and also a comparative system for mammalian germ cell development. Primary PGC lines will form the basis of an alternative technique for the preservation of avian germplasm and will be a valuable tool for transgenic technology, with both research and industrial applications.

  9. Derivation of completely cell culture-derived mice from early-passage embryonic stem cells.

    OpenAIRE

    Nagy, A.; Rossant, J.; Nagy, R.; Abramow-Newerly, W; Roder, J C

    1993-01-01

    Several newly generated mouse embryonic stem (ES) cell lines were tested for their ability to produce completely ES cell-derived mice at early passage numbers by ES cell tetraploid embryo aggregation. One line, designated R1, produced live offspring which were completely ES cell-derived as judged by isoenzyme analysis and coat color. These cell culture-derived animals were normal, viable, and fertile. However, prolonged in vitro culture negatively affected this initial totipotency of R1, and...

  10. Suspension culture of pluripotent stem cells: effect of shear on stem cell fate.

    Science.gov (United States)

    Keller, Kevin C; Rodrigues, Beatriz; zur Nieden, Nicole I

    2014-01-01

    Despite significant promise, the routine usage of suspension cell culture to manufacture stem cell-derived differentiated cells has progressed slowly. Suspension culture is an innovative way of either expanding or differentiating cells and sometimes both are combined into a single bioprocess. Its advantages over static 2D culturing include a homogeneous and controllable culture environment and producing a large quantity of cells in a fraction of time. This feature makes suspension cell culture ideal for use in stem cell research and eventually ideal in the large-scale production of differentiated cells for regenerative medicine. Because of their tremendous differentiation capacities and unlimited growth properties, pluripotent stem cells (PSCs) in particular are considered potential sources for future cell-replacement therapies. Currently, expansion of PSCs is accomplished in 2D, which only permits a limited amount of cell growth per culture flask before cells need to be passaged. However, before stem cells can be applied clinically, several aspects of their expansion, such as directed growth, but also differentiation, need to be better controlled. This review will summarize recent advantages in suspension culture of PSCs, while at the same time highlighting current challenges.

  11. Imprinting of confining sites for cell cultures on thermoplastic substrates

    Science.gov (United States)

    Cone, C. D.; Fleenor, E. N.

    1969-01-01

    Prevention of test cell migration beyond the field of observation involves confining cells or cultures in microlagoons made in either a layer of grease or a thermoplastic substrate. Thermoplastic films or dishes are easily imprinted with specifically designed patterns of microlagoons.

  12. Effect of Nanoparticles on Complement System in Cell Culture Model

    Science.gov (United States)

    2006-09-15

    7 Primary endothelial cells isolated from human umbilical veins Aseptically taken umbilical cord, preferably abort 20...suspended in cell culture medium. Prepared suspensions were agitated with ultrasounds for 20 minutes at 37C, then stored at +4C and used for testing on the

  13. Animal-cell culture in aqueous two-phase systems.

    NARCIS (Netherlands)

    Zijlstra, G.M.

    1998-01-01

    In current industrial biotechnology, animal-cell culture is an important source of therapeutic protein products. The conventional animal-cell production processes, however, include many unit operations as part of the fermentation and downstream processing strategy. The research described in this the

  14. Process validation for cell culture-derived pharmaceutical proteins.

    Science.gov (United States)

    Lubiniecki, A S; Wiebe, M E; Builder, S E

    1990-01-01

    Principles of process validation are extremely powerful tools in assurance of product quality. They are especially useful for reducing those risks not easily measured routinely during production. When combined with effective process and facility design principles, characterization of cell banks and products, appropriate lot release tests, and adherence to cGMP, safe cell culture biologicals can be prepared in a reliable manner.

  15. Isolation and Characterization of Poliovirus in Cell Culture Systems.

    Science.gov (United States)

    Thorley, Bruce R; Roberts, Jason A

    2016-01-01

    The isolation and characterization of enteroviruses by cell culture was accepted as the "gold standard" by clinical virology laboratories. Methods for the direct detection of all enteroviruses by reverse transcription polymerase chain reaction, targeting a conserved region of the genome, have largely supplanted cell culture as the principal diagnostic procedure. However, the World Health Organization's Global Polio Eradication Initiative continues to rely upon cell culture to isolate poliovirus due to the lack of a reliable sensitive genetic test for direct typing of enteroviruses from clinical specimens. Poliovirus is able to infect a wide range of mammalian cell lines, with CD155 identified as the primary human receptor for all three seroytpes, and virus replication leads to an observable cytopathic effect. Inoculation of cell lines with extracts of clinical specimens and subsequent passaging of the cells leads to an increased virus titre. Cultured isolates of poliovirus are suitable for testing by a variety of methods and remain viable for years when stored at low temperature.This chapter describes general procedures for establishing a cell bank and routine passaging of cell lines. While the sections on specimen preparation and virus isolation focus on poliovirus, the protocols are suitable for other enteroviruses.

  16. Quantitative phase imaging for cell culture quality control.

    Science.gov (United States)

    Kastl, Lena; Isbach, Michael; Dirksen, Dieter; Schnekenburger, Jürgen; Kemper, Björn

    2017-05-01

    The potential of quantitative phase imaging (QPI) with digital holographic microscopy (DHM) for quantification of cell culture quality was explored. Label-free QPI of detached single cells in suspension was performed by Michelson interferometer-based self-interference DHM. Two pancreatic tumor cell lines were chosen as cellular model and analyzed for refractive index, volume, and dry mass under varying culture conditions. Firstly, adequate cell numbers for reliable statistics were identified. Then, to characterize the performance and reproducibility of the method, we compared results from independently repeated measurements and quantified the cellular response to osmolality changes of the cell culture medium. Finally, it was demonstrated that the evaluation of QPI images allows the extraction of absolute cell parameters which are related to cell layer confluence states. In summary, the results show that QPI enables label-free imaging cytometry, which provides novel complementary integral biophysical data sets for sophisticated quantification of cell culture quality with minimized sample preparation. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

  17. Cell cultures from the symbiotic soft coral Sinularia flexibilis

    NARCIS (Netherlands)

    Khalesi, M.K.; Vera-Jimenez, N.I.; Aanen, D.K.; Beeftink, H.H.; Wijffels, R.H.

    2008-01-01

    The symbiotic octocoral Sinularia flexibilis is a producer of potential pharmaceuticals. Sustainable mass production of these corals as a source of such compounds demands innovative approaches, including coral cell culture. We studied various cell dissociation methodologies and the feasibility of cu

  18. Animal-cell culture in aqueous two-phase systems

    NARCIS (Netherlands)

    Zijlstra, G.M.

    1998-01-01

    In current industrial biotechnology, animal-cell culture is an important source of therapeutic protein products. The conventional animal-cell production processes, however, include many unit operations as part of the fermentation and downstream processing strategy. The research described in

  19. Replica-moulded polydimethylsiloxane culture vessel lids attenuate osmotic drift in long-term cell cultures

    Indian Academy of Sciences (India)

    Axel Blau; Tanja Neumann; Christiane Ziegler; Fabio Benfenati

    2009-03-01

    An imbalance in medium osmolarity is a determinant that affects cell culture longevity. Even in humidified incubators, evaporation of water leads to a gradual increase in osmolarity overtime. We present a simple replica-moulding strategy for producing self-sealing lids adaptable to standard, small-size cell-culture vessels. They are made of polydimethylsiloxane (PDMS), a flexible, transparent and biocompatible material, which is gas-permeable but largely impermeable to water. Keeping cell cultures in a humidified 5% CO2 incubator at 37°C, medium osmolarity increased by +6.86 mosmol/kg/day in standard 35 mm Petri dishes, while PDMS lids attenuated its rise by a factor of four to changes of +1.72 mosmol/kg/ day. Depending on the lid membrane thickness, pH drifts at ambient CO2 levels were attenuated by a factor of 4 to 9. Comparative evaporation studies at temperatures below 60°C yielded a 10-fold reduced water vapour flux of 1.75 g/day/dm2 through PDMS lids as compared with 18.69 g/day/dm2 with conventional Petri dishes. Using such PDMS lids, about 2/3 of the cell cultures grew longer than 30 days in vitro. Among these, the average survival time was 69 days with the longest survival being 284 days under otherwise conventional cell culture conditions.

  20. Radiosensitivity of cultured insect cells: I. Lepidoptera

    Energy Technology Data Exchange (ETDEWEB)

    Koval, T.M.

    1983-10-01

    The radiosensitivity of five lepidopteran insect cell lines representing five different genera has been investigated. These lines are: (1) TN-368, Trichoplusia ni; (2) IPLB-SF-1254, Spodoptera frugiperda; (3) IPLB-1075, Heliothis zea; (4) MRRL-CHl, clone GVl, Manduca sexta; and (5) IAL-PID2, Plodia interpunctella. The cell lines grew at different rates and had population doubling times that ranged from 19 to 52 hr. All of the lines are highly heteroploid and have approximate chromosome numbers near or above 100. The chromosomes are very small. All of the lines are extremely radioresistant; cell populations are able to recover from 260 kVp X-ray exposures up to and including 400 Gy, the highest dose examined. Cell survival curves were obtainable for only the TN-368 and IPLB-SF-1254 lines. The TN-368 cells displayed a biphasic survival response with D/sub 0/, d/sub q/, and n values of 65.7 and 130.2 Gy, 9.0 and -36.1 Gy, and 1.2 and 0.8, respectively, for the steep and shallow portions of the curve. The IPLB-SF-1254 cells had a D/sub 0/ of 63.9 Gy. D/sub q/ of 19.0 Gy, and n value of 1.4. These studies provide definitive evidence of the radioresistance of lepidopteran cells, and suggest that this radioresistance is a characteristic of lepidopteran insects.

  1. Phenotypic changes in satellite glial cells in cultured trigeminal ganglia.

    Science.gov (United States)

    Belzer, Vitali; Shraer, Nathanael; Hanani, Menachem

    2010-11-01

    Satellite glial cells (SGCs) are specialized cells that form a tight sheath around neurons in sensory ganglia. In recent years, there is increasing interest in SGCs and they have been studied in both intact ganglia and in tissue culture. Here we studied phenotypic changes in SGCs in cultured trigeminal ganglia from adult mice, containing both neurons and SGCs, using phase optics, immunohistochemistry and time-lapse photography. Cultures were followed for up to 14 days. After isolation virtually every sensory neuron is ensheathed by SGCs, as in the intact ganglia. After one day in culture, SGCs begin to migrate away from their parent neurons, but in most cases the neurons still retain an intact glial cover. At later times in culture, there is a massive migration of SGCs away from the neurons and they undergo clear morphological changes, and at 7 days they become spindle-shaped. At one day in culture SGCs express the glial marker glutamine synthetase, and also the purinergic receptor P2X7. From day 2 in culture the glutamine synthetase expression is greatly diminished, whereas that of P2X7 is largely unchanged. We conclude that SGCs retain most of their characteristics for about 24 h after culturing, but undergo major phenotypic changes at later times.

  2. Schwann cell cultures from human fetal dorsal root ganglia

    Institute of Scientific and Technical Information of China (English)

    Yaping Feng; Hui Zhu; Jiang Hao; Xinmin Wang; Shengping Wu; Li Bai; Xiangming Li; Yun Zha

    2009-01-01

    BACKGROUND:Previous studies have used many methods for in vitro Schwann cells (SCs) cul-tures and purification,such as single cell suspension and cytosine arabinoside.However,it has been difficult to obtain sufficient cellular density,and the procedures have been quite tedious.OBJECTIVE:To investigate the feasibility of culturing high-density SCs using fetal human dorsal root ganglion tissue explants.DESIGN,TIME AND SETTING:Cell culture and immunohistochemistry were performed at the Cen-tral Laboratory of Kunming General Hospital of Chinese PLA between March 2001 and October 2008.MATERIALS:Culture media containing 10% fetal bovine serum,as well as 0.2% collagenase and 0.25% trypsin were purchased from Gibco,USA;mouse anti-human S-100 monoclonal antibody and goat anti-mouse IgG labeled with horseradish peroxidase were provided by Beijing Institute of Bi-ological Products,China.METHODS:Primarily cultured SCs were dissociated from dorsal root ganglia of human aborted fe-tuses at 4-6 months pregnancy.Following removal of the dorsal root ganglion perineurium,the gan-glia were dissected into tiny pieces and digested with 0.2% collagenase and 0.25% trypsin (volume ratio 1:1),then explanted and cultured.SC purification was performed with 5 mL 10% fetal bovine serum added to the culture media,followed by differential adhesion.MAIN OUTCOME MEASURES:SCs morphology was observed under inverted phase contrast light microscopy.SC purity was evaluated according to percentage of S-100 immunostained cells.RESULTS:SCs were primarily cultured for 5-6 days and then subcultured for 4-5 passages.The highly enriched SC population reached > 95% purity and presented with normal morphology.CONCLUSION:A high purity of SCs was obtained with culture methods using human fetal dorsal root ganglion tissue explants.

  3. Growth and Plating of Cell Suspension Cultures of Datura Innoxia

    DEFF Research Database (Denmark)

    Engvild, Kjeld Christensen

    1974-01-01

    Suspension cultures of Datura innoxia Mill, were successfully grown on a modified Murashige and Skoog medium with 2,4–D, NAA or BAP as growth substances, provided the micronutrient levels were reduced to 1/10. Normal amounts of micronutrients were toxic. Attempts to identify the toxic elements did...... malate) or on NO3−-N alone. Dry weight yield was proportional to the amount of nitrate-N added (47 mg/mg N). Filtered suspension cultures containing single cells (plating cultures) could be grown in agar in petri dishes when NAA or 2,4-D were used as growth substances. Cells grew at densities above 500...... units/ml in the agar. Most colonies grew from cell aggregates but division in single cells was observed. The highest plating efficiency was about 50% on 10−6 M 2,4-D + 1 g/1 casein hydrolysate....

  4. Cloning higher plants from aseptically cultured tissues and cells

    Science.gov (United States)

    Krikorian, A. D.

    1982-01-01

    A review of aseptic culture methods for higher plants is presented, which focuses on the existing problems that limit or prevent the full realization of cloning plants from free cells. It is shown that substantial progress in clonal multiplication has been made with explanted stem tips or lateral buds which can be stimulated to produce numerous precocious axillary branches. These branches can then be separated or subdivided and induced to root in order to yield populations of genetically and phenotypically uniorm plantlets. Similarly, undifferentiated calluses can sometimes be induced to form shoots and/or roots adventitiously. Although the cell culture techniques required to produce somatic embryos are presently rudimentary, steady advances are being made in learning how to stimulate formation of somatic or adventive embryos from totipotent cells grown in suspension cultures. It is concluded that many problems exist in the producing and growing of totipotent or morphogenetically competent cell suspensions, but the potential benefits are great.

  5. Miniature Bioreactor System for Long-Term Cell Culture

    Science.gov (United States)

    Gonda, Steve R.; Kleis, Stanley J.; Geffert, Sandara K.

    2010-01-01

    A prototype miniature bioreactor system is designed to serve as a laboratory benchtop cell-culturing system that minimizes the need for relatively expensive equipment and reagents and can be operated under computer control, thereby reducing the time and effort required of human investigators and reducing uncertainty in results. The system includes a bioreactor, a fluid-handling subsystem, a chamber wherein the bioreactor is maintained in a controlled atmosphere at a controlled temperature, and associated control subsystems. The system can be used to culture both anchorage-dependent and suspension cells, which can be either prokaryotic or eukaryotic. Cells can be cultured for extended periods of time in this system, and samples of cells can be extracted and analyzed at specified intervals. By integrating this system with one or more microanalytical instrument(s), one can construct a complete automated analytical system that can be tailored to perform one or more of a large variety of assays.

  6. Culture of isolated single cells from Taxus suspensions for the propagation of superior cell populations.

    Science.gov (United States)

    Naill, Michael C; Roberts, Susan C

    2005-11-01

    Single cells isolated from aggregated Taxus cuspidata cultures via enzymatic digestion were grown in suspension culture. High seeding density (4 x 10(5 )cells/ml) and the addition of cell-free conditioned medium were essential for growth. Doubling the concentration of the nutrients [ascorbic acid (150 g/l), glutamine (6.25 mM: ), and citric acid (150 g/l)] had no effect on single cell growth or viability. A specific growth rate of 0.11 days(-1) was achieved, which is similar to the observed growth rate of aggregated Taxus suspensions. The biocide, Plant Preservative Mixture, added at 0.2% (v/v) to all single cell cultures to prevent microbial contamination, had no significant effect on growth or viability. Following cell sorting, single cell cultures can be used to establish new cell lines for biotechnology applications or provide cells for further study.

  7. Establishing a stem cell culture laboratory for clinical trials

    Directory of Open Access Journals (Sweden)

    Elíseo Joji Sekiya

    2012-01-01

    Full Text Available Adult stem/progenitor cells are found in different human tissues. An in vitro cell culture is needed for their isolation or for their expansion when they are not available in a sufficient quantity to regenerate damaged organs and tissues. The level of complexity of these new technologies requires adequate facilities, qualified personnel with experience in cell culture techniques, assessment of quality and clear protocols for cell production. The rules for the implementation of cell therapy centers involve national and international standards of good manufacturing practices. However, such standards are not uniform, reflecting the diversity of technical and scientific development. Here standards from the United States, the European Union and Brazil are analyzed. Moreover, practical solutions encountered for the implementation of a cell therapy center appropriate for the preparation and supply of cultured cells for clinical studies are described. Development stages involved the planning and preparation of the project, the construction of the facility, standardization of laboratory procedures and development of systems to prevent cross contamination. Combining the theoretical knowledge of research centers involved in the study of cells with the practical experience of blood therapy services that manage structures for cell transplantation is presented as the best potential for synergy to meet the demands to implement cell therapy centers.

  8. Cell death in mammalian cell culture: molecular mechanisms and cell line engineering strategies.

    Science.gov (United States)

    Krampe, Britta; Al-Rubeai, Mohamed

    2010-07-01

    Cell death is a fundamentally important problem in cell lines used by the biopharmaceutical industry. Environmental stress, which can result from nutrient depletion, by-product accumulation and chemical agents, activates through signalling cascades regulators that promote death. The best known key regulators of death process are the Bcl-2 family proteins which constitute a critical intracellular checkpoint of apoptosis cell death within a common death pathway. Engineering of several members of the anti-apoptosis Bcl-2 family genes in several cell types has extended the knowledge of their molecular function and interaction with other proteins, and their regulation of cell death. In this review, we describe the various modes of cell death and their death pathways at molecular and organelle level and discuss the relevance of the growing knowledge of anti-apoptotic engineering strategies to inhibit cell death and increase productivity in mammalian cell culture.

  9. Cell division and differentiation in protoplasts from cell cultures of Glycine species and leaf tissue of soybean.

    Science.gov (United States)

    Gamborg, O L; Davis, B P; Stahlhut, R W

    1983-08-01

    Protoplasts were isolated from cell cultures of G. soja and G. tabacina, respectively. The isolation procedure employed Percoll for the separation and concentration of protoplasts. The cultured protoplasts formed cells which developed into embryo-like structures. Protoplasts also were isolated from leaf tissue of soybean cv. Williams 82. Upon culture, the protoplasts regenerated cell walls and divided to form cell cultures.

  10. Cytopathogenicity of Naegleria for cultured neuroblastoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Fulford, D.E.

    1985-01-01

    The cytopathic activity of live Naegleria amoebae and cell-free lysates of Naegleria for B-103 rat neuroblastoma cells was investigated using a /sup 51/Cr release assay. Live amoebae and cell-free lysates of N. fowleri, N. australiensis, N. lovaniensis, and N. gruberi all induced sufficient damage to radiolabeled B-103 cells to cause a significant release of chromium. The cytotoxic activity present in the cell-free lysates of N. fowleri can be recovered in the supernatant fluid following centrifugation at 100,000xg and precipitation of the 100,000xg supernatant fluid with ammonium sulfate. Initial characterization of the cytotoxic factor indicates that it is a heat labile, pH sensitive, soluble protein. The cytotoxic activity is abolished by either extraction, unaffected by repeated freeze-thawing, and is not sensitive to inhibitors of proteolytic enzymes. Phospholipase A activity was detected in the cytotoxic ammonium sulfate precipitable material, suggesting that this enzyme activity may have a role in the cytotoxic activity of the cell-free lysates.

  11. The replacement of serum by hormones in cell culture media.

    Science.gov (United States)

    Sato, G; Hayashi, I

    1976-12-01

    The replacement of serum by hormones in cell culture media. (Reemplazo del suero por hormonas en el medio de cultivo de células). Arch. Biol. Med. Exper. 10: 120-121, 1976. The serum used in cell culture media can be replaced by a mixture of hormones and some accesory blood factors. The pituitary cell line GH3 can be grown in a medium in which serum is replaced by triiodothyronine, transferrin, parathormone, tyrotrophin releasing hormone and somatomedins. Hela and BHK cell strains can also be grown in serum free medium supplemented with hormones. Each cell type appears to have different hormonal requirements yet it may found that some hormones are required for most cell types.

  12. Foetal hepatic progenitor cells assume a cholangiocytic cell phenotype during two-dimensional pre-culture.

    Science.gov (United States)

    Anzai, Kazuya; Chikada, Hiromi; Tsuruya, Kota; Ida, Kinuyo; Kagawa, Tatehiro; Inagaki, Yutaka; Mine, Tesuya; Kamiya, Akihide

    2016-06-23

    Liver consists of parenchymal hepatocytes and other cells. Liver progenitor cell (LPC) is the origin of both hepatocytes and cholangiocytic cells. The analyses of mechanism regulating differentiation of LPCs into these functional cells are important for liver regenerative therapy using progenitor cells. LPCs in adult livers were found to form cysts with cholangiocytic characteristics in 3D culture. In contrast, foetal LPCs cannot form these cholangiocytic cysts in the same culture. Thus, the transition of foetal LPCs into cholangiocytic progenitor cells might occur during liver development. Primary CD45(-)Ter119(-)Dlk1(+) LPCs derived from murine foetal livers formed ALBUMIN (ALB)(+)CYTOKERATIN (CK)19(-) non-cholangiocytic cysts within 3D culture. In contrast, when foetal LPCs were pre-cultured on gelatine-coated dishes, they formed ALB(-)CK19(+) cholangiocytic cysts. When hepatocyte growth factor or oncostatin M, which are inducers of hepatocytic differentiation, was added to pre-culture, LPCs did not form cholangiocytic cysts. These results suggest that the pre-culture on gelatine-coated dishes changed the characteristics of foetal LPCs into cholangiocytic cells. Furthermore, neonatal liver progenitor cells were able to form cholangiocytic cysts in 3D culture without pre-culture. It is therefore possible that the pre-culture of mid-foetal LPCs in vitro functioned as a substitute for the late-foetal maturation step in vivo.

  13. Differential heat shock response of primary human cell cultures and established cell lines

    DEFF Research Database (Denmark)

    Richter, W W; Issinger, O G

    1986-01-01

    degrees C treatment, whereas in immortalized cell lines usually 90% of the cells were found in suspension. Enhanced expression of the major heat shock protein (hsp 70) was found in all heat-treated cells. In contrast to the primary cell cultures, established and transformed cell lines synthesized...

  14. Wave characterization for mammalian cell culture: residence time distribution.

    Science.gov (United States)

    Rodrigues, Maria Elisa; Costa, Ana Rita; Henriques, Mariana; Azeredo, Joana; Oliveira, Rosário

    2012-02-15

    The high dose requirements of biopharmaceutical products led to the development of mammalian cell culture technologies that increase biomanufacturing capacity. The disposable Wave bioreactor is one of the most promising technologies, providing ease of operation and no cross-contamination, and using an innovative undulation movement that ensures good mixing and oxygen transfer without cell damage. However, its recentness demands further characterization. This study evaluated the residence time distribution (RTD) in Wave, allowing the characterization of mixing and flow and the comparison with ideal models and a Stirred tank reactor (STR) used for mammalian cell culture. RTD was determined using methylene blue with pulse input methodology, at three flow rates common in mammalian cell culture (3.3×10(-5)m(3)/h, 7.9×10(-5)m(3)/h, and 1.25×10(-4)m(3)/h) and one typical of microbial culture (5×10(-3)m(3)/h). Samples were taken periodically and the absorbance read at 660nm. It was observed that Wave behavior diverted from ideal models, but was similar to STR. Therefore, the deviations are not related to the particular Wave rocking mechanism, but could be associated with the inadequacy of these reactors to operate in continuous mode or to a possible inability of the theoretical models to properly describe the behavior of reactors designed for mammalian cell culture. Thus, the development of new theoretical models could better characterize the performance of these reactors.

  15. Isolation and Culture of Postnatal Stem Cells from Deciduous Teeth

    OpenAIRE

    Olávez, Daniela; Facultad de Odontología Universidad de Los Andes; Salmen, Siham; Instituto de Inmunología Clínica, Universidad de Los Andes.; Padrón, Karla; Facultad de Odontología. Univerisdad de Los Andes.; Lobo, Carmine; Facultad de Odontología. Univerisdad de Los Andes.; Díaz, Nancy; Facultad de Odontología, Universidad de Los Andes.; Berrueta, Lisbeth; Doctora en Inmunología por Instituto Venezolano de Investigaciones Científicas (IVIC). Instituto de Inmunología Clínica, Facultad de Medicina, Universidad de Los Andes, Venezuela.; Solorzanio, Eduvigis; Facultad de Odontología, Universidad de Los Andes.

    2014-01-01

    Background: Currently, degenerative diseases represent a public health problem; therefore, the development and implementation of strategies to fully or partially recover of damaged tissues has a special interest in the biomedical field. Therapeutic strategies based on mesenchymal stem cells transplantation from dental pulp have been proposed as an alternative. Purpose: To develop a mesenchymal stem cells culture isolated from dental pulp of deciduous teeth. Methods: The mesenchymal stem cells...

  16. Culture of Neural Stem Cells in Calcium-alginate Microbeads

    Institute of Scientific and Technical Information of China (English)

    Li-Song YAO; Tian-Qing LIU; Dan GE; Xue-Hu MA; Zhan-Feng CUI

    2005-01-01

    @@ 1 Introduction Recent research shows that neural stem cells may play an important role in the nerve injury reparation and nerve disease treatment. The shortage of the source and the number of NSCs, however, is the main challenge for its clinic application. In this situation, expansion of NSCs in large scale and culture in three dimensional environment are very worth of exploration. Notablely, the shear stress existed in bioreactors can cause serious cell injury especially for the shear sensitive cells like NSCs.

  17. Isolation and Culture of Postnatal Stem Cells from Deciduous Teeth

    OpenAIRE

    Olávez, Daniela; Facultad de Odontología Universidad de Los Andes; Salmen, Siham; Instituto de Inmunología Clínica, Universidad de Los Andes.; Padrón, Karla; Facultad de Odontología. Univerisdad de Los Andes.; Lobo, Carmine; Facultad de Odontología. Univerisdad de Los Andes.; Díaz, Nancy; Facultad de Odontología, Universidad de Los Andes.; Berrueta, Lisbeth; Doctora en Inmunología por Instituto Venezolano de Investigaciones Científicas (IVIC). Instituto de Inmunología Clínica, Facultad de Medicina, Universidad de Los Andes, Venezuela.; Solorzanio, Eduvigis; Facultad de Odontología, Universidad de Los Andes.

    2014-01-01

    Background: Currently, degenerative diseases represent a public health problem; therefore, the development and implementation of strategies to fully or partially recover of damaged tissues has a special interest in the biomedical field. Therapeutic strategies based on mesenchymal stem cells transplantation from dental pulp have been proposed as an alternative. Purpose: To develop a mesenchymal stem cells culture isolated from dental pulp of deciduous teeth. Methods: The mesenchymal stem cells...

  18. A novel closed cell culture device for fabrication of corneal epithelial cell sheets.

    Science.gov (United States)

    Nakajima, Ryota; Kobayashi, Toyoshige; Moriya, Noboru; Mizutani, Manabu; Kan, Kazutoshi; Nozaki, Takayuki; Saitoh, Kazuo; Yamato, Masayuki; Okano, Teruo; Takeda, Shizu

    2015-11-01

    Automation technology for cell sheet-based tissue engineering would need to optimize the cell sheet fabrication process, stabilize cell sheet quality and reduce biological contamination risks. Biological contamination must be avoided in clinical settings. A closed culture system provides a solution for this. In the present study, we developed a closed culture device called a cell cartridge, to be used in a closed cell culture system for fabricating corneal epithelial cell sheets. Rabbit limbal epithelial cells were cultured on the surface of a porous membrane with 3T3 feeder cells, which are separate from the epithelial cells in the cell cartridges and in the cell-culture inserts as a control. To fabricate the stratified cell sheets, five different thicknesses of the membranes which were welded to the cell cartridge, were examined. Multilayered corneal epithelial cell sheets were fabricated in cell cartridges that were welded to a 25 µm-thick gas-permeable membrane, which was similar to the results with the cell-culture inserts. However, stratification of corneal epithelial cell sheets did not occur with cell cartridges that were welded to 100-300 µm-thick gas-permeable membranes. The fabricated cell sheets were evaluated by histological analyses to examine the expression of corneal epithelial-specific markers. Immunohistochemical analyses showed that a putative stem cell marker, p63, a corneal epithelial differentiation maker, CK3, and a barrier function marker, Claudin-1, were expressed in the appropriate position in the cell sheets. These results suggest that the cell cartridge is effective for fabricating corneal epithelial cell sheets.

  19. A biocompatible micro cell culture chamber (microCCC) for the culturing and on-line monitoring of eukaryote cells

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Petronis, Sarunas; Jørgensen, A M

    2006-01-01

    We have previously shown that a polymeric (PMMA) chip with medium perfusion and integrated heat regulation provides sufficiently precise heat regulation, pH-control and medium exchange to support cell growth for weeks. However, it was unclear how closely the cells cultured in the chip resembled c...

  20. Experiments on tissue culture in the genus Lycopersicon miller : Shoot formation from protoplasts of tomato long-term cell cultures.

    Science.gov (United States)

    Koblitz, H; Koblitz, D

    1982-06-01

    Callus cultures from cotyledon explants were established and maintained in culture for more than two years. After several months callus cultures were transferred into liquid medium and cultured as cell suspensions. Protoplasts were isolated from these cell suspension cultures and cultured in a liquid medium. After formation of new cell walls the cells were further cultured in liquid medium and afterwards transferred to an agar-solidified medium to give a vigorously growing callus culture. In the case of the cultivar 'Lukullus' shoots were recovered from callus. All efforts to root these shoots failed and this, in addition to variations in appearence, suggests that the shoots are changed genetically possibly due to the prolonged culture period.

  1. Comparison of human nasal epithelial cells grown as explant outgrowth cultures or dissociated tissue cultures in vitro.

    Science.gov (United States)

    Jiao, Jian; Meng, Na; Wang, Hong; Zhang, Luo

    2013-12-01

    The purpose of this study was to compare cell growth characteristics, ciliated cell differentiation, and function of human nasal epithelial cells established as explant outgrowth cultures or dissociated tissue cultures. Human nasal mucosa of the uncinate process was obtained by endoscopy and epithelial cell cultures were established by explant outgrowth or dissociated tissue culture methods. Epithelial cell growth characteristics were observed by inverted phase contrast microscopy. Ciliated cell differentiation was detected by β-tubulin IVand ZO-1 immunocytochemistry. Basal and ATP-stimulated ciliary beat frequency (CBF) was measured using a highspeed digital microscopic imaging system. Both the explant and dissociated tissue cultures established as monolayers with tight junctions and differentiated cell composition, with both types of cultures comprising ciliated and non-ciliated epithelial cells. Fibroblasts were also frequently found in explant cultures but rarely seen in dissociated tissue cultures. In both culture systems, the highest ciliated cell density appeared at 7th-10th culture day and declined with time, with the lifespan of ciliated cells ranging from 14 to 21 days. Overall, 10% of the cells in explant cultures and 20% of the cells in the dissociated tissue cultures were ciliated. These two cultures demonstrated similar ciliary beat frequency values at baseline (7.78 ± 1.99 Hz and 7.91 ± 2.52 Hz, respectively) and reacted equivalently following stimulation with 100 μM ATP. The results of this study indicate that both the explant outgrowth and dissociated tissue culture techniques are suitable for growing well-differentiated nasal ciliated and non-ciliated cells, which have growth characteristics and ciliary activity similar to those of nasal epithelial cells in vivo.

  2. Metabolic measurements in cell culture and tissue constructs

    Science.gov (United States)

    Rolfe, P.

    2008-10-01

    This paper concerns the study and use of biological cells in which there is a need for sensors and assemblies for the measurement of a diverse range of physical and chemical variables. In this field cell culture is used for basic research and for applications such as protein and drug synthesis, and in cell, tissue and organ engineering. Metabolic processes are fundamental to cell behaviour and must therefore be monitored reliably. Basic metabolic studies measure the transport of oxygen, glucose, carbon dioxide, lactic acid to, from, or within cells, whilst more advanced research requires examination of energy storage and utilisation. Assemblies are designed to incorporate bioreactor functions for cell culture together with appropriate sensing devices. Oxygen consumption by populations of cells is achieved in a flowthrough assembly that incorporates O2 micro-sensors based on either amperometry or fluorescence. Measurements in single cell are possible with intra-cellular fluorophores acting as biosensors together with optical stimulation and detection. Near infra-red spectroscopy (NIRS) is used for analysis within culture fluid, for example for estimation of glucose levels, as well as within cell populations, for example to study the respiratory enzymes.Â#

  3. Cellular interactions regulate stem cell differentiation in tri-culture.

    Science.gov (United States)

    Wang, I-Ning E; Bogdanowicz, Danielle R; Mitroo, Siddarth; Shan, Jing; Kala, Sonam; Lu, Helen H

    2016-11-01

    Currently, the mechanism governing the regeneration of the soft tissue-to-bone interface, such as the transition between the anterior cruciate ligament (ACL) and bone, is not known. Focusing on the ACL-to-bone insertion, this study tests the novel hypothesis that interactions between cells from the ligament (fibroblasts) and bone (osteoblasts) initiate interface regeneration. Specifically, these heterotypic cell interactions direct the fibrochondrogenic differentiation of interface-relevant cell populations, defined here as ligament fibroblasts and bone marrow stromal cells (BMSC). The objective of this study is to examine the effects of heterotypic cellular interactions on BMSC or fibroblast growth and biosynthesis, as well as expression of fibrocartilage-relevant markers in tri-culture. The effects of cell-cell physical contact and paracrine interactions between fibroblasts and osteoblasts were also determined. It was found that, in tri-culture with fibroblasts and osteoblasts, BMSC exhibited greater fibrochondrogenic potential than ligament fibroblasts. The growth of BMSC decreased while proteoglycan production and TGF-β3 expression increased. Moreover, tri-culture regulated BMSC response via paracrine factors, and interestingly, fibroblast-osteoblast contact further promoted proteoglycan and TGF-β1 synthesis as well as induced SOX9 expression in BMSC. Collectively, the findings of this study suggest that fibroblast-osteoblast interactions play an important role in regulating the stem cell niche for fibrocartilage regeneration, and the mechanisms of these interactions are directed by paracrine factors and augmented with direct cell-cell contact.

  4. Hydroxyapatite incorporated into collagen gels for mesenchymal stem cell culture.

    Science.gov (United States)

    Laydi, F; Rahouadj, R; Cauchois, G; Stoltz, J-F; de Isla, N

    2013-01-01

    Collagen gels could be used as carriers in tissue engineering to improve cell retention and distribution in the defect. In other respect hydroxyapatite could be added to gels to improve mechanical properties and regulate gel contraction. The aim of this work was to analyze the feasibility to incorporate hydroxyapatite into collagen gels and culture mesenchymal stem cells inside it. Human bone marrow mesenchymal stem cells (hMSC-BM) were used in this study. Gels were prepared by mixing rat tail type I collagen, hydroxyapatite microparticles and MSCs. After polymerization gels were kept in culture while gel contraction and mechanical properties were studied. In parallel, cell viability and morphology were analyzed. Gels became free-floating gels contracted from day 3, only in the presence of cells. A linear rapid contraction phase was observed until day 7, then a very slow contraction phase took place. The incorporation of hydroxyapatite improved gel stability and mechanical properties. Cells were randomly distributed on the gel and a few dead cells were observed all over the experiment. This study shows the feasibility and biocompatibility of hydroxyapatite supplemented collagen gels for the culture of mesenchymal stem cells that could be used as scaffolds for cell delivery in osteoarticular regenerative medicine.

  5. Specimen Sample Preservation for Cell and Tissue Cultures

    Science.gov (United States)

    Meeker, Gabrielle; Ronzana, Karolyn; Schibner, Karen; Evans, Robert

    1996-01-01

    The era of the International Space Station with its longer duration missions will pose unique challenges to microgravity life sciences research. The Space Station Biological Research Project (SSBRP) is responsible for addressing these challenges and defining the science requirements necessary to conduct life science research on-board the International Space Station. Space Station will support a wide range of cell and tissue culture experiments for durations of 1 to 30 days. Space Shuttle flights to bring experimental samples back to Earth for analyses will only occur every 90 days. Therefore, samples may have to be retained for periods up to 60 days. This presents a new challenge in fresh specimen sample storage for cell biology. Fresh specimen samples are defined as samples that are preserved by means other than fixation and cryopreservation. The challenge of long-term storage of fresh specimen samples includes the need to suspend or inhibit proliferation and metabolism pending return to Earth-based laboratories. With this challenge being unique to space research, there have not been any ground based studies performed to address this issue. It was decided hy SSBRP that experiment support studies to address the following issues were needed: Fixative Solution Management; Media Storage Conditions; Fresh Specimen Sample Storage of Mammalian Cell/Tissue Cultures; Fresh Specimen Sample Storage of Plant Cell/Tissue Cultures; Fresh Specimen Sample Storage of Aquatic Cell/Tissue Cultures; and Fresh Specimen Sample Storage of Microbial Cell/Tissue Cultures. The objective of these studies was to derive a set of conditions and recommendations that can be used in a long duration microgravity environment such as Space Station that will permit extended storage of cell and tissue culture specimens in a state consistent with zero or minimal growth, while at the same time maintaining their stability and viability.

  6. 40 CFR 798.5300 - Detection of gene mutations in somatic cells in culture.

    Science.gov (United States)

    2010-07-01

    ... cells in culture. 798.5300 Section 798.5300 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY....5300 Detection of gene mutations in somatic cells in culture. (a) Purpose. Mammalian cell culture... selected by resistance to ouabain. (2) Description. Cells in suspension or monolayer culture are exposed to...

  7. 21 CFR 864.2240 - Cell and tissue culture supplies and equipment.

    Science.gov (United States)

    2010-04-01

    ... SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture Products § 864.2240 Cell and tissue culture supplies and equipment. (a) Identification. Cell and tissue culture... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Cell and tissue culture supplies and...

  8. Effects of diluents on cell culture viability measured by automated cell counter

    National Research Council Canada - National Science Library

    Aaron Chen; Matthew Leith; Roger Tu; Gurpreet Tahim; Anish Sudra; Swapnil Bhargava

    2017-01-01

      Commercially available automated cell counters based on trypan blue dye-exclusion are widely used in industrial cell culture process development and manufacturing to increase throughput and eliminate...

  9. Hollow fiber clinostat for simulating microgravity in cell culture

    Science.gov (United States)

    Rhodes, Percy H. (Inventor); Miller, Teresa Y. (Inventor); Snyder, Robert S. (Inventor)

    1992-01-01

    A clinostat for simulating microgravity on cell systems carried in a fiber fixedly mounted in a rotatable culture vessel is disclosed. The clinostat is rotated horizontally along its longitudinal axis to simulate microgravity or vertically as a control response. Cells are injected into the fiber and the ends of the fiber are sealed and secured to spaced end pieces of a fiber holder assembly which consists of the end pieces, a hollow fiber, a culture vessel, and a tension spring with three alignment pins. The tension spring is positioned around the culture vessel with its ends abutting the end pieces for alignment of the spring. After the fiber is secured, the spring is decompressed to maintain tension on the fiber while it is being rotated. This assures that the fiber remains aligned along the axis of rotation. The fiber assembly is placed in the culture vessel and culture medium is added. The culture vessel is then inserted into the rotatable portion of the clinostat and subjected to rotate at selected rpms. The internal diameter of the hollow fiber determines the distance the cells are from the axis of rotation.

  10. Experimental study of bioartificial liver with cultured human liver cells

    Institute of Scientific and Technical Information of China (English)

    1999-01-01

    AIM To establish an extracorporeal bioartificial liver support system (EBLSS) using cultured human liver cells and to study its support effect for fulminant hepatic failure (FHF).METHODS The liver support experiment of EBLSS consisting of aggregates cultured human liver cells, hollow fiber bioreactor, and circulation unit was carried out in dizhepatic dogs.RESULTS The viability of isolated hepatocytes and nonparenchymal liver cells reached 96%. These cells were successfully cultured as multicellular spheroids with synthetic technique. The typical morphological appearance was retained up to the end of the artificial liver experiment. Compared with the control dogs treated with EBLSS without liver cells, the survival time of artificial liver support dogs was significantly prolonged. The changes of blood pressure, heart rate and ECG were slow. Both serum ammonia and lactate levels were significantly lowered at the 3rd h and 5th h. In addition, a good viability of human liver cells was noted after 5 h experiment.CONCLUSION EBLSS playing a metabolic role of cultured human hepatocytes, is capable of compensating the function of the liver, and could provide effective artificial liver support and therapy for patients with FHF.

  11. Adult human brain cell culture for neuroscience research.

    Science.gov (United States)

    Gibbons, Hannah M; Dragunow, Mike

    2010-06-01

    Studies of the brain have progressed enormously through the use of in vivo and in vitro non-human models. However, it is unlikely such studies alone will unravel the complexities of the human brain and so far no neuroprotective treatment developed in animals has worked in humans. In this review we discuss the use of adult human brain cell culture methods in brain research to unravel the biology of the normal and diseased human brain. The advantages of using adult human brain cells as tools to study human brain function from both historical and future perspectives are discussed. In particular, studies using dissociated cultures of adult human microglia, astrocytes, oligodendrocytes and neurons are described and the applications of these types of study are evaluated. Alternative sources of human brain cells such as adult neural stem cells, induced pluripotent stem cells and slice cultures of adult human brain tissue are also reviewed. These adult human brain cell culture methods could benefit basic research and more importantly, facilitate the translation of basic neuroscience research to the clinic for the treatment of brain disorders. Copyright 2009 Elsevier Ltd. All rights reserved.

  12. PKC activation induces inflammatory response and cell death in human bronchial epithelial cells.

    Directory of Open Access Journals (Sweden)

    Hyunhee Kim

    Full Text Available A variety of airborne pathogens can induce inflammatory responses in airway epithelial cells, which is a crucial component of host defence. However, excessive inflammatory responses and chronic inflammation also contribute to different diseases of the respiratory system. We hypothesized that the activation of protein kinase C (PKC is one of the essential mechanisms of inflammatory response in airway epithelial cells. In the present study, we stimulated human bronchial lung epithelial (BEAS-2B cells with the phorbol ester Phorbol 12, 13-dibutyrate (PDBu, and examined gene expression profile using microarrays. Microarray analysis suggests that PKC activation induced dramatic changes in gene expression related to multiple cellular functions. The top two interaction networks generated from these changes were centered on NFκB and TNF-α, which are two commonly known pathways for cell death and inflammation. Subsequent tests confirmed the decrease in cell viability and an increase in the production of various cytokines. Interestingly, each of the increased cytokines was differentially regulated at mRNA and/or protein levels by different sub-classes of PKC isozymes. We conclude that pathological cell death and cytokine production in airway epithelial cells in various situations may be mediated through PKC related signaling pathways. These findings suggest that PKCs can be new targets for treatment of lung diseases.

  13. "Humanized" stem cell culture techniques: the animal serum controversy.

    Science.gov (United States)

    Tekkatte, Chandana; Gunasingh, Gency Ponrose; Cherian, K M; Sankaranarayanan, Kavitha

    2011-01-01

    Cellular therapy is reaching a pinnacle with an understanding of the potential of human mesenchymal stem cells (hMSCs) to regenerate damaged tissue in the body. The limited numbers of these hMSCs in currently identified sources, like bone marrow, adipose tissue, and so forth, bring forth the need for their in vitro culture/expansion. However, the extensive usage of supplements containing xenogeneic components in the expansion-media might pose a risk to the post-transplantation safety of patients. This warrants the necessity to identify and develop chemically defined or "humanized" supplements which would make in vitro cultured/processed cells relatively safer for transplantation in regenerative medicine. In this paper, we outline the various caveats associated with conventionally used supplements of xenogenic origin and also portray the possible alternatives/additives which could one day herald the dawn of a new era in the translation of in vitro cultured cells to therapeutic interventions.

  14. [In vitro cell culture technology in cosmetology research].

    Science.gov (United States)

    Gojniczek, Katarzyna; Garncarczyk, Agnieszka; Pytel, Agata

    2005-01-01

    For ages the humanity has been looking for all kind of active substances, which could be used in improving the health and the appearance of our skin. People try to find out how to protect the skin from harmful, environmental factors. Every year a lot of new natural and synthetic, chemical substances are discovered. All of them potentially could be used as a cosmetic ingredient. In cosmetology research most of new xenobiotics were tested in vivo on animals. Alternative methods to in vivo tests are in vitro tests with skin cell culture system. The aim of this work was to describe two-dimensional and tree-dimensional skin cell cultures. Additionally, in this work we wanted to prove the usefulness of in vitro skin cell cultures in cosmetology research.

  15. Batch variation between branchial cell cultures: An analysis of variance

    DEFF Research Database (Denmark)

    Hansen, Heinz Johs. Max; Grosell, M.; Kristensen, L.

    2003-01-01

    We present in detail how a statistical analysis of variance (ANOVA) is used to sort out the effect of an unexpected batch-to-batch variation between cell cultures. Two separate cultures of rainbow trout branchial cells were grown on permeable filtersupports ("inserts"). They were supposed...... and introducing the observed difference between batches as one of the factors in an expanded three-dimensional ANOVA, we were able to overcome an otherwisecrucial lack of sufficiently reproducible duplicate values. We could thereby show that the effect of changing the apical medium was much more marked when...... the radioactive lipid precursors were added on the apical, rather than on the basolateral, side. Theinsert cell cultures were obviously polarized. We argue that it is not reasonable to reject troublesome experimental results, when we do not know a priori that something went wrong. The ANOVA is a very useful...

  16. A novel feeder-free culture system for human pluripotent stem cell culture and induced pluripotent stem cell derivation.

    Directory of Open Access Journals (Sweden)

    Sanna Vuoristo

    Full Text Available Correct interactions with extracellular matrix are essential to human pluripotent stem cells (hPSC to maintain their pluripotent self-renewal capacity during in vitro culture. hPSCs secrete laminin 511/521, one of the most important functional basement membrane components, and they can be maintained on human laminin 511 and 521 in defined culture conditions. However, large-scale production of purified or recombinant laminin 511 and 521 is difficult and expensive. Here we have tested whether a commonly available human choriocarcinoma cell line, JAR, which produces high quantities of laminins, supports the growth of undifferentiated hPSCs. We were able to maintain several human pluripotent stem cell lines on decellularized matrix produced by JAR cells using a defined culture medium. The JAR matrix also supported targeted differentiation of the cells into neuronal and hepatic directions. Importantly, we were able to derive new human induced pluripotent stem cell (hiPSC lines on JAR matrix and show that adhesion of the early hiPSC colonies to JAR matrix is more efficient than to matrigel. In summary, JAR matrix provides a cost-effective and easy-to-prepare alternative for human pluripotent stem cell culture and differentiation. In addition, this matrix is ideal for the efficient generation of new hiPSC lines.

  17. Biological Effects of Culture Substrates on Human Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Yohei Hayashi

    2016-01-01

    Full Text Available In recent years, as human pluripotent stem cells (hPSCs have been commonly cultured in feeder-free conditions, a number of cell culture substrates have been applied or developed. However, the functional roles of these substrates in maintaining hPSC self-renewal remain unclear. Here in this review, we summarize the types of these substrates and their effect on maintaining hPSC self-renewal. Endogenous extracellular matrix (ECM protein expression has been shown to be crucial in maintaining hPSC self-renewal. These ECM molecules interact with integrin cell-surface receptors and transmit their cellular signaling. We discuss the possible effect of integrin-mediated signaling pathways on maintaining hPSC self-renewal. Activation of integrin-linked kinase (ILK, which transmits ECM-integrin signaling to AKT (also known as protein kinase B, has been shown to be critical in maintaining hPSC self-renewal. Also, since naïve pluripotency has been widely recognized as an alternative pluripotent state of hPSCs, we discuss the possible effects of culture substrates and integrin signaling on naïve hPSCs based on the studies of mouse embryonic stem cells. Understanding the role of culture substrates in hPSC self-renewal and differentiation enables us to control hPSC behavior precisely and to establish scalable or microfabricated culture technologies for regenerative medicine and drug development.

  18. Aragonite precipitation by "proto-polyps" in coral cell cultures.

    Directory of Open Access Journals (Sweden)

    Tali Mass

    Full Text Available The mechanisms of coral calcification at the molecular, cellular and tissue levels are poorly understood. In this study, we examine calcium carbonate precipitation using novel coral tissue cultures that aggregate to form "proto-polyps". Our goal is to establish an experimental system in which calcification is facilitated at the cellular level, while simultaneously allowing in vitro manipulations of the calcifying fluid. This novel coral culturing technique enables us to study the mechanisms of biomineralization and their implications for geochemical proxies. Viable cell cultures of the hermatypic, zooxanthellate coral, Stylophora pistillata, have been maintained for 6 to 8 weeks. Using an enriched seawater medium with aragonite saturation state similar to open ocean surface waters (Ω(arag~4, the primary cell cultures assemble into "proto-polyps" which form an extracellular organic matrix (ECM and precipitate aragonite crystals. These extracellular aragonite crystals, about 10 µm in length, are formed on the external face of the proto-polyps and are identified by their distinctive elongated crystallography and X-ray diffraction pattern. The precipitation of aragonite is independent of photosynthesis by the zooxanthellae, and does not occur in control experiments lacking coral cells or when the coral cells are poisoned with sodium azide. Our results demonstrate that proto-polyps, aggregated from primary coral tissue culture, function (from a biomineralization perspective similarly to whole corals. This approach provides a novel tool for investigating the biophysical mechanism of calcification in these organisms.

  19. Isolated Cells of Porphyra yezoensis Cultured on Solid Medium

    Institute of Scientific and Technical Information of China (English)

    沈颂东; 戴继勋

    2001-01-01

    Vegetative cells of Porphyra yezoensis are isolated with sea snail enzyme and cultured on the solidified agar medium. The results of experiments show that the isolated cells can survive,divide and regenerate well on the medium solidified with agar. The first division on the solid medium starts after 7 days' culture, 4 days later than the liquid culture. The survival rate of isolated cells is 71.3% on the solid medium, lower than the 86.2% of that in seawater.Thalli, thalloids,conchocelis, spermatangia and multicellular masses are developed on the solid/medium in the first month, slowly but normally. Spermatangia sacs disappear within 4 weeks. Without adding nutrient liquid onto the surface of solid medium or injecting seawater under the agar layer in order to keep moisture, the thalli and cell groups release monospores to form new thalli instead of enlarging their areas after 5 weeks' culturing. Some monospores regenerate new thalli. Other monospores lose their pigments and minimize their volume and divide quickly to form light pink calli. After 16 weeks, numerous calli can be seen on the solid medium and after 24 weeks' culturing, almost only calli and conchocelis can be seen. If the calli are immersed in seawater, the monospores are released and may develop into young thallus.

  20. Differential oligonucleotide activity in cell culture versus mouse models.

    Science.gov (United States)

    Wickstrom, E; Tyson, F L

    1997-01-01

    The usual course of drug discovery begins with the demonstration of compound activity in cells and, usually, a lower level of activity in animals. Successive rounds of drug design may result in a compound with sufficient activity in animals to justify clinical trials. The basic endpoints of therapeutic oligonucleotide experiments include target antigen reduction, target messenger reduction and inhibition of transformed cell proliferation or viral replication. However, one should expect oligonucleotides to exhibit pleiotropic behaviour, as do all other drugs. In an animal oligonucleotides will necessarily bind to and dissociate from all macromolecules encountered in the blood, in tissues, on cell surfaces and within cellular compartments. Contrary to expectations, oligonucleotides designed to be complementary to certain transcripts have sometimes been found moderately effective in cell-free extracts, more effective in cell culture and most effective in animal models. If greater potency against standard endpoints is reported in mouse models than was observed in cell culture, critical examination must consider alternate modes of action in animals that may not apply in cell culture. This counterintuitive paradox will be examined, based on studies of Ha-ras expression in bladder cancer, Ki-ras expression in pancreatic cancer, erbB2 expression in ovarian cancer and c-myc expression in B cell lymphoma.

  1. Crude subcellular fractionation of cultured mammalian cell lines

    Directory of Open Access Journals (Sweden)

    Holden Paul

    2009-12-01

    Full Text Available Abstract Background The expression and study of recombinant proteins in mammalian culture systems can be complicated during the cell lysis procedure by contaminating proteins from cellular compartments distinct from those within which the protein of interest resides and also by solubility issues that may arise from the use of a single lysis buffer. Partial subcellular fractionation using buffers of increasing stringency, rather than whole cell lysis is one way in which to avoid or reduce this contamination and ensure complete recovery of the target protein. Currently published protocols involve time consuming centrifugation steps which may require expensive equipment and commercially available kits can be prohibitively expensive when handling large or multiple samples. Findings We have established a protocol to sequentially extract proteins from cultured mammalian cells in fractions enriched for cytosolic, membrane bound organellar, nuclear and insoluble proteins. All of the buffers used can be made inexpensively and easily and the protocol requires no costly equipment. While the method was optimized for a specific cell type, we demonstrate that the protocol can be applied to a variety of commonly used cell lines and anticipate that it can be applied to any cell line via simple optimization of the primary extraction step. Conclusion We describe a protocol for the crude subcellular fractionation of cultured mammalian cells that is both straightforward and cost effective and may facilitate the more accurate study of recombinant proteins and the generation of purer preparations of said proteins from cell extracts.

  2. Lingual Epithelial Stem Cells and Organoid Culture of Them.

    Science.gov (United States)

    Hisha, Hiroko; Tanaka, Toshihiro; Ueno, Hiroo

    2016-01-28

    As tongue cancer is one of the major malignant cancers in the world, understanding the mechanism of maintenance of lingual epithelial tissue, which is known to be the origin of tongue cancer, is unquestionably important. However, the actual stem cells that are responsible for the long-term maintenance of the lingual epithelium have not been identified. Moreover, a simple and convenient culture method for lingual epithelial stem cells has not yet been established. Recently, we have shown that Bmi1-positive cells, residing at the second or third layer of the epithelial cell layer at the base of the interpapillary pit (IPP), were slow-cycling and could supply keratinized epithelial cells for over one year, indicating that Bmi1-positive cells are long-term lingual epithelial stem cells. In addition, we have developed a novel lingual epithelium organoid culture system using a three-dimensional matrix and growth factors. Here, we discuss current progress in the identification of lingual stem cells and future applications of the lingual culture system for studying the regulatory mechanisms of the lingual epithelium and for regenerative medicine.

  3. Lingual Epithelial Stem Cells and Organoid Culture of Them

    Directory of Open Access Journals (Sweden)

    Hiroko Hisha

    2016-01-01

    Full Text Available As tongue cancer is one of the major malignant cancers in the world, understanding the mechanism of maintenance of lingual epithelial tissue, which is known to be the origin of tongue cancer, is unquestionably important. However, the actual stem cells that are responsible for the long-term maintenance of the lingual epithelium have not been identified. Moreover, a simple and convenient culture method for lingual epithelial stem cells has not yet been established. Recently, we have shown that Bmi1-positive cells, residing at the second or third layer of the epithelial cell layer at the base of the interpapillary pit (IPP, were slow-cycling and could supply keratinized epithelial cells for over one year, indicating that Bmi1-positive cells are long-term lingual epithelial stem cells. In addition, we have developed a novel lingual epithelium organoid culture system using a three-dimensional matrix and growth factors. Here, we discuss current progress in the identification of lingual stem cells and future applications of the lingual culture system for studying the regulatory mechanisms of the lingual epithelium and for regenerative medicine.

  4. A Simple Hydrophilic Treatment of SU-8 Surfaces for Cell Culturing and Cell Patterning

    DEFF Research Database (Denmark)

    Wang, Zhenyu; Stangegaard, Michael; Dufva, Hans Martin

    2005-01-01

    SU-8, an epoxy-based photoresist, widely used in constitution different mTAS systems, is incompatible with mammalian cell adhesion and culture in its native form. Here, we demonstrate a simple, cheap and robust two-step method to render a SU-8 surface hydrophilic and compatible with cell culture...

  5. Effects of rotational culture on morphology, nitric oxide production and cell cycle of endothelial cells.

    Science.gov (United States)

    Tang, Chaojun; Wu, Xue; Ye, Linqi; Xie, Xiang; Wang, Guixue

    2012-12-01

    Devices for the rotational culture of cells and the study of biological reactions have been widely applied in tissue engineering. However, there are few reports exploring the effects of rotational culture on cell morphology, nitric oxide (NO) production, and cell cycle of the endothelial cells from human umbilical vein on the stent surface. This study focuses on these parameters after the cells are seeded on the stents. Results showed that covering of stents by endothelial cells was improved by rotational culture. NO production decreased within 24 h in both rotational and static culture groups. In addition, rotational culture significantly increased NO production by 37.9% at 36 h and 28.9% at 48 h compared with static culture. Flow cytometry showed that the cell cycle was not obviously influenced by rotational culture. Results indicate that rotational culture may be helpful for preparation of cell-seeded vascular grafts and intravascular stents, which are expected to be the most frequently implanted materials in the future.

  6. A Simple Hydrophilic Treatment of SU-8 Surfaces for Cell Culturing and Cell Patterning

    DEFF Research Database (Denmark)

    Wang, Zhenyu; Stangegaard, Michael; Dufva, Hans Martin;

    2005-01-01

    SU-8, an epoxy-based photoresist, widely used in constitution different mTAS systems, is incompatible with mammalian cell adhesion and culture in its native form. Here, we demonstrate a simple, cheap and robust two-step method to render a SU-8 surface hydrophilic and compatible with cell culture...

  7. Distinguishing between linear and exponential cell growth during the division cycle: Single-cell studies, cell-culture studies, and the object of cell-cycle research

    OpenAIRE

    Cooper Stephen

    2006-01-01

    Abstract Background Two approaches to understanding growth during the cell cycle are single-cell studies, where growth during the cell cycle of a single cell is measured, and cell-culture studies, where growth during the cell cycle of a large number of cells as an aggregate is analyzed. Mitchison has proposed that single-cell studies, because they show variations in cell growth patterns, are more suitable for understanding cell growth during the cell cycle, and should be preferred over cultur...

  8. Cell sources for in vitro human liver cell culture models.

    Science.gov (United States)

    Zeilinger, Katrin; Freyer, Nora; Damm, Georg; Seehofer, Daniel; Knöspel, Fanny

    2016-09-01

    In vitro liver cell culture models are gaining increasing importance in pharmacological and toxicological research. The source of cells used is critical for the relevance and the predictive value of such models. Primary human hepatocytes (PHH) are currently considered to be the gold standard for hepatic in vitro culture models, since they directly reflect the specific metabolism and functionality of the human liver; however, the scarcity and difficult logistics of PHH have driven researchers to explore alternative cell sources, including liver cell lines and pluripotent stem cells. Liver cell lines generated from hepatomas or by genetic manipulation are widely used due to their good availability, but they are generally altered in certain metabolic functions. For the past few years, adult and pluripotent stem cells have been attracting increasing attention, due their ability to proliferate and to differentiate into hepatocyte-like cells in vitro However, controlling the differentiation of these cells is still a challenge. This review gives an overview of the major human cell sources under investigation for in vitro liver cell culture models, including primary human liver cells, liver cell lines, and stem cells. The promises and challenges of different cell types are discussed with a focus on the complex 2D and 3D culture approaches under investigation for improving liver cell functionality in vitro Finally, the specific application options of individual cell sources in pharmacological research or disease modeling are described.

  9. Polyamines in relation to growth in carrot cell cultures.

    Science.gov (United States)

    Fallon, K M; Phillips, R

    1988-09-01

    Changes in polyamine metabolism were investigated in relation to growth of cell suspension cultures of carrot (Daucus carota, cv Chantenay). Changes in levels of the major amines putrescine and spermidine throughout the culture period correlated poorly with changes in fresh weight, but a closer correlation with the minor component spermine was observed. The arginine decarboxylase (ADC) inhibitor difluoromethylarginine (DFMA) strongly and specifically inhibited ADC activity in the supernatant, reduced the major amine (putrescine) by 95% and the total amine content by 80%. It had no effect on cell number and stimulated fresh weight by over 25% through increased cell expansion. Spermine content, in contrast, increased with DFMA concentration in parallel with fresh weight increases. Difluoromethylornithine strongly inhibited ornithine decarboxylase activity in the pellet, but had little effect on either polyamine levels or culture growth. It was concluded that little evidence for a correlation between free polyamines and cell number in carrot cultures could be detected, but that a possible correlation between spermine content and cell expansion was observed.

  10. Polyamines in Relation to Growth in Carrot Cell Cultures 1

    Science.gov (United States)

    Fallon, Kevin M.; Phillips, Richard

    1988-01-01

    Changes in polyamine metabolism were investigated in relation to growth of cell suspension cultures of carrot (Daucus carota, cv Chantenay). Changes in levels of the major amines putrescine and spermidine throughout the culture period correlated poorly with changes in fresh weight, but a closer correlation with the minor component spermine was observed. The arginine decarboxylase (ADC) inhibitor difluoromethylarginine (DFMA) strongly and specifically inhibited ADC activity in the supernatant, reduced the major amine (putrescine) by 95% and the total amine content by 80%. It had no effect on cell number and stimulated fresh weight by over 25% through increased cell expansion. Spermine content, in contrast, increased with DFMA concentration in parallel with fresh weight increases. Difluoromethylornithine strongly inhibited ornithine decarboxylase activity in the pellet, but had little effect on either polyamine levels or culture growth. It was concluded that little evidence for a correlation between free polyamines and cell number in carrot cultures could be detected, but that a possible correlation between spermine content and cell expansion was observed. PMID:16666271

  11. A self-feeding roller bottle for continuous cell culture.

    Science.gov (United States)

    Berson, R Eric; Friederichs, Goetz

    2008-01-01

    The concept of a self-feeding roller bottle that delivers a continuous supply of fresh media to cells in culture, which is mechanically simplistic and works with existing roller apparatuses, is presented here. A conventional roller bottle is partitioned into two chambers; one chamber contains the fresh culture media reservoir, and the other contains the cell culture chamber. A spiroid of tubing inside the fresh media reservoir acts as a pump when the bottle rotates on its horizontal axis, continuously delivering fresh media through an opening in the partition to the cell culture chamber. The modified bottle proved capable of maintaining steady-state cell densities of a hybridoma cell line over the 10-day period tested, although at lower densities than reached during batch operation due to the continuous volume dilution. Steady-state density proved to be controllable by adjusting the perfusion rate, which changes with the rotation rate of the bottle. Specific antibody production rate is as much as 3.7 times the rate in conventional roller bottles operating with intermittent batch feeding.

  12. Cell culture systems for the hepatitis C virus

    Institute of Scientific and Technical Information of China (English)

    Gilles Duverlie; Czeslaw Wychowski

    2007-01-01

    Since the discovery of HCV in 1989, the lack of a cell culture system has hampered research progress on this important human pathogen. No robust system has been obtained by empiric approaches, and HCV cell culture remained hypothetical until 2005. The construction of functional molecular clones has served as a starting point to reconstitute a consensus infectious cDNA that was able to transcribe infectious HCV RNAs as shown by intrahepatic inoculation in a chimpanzee. Other consensus clones have been selected and established in a human hepatoma cell line as replicons, i.e. self-replicating subgenomic or genomic viral RNAs. However, these replicons did not support production of infectious virus. Interestingly, some full-length replicons could be established without adaptive mutations and one of them was able to replicate at very high levels and to release virus particles that are infectious in cell culture and in vivo. This new cell culture system represents a major breakthrough in the HCV field and should enable a broad range of basic and applied studies to be achieved.

  13. A single-cell and feeder-free culture system for monkey embryonic stem cells.

    Science.gov (United States)

    Ono, Takashi; Suzuki, Yutaka; Kato, Yosuke; Fujita, Risako; Araki, Toshihiro; Yamashita, Tomoko; Kato, Hidemasa; Torii, Ryuzo; Sato, Naoya

    2014-01-01

    Primate pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), hold great potential for research and application in regenerative medicine and drug discovery. To maximize primate PSC potential, a practical system is required for generating desired functional cells and reproducible differentiation techniques. Much progress regarding their culture systems has been reported to date; however, better methods would still be required for their practical use, particularly in industrial and clinical fields. Here we report a new single-cell and feeder-free culture system for primate PSCs, the key feature of which is an originally formulated serum-free medium containing FGF and activin. In this culture system, cynomolgus monkey ESCs can be passaged many times by single-cell dissociation with traditional trypsin treatment and can be propagated with a high proliferation rate as a monolayer without any feeder cells; further, typical PSC properties and genomic stability can be retained. In addition, it has been demonstrated that monkey ESCs maintained in the culture system can be used for various experiments such as in vitro differentiation and gene manipulation. Thus, compared with the conventional culture system, monkey ESCs grown in the aforementioned culture system can serve as a cell source with the following practical advantages: simple, stable, and easy cell maintenance; gene manipulation; cryopreservation; and desired differentiation. We propose that this culture system can serve as a reliable platform to prepare primate PSCs useful for future research and application.

  14. Identification of a long non-coding RNA gene, growth hormone secretagogue receptor opposite strand, which stimulates cell migration in non-small cell lung cancer cell lines.

    Science.gov (United States)

    Whiteside, Eliza J; Seim, Inge; Pauli, Jana P; O'Keeffe, Angela J; Thomas, Patrick B; Carter, Shea L; Walpole, Carina M; Fung, Jenny N T; Josh, Peter; Herington, Adrian C; Chopin, Lisa K

    2013-08-01

    The molecular mechanisms involved in non‑small cell lung cancer tumourigenesis are largely unknown; however, recent studies have suggested that long non-coding RNAs (lncRNAs) are likely to play a role. In this study, we used public databases to identify an mRNA-like, candidate long non-coding RNA, GHSROS (GHSR opposite strand), transcribed from the antisense strand of the ghrelin receptor gene, growth hormone secretagogue receptor (GHSR). Quantitative real-time RT-PCR revealed higher expression of GHSROS in lung cancer tissue compared to adjacent, non-tumour lung tissue. In common with many long non-coding RNAs, GHSROS is 5' capped and 3' polyadenylated (mRNA-like), lacks an extensive open reading frame and harbours a transposable element. Engineered overexpression of GHSROS stimulated cell migration in the A549 and NCI-H1299 non-small cell lung cancer cell lines, but suppressed cell migration in the Beas-2B normal lung-derived bronchoepithelial cell line. This suggests that GHSROS function may be dependent on the oncogenic context. The identification of GHSROS, which is expressed in lung cancer and stimulates cell migration in lung cancer cell lines, contributes to the growing number of non-coding RNAs that play a role in the regulation of tumourigenesis and metastatic cancer progression.

  15. Continuous perfusion microfluidic cell culture array for high-throughput cell-based assays.

    Science.gov (United States)

    Hung, Paul J; Lee, Philip J; Sabounchi, Poorya; Lin, Robert; Lee, Luke P

    2005-01-05

    We present for the first time a microfluidic cell culture array for long-term cellular monitoring. The 10 x 10 array could potentially assay 100 different cell-based experiments in parallel. The device was designed to integrate the processes used in typical cell culture experiments on a single self-contained microfluidic system. Major functions include repeated cell growth/passage cycles, reagent introduction, and real-time optical analysis. The single unit of the array consists of a circular microfluidic chamber, multiple narrow perfusion channels surrounding the main chamber, and four ports for fluidic access. Human carcinoma (HeLa) cells were cultured inside the device with continuous perfusion of medium at 37 degrees C. The observed doubling time was 1.4 +/- 0.1 days with a peak cell density of approximately 2.5*10(5) cells/cm(2). Cell assay was demonstrated by monitoring the fluorescence localization of calcein AM from 1 min to 10 days after reagent introduction. Confluent cell cultures were passaged within the microfluidic chambers using trypsin and successfully regrown, suggesting a stable culture environment suitable for continuous operation. The cell culture array could offer a platform for a wide range of assays with applications in drug screening, bioinformatics, and quantitative cell biology. (c) 2004 Wiley Periodicals, Inc.

  16. Acquired resistance to auranofin in cultured human cells.

    Science.gov (United States)

    Glennås, A; Rugstad, H E

    1985-01-01

    A substrain (HEAF) of cultured human epithelial cells, grown as monolayers, was selected for resistance to auranofin (AF), a gold-containing anti-arthritic drug, by growing the parental HE cells with stepwise increased concentrations of AF in the medium. HEAF cells acquired resistance to 2 mumol AF/l, twice the concentration tolerated by the sensitive HE cells. Resistance to AF was also demonstrated in another substrain (HE100) originally selected for by its cadmium resistance, and characterized by a high cytosolic metallothionein (MT) content. Following continuous exposure to 2 mumol AF/l for 4 days, 58% of the HEAF cells, 67% of the HE100 cells, and 16% of the HE cells remained adherent to the flasks, compared with non-treated controls. Following 24 h AF exposure to living cells, HEAF cells had one-half and HE100 cells twice the cellular and cytosolic gold concentration per mg protein, as compared with HE cells. Gel filtration of cell cytosols revealed gold-binding proteins with a mol. wt. of about 10 000 apparently occurring on AF exposure in HEAF and HE cells. They bound 10-15% of cytosolic gold. MT in HE100 cells bound AF-gold to about the same extent. We suggest that the ability of cells to maintain the gold concentration at a low level (HEAF) and trapping of gold by MT (HE100) or low molecular weight proteins occurring on AF treatment (HEAF) may be mechanisms contributing to the observed cellular resistance to AF.

  17. Allotransplantation of cultured parathyroid progenitor cells without immunosuppression: clinical results.

    Science.gov (United States)

    Nawrot, Ireneusz; Woźniewicz, Bogdan; Tołłoczko, Tadeusz; Sawicki, Andrzej; Górski, Andrzej; Chudziński, Witold; Wojtaszek, Mikołaj; Grzesiuk, Wiesław; Sladowski, Dariusz; Karwacki, Jerzy; Zawitkowska, Teresa; Szmidt, Jacek

    2007-03-27

    Hypoparathyroidism is a well-known consequence of extensive thyroid and parathyroid surgery. Allotransplantation of cultured parathyroid cells can be considered as an alternative to vitamin D3 and calcium supplementation in treatment of hypoparathyroidism. We present the long-term allotransplant activity in 85 patients who had undergone cellular allotransplantation for surgical hypoparathyroidism. Also, a modified technique to prepare parathyroid explants is described for obtaining a new nonimmunogenic cell population. From March 1990 to December 2004, 85 patients underwent 116 allotransplantations of cultured parathyroid cells. Mean recipient age was 46.2+/-11.1 years. Donors were selected from patients undergoing parathyroidectomy for secondary and tertiary hyperparathyroidism. After 6 weeks of cultivation and freezing, the parathyroid cells decreased their normal human leukocyte antigen (HLA) class I ABC expression and were free of HLA class II positive cells. The viability of cultured cells was 95.15+/-2.94%. Eighty-five patients underwent primary allotransplantation. Of these, 25 patients subsequently underwent a repeat procedure. In six cases, the parathyroid cells were obtained from the same donor and in 19 cases from a different donor. For all patients, the mean cellular allograft survival was 6.35+/-13.08 months. In 64 patients (55.1%), the allografts retained their endocrine function for more than 2 months. The present study has shown that in some patients parathyroid cell allotransplantation may be considered a method of treatment for permanent hypoparathyroidism after thyroid surgery. Graft function and/or survival did not depend on the baseline viability or secretory activity of cultured cells used for transplantation.

  18. Testing of serum atherogenicity in cell cultures: questionable data published

    Directory of Open Access Journals (Sweden)

    Sergei V. Jargin

    2012-01-01

    Full Text Available In a large series of studies was reported that culturing of smooth muscle cells with serum from atherosclerosis patients caused intracellular lipid accumulation, while serum from healthy controls had no such effect. Cultures were used for evaluation of antiatherogenic drugs. Numerous substances were reported to lower serum atherogenicity: statins, trapidil, calcium antagonists, garlic derivatives etc. On the contrary, beta-blockers, phenothiazines and oral hypoglycemics were reported to be pro-atherogenic. Known antiatherogenic agents can influence lipid metabolism and cholesterol synthesis, intestinal absorption or endothelium-related mechanisms. All these targets are absent in cell monocultures. Inflammatory factors, addressed by some antiatherogenic drugs, are also not reproduced. In vivo, relationship between cholesterol uptake by cells and atherogenesis must be inverse rather than direct: in familial hypercholesterolemia, inefficient clearance of LDL-cholesterol by cells predisposes to atherosclerosis. Accordingly, if a pharmacological agent reduces cholesterol uptake by cells in vitro, it should be expected to elevate cholesterol in vivo. Validity of clinical recommendations, based on serum atherogenicity testing in cell monocultures, is therefore questionable. These considerations pertain also to the drugs developed on the basis of the cell culture experiments.

  19. Cell culture media impact on drug product solution stability.

    Science.gov (United States)

    Purdie, Jennifer L; Kowle, Ronald L; Langland, Amie L; Patel, Chetan N; Ouyang, Anli; Olson, Donald J

    2016-07-08

    To enable subcutaneous administration of monoclonal antibodies, drug product solutions are often needed at high concentrations. A significant risk associated with high drug product concentrations is an increase in aggregate level over the shelf-life dating period. While much work has been done to understand the impact of drug product formulation on aggregation, there is limited understanding of the link between cell culture process conditions and soluble aggregate growth in drug product. During cell culture process development, soluble aggregates are often measured at harvest using cell-free material purified by Protein A chromatography. In the work reported here, cell culture media components were evaluated with respect to their impact on aggregate levels in high concentration solution drug product during accelerated stability studies. Two components, cysteine and ferric ammonium citrate, were found to impact aggregate growth rates in our current media (version 1) leading to the development of new chemically defined media and concentrated feed formulations. The new version of media and associated concentrated feeds (version 2) were evaluated across four cell lines producing recombinant IgG4 monoclonal antibodies and a bispecific antibody. In all four cell lines, the version 2 media reduced aggregate growth over the course of a 12 week accelerated stability study compared with the version 1 media, although the degree to which aggregate growth decreased was cell line dependent. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:998-1008, 2016.

  20. Testicular Sertoli cells influence the proliferation and immunogenicity of co-cultured endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Fan, Ping, E-mail: fanpinggoodluck@163.com [Department of Rheumatism and Immunity, The First Affiliated Hospital Xi' an Jiaotong University School of Medicine, Xi' an, Shaanxi 710061 (China); He, Lan; Pu, Dan; Lv, Xiaohong; Zhou, Wenxu; Sun, Yining; Hu, Nan [Department of Rheumatism and Immunity, The First Affiliated Hospital Xi' an Jiaotong University School of Medicine, Xi' an, Shaanxi 710061 (China)

    2011-01-21

    Research highlights: {yields} The proliferation of dramatic increased by co-cultured with Sertoli cells. {yields} VEGF receptor-2 expression of ECs was up-regulated by co-cultured with Sertoli cells. {yields} The MHC expression of ECs induced by INF-{gamma} and IL-6, IL-8 and sICAM induced by TNF-{alpha} decreased respectively after co-cultured with Sertoli cells. {yields} ECs co-cultured with Sertoli cells also didn't increase the stimulation index of spleen lymphocytes. -- Abstract: The major problem of the application of endothelial cells (ECs) in transplantation is the lack of proliferation and their immunogenicity. In this study, we co-cultured ECs with Sertoli cells to monitor whether Sertoli cells can influence the proliferation and immunogenicity of co-cultured ECs. Sertoli cells were isolated from adult testicular tissue. ECs were divided into the control group and the experimental group, which included three sub-groups co-cultured with 1 x 10{sup 3}, 1 x 10{sup 4} or 1 x 10{sup 5} cell/ml of Sertoli cells. The growth and proliferation of ECs were observed microscopically, and the expression of vascular endothelial growth factor (VEGF) receptor-2 (KDR) was examined by Western blotting. In another experiment, ECs were divided into the control group, the single culture group and the co-culture group with the optimal concentration of Sertoli cells. After INF-{gamma} and TNF-{alpha} were added to the culture medium, MHC II antigen expression was detected by immunofluorescence staining and western blotting; interleukin (IL)-6, IL-8 and soluble intercellular adhesion molecule (sICAM) were measured in the culture medium by ELISA. We demonstrated that 1 x 10{sup 4} cell/ml Sertoli cells promoted the proliferation of co-cultured ECs more dramatically than that in other groups (P < 0.05). Western blotting showed that 1 x 10{sup 4} cell/ml of the Sertoli cells was most effective in the up-regulation of KDR expression in the co-cultured ECs (P < 0.05). Sertoli

  1. Pathogenic Trichomonas vaginalis cytotoxicity to cell culture monolayers.

    Science.gov (United States)

    Alderete, J F; Pearlman, E

    1984-04-01

    Exposure of monolayer cultures of human urogenital and vaginal (HeLa), human epithelial (HEp-2), normal baboon testicular (NBT), and monkey kidney (Vero) cells to live pathogenic Trichomonas vaginalis resulted in extensive disruption of monolayers. Trypan blue was taken up by all host cells released from cell monolayers, which indicated irreversible damage of these cell types by trichomonads. Time and dose related data on cytotoxicity kinetics were obtained using increasing ratios of parasites to cells. All cell types were most sensitive to trichomonads at a multiplicity of infection of one. Release of tritiated thymidine (3H-thymidine) of the deoxyribonucleic acid (DNA) of prelabelled host cells after incubation with T vaginalis corroborated that extensive cytotoxicity was caused by pathogenic trichomonads in man. Only living parasites were cytotoxic, and no trichomonal toxic products were implicated in disruption of the cell monolayer cultures. A pathogenic bovine trichomonad, Tritrichomonas foetus KV-1, produced half as much cell damage as did T vaginalis. Trichomonas tenax, a non-pathogenic member of the normal flora of the oral cavity in man, produced no measurable cytotoxicity to HeLa cells when compared with the pathogenic human trichomonads.

  2. Effects of Visible Light on Cultured Bovine Trabecular Cells

    Institute of Scientific and Technical Information of China (English)

    姜发纲; 郝风芹; 魏厚仁; 许德胜

    2004-01-01

    To explore the biological effects of light on trabecular cells, cultured bovine trabecular cells were exposed to visible light of different wavelength with different energy. Cellular morphology, structure, proliferation, and phagocytosis were observed. The cells showed no remarkable changes when the energy was low. When the exposure energy reached 1. 12 mW/cm2 , the cytoplasm showed a rough appearance, and cell proliferation and phagocytosis decreased. This phototoxicity was strong with white light (compound chromatic light), moderate with violet light or yellow light, and mild with red light.

  3. Reactivity of alveolar epithelial cells in primary culture with type I cell monoclonal antibodies.

    Science.gov (United States)

    Danto, S I; Zabski, S M; Crandall, E D

    1992-03-01

    An understanding of the process of alveolar epithelial cell growth and differentiation requires the ability to trace and analyze the phenotypic transitions that the cells undergo. This analysis demands specific phenotypic probes to type II and, especially, type I pneumocytes. To this end, monoclonal antibodies have been generated to type I alveolar epithelial cells using an approach designed to enhance production of lung-specific clones from a crude lung membrane preparation. The monoclonal antibodies were screened by a combination of enzyme-linked immunosorbent assay and immunohistochemical techniques, with the determination of type I cell specificity resting primarily on immunoelectron microscopic localization. Two of these new markers of the type I pneumocyte phenotype (II F1 and VIII B2) were used to analyze primary cultures of type II cells growing on standard tissue culture plastic and on a variety of substrata reported to affect the morphology of these cells in culture. On tissue culture plastic, the antibodies fail to react with early (days 1 to 3) type II cell cultures. The cells become progressively more reactive with time in culture to a plateau of approximately 6 times background by day 8, with a maximum rate of increase between days 3 and 5. This finding is consistent with the hypothesis that type II cells in primary culture undergo at least partial differentiation into type I cells. Type II cells grown on laminin, which reportedly delays the loss of type II cell appearance, and on fibronectin, which has been reported to facilitate cell spreading and loss of type II cell features, develop the type I cell markers during cultivation in vitro with kinetics similar to those on uncoated tissue culture plastic. Cells on type I collagen and on tissue culture-treated Nuclepore filters, which have been reported to support monolayers with type I cell-like morphology, also increase their expression of the II F1 and VIII B2 epitopes around days 3 to 5. Taken

  4. Xeno-free culture of human pluripotent stem cells.

    Science.gov (United States)

    Bergström, Rosita; Ström, Susanne; Holm, Frida; Feki, Anis; Hovatta, Outi

    2011-01-01

    Stem cell culture systems that rely on undefined animal-derived components introduce variability to the cultures and complicate their therapeutic use. The derivation of human embryonic stem cells and the development of methods to produce induced pluripotent stem cells combined with their potential to treat human diseases have accelerated the drive to develop xenogenic-free, chemically defined culture systems that support pluripotent self-renewal and directed differentiation. In this chapter, we describe four xeno-free culture systems that have been successful in supporting undifferentiated growth of hPSCs as well as methods for xeno-free subculture and cryopreservation of hPSCs. Each culture system consists of a xeno-free growth medium and xeno-free substratum: (1) TeSR2™ with human recombinant laminin (LN-511); (2) NutriStem™ with LN-511; (3) RegES™ with human foreskin fibroblasts (hFFs); (4) KO-SR Xeno-Free™/GF cocktail with CELLstart™ matrix.

  5. Cultured meat from stem cells: challenges and prospects.

    Science.gov (United States)

    Post, Mark J

    2012-11-01

    As one of the alternatives for livestock meat production, in vitro culturing of meat is currently studied. The generation of bio-artificial muscles from satellite cells has been ongoing for about 15 years, but has never been used for generation of meat, while it already is a great source of animal protein. In order to serve as a credible alternative to livestock meat, lab or factory grown meat should be efficiently produced and should mimic meat in all of its physical sensations, such as visual appearance, smell, texture and of course, taste. This is a formidable challenge even though all the technologies to create skeletal muscle and fat tissue have been developed and tested. The efficient culture of meat will primarily depend on culture conditions such as the source of medium and its composition. Protein synthesis by cultured skeletal muscle cells should further be maximized by finding the optimal combination of biochemical and physical conditions for the cells. Many of these variables are known, but their interactions are numerous and need to be mapped. This involves a systematic, if not systems, approach. Given the urgency of the problems that the meat industry is facing, this endeavor is worth undertaking. As an additional benefit, culturing meat may provide opportunities for production of novel and healthier products. Copyright © 2012 Elsevier Ltd. All rights reserved.

  6. On-line characterization of a hybridoma cell culture process.

    Science.gov (United States)

    Zhou, W; Hu, W S

    1994-06-20

    The on-line determination of the physiological state of a cell culture process requires reliable on-line measurements of various parameters and calculations of specific rates from these measurements. The cell concentration of a hybridoma culture was estimated on-line by measuring optical density (OD) with a laser turbidity probe. The oxygen uptake rate (OUR) was determined by monitoring dynamically dissolved oxygen concentration profiles and closing oxygen balances in the culture. The base addition for neutralizing lactate produced by cells was also monitored on-line via a balance. Using OD and OUR measurements, the specific growth and specific oxygen consumption rates were determined on-line. By combining predetermined stoichiometric relationships among oxygen and glucose consumption and lactate production, the specific glucose consumption and lactate production rates were also calculated on-line. Using these on-line measurements and calculations, the hybridoma culture process was characterized on-line by identifying the physiological states. They will also facilitate the implementation of nutrient feeding strategies for fed-batch and perfusion cultures. (c) 1994 John Wiley & Sons, Inc.

  7. Cell culture plastics with immobilized interleukin-4 for monocyte differentiation

    DEFF Research Database (Denmark)

    Hansen, Morten; Hjortø, Gertrud Malene; Met, Özcan;

    2011-01-01

    Standard cell culture plastic was surface modified by passive adsorption or covalent attachment of interleukin (IL)-4 and investigated for its ability to induce differentiation of human monocytes into mature dendritic cells, a process dose-dependently regulated by IL-4. Covalent attachment of IL-4...... proceeded via anthraquinone photochemistry to introduce amine functionalities at the surface followed by coupling of IL-4 through a bifunctional amine-reactive linker. X-ray photoelectron spectroscopy showed that undesirable multilayer formation of the photoactive compound could be avoided by reaction...... in water instead of phosphate-buffered saline. Passively adsorbed IL-4 was observed to induce differentiation to dendritic cells, but analysis of cell culture supernatants revealed that leakage of IL-4 into solution could account for the differentiation observed. Covalent attachment resulted in bound IL-4...

  8. Measurement and analysis of calcium signaling in heterogeneous cell cultures.

    Science.gov (United States)

    Richards, Gillian R; Jack, Andrew D; Platts, Amy; Simpson, Peter B

    2006-01-01

    High-content imaging platforms capable of studying kinetic responses at a single-cell level have elevated kinetic recording techniques from labor-intensive low-throughput experiments to potential high-throughput screening assays. We have applied this technology to the investigation of heterogeneous cell cultures derived from primary neural tissue. The neuronal cultures mature into a coupled network and display spontaneous oscillations in intracellular calcium, which can be modified by the addition of pharmacological agents. We have developed algorithms to perform Fourier analysis and quantify both the degree of synchronization and the effects of modulators on the oscillations. Functional and phenotypic experiments can be combined using this approach. We have used post-hoc immunolabeling to identify subpopulations of cells in cocultures and to dissect the calcium responses of these cells from the population response. The combination of these techniques represents a powerful tool for drug discovery.

  9. Chikungunya virus isolation using simplified cell culture technique in Mauritius.

    Science.gov (United States)

    Pyndiah, M N; Pursem, V; Meetoo, G; Daby, S; Ramuth, V; Bhinkah, P; Chuttoo, R; Paratian, U

    2012-03-01

    During the chikungunya outbreak of 2005 - 2006, the only laboratory facilities available in Mauritius were virus isolation in cell culture tubes and serology. The laboratory was submerged with large numbers of blood samples. Comparative isolation was made in human embryonic lung (HEL) and VERO cells grown in 96-well plate. Culture on HEL cells was found to be more sensitive and presence of cytopathic effect (CPE) was observed earlier than in VERO cells. Out of the 18 300 blood samples inoculated on HEL, 11 165 were positive. This virus isolation method was of great help for the surveillance and control of the vectors. In cases of an outbreak a cheap, rapid and simple method of isolating chikungunya virus is described.

  10. Microwell engineering characterization for mammalian cell culture process development.

    Science.gov (United States)

    Barrett, Timothy A; Wu, Andrew; Zhang, Hu; Levy, M Susana; Lye, Gary J

    2010-02-01

    Experimentation in shaken microplate formats offers a potential platform technology for the rapid evaluation and optimization of cell culture conditions. Provided that cell growth and antibody production kinetics are comparable to those found in currently used shake flask systems then the microwell approach offers the possibility to obtain early process design data more cost effectively and with reduced material requirements. This work describes a detailed engineering characterization of liquid mixing and gas-liquid mass transfer in microwell systems and their impact on suspension cell cultures. For growth of murine hybridoma cells producing IgG1, 24-well plates have been characterized in terms of energy dissipation (P/V) (via Computational Fluid Dynamics, CFD), fluid flow, mixing and oxygen transfer rate as a function of shaking frequency and liquid fill volume. Predicted k(L)a values varied between 1.3 and 29 h(-1); liquid-phase mixing time, quantified using iodine decolorization experiments, varied from 1.7 s to 3.5 h; while the predicted P/V ranged from 5 to 35 W m(-3). CFD simulations of the shear rate predicted hydrodynamic forces will not be detrimental to cells. For hybridoma cultures however, high shaking speeds (>250 rpm) were shown to have a negative impact on cell growth, while a combination of low shaking speed and high well fill volume (120 rpm, 2,000 microL) resulted in oxygen limited conditions. Based on these findings a first engineering comparison of cell culture kinetics in microwell and shake flask formats was made at matched average energy dissipation rates. Cell growth kinetics and antibody titer were found to be similar in 24-well microtiter plates and 250 mL shake flasks. Overall this work has demonstrated that cell culture performed in shaken microwell plates can provide data that is both reproducible and comparable to currently used shake flask systems while offering at least a 30-fold decrease in scale of operation and material

  11. Culture of Oral Mucosal Epithelial Cells for the Purpose of Treating Limbal Stem Cell Deficiency

    Directory of Open Access Journals (Sweden)

    Tor Paaske Utheim

    2016-03-01

    Full Text Available The cornea is critical for normal vision as it allows allowing light transmission to the retina. The corneal epithelium is renewed by limbal epithelial cells (LEC, which are located in the periphery of the cornea, the limbus. Damage or disease involving LEC may lead to various clinical presentations of limbal stem cell deficiency (LSCD. Both severe pain and blindness may result. Transplantation of cultured autologous oral mucosal epithelial cell sheet (CAOMECS represents the first use of a cultured non-limbal autologous cell type to treat this disease. Among non-limbal cell types, CAOMECS and conjunctival epithelial cells are the only laboratory cultured cell sources that have been explored in humans. Thus far, the expression of p63 is the only predictor of clinical outcome following transplantation to correct LSCD. The optimal culture method and substrate for CAOMECS is not established. The present review focuses on cell culture methods, with particular emphasis on substrates. Most culture protocols for CAOMECS used amniotic membrane as a substrate and included the xenogeneic components fetal bovine serum and murine 3T3 fibroblasts. However, it has been demonstrated that tissue-engineered epithelial cell sheet grafts can be successfully fabricated using temperature-responsive culture surfaces and autologous serum. In the studies using different substrates for culture of CAOMECS, the quantitative expression of p63 was generally poorly reported; thus, more research is warranted with quantification of phenotypic data. Further research is required to develop a culture system for CAOMECS that mimics the natural environment of oral/limbal/corneal epithelial cells without the need for undefined foreign materials such as serum and feeder cells.

  12. Culture of Oral Mucosal Epithelial Cells for the Purpose of Treating Limbal Stem Cell Deficiency.

    Science.gov (United States)

    Utheim, Tor Paaske; Utheim, Øygunn Aass; Khan, Qalb-E-Saleem; Sehic, Amer

    2016-03-01

    The cornea is critical for normal vision as it allows allowing light transmission to the retina. The corneal epithelium is renewed by limbal epithelial cells (LEC), which are located in the periphery of the cornea, the limbus. Damage or disease involving LEC may lead to various clinical presentations of limbal stem cell deficiency (LSCD). Both severe pain and blindness may result. Transplantation of cultured autologous oral mucosal epithelial cell sheet (CAOMECS) represents the first use of a cultured non-limbal autologous cell type to treat this disease. Among non-limbal cell types, CAOMECS and conjunctival epithelial cells are the only laboratory cultured cell sources that have been explored in humans. Thus far, the expression of p63 is the only predictor of clinical outcome following transplantation to correct LSCD. The optimal culture method and substrate for CAOMECS is not established. The present review focuses on cell culture methods, with particular emphasis on substrates. Most culture protocols for CAOMECS used amniotic membrane as a substrate and included the xenogeneic components fetal bovine serum and murine 3T3 fibroblasts. However, it has been demonstrated that tissue-engineered epithelial cell sheet grafts can be successfully fabricated using temperature-responsive culture surfaces and autologous serum. In the studies using different substrates for culture of CAOMECS, the quantitative expression of p63 was generally poorly reported; thus, more research is warranted with quantification of phenotypic data. Further research is required to develop a culture system for CAOMECS that mimics the natural environment of oral/limbal/corneal epithelial cells without the need for undefined foreign materials such as serum and feeder cells.

  13. Cannabinoids induce incomplete maturation of cultured human leukemia cells

    Energy Technology Data Exchange (ETDEWEB)

    Murison, G.; Chubb, C.B.H.; Maeda, S.; Gemmell, M.A.; Huberman, E.

    1987-08-01

    Monocyte maturation markers were induced in cultured human myeloblastic ML-2 leukemia cells after treatment for 1-6 days with 0.03-30 ..mu..M ..delta../sup 9/-tetrahydrocannabinol (THC), the major psychoactive component of marijuana. After a 2-day or longer treatment, 2- to 5-fold increases were found in the percentages of cells exhibiting reactivity with either the murine OKM1 monoclonal antibody of the Leu-M5 monoclonal antibody, staining positively for nonspecific esterase activity, and displaying a promonocyte morphology. The increases in these differentiation markers after treatment with 0.03-1 ..mu..M THC were dose dependent. At this dose range, THC did not cause an inhibition of cell growth. The THC-induced cell maturation was also characterized by specific changes in the patterns of newly synthesized proteins. The THC-induced differentiation did not, however, result in cells with a highly developed mature monocyte phenotype. However, treatment of these incompletely matured cells with either phorbol 12-myristate 13-acetate of 1..cap alpha..,25-dihydroxycholecalciferol, which are inducers of differentiation in myeloid leukemia cells (including ML-2 cells), produced cells with a mature monocyte morphology. The ML-2 cell system described here may be a useful tool for deciphering critical biochemical events that lead to the cannabinoid-induced incomplete cell differentiation of ML-2 cells and other related cell types. Findings obtained from this system may have important implications for studies of cannabinoid effects on normal human bone-marrow progenitor cells.

  14. Withaferin A from cell cultures of Withania somnifera

    Directory of Open Access Journals (Sweden)

    Ciddi Veeresham

    2006-01-01

    Full Text Available Suspension cultures of Withania somnifera cells were established and shown to produce withaferin A. The identification of withaferin A was done by TLC, UV absorption, HPLC and electron spray mass spectroscopy. These cultures could be strongly elicited by exposure to salacin. Addition of salacin at the concentration of 750 µM to the cultures in production medium enhanced production levels of withaferin A to 25±2.9 mg/l compared to 0.47±0.03 mg/l in unelicited controls. This report is the first to demonstrate withaferin A production in plant suspension cultures and provides prerequisites for commercial scale, controlled production of withaferin A.

  15. Crude subcellular fractionation of cultured mammalian cell lines

    OpenAIRE

    Holden Paul; Horton William A

    2009-01-01

    Abstract Background The expression and study of recombinant proteins in mammalian culture systems can be complicated during the cell lysis procedure by contaminating proteins from cellular compartments distinct from those within which the protein of interest resides and also by solubility issues that may arise from the use of a single lysis buffer. Partial subcellular fractionation using buffers of increasing stringency, rather than whole cell lysis is one way in which to avoid or reduce this...

  16. Isolation of Cultured Endothelial Progenitor Cells in vitro from PBMCs and CD133~+ Enriched Cells

    Institute of Scientific and Technical Information of China (English)

    郑伟红; 万亚峰; 马小鹏; 李兴睿; 杨志芳; 殷茜; 易继林

    2010-01-01

    Two isolation methods for sorting of endothelial progenitor cells(EPCs):from peripheral blood mononuclear cells(PBMCs)and CD133+ enriched cells were compared,by defining the cell morphology,phenotype,reproductive activities and function in vitro,to provide a reference for clinical application of EPCs.PBMCs from healthy subjects were used either directly for cell culture or for CD133+ sorting.The two groups of cells were cultured in complete medium 199(M199)for 7 to 14 days and the phenotypes of EPCs were an...

  17. Effects of viscoelastic ophthalmic solutions on cell cultures

    Directory of Open Access Journals (Sweden)

    Madhavan Hajib

    1998-01-01

    Full Text Available The development of mild but significant inflammation probably attributable to viscoelastic ophthalmic solutions in cataract surgery was recently brought to the notice of the authors, and hence a study of the effects of these solutions available in India, on cell cultures was undertaken. We studied the effects of 6 viscoelastic ophthalmic solutions (2 sodium hyaluronate designated as A and B, and 4 hydroxypropylmethylcellulose designated as C, D, E and F on HeLa, Vero and BHK-21 cell lines in tissue culture microtitre plates using undiluted, 1:10 and 1:100 dilutions of the solutions, and in cover slip cultures using undiluted solutions. Phase contrast microscopic examination of the solutions was also done to determine the presence of floating particles. The products D and F produced cytotoxic changes in HeLa cell line and these products also showed the presence of floating particles under phase contrast microscopy. Other products did not have any adverse effects on the cell lines nor did they show floating particles. The viscoelastic ophthalmic pharmaceutical products designated D and F have cytotoxic effects on HeLa cell line which appears to be a useful cell line for testing these products for their toxicity. The presence of particulate materials in products D and F indicates that the methods used for purification of the solution are not effective.

  18. Characterization of tendon cell cultures of the human rotator cuff.

    Science.gov (United States)

    Pauly, S; Klatte, F; Strobel, C; Schmidmaier, G; Greiner, S; Scheibel, M; Wildemann, B

    2010-07-26

    Rotator cuff tears are common soft tissue injuries of the musculoskeletal system that heal by formation of repair tissue and may lead to high retear rates and joint dysfunction. In particular, tissue from chronic, large tendon tears is of such degenerative nature that it may be prone to retear after surgical repair. Besides several biomechanical approaches, biologically based strategies such as application of growth factors may be promising for increasing cell activity and production of extracellular tendon matrix at the tendon-to-bone unit. As a precondition for subsequent experimental growth factor application, the aim of the present study was to establish and characterize a human rotator cuff tendon cell culture. Long head biceps (LHB)- and supraspinatus muscle (SSP)- tendon samples from donor patients undergoing shoulder surgery were cultivated and examined at the RNA level for expression of collagen type-I, -II and -III, biglycan, decorin, tenascin-C, aggrecan, osteocalcin, tenomodulin and scleraxis (by Real-time PCR). Finally, results were compared to chondrocytes and osteoblasts as control cells. An expression pattern was found which may reflect a human rotator cuff tenocyte-like cell culture. Both SSP and LHB tenocyte-like cells differed from chondrocyte cell cultures in terms of reduced expression of collagen type-II (ptendon matrix and osteofibroblastic integration at the tendon-bone unit following tendon repair.

  19. Complementation of mutant phenotypes and genotypes of cultured mammalian cells

    NARCIS (Netherlands)

    A.J.R. de Jonge

    1985-01-01

    textabstractThis dissertation describes experiments aimed at the complementation of a genetic mutation in cultured mammalian cells in order to investigate several aspects of the structure and functioning of the human genome. Complementation is indicated by the correction of a biochemical function in

  20. Preparation of crude rough microsomes from tissue culture cells.

    Science.gov (United States)

    Sabatini, David D

    2014-09-02

    There are various procedures for isolating microsomal fractions from tissue culture cells. The essential conditions for each step of one procedure are described here. Notes for special circumstances are included so that the procedure can be modified according to the experimental purpose.

  1. Disposable Bioreactors for Plant Micropropagation and Mass Plant Cell Culture

    Science.gov (United States)

    Ducos, Jean-Paul; Terrier, Bénédicte; Courtois, Didier

    Different types of bioreactors are used at Nestlé R&D Centre - Tours for mass propagation of selected plant varieties by somatic embryogenesis and for large scale culture of plants cells to produce metabolites or recombinant proteins. Recent studies have been directed to cut down the production costs of these two processes by developing disposable cell culture systems. Vegetative propagation of elite plant varieties is achieved through somatic embryogenesis in liquid medium. A pilot scale process has recently been set up for the industrial propagation of Coffea canephora (Robusta coffee). The current production capacity is 3.0 million embryos per year. The pre-germination of the embryos was previously conducted by temporary immersion in liquid medium in 10-L glass bioreactors. An improved process has been developed using a 10-L disposable bioreactor consisting of a bag containing a rigid plastic box ('Box-in-Bag' bioreactor), insuring, amongst other advantages, a higher light transmittance to the biomass due to its horizontal design. For large scale cell culture, two novel flexible plastic-based disposable bioreactors have been developed from 10 to 100 L working volumes, validated with several plant species ('Wave and Undertow' and 'Slug Bubble' bioreactors). The advantages and the limits of these new types of bioreactor are discussed, based mainly on our own experience on coffee somatic embryogenesis and mass cell culture of soya and tobacco.

  2. Morphological characteristics of cultured fresh and thawed pericardium cells.

    Science.gov (United States)

    Maslova, Olga; Fedevych, Oleg; Shuvalova, Nadiia; Deryabina, Olena; Zhovnir, Volodymyr; Novak, Miroslav; Kruzliak, Peter

    2016-06-01

    The need for selection of the optimal material for the manufacturing of cardio-patches can be resolved by the use of cryostored autologous pericardial tissue. This short communication is a concise fragment of a large-scale research and demonstrates only the efficiency of cell culturing before and after pericardial preservation in the low temperature conditions.

  3. CYTOTOXICITY TESTING OF WOUND DRESSINGS USING METHYLCELLULOSE CELL-CULTURE

    NARCIS (Netherlands)

    VANLUYN, MJA; VANWACHEM, PB; NIEUWENHUIS, P; JONKMAN, MF

    1992-01-01

    Wound dressings may induce cytotoxic effects. In this study, we check several, mostly commercially available, wound dressings for cytotoxicity. We used our previously described, newly developed and highly sensitive 7 d methylcellulose cell culture with fibroblasts as the test system. Cytotoxicity is

  4. Spontaneous calcium waves in granule cells in cerebellar slice cultures

    DEFF Research Database (Denmark)

    Apuschkin, Mia; Ougaard, Maria; Rekling, Jens C

    2013-01-01

    and establishment of synaptic transmission. Here, we used calcium imaging in slice cultures of the postnatal cerebellum, and observe spontaneous propagating calcium waves in NeuN-positive granule-like cells. Wave formation was blocked by TTX and the AMPA antagonist NBQX, but persisted after NMDA receptor blockade...

  5. Test chambers for cell culture in static magnetic field

    Energy Technology Data Exchange (ETDEWEB)

    Glinka, Marek, E-mail: mag@iq.pl [Research and Development Centre of Electrical Machines. 188 Rozdzienskiego Street, 40-203 Katowice (Poland); Gawron, Stanisław, E-mail: s.gawron@komel.katowice.pl [Research and Development Centre of Electrical Machines. 188 Rozdzienskiego Street, 40-203 Katowice (Poland); Sieroń, Aleksander, E-mail: sieron1@tlen.pl [Department of Internal Diseases, Angiology and Physical Medicine in Bytom. Medical University of Silesia in Katowice. 15 Batorego Street, 41-902 Bytom (Poland); Pawłowska–Góral, Katarzyna, E-mail: kgoral@sum.edu.pl [Department of Food and Nutrition in Sosnowiec. Medical University of Silesia in Katowice. 8 Jednosci Street, 41-200 Sosnowiec (Poland); Cieślar, Grzegorz, E-mail: cieslar1@tlen.pl [Department of Internal Diseases, Angiology and Physical Medicine in Bytom. Medical University of Silesia in Katowice. 15 Batorego Street, 41-902 Bytom (Poland); Sieroń–Stołtny, Karolina [Department of Internal Diseases, Angiology and Physical Medicine in Bytom. Medical University of Silesia in Katowice. 15 Batorego Street, 41-902 Bytom (Poland)

    2013-04-15

    Article presents a test chamber intended to be used for in vitro cell culture in homogenous constant magnetic field with parametrically variable magnitude. We constructed test chambers with constant parameters of control homeostasis of cell culture for the different parameters of static magnetic field. The next step was the computer calculation of 2D and 3D simulation of the static magnetic field distribution in the chamber. The analysis of 2D and 3D calculations of magnetic induction in the cells' exposition plane reveals, in comparison to the detection results, the greater accuracy of 2D calculations (Figs. 9 and 10). The divergence in 2D method was 2–4% and 8 to 10% in 3D method (reaching 10% only out of the cells′ cultures margins). -- Highlights: ► We present test chamber to be used for in vitro cell culture in static magnetic field. ► The technical data of the chamber construction was presented. ► 2D versus 3D simulation of static magnetic field distribution in chamber was reported. ► We report the accuracy of 2D calculation than 3D.

  6. Plant Cell Cultures as Source of Cosmetic Active Ingredients

    Directory of Open Access Journals (Sweden)

    Ani Barbulova

    2014-04-01

    Full Text Available The last decades witnessed a great demand of natural remedies. As a result, medicinal plants have been increasingly cultivated on a commercial scale, but the yield, the productive quality and the safety have not always been satisfactory. Plant cell cultures provide useful alternatives for the production of active ingredients for biomedical and cosmetic uses, since they represent standardized, contaminant-free and biosustainable systems, which allow the production of desired compounds on an industrial scale. Moreover, thanks to their totipotency, plant cells grown as liquid suspension cultures can be used as “biofactories” for the production of commercially interesting secondary metabolites, which are in many cases synthesized in low amounts in plant tissues and differentially distributed in the plant organs, such as roots, leaves, flowers or fruits. Although it is very widespread in the pharmaceutical industry, plant cell culture technology is not yet very common in the cosmetic field. The aim of the present review is to focus on the successful research accomplishments in the development of plant cell cultures for the production of active ingredients for cosmetic applications.

  7. Patterns of mitochondrial DNA instability in Brassica campestris cultured cells.

    Science.gov (United States)

    Shirzadegan, M; Palmer, J D; Christey, M; Earle, E D

    1991-01-01

    We previously showed that the mitochondrial DNA (mtDNA) of a Brassica campestris callus culture had undergone extensive rearrangements (i.e. large inversions and a duplication) relative to DNA of the control plant [54]. In this study we observed that after continued growth, the mtDNA of this culture continues to change, with rearranged forms amplifying and diminishing to varying proportions. Strikingly similar changes were detected in the mtDNA profiles of a variety of other long- and short-term callus and cell suspension lines. However, the proportions of parental ('unrearranged') and novel ('rearranged') forms varied in different cultured cell mtDNAs. To address the source of this heterogeneity, we compared the mtDNA organization of 28 individual plants from the parental seed stock. With the exception of one plant containing high levels of a novel plasmid-like mtDNA molecule, no significant variation was detected among individual plants and therefore source plant variation is unlikely to have contributed to the diversity of mitochondrial genomes observed in cultured cells. The source of this culture-induced heterogeneity was also investigated in 16 clones derived from single protoplasts. A mixed population of unrearranged and rearranged mtDNA molecules was apparent in each protoclone, suggesting that the observed heterogeneity in various cultures might reflect the genomic composition of each individual cell; however, the induction of an intercellular heterogeneity subsequent to the protoplast isolation was not tested and therefore cannot be ruled out. The results of this study support our earlier model that the rapid structural alteration of B. campestris mtDNA in vitro results from preferential amplification and reassortment of minor pre-existing forms of the genome rather than de novo rearrangement. Infrequent recombination between short dispersed repeated elements is proposed as the underlying mechanism for the formation of these minor mtDNA molecules.

  8. PDMS/glass microfluidic cell culture system for cytotoxicity tests and cells passage

    DEFF Research Database (Denmark)

    Ziolkowska, K.; Jedrych, E.; Kwapiszewski, R.

    2010-01-01

    microdevice was developed and successfully tested. The MCCS microdevice is fully reusable, i.e. it can be used several times for various cell culture and cytotoxic experiments. The suitability of designed MCCS for cell-based cytotoxicity assay application was verified using 1,4-dioxane as a model toxic agent....... The series of cytotoxicity tests in the microdevice as well as in classic way using 96-well cell culture plates were performed to compare results obtained in micro- and macroscale. Fluorescein dibutyrate (FDB) and iodide propidine (PI) were used as viable and dead cells' markers, respectively. Fabricated...... MCCS microdevices were reproducible and apart from cell culture for long period of time, including cell passaging, it allowed cell-based cytotoxicity assays performance. The MCCS can be applied in high-throughput cell-based assays providing important informations on potential drug targets, substances...

  9. Enzymatic Cell Isolation and Explant Cultures of Rat Calvarial Osteoblast Cells

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Osteoblast cells were isolated from the calvarial bones of newborn Wistar rats and cultured in vitro via both collagenase digestion method and explant technique, and a comparative study was carried out on the two culture methods. The biologic characteristics of tbs osteoblast cells were studied via cell number counting,morphology observation, alkaline phosphatase staining of the cells and alizarine- red staining of the calcified nodules. The results show that osteoblast cells can be cultured in vitro via collagenase digestion method and explant technique, and the obtained cells are of good biologic characteristics. In comparison with the explant techniqne,the operative procedure of the enzymatic digestion method is more complicated. The digestion time must be carefully controlled. However, with this method, one can obtain a lager number of cells in a short time. The operative procedure of the explant technique is simpler, but it usually takes longer time to obtain cells of desirable number.

  10. Differential effect of culture temperature and specific growth rate on CHO cell behavior in chemostat culture.

    Science.gov (United States)

    Vergara, Mauricio; Becerra, Silvana; Berrios, Julio; Osses, Nelson; Reyes, Juan; Rodríguez-Moyá, María; Gonzalez, Ramon; Altamirano, Claudia

    2014-01-01

    Mild hypothermia condition in mammalian cell culture technology has been one of the main focuses of research for the development of breeding strategies to maximize productivity of these production systems. Despite the large number of studies that show positive effects of mild hypothermia on specific productivity of r-proteins, no experimental approach has addressed the indirect effect of lower temperatures on specific cell growth rate, nor how this condition possibly affects less specific productivity of r-proteins. To separately analyze the effects of mild hypothermia and specific growth rate on CHO cell metabolism and recombinant human tissue plasminogen activator productivity as a model system, high dilution rate (0.017 h(-1)) and low dilution rate (0.012 h(-1)) at two cultivation temperatures (37 and 33 °C) were evaluated using chemostat culture. The results showed a positive effect on the specific productivity of r-protein with decreasing specific growth rate at 33 °C. Differential effect was achieved by mild hypothermia on the specific productivity of r-protein, contrary to the evidence reported in batch culture. Interestingly, reduction of metabolism could not be associated with a decrease in culture temperature, but rather with a decrease in specific growth rate.

  11. Mefloquine damage vestibular hair cells in organotypic cultures.

    Science.gov (United States)

    Yu, Dongzhen; Ding, Dalian; Jiang, Haiyan; Stolzberg, Daniel; Salvi, Richard

    2011-07-01

    Mefloquine is an effective and widely used anti-malarial drug; however, some clinical reports suggest that it can cause dizziness, balance, and vestibular disturbances. To determine if mefloquine might be toxic to the vestibular system, we applied mefloquine to organotypic cultures of the macula of the utricle from postnatal day 3 rats. The macula of the utricle was micro-dissected out as a flat surface preparation and cultured with 10, 50, 100, or 200 μM mefloquine for 24 h. Specimens were stained with TRITC-conjugated phalloidin to label the actin in hair cell stereocilia and TO-PRO-3 to visualize cell nuclei. Some utricles were also labeled with fluorogenic caspase-3, -8, or -9 indicators to evaluate the mechanism of programmed cell death. Mefloquine treatment caused a dose-dependent loss of utricular hair cells. Treatment with 10 μM caused a slight reduction, 50 μM caused a significant reduction, and 200 μM destroyed nearly all the hair cells. Hair cell nuclei in mefloquine-treated utricles were condensed and fragmented, morphological features of apoptosis. Mefloquine-treated utricles were po