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Sample records for vitro bewo cell

  1. Modeling placental transport: correlation of in vitro BeWo cell permeability and ex vivo human placental perfusion

    DEFF Research Database (Denmark)

    Poulsen, Marie Sønnegaard; Rytting, Erik; Mose, Tina

    2009-01-01

    across the BeWo cells was observed in the rank order of caffeine>antipyrine>benzoic acid>glyphosate in terms of both the apparent permeability coefficient and the initial slope, defined as the linear rate of substance transferred to the fetal compartment as percent per time, a parameter used to compare...

  2. Plasmodium falciparum parasites expressing pregnancy-specific variant surface antigens adhere strongly to the choriocarcinoma cell line BeWo

    DEFF Research Database (Denmark)

    Haase, Rikke N; Megnekou, Rosette; Lundquist, Maja

    2006-01-01

    Placenta-sequestering Plasmodium falciparum parasites causing pregnancy-associated malaria express pregnancy-specific variant surface antigens (VSA(PAM)). We report here that VSA(PAM)-expressing patient isolates adhere strongly to the choriocarcinoma cell line BeWo and that the BeWo line can...... be used to efficiently select for VSA(PAM) expression in vitro....

  3. Iron metabolism in BeWo chorion carcinoma cells. Transferrin-mediated uptake and release of iron

    NARCIS (Netherlands)

    van der Ende, A.; du Maine, A.; Simmons, C. F.; Schwartz, A. L.; Strous, G. J.

    1987-01-01

    Growing human choriocarcinoma BeWo b24 cells contain 1.5 X 10(6) functional cell surface transferrin binding sites and 2.0 X 10(6) intracellular binding sites. These cells rapidly accumulate iron at a rate of 360,000 iron atoms/min/cell. During iron uptake the transferrin and its receptor recycle at

  4. Relevance of Wnt10b and activation of β-catenin/GCMa/syncytin-1 pathway in BeWo cell fusion.

    Science.gov (United States)

    Malhotra, Sudha Saryu; Banerjee, Priyanka; Chaudhary, Piyush; Pal, Rahul; Gupta, Satish Kumar

    2017-10-01

    To study the involvement of specific Wnt(s) ligand during trophoblastic BeWo cell differentiation. BeWo cells on treatment with forskolin/human chorionic gonadotropin (hCG) were studied for cell fusion by desmoplakin I+II staining and/or hCG secretion by ELISA. Levels of Wnt10b/β-catenin/glial cell missing a (GCMa)/syncytin-1 were studied by qPCR/Western blotting in forskolin-/hCG-treated control siRNA and Wnt10b silenced BeWo cells. BeWo cells on treatment with hCG (5 IU/mL) led to a 94-fold increase in Wnt10b transcript. Wnt10b silencing showed significant decrease in forskolin-/hCG-mediated BeWo cell fusion and/or hCG secretion. It led to down-regulation of β-catenin (nuclear and cytoplasmic), GCMa and syncytin-1 expression. Treatment of BeWo cells with H89, protein kinase A (PKA) signaling inhibitor, significantly reduced forskolin-/hCG-induced Wnt10b, β-catenin, and syncytin-1 expression, which also resulted in reduced cell fusion. Wnt10b is involved in forskolin/hCG-mediated BeWo cell fusion via β-catenin/GCMa/syncytin pathway, which may also involve activation of PKA. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  5. Relevance of the NR4A sub-family of nuclear orphan receptors in trophoblastic BeWo cell differentiation.

    Science.gov (United States)

    Malhotra, Sudha Saryu; Gupta, Satish Kumar

    2017-01-01

    Nur-77, a member of the NR4A sub-family of nuclear orphan receptors, is downregulated in the placentae of pre-eclamptic women. Here, we investigate the relevance of Nor-1, Nurr-1 and Nur-77 in trophoblastic cell differentiation. Their transcript levels were found to be significantly upregulated in BeWo cells treated with forskolin. The maximum increase was observed after 2 h, with a second peak in the expression levels after 48 h. The expression of NR4A sub-family members was also found to be upregulated in BeWo cells after treatment with hCG and GnRH. A similar significant increase was observed at the respective protein levels after 2 and 48 h of treatment with forskolin, hCG or GnRH. Silencing Nor-1, Nurr-1 or Nur-77 individually did not show any effect on forskolin-, hCG- and/or GnRH-mediated BeWo cell fusion and/or hCG secretion. After silencing any one member of the NR4A sub-family, an increase in the transcript levels of the other sub-family members was observed, indicating a compensatory effect due to their functional redundancy. Simultaneously silencing all three NR4A sub-family members significantly downregulated forskolin- and hCG-mediated BeWo cell fusion and/or hCG secretion. However, a considerable amount of cell death occurred after forskolin or hCG treatment as compared to the control siRNA-transfected cells. These results suggest that the NR4A sub-family of nuclear orphan receptors has a role in trophoblastic cell differentiation.

  6. Isolation of basal membrane proteins from BeWo cells and their expression in placentas from fetal growth-restricted pregnancies.

    Science.gov (United States)

    Oh, Soo-Young; Hwang, Jae Ryoung; Lee, Yoonna; Choi, Suk-Joo; Kim, Jung-Sun; Kim, Jong-Hwa; Sadovsky, Yoel; Roh, Cheong-Rae

    2016-03-01

    The syncytiotrophoblast, a key barrier between the mother and fetus, is a polarized epithelium composed of a microvillus and basal membrane (BM). We sought to characterize BM proteins of BeWo cells in relation to hypoxia and to investigate their expression in placentas from pregnancies complicated by fetal growth restriction (FGR). We isolated the BM fraction of BeWo cells by the cationic colloidal silica method and identified proteins enriched in this fraction by mass spectrometry. We evaluated the effect of hypoxia on the expression and intracellular localization of identified proteins and compared their expression in BM fractions of FGR placentas to those from normal pregnancies. We identified BM proteins from BeWo cells. Among BM proteins, we further characterized heme oxygenase-1 (HO-1), voltage-dependent anion channel-1 (VDAC1), and ribophorin II (RPN2), based on their relevance to placental biology. Hypoxia enhanced the localization of these proteins to the BM of BeWo cells. HO-1, VDAC1, and RPN2 were selectively expressed in the human placental BM fraction. C-terminally truncated HO-1 was identified in placental BM fractions, and its BM expression was significantly reduced in FGR placentas than in normal placentas. Interestingly, a truncated HO-1 construct was predominantly localized in the BM in response to hypoxia and co-localized with VDAC1 in BeWo cells. Hypoxia increased the BM localization of HO-1, VDAC1, and RPN2 proteins. FGR significantly reduced the expression of truncated HO-1, which was surmised to co-localize with VDAC1 in hypoxic BeWo cells. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Enrofloxacin and Toltrazuril Are Able to Reduce Toxoplasma gondii Growth in Human BeWo Trophoblastic Cells and Villous Explants from Human Third Trimester Pregnancy

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    Rafaela J. da Silva

    2017-07-01

    Full Text Available Classical treatment for congenital toxoplasmosis is based on combination of sulfadiazine and pyrimethamine plus folinic acid. Due to teratogenic effects and bone marrow suppression caused by pyrimethamine, the establishment of new therapeutic strategies is indispensable to minimize the side effects and improve the control of infection. Previous studies demonstrated that enrofloxacin and toltrazuril reduced the incidence of Neospora caninum and Toxoplasma gondii infection. The aim of the present study was to evaluate the efficacy of enrofloxacin and toltrazuril in the control of T. gondii infection in human trophoblast cells (BeWo line and in human villous explants from the third trimester. BeWo cells and villous were treated with several concentrations of enrofloxacin, toltrazuril, sulfadiazine, pyrimethamine, or combination of sulfadiazine+pyrimethamine, and the cellular or tissue viability was verified. Next, BeWo cells were infected by T. gondii (2F1 clone or the ME49 strain, whereas villous samples were only infected by the 2F1 clone. Then, infected cells and villous were treated with all antibiotics and the T. gondii intracellular proliferation as well as the cytokine production were analyzed. Finally, we evaluated the direct effect of enrofloxacin and toltrazuril in tachyzoites to verify possible changes in parasite structure. Enrofloxacin and toltrazuril did not decrease the viability of cells and villous in lower concentrations. Both drugs were able to significantly reduce the parasite intracellular proliferation in BeWo cells and villous explants when compared to untreated conditions. Regardless of the T. gondii strain, BeWo cells infected and treated with enrofloxacin or toltrazuril induced high levels of IL-6 and MIF. In villous explants, enrofloxacin induced high MIF production. Finally, the drugs increased the number of unviable parasites and triggered damage to tachyzoite structure. Taken together, it can be concluded that

  8. Enrofloxacin and Toltrazuril Are Able to Reduce Toxoplasma gondii Growth in Human BeWo Trophoblastic Cells and Villous Explants from Human Third Trimester Pregnancy.

    Science.gov (United States)

    da Silva, Rafaela J; Gomes, Angelica O; Franco, Priscila S; Pereira, Ariane S; Milian, Iliana C B; Ribeiro, Mayara; Fiorenzani, Paolo; Dos Santos, Maria C; Mineo, José R; da Silva, Neide M; Ferro, Eloisa A V; de Freitas Barbosa, Bellisa

    2017-01-01

    Classical treatment for congenital toxoplasmosis is based on combination of sulfadiazine and pyrimethamine plus folinic acid. Due to teratogenic effects and bone marrow suppression caused by pyrimethamine, the establishment of new therapeutic strategies is indispensable to minimize the side effects and improve the control of infection. Previous studies demonstrated that enrofloxacin and toltrazuril reduced the incidence of Neospora caninum and Toxoplasma gondii infection. The aim of the present study was to evaluate the efficacy of enrofloxacin and toltrazuril in the control of T. gondii infection in human trophoblast cells (BeWo line) and in human villous explants from the third trimester. BeWo cells and villous were treated with several concentrations of enrofloxacin, toltrazuril, sulfadiazine, pyrimethamine, or combination of sulfadiazine+pyrimethamine, and the cellular or tissue viability was verified. Next, BeWo cells were infected by T. gondii (2F1 clone or the ME49 strain), whereas villous samples were only infected by the 2F1 clone. Then, infected cells and villous were treated with all antibiotics and the T. gondii intracellular proliferation as well as the cytokine production were analyzed. Finally, we evaluated the direct effect of enrofloxacin and toltrazuril in tachyzoites to verify possible changes in parasite structure. Enrofloxacin and toltrazuril did not decrease the viability of cells and villous in lower concentrations. Both drugs were able to significantly reduce the parasite intracellular proliferation in BeWo cells and villous explants when compared to untreated conditions. Regardless of the T. gondii strain, BeWo cells infected and treated with enrofloxacin or toltrazuril induced high levels of IL-6 and MIF. In villous explants, enrofloxacin induced high MIF production. Finally, the drugs increased the number of unviable parasites and triggered damage to tachyzoite structure. Taken together, it can be concluded that enrofloxacin and

  9. BeWo cells stimulate smooth muscle cell apoptosis and elastin breakdown in a model of spiral artery transformation

    OpenAIRE

    Harris, L. K.; Keogh, R. J.; Wareing, M.; Baker, P. N.; Cartwright, J. E.; Whitley, G. S.; Aplin, J. D.

    2007-01-01

    BACKGROUND: During pregnancy, extravillous trophoblast invades the uterine wall and enters the spiral arteries. Remodelling ensues, with loss of vascular smooth muscle cells (SMCs) to create high flow, low resistance vessels. Pregnancies complicated by pre-eclampsia are characterized by incomplete arterial remodelling. Endovascular trophoblast is not easily accessible for studies to establish the pathogenesis of pre-eclampsia, so we have developed a model appropriate to carry out mechanistic ...

  10. Regulation of pregnancy-associated plasma protein A2 (PAPPA2 in a human placental trophoblast cell line (BeWo

    Directory of Open Access Journals (Sweden)

    Christians Julian K

    2011-04-01

    Full Text Available Abstract Background Pregnancy-associated plasma protein A2 (PAPPA2 is an insulin-like growth factor-binding protein (IGFBP protease expressed at high levels in the placenta and upregulated in pregnancies complicated by preeclampsia and HELLP (Hemolytic anemia, Elevated Liver enzymes, and Low Platelet count syndrome. However, it is unclear whether elevated PAPPA2 expression causes abnormal placental development, or whether upregulation compensates for placental pathology. In the present study, we investigate whether PAPPA2 expression is affected by hypoxia, oxidative stress, syncytialization factors or substances known to affect the expression of PAPPA2's paralogue, PAPPA. Methods BeWo cells, a model of placental trophoblasts, were treated with one of the following: hypoxia (2% O2, oxidative stress (20 microM hydrogen peroxide, forskolin (10 microM and 100 microM, TGF-beta (10 and 50 ng/mL, TNF-alpha (100 ng/mL, IL-1beta (100 ng/mL or PGE2 (1 microM. We used quantitative RT-PCR (qRT-PCR to quantify the mRNA levels of PAPPA2, as well as those of PAPPA and ADAM12 since these proteases have similar substrates and are also highly expressed in the placenta. Where we observed significant effects on PAPPA2 mRNA levels, we tested for effects at the protein level using an in-cell Western assay. Results Hypoxia, but not oxidative stress, caused a 47-fold increase in PAPPA2 mRNA expression, while TNF-alpha resulted in a 6-fold increase, and both of these effects were confirmed at the protein level. PGE2 resulted in a 14-fold upregulation of PAPPA2 mRNA but this was not reflected at the protein level. Forskolin, TGF-beta and IL-1beta had no significant effect on PAPPA2 mRNA expression. We observed no effects of any treatment on PAPPA or ADAM12 expression. Conclusion Our study demonstrates that factors previously known to be highly expressed in preeclamptic placentae (PGE2 and TNF-alpha, contribute to the upregulation of PAPPA2. Hypoxia, known to occur in

  11. Progranulin shows cytoprotective effects on trophoblast cells in vitro but does not antagonize TNF-α-induced apoptosis.

    Science.gov (United States)

    Stubert, Johannes; Waldmann, Kathrin; Dieterich, Max; Richter, Dagmar-Ulrike; Briese, Volker

    2014-11-01

    The glycoprotein progranulin directly binds to TNF-receptors and thereby can antagonize the inflammatory effects of TNF-α. Here we analyzed the impact of both cytokines on cytotoxicity and viability of trophoblast cells. Isolated villous first trimester human trophoblast cells and the human choriocarcinoma cell line BeWo were treated with recombinant human progranulin and TNF-α. Analyses were performed by LDH- and MTT-assay and measurement of caspase-8-activity. Progranulin treatment showed some cytoprotective effects on isolated trophoblast cells. However, TNF-α-induced apoptosis was not antagonized by addition of progranulin. Effects were similar, but more pronounced in BeWo cells. The cytoprotective activity of progranulin on trophoblast cells in vitro was only weak and of doubtful biologic relevance. It was not able to antagonize TNF-α. Future studies should focus on possible paracrine activities of progranulin.

  12. A comparison of the embryonic stem cell test and whole embryo culture assay combined with the BeWo placental passage model for predicting the embryotoxicity of azoles.

    NARCIS (Netherlands)

    Dimopoulou, Myrto; Verhoef, Aart; Gomes, Caroline A; van Dongen, Catharina W; Rietjens, Ivonne M C M; Piersma, Aldert H; van Ravenzwaay, Bennard

    2018-01-01

    In the present study, we show the value of combining toxico-dynamic and -kinetic in vitro approaches for embryotoxicity testing of azoles. Both the whole embryo culture (WEC) and the embryonic stem cells test (EST) predicted the in vivo potency ranking of twelve tested azoles with moderate accuracy.

  13. Development of a model for functional studies of ABCG2 (breast cancer resistance protein) efflux employing a standard BeWo clone (B24).

    Science.gov (United States)

    Crowe, Andrew; Keelan, Jeffrey A

    2012-10-01

    Human choriocarcinoma-derived BeWo cells express high levels of breast cancer resistance protein (BCRP/ABCG2) with no functional P-glycoprotein (P-gp) (ABCB1) activity, making them a potential model to study bidirectional ABCG2-mediated drug transport. However, the original BeWo clone (B24) available to researchers does not form confluent monolayers with tight junctions required by the model. Our aim was to adapt culture conditions to attempt to generate confluent BeWo monolayers for drug transport studies using the standard B24 clone. BeWo cells (B24; American Type Culture collection [ATCC]) were cultured in six-well plates or polycarbonate millicell inserts in a number of media formulations, growth supplements, and basement membrane substitutes. Cells were examined for confluence by microscopy, and transepithelial electrical resistance (TEER) was measured daily; monolayer permeability was assessed when TEER had stabilized. Optimal growth rates were achieved in culture conditions consisting of Medium 199 (M199) supplemented with epidermal growth factor (EGF; 20 ng/mL), vitamin supplements, and 10% fetal calf serum (FCS) with collagen coating. A TEER of 170 Ω in 0.6 cm(2) inserts was achieved 2 weeks after seeding under optimal conditions. The cell-impermeable diffusion marker 5(6) carboxy-2,7dichlorodihydrofluorescein (C-DCDHF) had a permeability coefficient of 3.5×10(-6) cm/s, indicative of minimal paracellular permeability. ABCG2 expression, as determined by immunoblotting, remained unaffected by confluency. In conclusion, we describe culture conditions for the B24 BeWo clone that facilitate the formation of monolayers with tighter junctions and reduced paracellular transport compared to previously published models. These growth conditions provide a good model of ABCG2-mediated drug transport in a human placental cell line.

  14. Human parvovirus B19 antibodies induce altered membrane protein expression and apoptosis of BeWo trophoblasts.

    Science.gov (United States)

    Tzang, Bor-Show; Chiang, Szu-Yi; Chan, Hsu-Chin; Liu, Chung-Hsien; Hsu, Tsai-Ching

    2016-11-01

    Human parvovirus B19 (B19) is harmful during pregnancy since it can be vertically transmitted to the developing fetus. In addition, the anti‑B19 antibodies induced by B19 infection are believed to have a cytopathic role in B19 transmission; however, knowledge regarding the effects of anti‑B19 antibodies during pregnancy is limited. To investigate the possible roles of anti‑B19 antibodies during pregnancy, the present study examined the effects of anti‑B19‑VP1 unique region (VP1u), anti‑B19‑VP2 and anti‑B19‑nonstructural protein 1 (NS1) immunoglobulin G (IgG) antibodies on BeWo trophoblasts. Briefly, BeWo trophoblasts were incubated with purified IgG against B19‑VP1u, B19‑VP2 and B19‑NS1. Subsequently, the expression of surface proteins and apoptotic molecules were assessed in BeWo trophoblasts using flow cytometry, ELISA and western blotting. The expression levels of human leukocyte antigen (HLA)‑G were significantly increased on BeWo trophoblasts treated with rabbit anti‑B19‑VP1u IgG, and were unchanged in those treated with rabbit anti‑B19‑NS1 and anti‑B19‑VP2 IgG, as compared with the control group. Furthermore, the expression levels of globoside (P blood group antigen) and cluster of differentiation (CD)29 (β1 integrin) were significantly increased in BeWo trophoblasts treated with rabbit anti‑B19‑NS1 and anti‑B19‑VP2 IgG, whereas only CD29 was also significantly increased in cells treated with anti‑B19‑VP1u IgG. In addition, the number of cells at sub‑G1 phase; caspase‑3 activity; and the expression of intrinsic and extrinsic apoptotic molecules, including Fas‑associated death domain protein, activated caspase‑8, activated caspase‑3, B‑cell lymphoma 2‑associated X protein, cytochrome c, apoptotic peptidase activating factor 1 and activated caspase‑9, were significantly increased in BeWo trophoblasts treated with anti‑B19‑VP1u and anti‑B19‑NS1 IgG. In conclusion, the present

  15. In vitro placental model optimization for nanoparticle transport studies

    Directory of Open Access Journals (Sweden)

    Cartwright L

    2012-01-01

    Full Text Available Laura Cartwright1, Marie Sønnegaard Poulsen2, Hanne Mørck Nielsen3, Giulio Pojana4, Lisbeth E Knudsen2, Margaret Saunders1, Erik Rytting2,51Bristol Initiative for Research of Child Health (BIRCH, Biophysics Research Unit, St Michael's Hospital, UH Bristol NHS Foundation Trust, Bristol, UK; 2University of Copenhagen, Faculty of Health Sciences, Department of Public Health, 3University of Copenhagen, Faculty of Pharmaceutical Sciences, Department of Pharmaceutics and Analytical Chemistry, Copenhagen, Denmark; 4Department of Environmental Sciences, Informatics and Statistics, University Ca' Foscari Venice, Venice, Italy; 5Department of Obstetrics and Gynecology, University of Texas Medical Branch, Galveston, Texas, USABackground: Advances in biomedical nanotechnology raise hopes in patient populations but may also raise questions regarding biodistribution and biocompatibility, especially during pregnancy. Special consideration must be given to the placenta as a biological barrier because a pregnant woman's exposure to nanoparticles could have significant effects on the fetus developing in the womb. Therefore, the purpose of this study is to optimize an in vitro model for characterizing the transport of nanoparticles across human placental trophoblast cells.Methods: The growth of BeWo (clone b30 human placental choriocarcinoma cells for nanoparticle transport studies was characterized in terms of optimized Transwell® insert type and pore size, the investigation of barrier properties by transmission electron microscopy, tight junction staining, transepithelial electrical resistance, and fluorescein sodium transport. Following the determination of nontoxic concentrations of fluorescent polystyrene nanoparticles, the cellular uptake and transport of 50 nm and 100 nm diameter particles was measured using the in vitro BeWo cell model.Results: Particle size measurements, fluorescence readings, and confocal microscopy indicated both cellular uptake of

  16. Live cell imaging of in vitro human trophoblast syncytialization.

    Science.gov (United States)

    Wang, Rui; Dang, Yan-Li; Zheng, Ru; Li, Yue; Li, Weiwei; Lu, Xiaoyin; Wang, Li-Juan; Zhu, Cheng; Lin, Hai-Yan; Wang, Hongmei

    2014-06-01

    Human trophoblast syncytialization, a process of cell-cell fusion, is one of the most important yet least understood events during placental development. Investigating the fusion process in a placenta in vivo is very challenging given the complexity of this process. Application of primary cultured cytotrophoblast cells isolated from term placentas and BeWo cells derived from human choriocarcinoma formulates a biphasic strategy to achieve the mechanism of trophoblast cell fusion, as the former can spontaneously fuse to form the multinucleated syncytium and the latter is capable of fusing under the treatment of forskolin (FSK). Live-cell imaging is a powerful tool that is widely used to investigate many physiological or pathological processes in various animal models or humans; however, to our knowledge, the mechanism of trophoblast cell fusion has not been reported using a live- cell imaging manner. In this study, a live-cell imaging system was used to delineate the fusion process of primary term cytotrophoblast cells and BeWo cells. By using live staining with Hoechst 33342 or cytoplasmic dyes or by stably transfecting enhanced green fluorescent protein (EGFP) and DsRed2-Nuc reporter plasmids, we observed finger-like protrusions on the cell membranes of fusion partners before fusion and the exchange of cytoplasmic contents during fusion. In summary, this study provides the first video recording of the process of trophoblast syncytialization. Furthermore, the various live-cell imaging systems used in this study will help to yield molecular insights into the syncytialization process during placental development. © 2014 by the Society for the Study of Reproduction, Inc.

  17. In vitro toxicological effects of estrogenic mycotoxins on human placental cells: Structure activity relationships

    Energy Technology Data Exchange (ETDEWEB)

    Prouillac, Caroline, E-mail: c.prouillac@vetagro-sup.fr [Université Lyon, US/C 1233 INRA VetAgroSup, Métabolisme et Toxicologie Comparée des Xénobiotiques, 1 avenue Bourgelat, BP 83, 69280 Marcy l' Etoile (France); Koraichi, Farah; Videmann, Bernadette; Mazallon, Michelle [Université Lyon, US/C 1233 INRA VetAgroSup, Métabolisme et Toxicologie Comparée des Xénobiotiques, 1 avenue Bourgelat, BP 83, 69280 Marcy l' Etoile (France); Rodriguez, Frédéric; Baltas, Michel [Université Paul Sabatier, SPCMIB-UMR5068, Laboratoire de Synthèse et de Physicochimie des Molécules d' Intérêt Biologique, 118 route de Narbonne, 31062 TOULOUSE cedex 9 (France); Lecoeur, Sylvaine [Université Lyon, US/C 1233 INRA VetAgroSup, Métabolisme et Toxicologie Comparée des Xénobiotiques, 1 avenue Bourgelat, BP 83, 69280 Marcy l' Etoile (France)

    2012-03-15

    Zearalenone (ZEN) is a non-steroid estrogen mycotoxin produced by numerous strains of Fusarium which commonly contaminate cereals. After oral administration, ZEN is reduced via intestinal and hepatic metabolism to α- and β-zearalenol (αZEL and βZEL). These reduced metabolites possess estrogenic properties, αZEL showing the highest affinity for ERs. ZEN and reduced metabolites cause hormonal effects in animals, such as abnormalities in the development of the reproductive tract and mammary gland in female offspring, suggesting a fetal exposure to these contaminants. In our previous work, we have suggested the potential impact of ZEN on placental cells considering this organ as a potential target of xenobiotics. In this work, we first compared the in vitro effects of αZEL and βΖΕL on cell differentiation to their parental molecule on human trophoblast (BeWo cells). Secondly, we investigated their molecular mechanisms of action by investigating the expression of main differentiation biomarkers and the implication of nuclear receptor by docking prediction. Conversely to ZEN, reduced metabolites did not induce trophoblast differentiation. They also induced significant changes in ABC transporter expression by potential interaction with nuclear receptors (LXR, PXR, PR) that could modify the transport function of placental cells. Finally, the mechanism of ZEN differentiation induction seemed not to involve nuclear receptor commonly involved in the differentiation process (PPARγ). Our results demonstrated that in spite of structure similarities between ZEN, αZEL and βZEL, toxicological effects and toxicity mechanisms were significantly different for the three molecules. -- Highlights: ► ZEN and metabolites have differential effect on trophoblast differentiation. ► ZEN and metabolites have differential effect on ABC transporter expression. ► ZEN and metabolites effects involved nuclear receptors interaction.

  18. In vitro toxicological effects of estrogenic mycotoxins on human placental cells: Structure activity relationships

    International Nuclear Information System (INIS)

    Prouillac, Caroline; Koraichi, Farah; Videmann, Bernadette; Mazallon, Michelle; Rodriguez, Frédéric; Baltas, Michel; Lecoeur, Sylvaine

    2012-01-01

    Zearalenone (ZEN) is a non-steroid estrogen mycotoxin produced by numerous strains of Fusarium which commonly contaminate cereals. After oral administration, ZEN is reduced via intestinal and hepatic metabolism to α- and β-zearalenol (αZEL and βZEL). These reduced metabolites possess estrogenic properties, αZEL showing the highest affinity for ERs. ZEN and reduced metabolites cause hormonal effects in animals, such as abnormalities in the development of the reproductive tract and mammary gland in female offspring, suggesting a fetal exposure to these contaminants. In our previous work, we have suggested the potential impact of ZEN on placental cells considering this organ as a potential target of xenobiotics. In this work, we first compared the in vitro effects of αZEL and βΖΕL on cell differentiation to their parental molecule on human trophoblast (BeWo cells). Secondly, we investigated their molecular mechanisms of action by investigating the expression of main differentiation biomarkers and the implication of nuclear receptor by docking prediction. Conversely to ZEN, reduced metabolites did not induce trophoblast differentiation. They also induced significant changes in ABC transporter expression by potential interaction with nuclear receptors (LXR, PXR, PR) that could modify the transport function of placental cells. Finally, the mechanism of ZEN differentiation induction seemed not to involve nuclear receptor commonly involved in the differentiation process (PPARγ). Our results demonstrated that in spite of structure similarities between ZEN, αZEL and βZEL, toxicological effects and toxicity mechanisms were significantly different for the three molecules. -- Highlights: ► ZEN and metabolites have differential effect on trophoblast differentiation. ► ZEN and metabolites have differential effect on ABC transporter expression. ► ZEN and metabolites effects involved nuclear receptors interaction.

  19. T Cell Genesis: In Vitro Veritas Est?

    OpenAIRE

    Brauer, Patrick M.; Singh, Jastaranpreet; Xhiku, Sintia; Zúñiga-Pflücker, Juan Carlos

    2016-01-01

    T cells, as orchestrators of the adaptive immune response, serve important physiological and potentially therapeutic roles, for example in cancer immunotherapy. T cells are readily isolated from patients; however, the yield of antigen-specific T cells is limited, thus making their clinical use challenging. Therefore, the generation of T lymphocytes from hematopoietic stem/progenitor cells (HSPCs) and human pluripotent stem cells (PSCs) in vitro provides an attractive method for large-scale pr...

  20. 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

    2017-12-01

    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.

  1. Responses of Cells to Flow in Vitro

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    Shigehiro Hashimoto

    2013-06-01

    Full Text Available The response of cells to a flow has been studied in vitro. The response of cells was examined in two types of flow channels: a circumnutating flow in a donut-shaped open channel in a culture dish, and a one-way flow in a parallelepiped rhombus flow channel. Variation was made on the material of the parallelepiped channel to study on adhesion of cells to the plates: glass and polydimethylsiloxane. Behavior of cells on the plate was observed under a flow of a medium with an inverted phase-contrast-microscope. The shear stress on the plate is calculated with an estimated parabolic distribution of the velocity between the parallel plates. The adhesion of cells was evaluated with the cumulated shear, which is a product of the shear stress and the exposure time. The experimental results show that cells are responsive to the flow, which governs orientation, exfoliation, and differentiation. The response depends on the kinds of cells: endothelial cells orient along the stream line, although myocytes orient perpendicular to the stream line. The adhesion depends on the combination between scaffold and cell: myocytes are more adhesive to glass than cartilage cells, and fibroblasts are more adhesive to oxygenated polydimethylsiloxane than glass.

  2. Assessing nanotoxicity in cells in vitro.

    Science.gov (United States)

    Hillegass, Jedd M; Shukla, Arti; Lathrop, Sherrill A; MacPherson, Maximilian B; Fukagawa, Naomi K; Mossman, Brooke T

    2010-01-01

    Nanomaterials are commonly defined as particles or fibers of less than 1 microm in diameter. For these reasons, they may be respirable in humans and have the potential, based upon their geometry, composition, size, and transport or durability in the body, to cause adverse effects on human health, especially if they are inhaled at high concentrations. Rodent inhalation models to predict the toxicity and pathogenicity of nanomaterials are prohibitive in terms of time and expense. For these reasons, a panel of in vitro assays is described below. These include cell culture assays for cytotoxicity (altered metabolism, decreased growth, lytic or apoptotic cell death), proliferation, genotoxicity, and altered gene expression. The choice of cell type for these assays may be dictated by the procedure or endpoint selected. Most of these assays have been standardized in our laboratory using pathogenic minerals (asbestos and silica) and non-pathogenic particles (fine titanium dioxide or glass beads) as negative controls. The results of these in vitro assays should predict whether testing of selected nanomaterials should be pursued in animal inhalation models that simulate physiologic exposure to inhaled nanomaterials. Conversely, intrathoracic or intrapleural injection of nanomaterials into rodents can be misleading because they bypass normal clearance mechanisms, and non-pathogenic fibers and particles can test positively in these assays.

  3. Cell-to-Cell Contact Results in a Selective Translocation of Maternal Human Immunodeficiency Virus Type 1 Quasispecies across a Trophoblastic Barrier by both Transcytosis and Infection

    Science.gov (United States)

    Lagaye, S.; Derrien, M.; Menu, E.; Coïto, C.; Tresoldi, E.; Mauclère, P.; Scarlatti, G.; Chaouat, G.; Barré-Sinoussi, F.; Bomsel, M.

    2001-01-01

    Mother-to-child transmission can occur in utero, mainly intrapartum and postpartum in case of breastfeeding. In utero transmission is highly restricted and results in selection of viral variant from the mother to the child. We have developed an in vitro system that mimics the interaction between viruses, infected cells present in maternal blood, and the trophoblast, the first barrier protecting the fetus. Trophoblastic BeWo cells were grown as a tight polarized monolayer in a two-chamber system. Cell-free virions applied to the apical pole neither crossed the barrier nor productively infected BeWo cells. In contrast, apical contact with human immunodeficiency virus (HIV)-infected peripheral blood mononuclear cells (PBMCs) resulted in transcytosis of infectious virus across the trophoblastic monolayer and in productive infection correlating with the fusion of HIV-infected PBMCs with trophoblasts. We showed that viral variants are selected during these two steps and that in one case of in utero transmission, the predominant maternal viral variant characterized after transcytosis was phylogenetically indistinguishable from the predominant child's virus. Hence, the first steps of transmission of HIV-1 in utero appear to involve the interaction between HIV type 1-infected cells and the trophoblastic layer, resulting in the passage of infectious HIV by transcytosis and by fusion/infection, both leading to a selection of virus quasispecies. PMID:11312350

  4. In vitro differentiation of mouse embryonic stem cells into functional ...

    African Journals Online (AJOL)

    Jane

    2011-08-22

    Aug 22, 2011 ... hepatocyte transplantation therapy and toxicity screening in drug discovery. Key words: Embryonic stem cells, hepatic-like cells, in vitro differentiation, sodium butyrate, ... from embryonic stem (ES) cell or induced pluripotent.

  5. Placental transport and in vitro effects of Bisphenol A

    DEFF Research Database (Denmark)

    Mørck, Thit J; Sorda, Giuseppina; Bechi, Nicoletta

    2010-01-01

    Bisphenol A (BPA), an estrogen-like chemical, leaches from consumer products potentially causing human exposure. To examine the effects of BPA exposure during pregnancy, we performed studies using the BeWo trophoblast cell line, placental explant cultures, placental perfusions and skin diffusion...... transfer of BPA was observed across the term placentae and the BeWo cell monolayer. Further, transdermal transport of BPA was observed. These results indicate that fetal BPA exposure through placental exchange occurs with potential adverse implications for placental and fetal development. This battery...

  6. In Vitro Cardiomyogenic Potential of Human Amniotic Fluid Stem Cells

    OpenAIRE

    Guan, Xuan; Delo, Dawn M.; Atala, Anthony; Soker, Shay

    2011-01-01

    Stem cell therapy for damaged cardiac tissue is currently limited by a number of factors, including the inability to obtain sufficient cell numbers, the potential tumorigenicity of certain types of stem cells, and the possible link between stem cell therapy and the development of malignant arrhythmias. In this study, we investigated whether human amniotic fluid-derived stem (hAFS) cells could be a potential source of cells for cardiac cell therapy by testing the in vitro differentiation capab...

  7. In vitro repopulation of haemopoietic stem cells after irradiation

    International Nuclear Information System (INIS)

    Mori, K.J.; Kumagai, Keiko; Seto, Akira; Ito, Yohei

    1981-01-01

    A culture system was designed in which proliferation of the haemopoietic stem cells was supported by adherent 'stromal' cell colonies. Application of the culture system to studies on kinetic behaviour of the haemopoietic stem cells after irradiation revealed; i) bone marrow stromal cells were radiosensitive with D 0 = 95R, when measured as the capability to proliferate and form adherent cell colonies in vitro, ii) radiosensitivity of the pluripotent stem cells (CFUs) in vitro was within the range of the in vivo sensitivity, iii) irradiated bone marrow cells under in vitro condition could repopulate at the same rate as those under in vivo condition, thereby suggesting that the function related to the support of haemopoiesis was radioresistant, iv) concentrations of both CFUs and granulocyte-macrophage precursor cells (CFUc) were higher in the irradiated cultures than those in unirradiated control culture at 3 weeks after irradiation. (author)

  8. X-ray sensitivity of human tumor cells in vitro

    International Nuclear Information System (INIS)

    Weichselbaum, R.R.; Nove, J.; Little, J.B.

    1980-01-01

    Clonally-derived cells from ten human malignant tumors considered radiocurable (breast, neuroblastoma, medulloblastoma) or non-radiocurable (osteosarcoma, hypernephroma, glioblastoma, melanoma) were studied in cell culture and their in vitro x-ray survival curve parameters determined (anti n, D 0 ). There were no significant differences among the tumor cell lines suggesting that survival parameters in vitro do not explain differences in clinical radiocurability. Preliminary investigation with density inhibited human tumor cells indicate that such an approach may yield information regarding inherent cellular differences in radiocurability

  9. Immunogenicity of ascites tumor cells following in vitro hyperthermia

    International Nuclear Information System (INIS)

    Dickson, J.A.; Jasiewicz, M.L.; Simpson, A.C.

    1982-01-01

    The concept that host immunization may be achieved by heat-induced antigenic modifications of cancer cells and/or the release of immunogenic products by dead or dying tumor cells following in vitro heating was examined. Ehrlich ascites cells were used, inasmuch as it was claimed that in vitro hyperthermia increased the immunogenicity of these cells. Tumor cell populations of different viability were obtained by heating Ehrlich cells at 42.5 degrees, 45 degrees, or 60 degrees C. Viable and nonviable cells were separated by Ficoll-Hypaque density centrifugation; viable nonreplicating cells were obtained by treatment with mitomycin C. Cell populations of different viability after heating were left to die slowly over 3 days at 37 degrees C. Swiss TO mice were then given injections of the treated cells and/or medium. No survival benefit occurred in mice inoculated with any of these different components and then challenged with viable tumor cells. Injection of irradiated cells, however, did produce host immunity. Similarly, D23 rat hepatoma ascites cells produced host immunity after 15,000 rad but not after heating. The claim that in vitro hyperthermia increases the immunogenicity of tumor cells was not confirmed

  10. Propagation of bovine spermatogonial stem cells in vitro

    NARCIS (Netherlands)

    Aponte, Pedro M.; Soda, Takeshi; Teerds, Katja J.; Mizrak, S. Canan; van de Kant, Henk J. G.; de Rooij, Dirk G.

    2008-01-01

    The access to sufficient numbers of spermatogonial stem cells (SSCs) is a prerequisite for the study of their regulation and further biomanipulation. A specialized medium and several growth factors were tested to study the in vitro behavior of bovine type A spermatogonia, a cell population that

  11. Pluripotent stem cells: An in vitro model for nanotoxicity assessments.

    Science.gov (United States)

    Handral, Harish K; Tong, Huei Jinn; Islam, Intekhab; Sriram, Gopu; Rosa, Vinicus; Cao, Tong

    2016-10-01

    The advent of technology has led to an established range of engineered nanoparticles that are used in diverse applications, such as cell-cell interactions, cell-material interactions, medical therapies and the target modulation of cellular processes. The exponential increase in the utilization of nanomaterials and the growing number of associated criticisms has highlighted the potential risks of nanomaterials to human health and the ecosystem. The existing in vivo and in vitro platforms show limitations, with fluctuations being observed in the results of toxicity assessments. Pluripotent stem cells (PSCs) are viable source of cells that are capable of developing into specialized cells of the human body. PSCs can be efficiently used to screen new biomaterials/drugs and are potential candidates for studying impairments of biophysical morphology at both the cellular and tissue levels during interactions with nanomaterials and for diagnosing toxicity. Three-dimensional in vitro models obtained using PSC-derived cells would provide a realistic, patient-specific platform for toxicity assessments and in drug screening applications. The current review focuses on PSCs as an alternative in vitro platform for assessing the hazardous effects of nanomaterials on health systems and highlights the importance of PSC-derived in vitro platforms. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  12. Propagation of human spermatogonial stem cells in vitro.

    Science.gov (United States)

    Sadri-Ardekani, Hooman; Mizrak, Sefika C; van Daalen, Saskia K M; Korver, Cindy M; Roepers-Gajadien, Hermien L; Koruji, Morteza; Hovingh, Suzanne; de Reijke, Theo M; de la Rosette, Jean J M C H; van der Veen, Fulco; de Rooij, Dirk G; Repping, Sjoerd; van Pelt, Ans M M

    2009-11-18

    Young boys treated with high-dose chemotherapy are often confronted with infertility once they reach adulthood. Cryopreserving testicular tissue before chemotherapy and autotransplantation of spermatogonial stem cells at a later stage could theoretically allow for restoration of fertility. To establish in vitro propagation of human spermatogonial stem cells from small testicular biopsies to obtain an adequate number of cells for successful transplantation. Study performed from April 2007 to July 2009 using testis material donated by 6 adult men who underwent orchidectomy as part of prostate cancer treatment. Testicular cells were isolated and cultured in supplemented StemPro medium; germline stem cell clusters that arose were subcultured on human placental laminin-coated dishes in the same medium. Presence of spermatogonia was determined by reverse transcriptase polymerase chain reaction and immunofluorescence for spermatogonial markers. To test for the presence of functional spermatogonial stem cells in culture, xenotransplantation to testes of immunodeficient mice was performed, and migrated human spermatogonial stem cells after transplantation were detected by COT-1 fluorescence in situ hybridization. The number of colonized spermatogonial stem cells transplanted at early and later points during culture were counted to determine propagation. Propagation of spermatogonial stem cells over time. Testicular cells could be cultured and propagated up to 15 weeks. Germline stem cell clusters arose in the testicular cell cultures from all 6 men and could be subcultured and propagated up to 28 weeks. Expression of spermatogonial markers on both the RNA and protein level was maintained throughout the entire culture period. In 4 of 6 men, xenotransplantation to mice demonstrated the presence of functional spermatogonial stem cells, even after prolonged in vitro culture. Spermatogonial stem cell numbers increased 53-fold within 19 days in the testicular cell culture and

  13. 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. © 2015 International Federation for Cell Biology.

  14. Selective eradication of cancer cells in vitro

    International Nuclear Information System (INIS)

    Schneiderman, M.H.; Schneiderman, G.S.

    1984-01-01

    A simple system consisting of cultured HeLa (human cancer) and WI38 (normal human fetal lung) cells and the control cultures of the individual cells were set up to test and compare the effects of the cell cycle-active agents /sup 125/I-iododeoxyuridine (/sup 125/IUdR) and hydroxyurea (HU) on cell survival. The presence of cells and growth after treatment were used as a positive indication of survival. The experimental cultures were first seeded with WI38 cells and allowed to grow to confluency before adding 1.0 x 10/sup 5/ HeLa cells. After two days of treatment-free growth, the co-cultures were continuously treated with /sup 125/IUdR (0.5-2.0 μCi/ml, carrier free) or HU (1.0 x 10/sup -9/ and 1.0 x 10/sup -3/M). At the termination of treatment the co-cultures were split 3 to 1 and incubated for seven days. As expected, there was little or no detectable effect on the growth of WI38 cells treated with HU or /sup 125/IUdR while the cells were confluent. However, HeLa cells were reduced by 1.0 x 10/sup -3/M HU and were eradicated after all concentrations of /sup 125/IUdR

  15. Properties of acatalasic cells growing in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Krooth, R S; Howell, R R; Hamilton, H B

    1961-07-19

    Acatalasia, a disease due to homozygosity for a Mendelian gene, is characterized by the absence of the enzyme catalase from the tissues of the human body. Red cells from heterozygotes have enzyme activities about one-half normal. In this report, the development of cell lines from skin biopsies of an affected homozygote, a heterozygote and eight control patients is described. The cell type is the euploid fibroblast. It was found that acatalasic cells lacked the enzyme, even after growing for many months in a medium rich in catalase. The control lines all had mean catalase activity double or more that of the heterozygous line. Selection experiments, measuring growth of cells exposed for 20 minutes to varying concentrations of hydrogen peroxide, did not provide a system for preferentially eliminating acatalastic cells. Certain other experiments were performed bearing on the enzymatic defect in this disease. 23 references, 7 figures, 6 tables.

  16. Individual fates of mesenchymal stem cells in vitro

    Directory of Open Access Journals (Sweden)

    Drasdo Dirk

    2010-05-01

    Full Text Available Abstract Background In vitro cultivated stem cell populations are in general heterogeneous with respect to their expression of differentiation markers. In hematopoietic progenitor populations, this heterogeneity has been shown to regenerate within days from isolated subpopulations defined by high or low marker expression. This kind of plasticity has been suggested to be a fundamental feature of mesenchymal stem cells (MSCs as well. Here, we study MSC plasticity on the level of individual cells applying a multi-scale computer model that is based on the concept of noise-driven stem cell differentiation. Results By simulation studies, we provide detailed insight into the kinetics of MSC organisation. Monitoring the fates of individual cells in high and low oxygen culture, we calculated the average transition times of individual cells into stem cell and differentiated states. We predict that at low oxygen the heterogeneity of a MSC population with respect to differentiation regenerates from any selected subpopulation in about two days. At high oxygen, regeneration becomes substantially slowed down. Simulation results on the composition of the functional stem cell pool of MSC populations suggest that most of the cells that constitute this pool originate from more differentiated cells. Conclusions Individual cell-based models are well-suited to provide quantitative predictions on essential features of the spatio-temporal organisation of MSC in vitro. Our predictions on MSC plasticity and its dependence on the environment motivate a number of in vitro experiments for validation. They may contribute to a better understanding of MSC organisation in vitro, including features of clonal expansion, environmental adaptation and stem cell ageing.

  17. In vitro senescence of immune cells.

    Science.gov (United States)

    Effros, Rita B; Dagarag, Mirabelle; Valenzuela, Hector F

    2003-01-01

    Immune cells are eminently suitable model systems in which to address the possible role of replicative senescence during in vivo aging. Since there are more than 10(8) unique antigen specificities present within the total T lymphocyte population of each individual, the immune response to any single antigen requires massive clonal expansion of the small proportion of T cells whose receptors recognize that antigen. The Hayflick Limit may, therefore, constitute a barrier to effective immune function, at least for those T cells that encounter their specific antigen more than once over the life course. Application of the fibroblast replicative senescence model to the so-called cytotoxic or CD8 T cell, the class of T cells that controls viral infection and cancer, has revealed certain features in common with other cell types as well as several characteristics that are unique to T cells. One senescence-associated change that is T cell-specific is the complete loss of expression of the activation signaling surface molecule, CD28, an alteration that enabled the documentation of high proportions of senescent T cells in vivo. The T cell model has also provided the unique opportunity to analyze telomere dynamics in a cell type that has the ability to upregulate telomerase yet nevertheless undergoes senescence. The intimate involvement of the immune system in the control of pathogens and cancer as well as in modulation of bone homeostasis suggests that more extensive analysis of the full range of characteristics of senescent T cells may help elucidate a broad spectrum of age-associated physiological changes.

  18. Cell sources for in vitro human liver cell culture models

    Science.gov (United States)

    Freyer, Nora; Damm, Georg; Seehofer, Daniel; Knöspel, Fanny

    2016-01-01

    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. PMID:27385595

  19. Quercetin inhibits adipogenesis of muscle progenitor cells in vitro

    Directory of Open Access Journals (Sweden)

    Tomoko Funakoshi

    2018-03-01

    Full Text Available Muscle satellite cells are committed myogenic progenitors capable of contributing to myogenesis to maintain adult muscle mass and function. Several experiments have demonstrated that muscle satellite cells can differentiate into adipocytes in vitro, supporting the mesenchymal differentiation potential of these cells. Moreover, muscle satellite cells may be a source of ectopic muscle adipocytes, explaining the lipid accumulation often observed in aged skeletal muscle (sarcopenia and in muscles of patients` with diabetes. Quercetin, a polyphenol, is one of the most abundant flavonoids distributed in edible plants, such as onions and apples, and possesses antioxidant, anticancer, and anti-inflammatory properties. In this study, we examined whether quercetin inhibited the adipogenesis of muscle satellite cells in vitro with primary cells from rat limbs by culture in the presence of quercetin under adipogenic conditions. Morphological observations, Oil Red-O staining results, triglyceride content analysis, and quantitative reverse transcription polymerase chain reaction revealed that quercetin was capable of inhibiting the adipogenic induction of muscle satellite cells into adipocytes in a dose-dependent manner by suppressing the transcript levels of adipogenic markers, such as peroxisome proliferator-activated receptor-γ and fatty acid binding protein 4. Our results suggested that quercetin inhibited the adipogenesis of muscle satellite cells in vitro by suppressing the transcription of adipogenic markers. Keywords: Quercetin, Muscle satellite cell, Differentiation, Intramuscular lipid

  20. Lactobacilli Modulate Natural Killer Cell Responses In Vitro

    DEFF Research Database (Denmark)

    Fink, Lisbeth Nielsen; Christensen, Hanne Risager; Frøkiær, Hanne

    of certain lactic acid bacteria has been shown to increase in vivo NK cytotoxicity. Here, we investigated how human gut flora-derived lactobacilli affect NK cells in vitro, by measuring proliferation and IFN-gamma production of human NK cells upon bacterial stimulation. CD3-CD56+ NK cells were isolated from...... monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the NK cells. In contrast, a Lactobacillus paracasei strain caused the NK cells to proliferate only in the presence of monocytes. These results demonstrate that various strains of lactobacilli have...

  1. Radiation effects in mammalian cells in vitro

    International Nuclear Information System (INIS)

    Hill, C.K.; Han, A.; Elkind, M.M.; Wells, R.L.; Buess, E.M.; Lin, C.M.

    1985-01-01

    The purpose of this research effort is to elucidate the mechanisms for the radiation-induced changes in mammalian cells that lead to cell death, mutation, neoplastic transformation, DNA damage, and chromosomal alterations. Of particular interest are the effects of low-dose-rate and fractionated irradiation on these end points with respect to the mechanisms whereby these effects are influenced by cellular repair processes, inhibitors, and promoters that act at the genetic or biochemical level. 17 refs

  2. Induction of suppressor cells in vitro by Candida albicans.

    Science.gov (United States)

    Cuff, C F; Rogers, C M; Lamb, B J; Rogers, T J

    1986-06-01

    Normal splenocytes cultured with Formalin-killed Candida albicans were shown to acquire significant suppressor cell activity in a period of 3 days. These cells were found to suppress both the phytohemagglutinin-induced mitogen response as well as the anti-sheep erythrocyte antibody response. Experiments were carried out to determine the nature of the suppressor cell population. Results showed that these cells were not susceptible to treatment with anti-Thy 1 antibody and complement. Panning experiments showed that the suppressor cells were not plastic-adherent or Mac-1 antigen-positive. The suppressor cells were, however, adherent to anti-mouse immunoglobulin (F(ab')2-fragment)-coated dishes. Additional experiments showed that the suppressor cell activity was susceptible to treatment with monoclonal anti-Lyb 2.1 antibody and complement. These results suggest that the suppressor cell induced in vitro by Candida is a member of the B-lymphocyte lineage.

  3. SOMATIC MITOSES IN CELLS OF PICEA GLUCA CULTIVATED IN VITRO.

    Science.gov (United States)

    RISSER, P G

    1964-02-07

    The cytology of one strain of tumor tissue from the spruce tree, Picea glauca, grown in vitro for more than a year was examined. The cells of this strain are characterized by a uniform chromosome number of 24, and the strain appears to be quite stable. The implications of the results of this study and previous studies on similar material are discussed.

  4. Mechanotransduction by bone cells in vitro: mechanobiology of bone tissue

    NARCIS (Netherlands)

    Mullender, M.; El Haj, A.J.; Yang, Y.; van Duin, M.A.; Burger, E.H.; Klein-Nulend, J.

    2004-01-01

    Mechanical force plays an important role in the regulation of bone remodelling in intact bone and bone repair. In vitro, bone cells demonstrate a high responsiveness to mechanical stimuli. Much debate exists regarding the critical components in the load profile and whether different components, such

  5. In vitro germ cell differentiation from cynomolgus monkey embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Kaori Yamauchi

    Full Text Available BACKGROUND: Mouse embryonic stem (ES cells can differentiate into female and male germ cells in vitro. Primate ES cells can also differentiate into immature germ cells in vitro. However, little is known about the differentiation markers and culture conditions for in vitro germ cell differentiation from ES cells in primates. Monkey ES cells are thus considered to be a useful model to study primate gametogenesis in vitro. Therefore, in order to obtain further information on germ cell differentiation from primate ES cells, this study examined the ability of cynomolgus monkey ES cells to differentiate into germ cells in vitro. METHODS AND FINDINGS: To explore the differentiation markers for detecting germ cells differentiated from ES cells, the expression of various germ cell marker genes was examined in tissues and ES cells of the cynomolgus monkey (Macaca fascicularis. VASA is a valuable gene for the detection of germ cells differentiated from ES cells. An increase of VASA expression was observed when differentiation was induced in ES cells via embryoid body (EB formation. In addition, the expression of other germ cell markers, such as NANOS and PIWIL1 genes, was also up-regulated as the EB differentiation progressed. Immunocytochemistry identified the cells expressing stage-specific embryonic antigen (SSEA 1, OCT-4, and VASA proteins in the EBs. These cells were detected in the peripheral region of the EBs as specific cell populations, such as SSEA1-positive, OCT-4-positive cells, OCT-4-positive, VASA-positive cells, and OCT-4-negative, VASA-positive cells. Thereafter, the effect of mouse gonadal cell-conditioned medium and growth factors on germ cell differentiation from monkey ES cells was examined, and this revealed that the addition of BMP4 to differentiating ES cells increased the expression of SCP1, a meiotic marker gene. CONCLUSION: VASA is a valuable gene for the detection of germ cells differentiated from ES cells in monkeys, and the

  6. Cylindromatosis mediates neuronal cell death in vitro and in vivo.

    Science.gov (United States)

    Ganjam, Goutham K; Terpolilli, Nicole Angela; Diemert, Sebastian; Eisenbach, Ina; Hoffmann, Lena; Reuther, Christina; Herden, Christiane; Roth, Joachim; Plesnila, Nikolaus; Culmsee, Carsten

    2018-01-19

    The tumor-suppressor cylindromatosis (CYLD) is a deubiquitinating enzyme and key regulator of cell proliferation and inflammation. A genome-wide siRNA screen linked CYLD to receptor interacting protein-1 (RIP1) kinase-mediated necroptosis; however, the exact mechanisms of CYLD-mediated cell death remain unknown. Therefore, we investigated the precise role of CYLD in models of neuronal cell death in vitro and evaluated whether CYLD deletion affects brain injury in vivo. In vitro, downregulation of CYLD increased RIP1 ubiquitination, prevented RIP1/RIP3 complex formation, and protected neuronal cells from oxidative death. Similar protective effects were achieved by siRNA silencing of RIP1 or RIP3 or by pharmacological inhibition of RIP1 with necrostatin-1. In vivo, CYLD knockout mice were protected from trauma-induced brain damage compared to wild-type littermate controls. These findings unravel the mechanisms of CYLD-mediated cell death signaling in damaged neurons in vitro and suggest a cell death-mediating role of CYLD in vivo.

  7. A review of human cell radiosensitivity in vitro

    International Nuclear Information System (INIS)

    Deschavanne, Patrick J.; Fertil, Bernard

    1996-01-01

    The survival curves of 694 human cell lines irradiated in exponentially growing phase in vitro were collected from the literature. Among them, 271 were derived from tumors, 423 were nontransformed fibroblasts and other normal cell strains from healthy people or people with some genetic disorders. Seventy-six different cell types are identified, and a specific radiosensitivity could be associated with each, using D-bar and surviving fraction at 2 Gy. Technical factors such as culture medium, feeder cells, and scoring method were found to affect intrinsic radiosensitivity. In particular, the cell type is not a discriminating factor when cells are studied in agar. Results obtained with cells irradiated in agar must be used cautiously, depending on how the cells were prepared for the experiments. The use of feeder cells narrows the range of radiosensitivity of human cells. For cells irradiated as monolayer, it was possible to build a scale of radiosensitivity according to cell type, ranging, in terms of D-bar from 0.6 Gy for the most sensitive cell lines to more than 4 Gy for the most resistant. Considering that, in most cases, we could estimate the variation of radiosensitivity within each cell type, our classification among cell types can be used by researchers to place their results in the context of the literature

  8. In vitro proliferation of adult human beta-cells.

    Directory of Open Access Journals (Sweden)

    Sabine Rutti

    Full Text Available A decrease in functional beta-cell mass is a key feature of type 2 diabetes. Glucagon-like peptide 1 (GLP-1 analogues induce proliferation of rodent beta-cells. However, the proliferative capacity of human beta-cells and its modulation by GLP-1 analogues remain to be fully investigated. We therefore sought to quantify adult human beta-cell proliferation in vitro and whether this is affected by the GLP-1 analogue liraglutide.Human islets from 7 adult cadaveric organ donors were dispersed into single cells. Beta-cells were purified by FACS. Non-sorted cells and the beta-cell enriched ("beta-cells" population were plated on extracellular matrix from rat (804G and human bladder carcinoma cells (HTB9 or bovine corneal endothelial ECM (BCEC. Cells were maintained in culture+/-liraglutide for 4 days in the presence of BrdU.Rare human beta-cell proliferation could be observed either in the purified beta-cell population (0.051±0.020%; 22 beta-cells proliferating out of 84'283 beta-cells counted or in the non-sorted cell population (0.055±0.011%; 104 proliferating beta-cells out of 232'826 beta-cells counted, independently of the matrix or the culture conditions. Liraglutide increased human beta-cell proliferation on BCEC in the non-sorted cell population (0.082±0.034% proliferating beta-cells vs. 0.017±0.008% in control, p<0.05.These results indicate that adult human beta-cell proliferation can occur in vitro but remains an extremely rare event with these donors and particular culture conditions. Liraglutide increases beta-cell proliferation only in the non-sorted cell population and only on BCEC. However, it cannot be excluded that human beta-cells may proliferate to a greater extent in situ in response to natural stimuli.

  9. Veratridine increases the survival of retinal ganglion cells in vitro

    Directory of Open Access Journals (Sweden)

    S.P.F. Pereira

    1997-12-01

    Full Text Available Neuronal cell death is an important phenomenon involving many biochemical pathways. This degenerative event has been studied to understand how the cells activate the mechanisms that lead to self-destruction. Target cells and afferent cells play a relevant role in the regulation of natural cell death. We studied the effect of veratridine (1.5, 3.0, 4.5 and 6.0 µM on the survival of neonatal rat retinal ganglion cells in vitro. Veratridine (3.0 µM, a well-known depolarizing agent that opens the Na+ channel, promoted a two-fold increase in the survival of retinal ganglion cells kept in culture for 48 h. This effect was dose-dependent and was blocked by 1.0 µM tetrodotoxin (a classical voltage-dependent Na+ channel blocker and 30.0 µM flunarizine (a Na+ and Ca2+ channel blocker. These results indicate that electrical activity is also important for the maintenance of retinal ganglion cell survival in vitro

  10. Isolation and In Vitro Characterization of Epidermal Stem Cells

    DEFF Research Database (Denmark)

    Moestrup, Kasper S; Andersen, Marianne Stemann; Jensen, Kim Bak

    2017-01-01

    flow cytometry. Using markers that define the spatial origin of epidermal cells, it is possible to interrogate the specific characteristics of subpopulations of cells based on their in vivo credentials. Here, we describe how to isolate, culture, and characterize keratinocytes from murine back and tail......Colony-forming assays represent prospective methods, where cells isolated from enzymatically dissociated tissues or from tissue cultures are assessed for their proliferative capacity in vitro. Complex tissues such as the epithelial component of the skin (the epidermis) are characterized...

  11. In vitro regeneration of kidney from pluripotent stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Osafune, Kenji, E-mail: osafu@cira.kyoto-u.ac.jp [Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507 (Japan); PRESTO, Japan Science and Technology Agency (JST), 4-1-8 Honcho, Kawaguchi, Saitama 332-0012 (Japan); JST Yamanaka iPS Cell Special Project, Japan Science and Technology Agency (JST), 4-1-8 Honcho, Kawaguchi, Saitama 332-0012 (Japan)

    2010-10-01

    Although renal transplantation has proved a successful treatment for the patients with end-stage renal failure, the therapy is hampered by the problem of serious shortage of donor organs. Regenerative medicine using stem cells, including cell transplantation therapy, needs to be developed to solve the problem. We previously identified the multipotent progenitor cells in the embryonic mouse kidney that can give rise to several kinds of epithelial cells found in adult kidney, such as glomerular podocytes and renal tubular epithelia. Establishing the method to generate the progenitors from human pluripotent stem cells that have the capacity to indefinitely proliferate in vitro is required for the development of kidney regeneration strategy. We review the current status of the research on the differentiation of pluripotent stem cells into renal lineages and describe cues to promote this research field.

  12. In vitro regeneration of kidney from pluripotent stem cells

    International Nuclear Information System (INIS)

    Osafune, Kenji

    2010-01-01

    Although renal transplantation has proved a successful treatment for the patients with end-stage renal failure, the therapy is hampered by the problem of serious shortage of donor organs. Regenerative medicine using stem cells, including cell transplantation therapy, needs to be developed to solve the problem. We previously identified the multipotent progenitor cells in the embryonic mouse kidney that can give rise to several kinds of epithelial cells found in adult kidney, such as glomerular podocytes and renal tubular epithelia. Establishing the method to generate the progenitors from human pluripotent stem cells that have the capacity to indefinitely proliferate in vitro is required for the development of kidney regeneration strategy. We review the current status of the research on the differentiation of pluripotent stem cells into renal lineages and describe cues to promote this research field.

  13. In vitro propagation of male germline stem cells from piglets.

    Science.gov (United States)

    Zheng, Yi; Tian, Xiue; Zhang, Yaqing; Qin, Jinzhou; An, Junhui; Zeng, Wenxian

    2013-07-01

    To study the effects of serum and growth factors on propagation of porcine male germline stem cells (MGSCs) in vitro and develop a culture system for these stem cells. Fresh testicular cells from neonatal piglets were obtained by mechanical dissociation and collagenase-trypsin digestion. After differential plating, non-adhering cells were cultured in media supplemented with different concentrations of serum (0, 1 %, 2 %, 5 %, 10 %). After 10 days of primary culture, the cells were maintained in media supplemented with different concentrations of growth factors (basic fibroblast growth factor and epidermal growth factor at 1, 5, 10 ng/ml). The number of MGSC-derived colonies with different sizes was determined in each treatment to assess the effects of serum concentrations and growth factors. The number of MGSC-derived colonies was significantly higher in the presence of 1 % rather than 10 % fetal bovine serum (FBS). Basic fibroblast growth factor (bFGF) at 1, 5 ng/ml and epidermal growth factor (EGF) at 5, 10 ng/ml significantly promoted colony formation. Immunocytochemistry, reverse transcriptase-polymerase chain reaction (RT-PCR) and xenotransplantation assays demonstrated the presence of functional stem cells in cultured cell population. In vitro propagation of porcine MGSCs could be maintained in the presence of 1 % FBS and supplementation of growth factors for 1 month.

  14. Growing Arabidopsis in vitro: cell suspensions, in vitro culture, and regeneration.

    Science.gov (United States)

    Barkla, Bronwyn J; Vera-Estrella, Rosario; Pantoja, Omar

    2014-01-01

    An understanding of basic methods in Arabidopsis tissue culture is beneficial for any laboratory working on this model plant. Tissue culture refers to the aseptic growth of cells, organs, or plants in a controlled environment, in which physical, nutrient, and hormonal conditions can all be easily manipulated and monitored. The methodology facilitates the production of a large number of plants that are genetically identical over a relatively short growth period. Techniques, including callus production, cell suspension cultures, and plant regeneration, are all indispensable tools for the study of cellular biochemical and molecular processes. Plant regeneration is a key technology for successful stable plant transformation, while cell suspension cultures can be exploited for metabolite profiling and mining. In this chapter we report methods for the successful and highly efficient in vitro regeneration of plants and production of stable cell suspension lines from leaf explants of both Arabidopsis thaliana and Arabidopsis halleri.

  15. Radiation effects on human glia and glioma cells in vitro

    International Nuclear Information System (INIS)

    Nilsson, S.

    1983-01-01

    The radiosensitivity of human glia and glioma cells has been studied in vitro, and a new cloning method has been developed to overcome the difficulties due to the very low cloning efficiency of these cells. The cells were confined to small palladium areas surrounded by agarose, which increased the cell density, but kept the clones separated. Using this method, the glia cells were found to be very sensitive to gamma irradiation (D 0 =1.0-1.5 Gy and n=1) in comparision with the glioma cells (D 0 =1.5-2.5 Gy and n=3.5). The induction and repair of DNA strand breaks were studied with two DNA unwinding techniques. No differences between the two cell-lines were detected when induction and fast repair were studied with the single-labelling method, while the glioma cells showed less unrepaired DNA strand breaks than the glia cells after 1, 2 and 3 hours, when the double-labelling method was used. Detachment, attachment and growth kinetics were studied using the palladium-agarose cloning method. All of the glioma cell-lines studied, detached and attached themselves at rates higher than the normal diploid glia cell-lines. All of the cell-lines contained clones with different properties. Some clones were rapidly growing, others maintained a nearly constant number of cells or even decreased. The effects of chronic hypoxia were tested in a few experiments. Low oxygen tension in the culture medium reduced the rate of growth and the DNA synthesis of the glioma cells. The present study indicates that cultured human glioma cells are less radiosensitive than cultured glia cells. The palladium-agarose technique, enable studying growth kinetics detachment, attachment and radiosensitivity in a quantitative manner for cells with low cloning efficiency. (author)

  16. In vitro evaluation of a new nitrosourea, TCNU, against human small cell lung cancer cell lines

    DEFF Research Database (Denmark)

    Roed, H; Vindeløv, L L; Spang-Thomsen, M

    1987-01-01

    The cytotoxic activity of a new nitrosourea, TCNU, was compared with that of BCNU in five human small cell lung cancer cell lines in vitro. TCNU was found to be equivalent or inferior to BCNU when compared on a microgram to microgram basis. If the potential of in vitro phase II trials for selection...... of new drugs can be validated, it can be concluded that TCNU is not superior to other nitrosoureas for the treatment of SCCL....

  17. Induction of specific suppressor T cells in vitro

    International Nuclear Information System (INIS)

    Eardley, D.D.; Gershon, R.K.

    1976-01-01

    We describe conditions for generating sheep red blood cell-specific suppressor T cells in Mishell-Dutton cultures. The production of specific suppressor cells is favored by increasing antigen dose in the initial culture but can be produced by transferring more cells when lower doses of antigen are used. Transfer of small numbers of cells cultured with low doses of antigen leads to a specific helper effect. Transfer of large numbers of educated cells leads to nonspecific suppression. Suppression can be effected by the effluent cells from nylon wool columns which do not make detectable PFC. A fraction of these cells become resistant to treatment with anti-T cell sera and complement after culture. The suppressor cells are radiation sensitive and must be able to synthesize protein to suppress. They take 2 to 3 days of education to reach maximum suppressive efficiency and will not suppress cultures if added 2 to 3 days after culture initiation. Their production is favored by the absence of mercaptoethanol, suggesting that the observed suppression is not ''too much help.'' The ability to generate specific suppressor cells in vitro should be of great benefit in determining the factors that regulate their appearance in vivo

  18. In vitro cytotoxicity of chemical preservatives on human fibroblast cells

    Directory of Open Access Journals (Sweden)

    Daniel Gonsales Spindola

    2018-05-01

    Full Text Available ABSTRACT Preservatives are widely used substances that are commonly added to various cosmetic and pharmaceutical products to prevent or inhibit microbial growth. In this study, we compared the in vitro cytotoxicity of different types of currently used preservatives, including methylparaben, imidazolidinyl urea (IMU, and sodium benzoate, using the human newborn fibroblast cell line CCD1072Sk. Of the tested preservatives, only IMU induced a reduction in cell viability, as shown using the MTT assay and propidium iodide staining (IMU>methylparaben>sodium benzoate. IMU was shown to promote homeostatic alterations potentially related to the initiation of programed cell death, such as decreased mitochondrial membrane potential and caspase-3 activation, in the treated cells. Methylparaben and sodium benzoate were shown to have a very low cytotoxic activity. Taken together, our results suggest that IMU induces programed cell death in human fibroblasts by a canonical intrinsic pathway via mitochondrial perturbation and subsequent release of proapoptotic factors.

  19. In vitro cytotoxicity of nanoparticles in mammalian germline stem cells.

    Science.gov (United States)

    Braydich-Stolle, Laura; Hussain, Saber; Schlager, John J; Hofmann, Marie-Claude

    2005-12-01

    Gametogenesis is a complex biological process that is particularly sensitive to environmental insults such as chemicals. Many chemicals have a negative impact on the germline, either by directly affecting the germ cells, or indirectly through their action on the somatic nursing cells. Ultimately, these effects can inhibit fertility, and they may have negative consequences for the development of the offspring. Recently, nanomaterials such as nanotubes, nanowires, fullerene derivatives (buckyballs), and quantum dots have received enormous national attention in the creation of new types of analytical tools for biotechnology and the life sciences. Despite the wide application of nanomaterials, there is a serious lack of information concerning their impact on human health and the environment. Thus, there are limited studies available on toxicity of nanoparticles for risk assessment of nanomaterials. The purpose of this study was to assess the suitability of a mouse spermatogonial stem cell line as a model to assess nanotoxicity in the male germline in vitro. The effects of different types of nanoparticles on these cells were evaluated by light microscopy, and by cell proliferation and standard cytotoxicity assays. Our results demonstrate a concentration-dependent toxicity for all types of particles tested, whereas the corresponding soluble salts had no significant effect. Silver nanoparticles were the most toxic while molybdenum trioxide (MoO(3)) nanoparticles were the least toxic. Our results suggest that this cell line provides a valuable model with which to assess the cytotoxicity of nanoparticles in the germ line in vitro.

  20. Human CD8 T cells generated in vitro from hematopoietic stem cells are functionally mature

    Directory of Open Access Journals (Sweden)

    Zúñiga-Pflücker Juan

    2011-03-01

    Full Text Available Abstract Background T cell development occurs within the highly specialized thymus. Cytotoxic CD8 T cells are critical in adaptive immunity by targeting virally infected or tumor cells. In this study, we addressed whether functional CD8 T cells can be generated fully in vitro using human umbilical cord blood (UCB hematopoietic stem cells (HSCs in coculture with OP9-DL1 cells. Results HSC/OP9-DL1 cocultures supported the differentiation of CD8 T cells, which were TCR/CD3hi CD27hi CD1aneg and thus phenotypically resembled mature functional CD8 single positive thymocytes. These in vitro-generated T cells also appeared to be conventional CD8 cells, as they expressed high levels of Eomes and low levels of Plzf, albeit not identical to ex vivo UCB CD8 T cells. Consistent with the phenotypic and molecular characterization, upon TCR-stimulation, in vitro-generated CD8 T cells proliferated, expressed activation markers (MHC-II, CD25, CD38, secreted IFN-γ and expressed Granzyme B, a cytotoxic T-cell effector molecule. Conclusion Taken together, the ability to direct human hematopoietic stem cell or T-progenitor cells towards a mature functional phenotype raises the possibility of establishing cell-based treatments for T-immunodeficiencies by rapidly restoring CD8 effector function, thereby mitigating the risks associated with opportunistic infections.

  1. In Vitro Desensitization of Human Skin Mast Cells

    Science.gov (United States)

    Zhao, Wei; Gomez, Gregorio; Macey, Matthew; Kepley, Christopher L.

    2013-01-01

    Desensitization is a clinical procedure whereby incremental doses of a drug are administered over several hours to a sensitive patient until a therapeutic dose and clinical tolerance are achieved. Clinical tolerance may occur in part by attenuating the mast cell response. In the present study, primary human skin mast cells were used to establish and characterize an in vitro model of desensitization. Mast cells in culture were armed with allergen-specific (4-hydroxy-3-nitro-phenylacety and Der p2) and non-specific IgE antibodies, and then desensitized by incremental exposures to 4-hydroxy-3-nitrophenylacety-BSA. This desensitization procedure abrogated the subsequent degranulation response to the desensitizing allergen, to an unrelated allergen, and to IgG anti-FcεRI, but not to C5a, substance P, compound 48/80, and calcium ionophore. Desensitized cells regained their FcεRI-dependent degranulation capability by 24–48 h after free allergen had been removed. Therefore, sensitized human skin mast cells are reversibly desensitized in vitro by exposure to incremental doses of that allergen, which also cross-desensitizes them to an unrelated allergen. PMID:22009002

  2. Radiosensitization of hypoxic tumor cells in vitro by nitric oxide

    International Nuclear Information System (INIS)

    Griffin, Robert J.; Makepeace, Carol M.; Hur, Won-Joo; Song, Chang W.

    1996-01-01

    Purpose: The effects of nitric oxide (NO) on the radiosensitivity of SCK tumor cells in oxic and hypoxic environments in vitro were studied. Methods and Materials: NO was delivered to cell suspensions using the NO donors 2,2-diethyl-1-nitroso-oxyhydrazine sodium salt (DEA/NO), and a spermine/nitric oxide complex (SPER/NO), which release NO at half-lives of 2.1 min and 39 min at pH 7.4, respectively. The cells were suspended in media containing DEA/NO or SPER/NO for varying lengths of time under oxic or hypoxic conditions, irradiated, and the clonogenicity determined. Results: Both compounds markedly radiosensitized the hypoxic cells. The drug enhancement ratios (DER) for 0.1, 1.0, and 2.0 mM DEA/NO were 2.0, 2.3 and 3.0, respectively, and those for 0.1, 1.0, and 2.0 mM SPER/NO were 1.6, 2.3, and 2.8, respectively. Aerobic cells were not radiosensitized by DEA/NO or SPER/NO. When DEA/NO and SPER/NO were incubated in solution overnight to allow release of NO, they were found to have no radiosensitizing effect under hypoxic or oxic conditions indicating the sensitization by the NO donors was due to the NO molecule released from these drugs. At the higher concentrations, SPER/NO was found to be cytotoxic in aerobic conditions but not in hypoxic conditions. DEA/NO was only slightly toxic to the cells in both aerobic and hypoxic conditions. Conclusions: NO released from NO donors DEA/NO and SPER/NO is as effective as oxygen to radiosensitize hypoxic cells in vitro. Its application to the radiosensitization of hypoxic cells in solid tumors remains to be investigated

  3. Stimulation and support of haemopoietic stem cell proliferation by irradiated stroma cell colonies in bone marrow cell culture in vitro

    International Nuclear Information System (INIS)

    Mori, K.J.; Izumi, Hiroko; Seto, Akira

    1981-01-01

    A culture system was established in which haemopoietic stem cells can undergo a recovery proliferation after a depletion of the stem cells, completely in vitro. To elucidate the source of the stimulatory factors, normal bone marrow cells were overlayed on top of the irradiated adherent 'stromal' cell colonies in the bone marrow cell culture. This stimulated the proliferation of haemopoietic stem cells in the cultured cells in suspension. The present results indicate that the stromal cells produce factors which stimulate stem cell proliferation. Whether the stimulation is evoked by direct cell-cell interactions or by humoral factors is as yet to be studied. (author)

  4. Enteroantigen-presenting B cells efficiently stimulate CD4(+) T cells in vitro

    DEFF Research Database (Denmark)

    Schmidt, Esben Gjerløff Wedebye; Kristensen, Nanna Ny; Claesson, Mogens Helweg

    2011-01-01

    Presentation of enterobacterial antigens by antigen-presenting cells and activation of enteroantigen-specific CD4(+) T cells are considered crucial steps in inflammatory bowel disease (IBD) pathology. The detrimental effects of such CD4(+) T cells have been thoroughly demonstrated in models...... of colitis. Also, we have previously established an in vitro assay where murine enteroantigen-specific colitogenic CD4(+) CD25(-) T cells are activated by splenocytes pulsed with an enterobacterial extract....

  5. Stem Cells as In Vitro Model of Parkinson's Disease

    Directory of Open Access Journals (Sweden)

    Patricia L. Martínez-Morales

    2012-01-01

    Full Text Available Progress in understanding neurodegenerative cell biology in Parkinson's disease (PD has been hampered by a lack of predictive and relevant cellular models. In addition, the lack of an adequate in vitro human neuron cell-based model has been an obstacle for the uncover of new drugs for treating PD. The ability to generate induced pluripotent stem cells (iPSCs from PD patients and a refined capacity to differentiate these iPSCs into DA neurons, the relevant disease cell type, promises a new paradigm in drug development that positions human disease pathophysiology at the core of preclinical drug discovery. Disease models derived from iPSC that manifest cellular disease phenotypes have been established for several monogenic diseases, but iPSC can likewise be used for phenotype-based drug screens in complex diseases for which the underlying genetic mechanism is unknown. Here, we highlight recent advances as well as limitations in the use of iPSC technology for modelling PD “in a dish” and for testing compounds against human disease phenotypes in vitro. We discuss how iPSCs are being exploited to illuminate disease pathophysiology, identify novel drug targets, and enhance the probability of clinical success of new drugs.

  6. Generation of germ cells in vitro in the era of induced pluripotent stem cells.

    Science.gov (United States)

    Imamura, Masanori; Hikabe, Orie; Lin, Zachary Yu-Ching; Okano, Hideyuki

    2014-01-01

    Induced pluripotent stem cells (iPSCs) are stem cells that can be artificially generated via "cellular reprogramming" using gene transduction in somatic cells. iPSCs have enormous potential in stem-cell biology as they can give rise to numerous cell lineages, including the three germ layers. An evaluation of germ-line competency by blastocyst injection or tetraploid complementation, however, is critical for determining the developmental potential of mouse iPSCs towards germ cells. Recent studies have demonstrated that primordial germ cells obtained by the in vitro differentiation of iPSCs produce functional gametes as well as healthy offspring. These findings illustrate not only that iPSCs are developmentally similar to embryonic stem cells (ESCs), but also that somatic cells from adult tissues can produce gametes in vitro, that is, if they are reprogrammed into iPSCs. In this review, we discuss past and recent advances in the in vitro differentiation of germ cells using pluripotent stem cells, with an emphasis on ESCs and iPSCs. While this field of research is still at a stage of infancy, it holds great promises for investigating the mechanisms of germ-cell development, especially in humans, and for advancing reproductive and developmental engineering technologies in the future. © 2013 Wiley Periodicals, Inc.

  7. Lab on a chip automates in vitro cell culturing

    DEFF Research Database (Denmark)

    Perozziello, Gerardo; Møllenbach, Jacob; Laursen, Steen

    2012-01-01

    A novel in vitro fertilization system is presented based on an incubation chamber and a microfluidic device which serves as advanced microfluidic cultivation chamber. The flow is controlled by hydrostatic height differences and evaporation is avoided with help of mineral oil. Six patient compartm......A novel in vitro fertilization system is presented based on an incubation chamber and a microfluidic device which serves as advanced microfluidic cultivation chamber. The flow is controlled by hydrostatic height differences and evaporation is avoided with help of mineral oil. Six patient...... compartments allow six simultaneous temperature and pH controlled cultivations with 12 embryos with continuous logging of the monitoring data. Two media can be controlled with help of opening or closing of openings at the microfluidic disposable devices. The flow rates through the single cell compartments can...

  8. In vitro cell culture lethal dose submitted to gamma radiation

    Energy Technology Data Exchange (ETDEWEB)

    Moreno, Carolina S.; Rogero, Sizue O.; Rogero, Jose Roberto [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil)], e-mail: carolina_sm@hotmail.com; Ikeda, Tamiko I.; Cruz, Aurea S. [Instituto Adolfo Lutz, Sao Paulo, SP (Brazil)

    2009-07-01

    The present study was designed to evaluate the in vitro effect of gamma radiation in cell culture of mouse connective tissue exposed to different doses of gamma radiation and under several conditions. The cell viability was analyzed by neutral red uptake methodology. This assay was developed for establish a methodology to be used in the future in the study of resveratrol radioprotection. Resveratrol (3,4',5- trihydroxystilbene), a phenolic phytoalexin that occurs naturally in some spermatophytes, such as grapevines, in response to injury as fungal infections and exposure to ultraviolet light. In the wines this compound is found at high levels and is considered one of the highest antioxidant constituents. The intense antioxidant potential of resveratrol provides many pharmacological activities including cardioprotection, chemoprevention and anti-tumor effects. Our results demonstrated that {sup 60}Co gamma radiation lethal dose (LD50) on NCTC clone 929 cells was about 340Gy. (author)

  9. In vitro cell culture lethal dose submitted to gamma radiation

    International Nuclear Information System (INIS)

    Moreno, Carolina S.; Rogero, Sizue O.; Rogero, Jose Roberto; Ikeda, Tamiko I.; Cruz, Aurea S.

    2009-01-01

    The present study was designed to evaluate the in vitro effect of gamma radiation in cell culture of mouse connective tissue exposed to different doses of gamma radiation and under several conditions. The cell viability was analyzed by neutral red uptake methodology. This assay was developed for establish a methodology to be used in the future in the study of resveratrol radioprotection. Resveratrol (3,4',5- trihydroxystilbene), a phenolic phytoalexin that occurs naturally in some spermatophytes, such as grapevines, in response to injury as fungal infections and exposure to ultraviolet light. In the wines this compound is found at high levels and is considered one of the highest antioxidant constituents. The intense antioxidant potential of resveratrol provides many pharmacological activities including cardioprotection, chemoprevention and anti-tumor effects. Our results demonstrated that 60 Co gamma radiation lethal dose (LD50) on NCTC clone 929 cells was about 340Gy. (author)

  10. Effect of fractionated hyperthermia on hypoxic cells in vitro

    International Nuclear Information System (INIS)

    Nielson, O.S.

    1981-01-01

    The lethal response of asynchronous exponentially growing mouse lung (L1A2) cells heated to 42 0 C under hypoxic conditions was demonstrated in vitro. Acutely hypoxic cells (i.e. heated immediately after 30 min of N 2 +CO 2 gassing) and aerobic cells treated under the same extracellular pH were equally sensitive to a single hyperthermic treatment, and incubation under hypoxia for up to 24 hours prior to treatment did not influence cell survival. Similarly, under controlled pH conditions (pH within 7.0 to 7.4) recovery from hyperthermic damage demonstrated by two-dose hyperthermic fractionation (each of 1.5 hours at 42 0 C) was identical in hypoxic and aerobic cells, and the highest recovery was found at a 10-hour interval Preheating for 1.5 hours at 42 0 C induced thermal resistance. to a second treatment at 42 0 C (thermotolerance). At the 10-hour interval the degree of thermotolerance was not influenced by incubation under hypoxic conditions (thermotolerance ratio, TTR = 4.7 in both aerobic and hypoxic cells). The data indicate that hypoxic conditions do not influence the heat response in L1A2 cells to either a single or a two-dose fractionated hyperthermic treatment in which hypoxia or aerobic conditions were maintained in the interval between the heat treatments. (author)

  11. Metabolic and physiologic studies of nonimmune lymphoid cells cytotoxic for fibroblastic cells in vitro

    International Nuclear Information System (INIS)

    Mayhew, E.; Bennett, M.

    1974-01-01

    An in vitro reaction between mouse lymphoid cells and target fibroblastic cells in wells of microtest plates, which appears to simulate the in vivo rejection of hemopoietic allografts, has been analyzed for metabolic and physiologic requirements. Protein synthesis was required for only the first few hours of culture. Inhibition of RNA synthesis and alteration of cell surface charge with various agents were without obvious effects. Metabolic slowing at 4 0 C or deviation of the pH of the culture medium suppressed the reaction. Thymus cells, which are not cytotoxic in this system, significantly but not completely inhibited the cytotoxicity of lymph node cells. Antiserum directed against target cells specifically protected them from the cytotoxic lymphoid cells in the absence of complement. Precursors of cytotoxic lymphoid cells were radiosensitive, unlike the cytotoxic cells themselves. BALB/c anti-C57BL/6 spleen cell serum and 89 Sr both are able to prevent rejection of marrow allografts in vivo. Lymphoid cells incubated with this antiserum plus complement lost much of their cytotoxicity but were still effective at high ratios of aggressor to target cells. Lymphoid cells of mice treated with 89 Sr were effectively cytotoxic but lost practically all of their cytotoxicity after incubation with the antiserum plus complement. Thus, it appears that this reaction detects two different cytotoxic lymphoid cells, either of which can function in vitro. Both cell types may need to cooperate in vivo during marrow allograft rejections

  12. Instant magnetic labeling of tumor cells by ultrasound in vitro

    International Nuclear Information System (INIS)

    Mo Runyang; Yang Jian; Wu, Ed X.; Lin Shuyu

    2011-01-01

    Magnetic labeling of living cells creates opportunities for numerous biomedical applications. Here we describe an instantly cell magnetic labeling method based on ultrasound. We present a detailed study on the ultrasound performance of a simple and efficient labeling protocol for H-22 cells in vitro. High frequency focus ultrasound was investigated as an alternative method to achieve instant cell labeling with the magnetic particles without the need for adjunct agents or initiating cell cultures. Mean diameter of 168 nm dextran-T40 coated superparamagnetic iron oxide (SPIO) nanoparticles were prepared by means of classical coprecipitation in solution in our laboratory. H-22 tumor cells suspended in phosphate-buffered saline (PBS, pH=7.2) were exposed to ultrasound at 1.37 MHz for up to 120 s in the presence of SPIOs. The cellular uptake of iron oxide nanoparticles was detected by prussion blue staining. The viability of cells was determined by a trypan blue exclusion test. At 2 W power and 60 s ultrasound exposure in presence of 410 μg/ml SPIOs, H-22 cell labeling efficiency reached 69.4±6.3% and the labeled cells exhibited an iron content of 10.38±2.43 pg per cell. Furthermore, 95.2±3.2% cells remained viable. The results indicated that the ultrasound protocol could be potentially applied to label cells with large-sized magnetic particles. We also calculated the shear stress at the 2 W power and 1.37 MHz used in experiments. The results showed that the shear stress threshold for ultrasonically induced H-22 cell reparable sonoporation was 697 Pa. These findings provide a quantitative guidance in designing ultrasound protocols for cell labeling. - Highlights: → High frequency focus ultrasound can be used as a safe method for instant magnetic labeling of cells. → 8-16 times increased efficiency can be gained by ultrasound versus that by transfection agents. → Calculation of shear stress around cells provide a quantitative design for ultrasound protocols.

  13. Cell proliferation in vitro modulates fibroblast collagenase activity

    International Nuclear Information System (INIS)

    Lindblad, W.J.; Flood, L.

    1986-01-01

    Collagenase enzyme activity is regulated by numerous control mechanisms which prevent excessive release and activation of this protease. A primary mechanism for regulating enzyme extracellular activity may be linked to cell division, therefore they have examined the release of collagenase by fibroblasts in vitro in response to cellular proliferation. Studies were performed using fibroblasts derived from adult rat dermis maintained in DMEM containing 10% newborn calf serum, 25 mM tricine buffer, and antibiotics. Cells between subculture 10 and 19 were used with enzyme activity determined with a 14 C-labelled soluble Type I collagen substrate with and without trypsin activation. Fibroblasts, trypsinized and plated at low density secreted 8.5 fold more enzyme than those cells at confluence (975 vs. 115 dpm/μg DNA). This diminution occurred gradually as the cells went from logrithmic growth towards confluence. Confluent fibroblast monolayers were scraped in a grid arrangement, stimulating the remaining cells to divide, without exposure to trypsin. Within 24-48 hr postscraping enzyme levels had increased 260-400%, accompanied by enhanced incorporation of 3 H-thymidine and 3 H-uridine into cell macromolecules. The burst of enzyme release began to subside 12 hr later. These results support a close relationship between fibroblast proliferation and collagenase secretion

  14. In vitro differentiation of neural cells from human adipose tissue derived stromal cells.

    Science.gov (United States)

    Dave, Shruti D; Patel, Chetan N; Vanikar, Aruna V; Trivedi, Hargovind L

    2018-01-01

    Stem cells, including neural stem cells (NSCs), are endowed with self-renewal capability and hence hold great opportunity for the institution of replacement/protective therapy. We propose a method for in vitro generation of stromal cells from human adipose tissue and their differentiation into neural cells. Ten grams of donor adipose tissue was surgically resected from the abdominal wall of the human donor after the participants' informed consents. The resected adipose tissue was minced and incubated for 1 hour in the presence of an enzyme (collagenase-type I) at 37 0 C followed by its centrifugation. After centrifugation, the supernatant and pellets were separated and cultured in a medium for proliferation at 37 0 C with 5% CO2 for 9-10 days in separate tissue culture dishes for generation of mesenchymal stromal cells (MSC). At the end of the culture, MSC were harvested and analyzed. The harvested MSC were subjected for further culture for their differentiation into neural cells for 5-7 days using differentiation medium mainly comprising of neurobasal medium. At the end of the procedure, culture cells were isolated and studied for expression of transcriptional factor proteins: orthodenticle homolog-2 (OTX-2), beta-III-tubulin (β3-Tubulin), glial-fibrillary acid protein (GFAP) and synaptophysin-β2. In total, 50 neural cells-lines were generated. In vitro generated MSC differentiated neural cells' mean quantum was 5.4 ± 6.9 ml with the mean cell count being, 5.27 ± 2.65 × 10 3/ μl. All of them showed the presence of OTX-2, β3-Tubulin, GFAP, synaptophysin-β2. Neural cells can be differentiated in vitro from MSC safely and effectively. In vitro generated neural cells represent a potential therapy for recovery from spinal cord injuries and neurodegenerative disease.

  15. In vitro invasion of small-cell lung cancer cell lines correlates with expression of epidermal growth factor receptor

    DEFF Research Database (Denmark)

    Damstrup, L; Rude Voldborg, B; Spang-Thomsen, M

    1998-01-01

    receptor (EGFR) in a panel of 21 small-cell lung cancer (SCLC) cell lines. We have previously reported that ten of these cell lines expressed EGFR protein detected by radioreceptor and affinity labelling assays. In 11 small-cell lung cancer (SCLC) cell lines, EGFR mRNA was detected by Northern blot...... analysis. In vitro invasion in a Boyden chamber assay was found in all EGFR-positive cell lines, whereas no invasion was detected in the EGFR-negative cell lines. Quantification of the in vitro invasion in 12 selected SCLC cell lines demonstrated that, in the EGFR-positive cell lines, between 5% and 16......-PCR). However, in vitro invasive SCLC cell lines could not be distinguished from non-invasive cell lines based on the expression pattern of these molecules. In six SCLC cell lines, in vitro invasion was also determined in the presence of the EGFR-neutralizing monoclonal antibody mAb528. The addition...

  16. Derivation of corneal endothelial cell-like cells from rat neural crest cells in vitro.

    Directory of Open Access Journals (Sweden)

    Chengqun Ju

    Full Text Available The aim of this study was to investigate the feasibility of inducing rat neural crest cells (NCC to differentiate to functional corneal endothelial cell (CEC-like cells in vitro. Rat NCC were induced with adult CEC-derived conditioned medium. Immunofluorescence, flow cytometry and real time RT-PCR assay were used to detect expression of the corneal endothelium differentiation marker N-cadherin and transcription factors FoxC1 and Pitx2. CFDA SE-labeled CEC-like cells were transplanted to the corneal endothelium of a rat corneal endothelium deficiency model, and an eye-down position was maintained for 24 hours to allow cell attachment. The animals were observed for as long as 2 months after surgery and underwent clinical and histological examination. Spindle-like NCC turned to polygonal CEC-like after induction and expressed N-cadherin, FoxC1, Pitx2, zonula occludens-1 and sodium-potassium pump Na(+/K(+ ATPase. The corneas of the experimental group were much clearer than those of the control group and the mean corneal thickness in the experimental group was significantly less than in the control group7, 14, 21 and 28 days after surgery. Confocal microscopy through focusing and histological analysis confirmed that green fluorescence-positive CEC-like cells formed a monolayer covering the Descemet's membrane in the experimental group. In conclusion, CEC-like cells derived from NCCs displayed characters of native CEC, and the induction protocol provides guidance for future human CEC induction from NCC.

  17. Optimization of in vitro cell labeling methods for human umbilical cord-derived mesenchymal stem cells.

    Science.gov (United States)

    Tao, R; Sun, T-J; Han, Y-Q; Xu, G; Liu, J; Han, Y-F

    2014-01-01

    Human umbilical cord-derived mesenchymal stem cells (hUCMSCs) are a novel source of seed cells for cell therapy and tissue engineering. However, in vitro labeling methods for hUCMSCs need to be optimized for better detection of transplanted cells. To identify the most stable and efficient method for labeling hUCMSCs in vitro. hUCMSCs were isolated using a modified enzymatic digestion procedure and cultured. hUCMSCs of passage three (P3) were then labeled with BrdU, PKH26, or lentivirus-GFP and passaged further. Cells from the first labeled passage (LP1), the fourth labeled passage (LP4) and later passages were observed using a fluorescence microscope. The differentiation potential of LP4 cells was assessed by induction with adipogenic and osteogenic medium. Flow cytometry was used to measure the percentage of labeled cells and the percentage of apoptotic or dead cells. The labeling efficiencies of the three hUCMSC-labeling methods were compared in vitro. BrdU, PKH26, and lentivirus-GFP all labeled LP1 cells with high intensity and clarity. However, the BrdU labeling of the LP4 cells was vague and not localized to the cell nuclei; LP9 cells were not detected under a fluorescence microscope. There was also a significant decrease in the fluorescence intensity of PKH26-labeled LP4 cells, and LP11 cells were not detected under a fluorescence microscope. However, the fluorescence of LP4 cells labeled with lentivirus-GFP remained strong, and cells labeled with lentivirus-GFP were detected up to LP14 under a fluorescence microscope. Statistical analyses indicated that percentages of LP1 cells labeled with PKH26 and lentivirus-GFP were significantly higher than that of cells labeled with BrdU (p 0.05) was observed between the death rates of labeled and unlabeled cells. Lentivirus-GFP is a valid method for long-term in vitro labeling, and it may be used as a long-term hUCMSC tracker following transplantation in vivo.

  18. Tolerance induction to a thymus-dependent antigen in vitro: treatment of nonadherent cells with tolerogen biologically filtered in vitro

    International Nuclear Information System (INIS)

    Geyer, J.W.; Kong, Y.M.

    1974-01-01

    Highly tolerogenic bovine gamma globulin (BGG), a thymus-dependent antigen, was prepared by biologic filtration in vitro. It readily induced tolerance in vivo in BALB/c mice and also rendered their nonadherent lymph node cells tolerant after in vitro incubation. Biologic filtration in vitro was carried out by incubating 2.5 x 10 7 lymph node cells with 10 mg of nontolerogenic BGG in 10 ml of Eagle's medium containing 2 percent normal mouse serum at 37 0 C for 6 hr. The BGG-containing medium was clarified by centrifugation and was used without further dilution. For tolerance induction in vitro, lymph node cells were separated into adherent and nonadherent populations on Falcon plastic. These cells were incubated for 0 to 18 hr at 37 0 C with biologically filtered BGG (bBGG). After incubation, the cells were washed three times and (2 to 2.5) x 10 7 nonadherent or 4 x 10 6 adherent cells were injected iv with their untreated counterpart into lethally irradiated mice which had received 10 6 bone marrow cells. The recipients were then challenged with 300 μg of aggregated BGG, and tolerance was assayed by the elimination of labeled BGG, rosette formation, and passive hemagglutination. Spleen cells were similarly treated for comparison. Our findings show that tolerance was not induced in vitro in adherent lymph node cells. However, in the nonadherent populations, those from the lymph node but not the spleen were rendered tolerant. The acquisition of tolerance in vitro was gradual. It was dependent upon the length of exposure to bBGG and required at least 6 hr

  19. X-ray-induced in vitro neoplastic transformation of human diploid cells

    International Nuclear Information System (INIS)

    Borek, C.

    1980-01-01

    The neoplastic transformation, in vitro, of human diploid cells by x-ray irradiation into cells which can progress, in vitro, into advanced stages of neoplastic development is described. The cells are shown to form colonies in agar and to give rise to tumours when injected into nude mice. (U.K.)

  20. Differentiation of bone marrow cells with irradiated bone in vitro

    International Nuclear Information System (INIS)

    Toshiyuki Tominaga; Moritoshi Itoman; Izumi, T.; Wakita, R.; Uchino, M.

    1999-01-01

    , while in the groups of 40 and 50 kGy ALP staining increased slowly. The in vivo studies showed that the osteoinductive activity of the irradiated bones at 20-35 kGy decreased by 50-80 %. Our in vitro study revealed that ALP staining decreased by 1 0 % at 20-30 kGy on day 7. Since our system employed cell culture inserts which allow the action of humoral factors alone, other factors may explain the discrepancy between in vivo and in vitro studies

  1. In vitro assessment of curcumin against murine neuroblastoma cells.

    Science.gov (United States)

    Vanisree, Arambakkam Janardhanam; Ramanan, Ramya

    2007-04-01

    Neuroblastoma (NB) is a well-known malignant disease in infants, which comprises 10% of childhood malignancies. Despite recent advances in understanding the neuro-oncology, NB still accounts for more death in childhood than any other cancer. Research in childhood tumors should not only be focused on the malignant signatures of cancer cells but also novel drug prototypes using phytochemicals. The present study was aimed to determine the role of curcumin against murine neuroblastoma cell line (N2a). The in vitro assessment of curcumin against was made in N2a cell line in a dose-dependent manner (group I (control) and group II - IX (10 microM-80 microM). The efficacy of the drug was evaluated by estimating the levels of protein bound carbohydrates, glycoprotein, genomic DNA, total RNA levels, and inhibition of MMP-9 were studied. The gap junctional communication in the cells was also assessed. The levels of protein bound carbohydrates, DNA, RNA levels, glycoprotein were found to be altered on drug supplementation in NB cells. Inhibition of MMP-9 in curcumin-supplemented N2a cells was revealed by zymographic analysis. Assessment of Lucifer yellow dye uptake in curcumin-supplemented N2a cells showed the up-regulation of GJIC. These observations suggest that the curcumin, the active principle of curcuma longa, could be developed into an effective chemo preventive and chemotherapeutic agent. This selected concentration range needs further studies at molecular level, for conforming its role and its action against uncontrolled proliferation of NB.

  2. Ethane dimethanesulfonate (EDS) perturbs epididymal epithelial cell function in vitro

    International Nuclear Information System (INIS)

    Klinefelter, G.

    1990-01-01

    The formation of sperm granulomas in the epididymis following exposure to EDS, a Leydig cell toxicant, was reported by Cooper and Jackson in 1970. Recent work suggests that EDS may effect the epididymis directly. An in vitro system was developed to determine the nature of any direct effect. The caput epididymis from adult rats was dissected free of connective tissue and small pieces of the tissue were enzymatically digested until plaques of epididymal epithelial cells were obtained. Plaques were cultured on an extracellular matrix gelled on top of a semipermeable filter creating dual-compartment environments. The epithelial cells maintained typical morphology and protein secretion in this culture system for several days. Beginning on day 3, EDS (1 mM) was added to the basal compartment, with or without 35 S-methionine. After 24 hours, 35 S-labelled culture medium was taken from the apical compartment and analyzed by SDS-PAGE and fluorography. EDS caused decreased secretion of several proteins, including a 39 Kd molecule. Interestingly, a 39 Kd protein was also shown to disappear from sperm taken from the caput epididymidis following in vivo exposure to EDS. Unlabelled cultures were fixed and processed for light microscopy. No alterations in morphological integrity were observed. Thus, epididymal epithelial cell function is directly altered by EDS exposure

  3. A signal processing analysis of Purkinje cells in vitro

    Directory of Open Access Journals (Sweden)

    Ze'ev R Abrams

    2010-05-01

    Full Text Available Cerebellar Purkinje cells in vitro fire recurrent sequences of Sodium and Calcium spikes. Here, we analyze the Purkinje cell using harmonic analysis, and our experiments reveal that its output signal is comprised of three distinct frequency bands, which are combined using Amplitude and Frequency Modulation (AM/FM. We find that the three characteristic frequencies - Sodium, Calcium and Switching – occur in various combinations in all waveforms observed using whole-cell current clamp recordings. We found that the Calcium frequency can display a frequency doubling of its frequency mode, and the Switching frequency can act as a possible generator of pauses that are typically seen in Purkinje output recordings. Using a reversibly photo-switchable kainate receptor agonist, we demonstrate the external modulation of the Calcium and Switching frequencies. These experiments and Fourier analysis suggest that the Purkinje cell can be understood as a harmonic signal oscillator, enabling a higher level of interpretation of Purkinje signaling based on modern signal processing techniques.

  4. An in vitro clonogenic assay to assess radiation damage in rat CNS glial progenitor cells

    International Nuclear Information System (INIS)

    Maazen, R.W.M. van der; Verhagen, I.; Kogel, A.J. van der

    1990-01-01

    Normal glial progenitor cells can be isolated from the rat central nervous system (CNS) and cultured in vitro on a monolayer of type-1 astrocytes. These monolayers are able to support and stimulate explanted glial progenitor cells to proliferate. Employing these in vitro interactions of specific glial cell types, an in vivo-in vitro clonogenic assay has been developed. This method offers the possibility to study the intrinsic radiosensitivity, repair and regeneration of glial progenitor cells after in vitro or in vivo irradiation. (author)

  5. [Autologous regulatory T cells can suppress the proliferation of lymphoma cell line in vitro].

    Science.gov (United States)

    Ying, Zhi-Tao; Guo, Jun; Ren, Jun; Kong, Yan; Yuan, Zhi-Hong; Liu, Xi-Juan; Zhang, Chen; Zheng, Wen; Song, Yu-Qin; Zhang, Yun-Tao; Zhu, Jun

    2009-06-01

    This study was aimed to investigate the suppressive effect of regulatory T (Treg) cells on the T cell lymphoma EL4 cell line and to explore its mechanism. C57BL/6 Mouse Treg cells were isolated by MACS (magnetic cell sorting). The purity and the expression of Foxp3 were detected by flow cytometry. The suppressive effect of sorted Treg cells on EL4 cells was detected by MTT assay. The secretion of TGF-beta1 and IL-10 was examined by enzyme-linked immunosorbent assay (ELISA). The results showed that CD4(+)CD25(+) T cells could be successfully isolated by MACS with the purity reaching 91.6% and the expression level of Foxp3 was 78.9%. The ratio of viable cells was more than 95%. Regulatory T cells could suppress the proliferation of EL4 cells effectively in the presence of antigen presenting cells (APCs). And the suppressive effect was most significant at 1:1 ratio. In addition, the suppression still existed without APCs. TGF-beta1 and IL-10 could not be detected by ELISA. It is concluded that the Treg cells can suppress T lymphoma cell in vitro. The suppressive effect of Treg cells works in dose-dependent manner, but not in cytokine-dependent manner. The mechanism of this suppression may take effect through cell-cell contact.

  6. In vitro analysis of human periodontal microvascular endothelial cells.

    Science.gov (United States)

    Tsubokawa, Mizuki; Sato, Soh

    2014-08-01

    Endothelial cells (ECs) participate in key aspects of vascular biology, such as maintenance of capillary permeability, initiation of coagulation, and regulation of inflammation. According to previous reports, ECs have revealed highly specific characteristics depending on the organs and tissues. However, some reports have described the characteristics of the capillaries formed by human periodontal ECs. Therefore, the aim of the present study is to examine the functional characteristics of the periodontal microvascular ECs in vitro. Human periodontal ligament-endothelial cells (HPDL-ECs) and human gingiva-endothelial cells (HG-ECs) were isolated by immunoprecipitation with magnetic beads conjugated to a monoclonal anti-CD31 antibody. The isolated HPDL-ECs and HG-ECs were characterized to definitively demonstrate that these cell cultures represented pure ECs. Human umbilical-vein ECs and human dermal microvascular ECs were used for comparison. These cells were compared according to the proliferation potential, the formation of capillary-like tubes, the transendothelial electric resistance (TEER), and the expression of tight junction proteins. HPDL-ECs and HG-ECs with characteristic cobblestone monolayer morphology were obtained, as determined by light microscopy at confluence. Furthermore, the HPDL-ECs and HG-ECs expressed the EC markers platelet endothelial cell adhesion molecule-1 (also known as CD31), von Willebrand factor, and Ulex europaeus agglutinin 1, and the cells stained strongly positive for CD31 and CD309. In addition, the HPDL-ECs and HG-ECs were observed to form capillary-like tubes, and they demonstrated uptake of acetylated low-density lipoprotein. Functional analyses of the HPDL-ECs and HG-ECs showed that, compared to the control cells, tube formation persisted for only a brief period of time, and TEER was substantially reduced at confluence. Furthermore, the cells exhibited delocalization of zonula occludens-1 and occludin at cell-cell contact sites

  7. [Regulatory T cells inhibit proliferation of mouse lymphoma cell line EL4 in vitro].

    Science.gov (United States)

    Zhang, Chen; Kong, Yan; Guo, Jun; Ying, Zhi-Tao; Yuan, Zhi-Hong; Zhang, Yun-Tao; Zheng, Wen; Song, Yu-Qin; Li, Ping-Ping; Zhu, Jun

    2010-10-01

    This study was aimed to investigate the effect of regulatory T (Treg) cells on the T cell lymphoma EL4 cells and its mechanism in vitro. C57BL/6 mouse Treg cells were isolated by magnetic cell sorting (MACS). The purity of Treg cells and their expression of Foxp3 were identified by flow cytometry (FCM) and PT-PCR respectively. The suppression of Treg cells on EL4 cells was detected by 3H-TdR method. At the same time, enzyme-linked immunosorbent assay (ELISA) was used to detect the secretion of cytokine TGF-β1 and IL-10. The results showed that CD4+CD25+ T cells could be successfully isolated by MACS with the purity reaching 94.52% and the expression of Foxp3 reaching 84.72%. After sorting, the expression of Foxp3 mRNA could be detected by RT-PCR. 3H-TdR assay confirmed that regulatory T cells could suppress the proliferation of EL4 cells with or without antigen presenting cells (APC) or dendritic cells (DC), APC or DC might effectively enhance the suppression. In addition, DC alone also suppressed the proliferation. TGF-β1 and IL-10 could be detected in the supernatant by ELISA. It is concluded that the Treg cells can obviously suppress the proliferation of T cell lymphoma cells in vitro, APC or DC can enhance this suppressive effect, while the DC alone also can suppress the proliferation of EL4 cells, the TGF-β1 and IL-10 cytokine pathway may be one of the mechanisms of suppression.

  8. Determination of in vitro oxygen consumption rates for tumor cells

    International Nuclear Information System (INIS)

    Cardenas-Navia, L.I.; Moeller, B.J.; Kirkpatrick, J.P.; Laursen, T.A.; Dewhirst, M.W.

    2003-01-01

    To determine pO 2 at the surface of a monolayer of confluent HCT 116 cells, and to then determine consumption rate in vitro by examining the pO 2 profile in media above the cells. Materials and Methods: A recessed-tip polarographic oxygen microelectrode (diameter ∼10μm) was used to measure pO 2 profiles of media above a confluent monolayer of HCT 116 human colon adenocarcinoma cells in a T25 flask exposed to a 95% air, 5% CO 2 mixture. A two-dimensional finite element analysis of the diffusion equation was used to fit the data, thereby extracting a steady-state O 2 consumption rate. The diffusion equation was solved for zeroth and first-order expressions. No-flux boundary conditions were imposed on its bottom and side boundaries and experimental data was used for boundary conditions at the gas-media boundary. All flasks show an O 2 gradient in the media, with a mean (SE) media layer of 1677 (147) μm and a mean pO 2 at the cell layer/media interface of 44 (8) mm Hg (n=9). pO 2 gradient over the entire media layer is 630 (90) mm Hg/cm, equivalent to a consumption rate of 6.3 x 10 -4 (9.0 x 10 -5 ) mm Hg/s. The mean values for the zeroth and first order rate constants are 8.1 x 10 -9 (1.3 x 10 -9 ) g mol O 2 /cm 3 s and 1.0 x 10 3 (0.46 x 10 3 ) /s, respectively. Control experiments in flasks containing no cells show slight gradients in pO 2 of 38 (12) mm Hg/cm, resulting from some O 2 diffusion through the flask into the surrounding water bath. An addition of 10 -3 M NaCN to the media results in a dramatic increase in pO 2 at the cell layer, consistent with a shut-down in respiration. Under normal cell culture conditions there is an O 2 gradient present in the media of cull culture systems, resulting in physiologic O 2 concentrations at the cell layer, despite the non-physiologic O 2 concentration of the gas mixture to which the cell culture system is exposed. This significant (p -6 ) O 2 gradient in the media of cell culture systems is a result of cell O 2

  9. Neuromelanin is an immune stimulator for dendritic cells in vitro

    Directory of Open Access Journals (Sweden)

    Oberländer Uwe

    2011-11-01

    Full Text Available Abstract Background Parkinson's disease (PD is characterized at the cellular level by a destruction of neuromelanin (NM-containing dopaminergic cells and a profound reduction in striatal dopamine. It has been shown recently that anti-melanin antibodies are increased in sera of Parkinson patients, suggesting that NM may act as an autoantigen. In this study we tested whether NM is being recognized by dendritic cells (DCs, the major cell type for inducing T- and B-cell responses in vivo. This recognition of NM by DCs is a prerequisite to trigger an adaptive autoimmune response directed against NM-associated structures. Results Murine DCs were treated with NM of substantia nigra (SN from human subjects or with synthetic dopamine melanin (DAM. DCs effectively phagocytized NM and subsequently developed a mature phenotype (CD86high/MHCIIhigh. NM-activated DCs secreted the proinflammatory cytokines IL-6 and TNF-α. In addition, they potently triggered T cell proliferation in a mixed lymphocyte reaction, showing that DC activation was functional to induce a primary T cell response. In contrast, DAM, which lacks the protein and lipid components of NM but mimics the dopamine-melanin backbone of NM, had only very little effect on DC phenotype and function. Conclusions NM is recognized by DCs in vitro and triggers their maturation. If operative in vivo, this would allow the DC-mediated transport and presentation of SN antigens to the adaptive immune system, leading to autoimmmunity in susceptible individuals. Our data provide a rationale for an autoimmune-based pathomechanism of PD with NM as the initial trigger.

  10. In vitro neutron irradiation of glioma and endothelial cultured cells

    Energy Technology Data Exchange (ETDEWEB)

    Menichetti, L. [Department of PET and Radiopharmaceutical Chemistry, C.N.R. Institute of Clinical Physiology, Pisa (Italy)], E-mail: luca.menichetti@ifc.cnr.it; Gaetano, L. [University Scuola Superiore Sant' Anna, Pisa (Italy); Zampolli, A.; Del Turco, S. [Department of PET and Radiopharmaceutical Chemistry, C.N.R. Institute of Clinical Physiology, Pisa (Italy); Ferrari, C. [University of Pavia, Department of Surgery, Laboratory of experimental Surgery, Pavia (Italy); Bortolussi, S.; Stella, S.; Altieri, S. [University of Pavia, Department of Nuclear Physics, Pavia (Italy); National Institute for Nuclear Physics (INFN), Section of Pavia (Italy); Salvadori, P.A. [Department of PET and Radiopharmaceutical Chemistry, C.N.R. Institute of Clinical Physiology, Pisa (Italy); Cionini, L. [Unit of Radiotherapy, AOUP-University Hospital, Pisa (Italy)

    2009-07-15

    To fully develop its potential boron neutron capture therapy (BNCT) requires the combination of a suitable thermal/epithermal neutron flux together with a selective intake of {sup 10}B-boron nuclei in the target tissue. The latter condition is the most critical to be realized as none of the boron carriers used for experimental or clinical purposes proved at the moment an optimal selectivity for cancer cells compared to normal cells. In addition to complex physical factors, the assessment of the intracellular concentration of boron represent a crucial parameter to predict the dose delivered to the cancer cells during the treatment. Nowadays the dosimetry calculation and then the prediction of the treatment effectiveness are made using Monte Carlo simulations, but some of the model assumption are still uncertain: the radiobiological dose efficacy and the probability of tumour cell survival are crucial parameters that needs a more reliable experimental approach. The aim of this work was to evaluate the differential ability of two cell lines to selectively concentrate the boron-10 administered as di-hydroxyboryl-phenylalanine (BPA)-fructose adduct, and the effect of the differential boron intake on the damage produced by the irradiation with thermal neutrons; the two cell lines were selected to be representative one of normal tissues involved in the active/passive transport of boron carriers, and one of the tumour. Recent in vitro studies demonstrated how BPA is taken by proliferating cells, however the mechanism of BPA uptake and the parameters driving the kinetics of influx and the elimination of BPA are still not clarified. In these preliminary studies we analysed the survival of F98 and human umbilical vein endothelial cells (HUVEC) cells line after irradiation, using different thermal fluencies at the same level of density population and boron concentration in the growing medium prior the irradiation. This is first study performed on endothelium model obtained by

  11. Proteasome-independent degradation of HIV-1 in naturally non-permissive human placental trophoblast cells

    Directory of Open Access Journals (Sweden)

    Barré-Sinoussi Françoise

    2009-05-01

    Full Text Available Abstract Background The human placenta-derived cell line BeWo has been demonstrated to be restrictive to cell-free HIV-1 infection. BeWo cells are however permissive to infection by VSV-G pseudotyped HIV-1, which enters cells by a receptor-independent mechanism, and to infection by HIV-1 via a cell-to-cell route. Results Here we analysed viral entry in wild type BeWo (CCR5+, CXCR4+ and BeWo-CD4+ (CD4+, CCR5+, CXCR4+ cells. We report that HIV-1 internalisation is not restricted in either cell line. Levels of internalised p24 antigen between VSV-G HIV-1 pseudotypes and R5 or X4 virions were comparable. We next analysed the fate of internalised virions; X4 and R5 HIV-1 virions were less stable over time in BeWo cells than VSV-G HIV-1 pseudotypes. We then investigated the role of the proteasome in restricting cell-free HIV-1 infection in BeWo cells using proteasome inhibitors. We observed an increase in the levels of VSV-G pseudotyped HIV-1 infection in proteasome-inhibitor treated cells, but the infection by R5-Env or X4-Env pseudotyped virions remains restricted. Conclusion Collectively these results suggest that cell-free HIV-1 infection encounters a surface block leading to a non-productive entry route, which either actively targets incoming virions for non-proteasomal degradation, and impedes their release into the cytoplasm, or causes the inactivation of mechanisms essential for viral replication.

  12. In vitro Biological Evaluation of Graphene on Neuronal Cells

    Institute of Scientific and Technical Information of China (English)

    YI Jiling; ZHAO Zheng; LI Shipu; YIN Yixia; WANG Xinyu

    2016-01-01

    We prepared graphene(GE) with a mean size of 3087 nm. The transition of graphene oxide (GO) to GE was conifrmed by UV-visible spectroscopy, Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD). The experimental results of optical microscopic observation indicated that the GE ranged from 5 to 20 μg /mL did not affect the cell morphologies of the PC12 cells. The results of cell viability and membrane integrity assay supported that of optical microscopic observation and demonstrated that the GE ranged from 5 to 20 μg/ mL presented no obvious cytotoxicity. However, reactive oxygen species (ROS) assay suggested that an elevation of ROS level could be detected when the GE ranged from 20 to 100 μg/ mL. These results showed that the GE ranged from 5 to 10 μg/ mL presented an excellentin vitro biocompatibility and was one kind of potential biomaterials for neural tissue engineering.

  13. Immunoprotective capability of somatic hybrid cells in comparison with parental tumor cells maintained in vitro

    International Nuclear Information System (INIS)

    Mizushima, Yutaka; Cohen, E.P.

    1985-01-01

    The immunogenicity of X-irradiated hybrid cells derived from fusion of ASL-1 leukemia (A origin) and LM (TK - ) fibroblasts (C3H origin) was compared to X-irradiated parental ASL-1 leukemia cells maintained in vivo (V-ASL-1) and to X-irradiated ASL-1 leukemia cells maintained in vitro (C-ASL-1). Immunization with hybrid cells induced transplantation resistance against tumor rechallenge with V-ASL-1 more effectively than did immunization with V-ASL-1 tumor cells. Immunization with X-irradiated C-ASL-1 cells produced the same, or slightly stronger level of transplantation resistance than that with X-irradiated hybrid cells. These findings were observed both in A/J and in (C3H/HeJxA/J) F 1 mice. These results raise a question about whether the apparent increased immunogenicity of hybrid cells is due to a result of cell fusion or a result of their growth in vitro. (author)

  14. An In Vitro Study of Differentiation of Hematopoietic Cells to Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Qi Ru Wang

    2011-01-01

    medium (ECCM. BM-EPCs were characterized in terms of phenotype, lineage potential, and their functional properties. Endothelial cell colonies derived from BM-EPC were cultured with ECCM for 3 months. Cultured EPC colony cells expressed endothelial cell markers and formed the capillary-like network in vitro. EPC colony cells expressed differential proliferative capacity; some of the colonies exhibited a high proliferative potential (HPP capacity up to 20 population doublings. More importantly, these HPP-EPCs expressed hematopoietic marker CD45, exhibited endocytic activities, and preserved some of the myeloid cell activity. In addition, the HPP-EPCs secrete various growth factors including VEGF and GM-CSF into the culture medium. The results demonstrate that these EPCs were primarily derived from hematopoietic origin of early precursor cells and maintained high proliferative potential capacity, a feature with a significant potential in the application of cell therapy in ischemic diseases.

  15. In vitro ochratoxin A adsorption by commercial yeast cell walls

    Directory of Open Access Journals (Sweden)

    Carina Maricel Pereyra

    2015-03-01

    Full Text Available ABSTRACT. Pereyra C.M., Cavaglieri L.R., Keller K.M., Chiacchiera, S.M., Rosa C.A.R. & Dalcero A.M. In vitro ochratoxin A adsorption by commercial yeast cell walls. [Adsorção in vitro de ocratoxina A por paredes celulares de levedura comercial.] Revista Brasileira de Medicina Veterinária, 37(1:25-28, 2015. Departamento de Microbiología e Inmunología, Universidad Nacional de Río Cuarto, Ruta 36 Km 601, (5800 Río Cuarto, Córdoba, Argentina. E-mail: lcavaglieri@exa.unrc.edu.ar The aim of the present study was to evaluate the ochratoxin A (OTA adsorption capacity by two kinds of commercial yeast cell walls (YCW1 and YCW2 used as dietary supplements. An in vitro test was designed to mimic the temperature (37ºC, pH (2 and passage time (30 min through the stomach of a monogastric animal. The test was performed using different concentrations of YWC (100 and 200 µg/mL and OTA (10; 80; 160 and 1000 ng/mL and extracts were quantified by HPLC. For each OTA concentration, two independent trials varying the concentration of each YCW were performed. The two YCW assayed containing different percentages of polysaccharides, were able to adsorb similar amounts of OTA. Furthermore, the relationship mannans/β-glucans does not influence the rate of adsorption of OTA. In general, it was observed that the adsorption capacity increased with decreasing OTA concentration. Results from this work related to adsorption capacity of OTA with YCW allow predicting that other factor than 3D-structure and β-glucans and mannans could be involved. Future studies could be conducted to test the in vivo binding ability to alleviate the toxic effects of OTA under field conditions. Both YCW are a promising mycotoxin adsorbent to be used in animal feed to prevent mycotoxicoses.

  16. Boron neutron capture therapy induces cell cycle arrest and cell apoptosis of glioma stem/progenitor cells in vitro

    International Nuclear Information System (INIS)

    Sun, Ting; Zhang, Zizhu; Li, Bin; Chen, Guilin; Xie, Xueshun; Wei, Yongxin; Wu, Jie; Zhou, Youxin; Du, Ziwei

    2013-01-01

    Glioma stem cells in the quiescent state are resistant to clinical radiation therapy. An almost inevitable glioma recurrence is due to the persistence of these cells. The high linear energy transfer associated with boron neutron capture therapy (BNCT) could kill quiescent and proliferative cells. The present study aimed to evaluate the effects of BNCT on glioma stem/progenitor cells in vitro. The damage induced by BNCT was assessed using cell cycle progression, apoptotic cell ratio and apoptosis-associated proteins expression. The surviving fraction and cell viability of glioma stem/progenitor cells were decreased compared with differentiated glioma cells using the same boronophenylalanine pretreatment and the same dose of neutron flux. BNCT induced cell cycle arrest in the G2/M phase and cell apoptosis via the mitochondrial pathway, with changes in the expression of associated proteins. Glioma stem/progenitor cells, which are resistant to current clinical radiotherapy, could be effectively killed by BNCT in vitro via cell cycle arrest and apoptosis using a prolonged neutron irradiation, although radiosensitivity of glioma stem/progenitor cells was decreased compared with differentiated glioma cells when using the same dose of thermal neutron exposure and boronophenylalanine pretreatment. Thus, BNCT could offer an appreciable therapeutic advantage to prevent tumor recurrence, and may become a promising treatment in recurrent glioma

  17. Gammadelta receptor bearing T cells in scleroderma: enhanced interaction with vascular endothelial cells in vitro.

    Science.gov (United States)

    Kahaleh, M B; Fan, P S; Otsuka, T

    1999-05-01

    In view of the documented perivascular mononuclear cell infiltration in the involved organs in scleroderma (SSc) and the reported accumulation of gammadelta-T cells in SSc skin and lung, we evaluated gammadelta-T cell interaction with endothelial cells (EC) in vitro. gammadelta- and alphabeta-T cells were isolated from BPMN of SSc patients with early diffuse disease and of matched control subjects by an immunomagnetic method after stimulation with mycobacterium lysate and interleukin-2 for 2 weeks. Lymphocyte adhesion, proliferation, and cytotoxicity to EC were investigated. SSc gammadelta-T cells adhered to cultured EC and proliferated at higher rates than control cells. Furthermore, significant EC cytotoxicity by SSc gammadelta was seen. The cytotoxicity was blocked by addition of anti-gammadelta-TCR antibody and by anti-granzyme A antibody but not by anti-MHC class I and II antibodies. Expression of granzyme A mRNA was seen in five/five SSc gammadelta-T cells and in one/five control cells. alphabeta-T cells from both SSc and control subjects were significantly less interactive with EC than gammadelta-T cells. The data demonstrate EC recognition by SSc gammadelta-T cells and propose gammadelta-T cells as a possible effector cell type in the immune pathogenesis of SSc. Copyright 1999 Academic Press.

  18. Expression analysis of Tsga10 during in vitro differentiation of germ cells from mouse embryonic stem cell

    Directory of Open Access Journals (Sweden)

    Mohammad Miryounesi

    2015-02-01

    Conclusion: Expression pattern of Tsga10, as a gene with critical function in spermatogenesis, is similar during in vitro and in vivo germ cell generation. The results suggest that in vitro derived germ cells could be a trusted model to study genes behavior during spermatogenesis.

  19. Cell in situ zymography: an in vitro cytotechnology for localization of enzyme activity in cell culture.

    Science.gov (United States)

    Chhabra, Aastha; Jaiswal, Astha; Malhotra, Umang; Kohli, Shrey; Rani, Vibha

    2012-09-01

    In situ zymography is a unique technique for detection and localization of enzyme-substrate interactions majorly in histological sections. Substrate with quenched fluorogenic molecule is incorporated in gel over which tissue sections are mounted and then incubated in buffer. The enzymatic activity is observed in the form of fluorescent signal. With the advancements in the field of biological research, use of in vitro cell culture has become very popular and holds great significance in multiple fields including inflammation, cancer, stem cell biology and the still emerging 3-D cell cultures. The information on analysis of enzymatic activity in cell lines is inadequate presently. We propose a single-step methodology that is simple, sensitive, cost-effective, and functional to perform and study the 'in position' activity of enzyme on substrate for in vitro cell cultures. Quantification of enzymatic activity to carry out comparative studies on cells has also been illustrated. This technique can be applied to a variety of enzyme classes including proteases, amylases, xylanases, and cellulases in cell cultures.

  20. In vitro cell transformation induced by synthetic amorphous silica nanoparticles.

    Science.gov (United States)

    Fontana, Caroline; Kirsch, Anaïs; Seidel, Carole; Marpeaux, Léa; Darne, Christian; Gaté, Laurent; Remy, Aurélie; Guichard, Yves

    2017-11-01

    Synthetic amorphous silica nanoparticles (SAS) are among the most widely produced and used nanomaterials, but little is known about their carcinogenic potential. This study aims to evaluate the ability of four different SAS, two precipitated, NM-200 and NM-201, and two pyrogenic, NM-202 and NM-203, to induce the transformation process. For this, we used the recently developed in vitro Bhas 42 cell transformation assay (CTA). The genome of the transgenic Bhas 42 cells contains several copies of the v-Ha-ras gene, making them particularly sensitive to tumor-promoter agents. The Bhas 42 CTA, which includes an initiation assay and a promotion assay, was validated in our laboratory using known soluble carcinogenic substances. Its suitability for particle-type substances was verified by using quartz Min-U-Sil 5 (Min-U-Sil) and diatomaceous earth (DE) microparticles. As expected given their known transforming properties, Min-U-Sil responded positively in the Bhas 42 CTA and DE responded negatively. Transformation assays were performed with SAS at concentrations ranging from 2μg/cm 2 to 80μg/cm 2 . Results showed that all SAS have the capacity to induce transformed foci, interestingly only in the promotion assay, suggesting a mode of action similar to tumor-promoter substances. NM-203 exhibited transforming activity at a lower concentration than the other SAS. In conclusion, this study showed for the first time the transforming potential of different SAS, which act as tumor-promoter substances in the Bhas 42 model of cell transformation. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. THP-1 cell line: an in vitro cell model for immune modulation approach.

    Science.gov (United States)

    Chanput, Wasaporn; Mes, Jurriaan J; Wichers, Harry J

    2014-11-01

    THP-1 is a human leukemia monocytic cell line, which has been extensively used to study monocyte/macrophage functions, mechanisms, signaling pathways, and nutrient and drug transport. This cell line has become a common model to estimate modulation of monocyte and macrophage activities. This review attempts to summarize and discuss recent publications related to the THP-1 cell model. An overview on the biological similarities and dissimilarities between the THP-1 cell line and human peripheral blood mononuclear cell (PBMC) derived-monocytes and macrophages, as well as the advantages and disadvantages of the use of THP-1 cell line, is included. The review summarizes different published co-cultivation studies of THP-1 cells with other cell types, for instance, intestinal cells, adipocytes, T-lymphocytes, platelets, and vascular smooth muscle cells, which can be an option to study cell-cell interaction in vitro and can be an approach to better mimic in vivo conditions. Macrophage polarization is a relatively new topic which gains interest for which the THP-1 cell line also may be relevant. Besides that an overview of newly released commercial THP-1 engineered-reporter cells and THP-1 inflammasome test-cells is also given. Evaluation of recent papers leads to the conclusion that the THP-1 cell line has unique characteristics as a model to investigate/estimate immune-modulating effects of compounds in both activated and resting conditions of the cells. Although the THP-1 response can hint to potential responses that might occur ex vivo or in vivo, these should be, however, validated by in vivo studies to draw more definite conclusions. Copyright © 2013. Published by Elsevier B.V.

  2. In Vitro Modeling of Human Germ Cell Development Using Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Yuncheng Zhao

    2018-02-01

    Full Text Available Summary: Due to differences across species, the mechanisms of cell fate decisions determined in mice cannot be readily extrapolated to humans. In this study, we developed a feeder- and xeno-free culture protocol that efficiently induced human pluripotent stem cells (iPSCs into PLZF+/GPR125+/CD90+ spermatogonium-like cells (SLCs. These SLCs were enriched with key genes in germ cell development such as MVH, DAZL, GFRα1, NANOS3, and DMRT1. In addition, a small fraction of SLCs went through meiosis in vitro to develop into haploid cells. We further demonstrated that this chemically defined induction protocol faithfully recapitulated the features of compromised germ cell development of PSCs with NANOS3 deficiency or iPSC lines established from patients with non-obstructive azoospermia. Taken together, we established a powerful experimental platform to investigate human germ cell development and pathology related to male infertility. : In this article, Wang and colleagues established a feeder- and xeno-free system to robustly induce human pluripotent stem cells (PSCs into spermatogonia-like cells. This chemically defined induction protocol faithfully recapitulated the features of compromised germ cell development of PSCs with NANOS3 deficiency or iPSC lines established from patients with non-obstructive azoospermia. Keywords: pluripotent stem cells, spermatogonia, infertility, non-obstructive azoospermia

  3. In Vitro Differentiation and Propagation of Urothelium from Pluripotent Stem Cell Lines.

    Science.gov (United States)

    Osborn, Stephanie L; Kurzrock, Eric A

    2018-01-01

    Bioengineering of bladder tissue, particularly for those patients who have advanced bladder disease, requires a source of urothelium that is healthy, capable of significant proliferation in vitro and immunologically tolerated upon transplant. As pluripotent stem cells have the potential to fulfill such criteria, they provide a critical cell source from which urothelium might be derived in vitro and used clinically. Herein, we describe the in vitro differentiation of urothelium from the H9 human embryonic stem cell (hESC) line through the definitive endoderm (DE) phase via selective culture techniques. The protocol can be used to derive urothelium from other hESCs or human-induced pluripotent stem cells.

  4. Agent-Based Computational Modeling of Cell Culture: Understanding Dosimetry In Vitro as Part of In Vitro to In Vivo Extrapolation

    Science.gov (United States)

    Quantitative characterization of cellular dose in vitro is needed for alignment of doses in vitro and in vivo. We used the agent-based software, CompuCell3D (CC3D), to provide a stochastic description of cell growth in culture. The model was configured so that isolated cells assu...

  5. Differentiated NSC-34 cells as an in vitro Cell Model for VX

    Science.gov (United States)

    2014-09-11

    principally as potent cholinesterase inhibitors . The toxicity of these compounds and their mode of action are attributed to the inhibition of the enzyme...necrosis, with more cells undergoing apoptosis. Acetylcholinesterase inhibition by VX Since VX is an AChE inhibitor , the amount of VX-induced AChE...acetyl- cholinesterase by in vitro methods. Acta Medica (Hradec Kralove) 48:81–6. Landshamer S, Hoehn M, Barth N, et al. (2008). Bid-induced release of

  6. Benzo(a)pyrene induced cell cycle arrest and apoptosis in human choriocarcinoma cancer cells through reactive oxygen species-induced endoplasmic reticulum-stress pathway.

    Science.gov (United States)

    Kim, Soo-Min; Lee, Hae-Miru; Hwang, Kyung-A; Choi, Kyung-Chul

    2017-09-01

    Cigarette smoke (CS) contains over 60 well established carcinogens. In this study, we examined the effects of benzo(a)pyrene (B(a)P), a main CS component, on the viability and apoptosis of JEG-3 and BeWo human choriocarcinoma cancer cell lines. An MTT assay confirmed that B(a)P decreased the cell viability of JEG-3 and BeWo cells in a dose-dependent manner. Additionally, Western blot (WB) assay revealed that protein expression of cyclin D and cyclin E decreased, while protein expression of p21 and p27 was increased in response to B(a)P treatment for 48 h. The changes in reactive oxygen species (ROS) levels in JEG-3 and BeWo cells exposed to B(a)P were also measured by a dichlorofluorescein diacetate (DCF-DA) assay, which revealed that ROS levels increased in response to B(a)P treatment for 48 h. WB assay also confirmed that each B(a)P treatment of JEG-3 and BeWo cells for 4 h promoted the expression of phosphorylated eukaryotic initiation factor 2 alpha protein (p-eIF2α) and C/EBP homologous protein (CHOP), which are known to be involved in ROS-mediated endoplasmic reticulum stress (ER-stress) related apoptosis. Overall, the protein expression of Bax (a pro-apoptosis marker) increased, while the expression of Bcl-xl (an anti-apoptotic marker) decreased and the number of apoptotic cells increased in response to B(a)P treatment for 48 h. Taken together, these results suggest that B(a)P has the potential to induce apoptosis of JEG-3 and BeWo human choriocarcinoma cancer cells by increasing the ROS level and simultaneously activating ER-stress. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Redifferentiation of insulin-secreting cells after in vitro expansion of adult human pancreatic islet tissue

    International Nuclear Information System (INIS)

    Lechner, Andreas; Nolan, Anna L.; Blacken, Robyn A.; Habener, Joel F.

    2005-01-01

    Cellular replacement therapy holds promise for the treatment of diabetes mellitus but donor tissue is severely limited. Therefore, we investigated whether insulin-secreting cells could be differentiated in vitro from a monolayer of cells expanded from human donor pancreatic islets. We describe a three-step culture protocol that allows for the efficient generation of insulin-producing cell clusters from in vitro expanded, hormone-negative cells. These clusters express insulin at levels of up to 34% that of average freshly isolated human islets and secrete C-peptide upon membrane depolarization. They also contain cells expressing the other major islet hormones (glucagon, somatostatin, and pancreatic polypeptide). The source of the newly differentiated endocrine cells could either be indigenous stem/progenitor cells or the proliferation-associated dedifferentiation and subsequent redifferentiation of mature endocrine cells. The in vitro generated cell clusters may be efficacious in providing islet-like tissue for transplantation into diabetic recipients

  8. Lovastatin enhances in vitro radiation-induced apoptosis of rat B-cell lymphoma cells

    International Nuclear Information System (INIS)

    Rozados, V.R.; Hinrichsen, L.I.; Scharovsky, O.G.; Rosario Univ., Rosario; McDonnel, J.

    2005-01-01

    Our previous demonstration of an antimetastatic effect of lovastatin, both in rat sarcoma and lymphoma tumor-models, as well as the fact that lovastatin and radiation are able to stop the cell cycle in different phases, suggested the feasibility of a combined treatment. We studied the effect of the in vitro combined treatment of a B-cell rat lymphoma (L-TACB) with lovastatin and irradiation. The results herein obtained provide new information about the role of statins as radiosensitizers. The antitumor effect of the combined treatment was higher than that elicited by either treatment alone. This effect could be a consequence, at least in part, of an enhanced apoptosis

  9. X-ray-induced in vitro neoplastic transformation of human diploid cells

    International Nuclear Information System (INIS)

    Borek, C.

    1980-01-01

    Techniques have recently been developed to identify and score quantitatively neoplastic transformation caused by x-rays in cultured cells derived from rodents. The present report describes for the first time the neoplastic transformation in vitro of human diploid cells by x-ray irradiation into cells which can progress in vitro into advanced stages of neoplastic development, namely, to form colonies in agar and give rise to tumors when injected into nude mice

  10. Toxicity of diuron in human cancer cells.

    Science.gov (United States)

    Huovinen, Marjo; Loikkanen, Jarkko; Naarala, Jonne; Vähäkangas, Kirsi

    2015-10-01

    Diuron is a substituted phenylurea used as a herbicide to control broadleaf and grass weeds and as a biocidal antifouling agent. Diuron is carcinogenic in rat urinary bladder and toxic to the reproductive system of oysters, sea urchins and lizards. The few studies carried out in human cells do not include the genotoxicity of diuron. We have investigated the toxicity of diuron in human breast adenocarcinoma (MCF-7) and human placental choriocarcinoma (BeWo) cells. The production of reactive oxygen species (ROS) was statistically significantly increased in both cell lines but only at the highest 200 μM concentration. Diuron clearly reduced the viability of BeWo, but not MCF-7 cells. The relative cell number was decreased in both cell lines indicative of inhibition of cell proliferation. In the Comet assay, diuron increased DNA fragmentation in MCF-7 but not in BeWo cells. The expressions of p53 protein, a marker for cell stress, and p21 protein, a transcriptional target of p53, were increased, but only in MCF-7 cells. In conclusion, our results suggest that diuron is cytotoxic and potentially genotoxic in a tissue-specific manner and that ROS play a role in its toxicity. Thus, exposure to diuron may exert harmful effects on fetal development and damage human health. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. Factors affecting the loss of MED12-mutated leiomyoma cells during in vitro growth

    OpenAIRE

    Bloch, Jeannine; Holzmann, Carsten; Koczan, Dirk; Helmke, Burkhard Maria; Bullerdiek, J?rn

    2017-01-01

    Uterine leiomyomas (UL) are the most prevalent symptomatic human tumors at all and somatic mutations of the gene encoding mediator subcomplex 12 (MED12) constitute the most frequent driver mutations in UL. Recently, a rapid loss of mutated cells during in vitro growth of UL-derived cell cultures was reported, resulting in doubts about the benefits of UL-derived cell cultures. To evaluate if the rapid loss of MED12-mutated cells in UL cell cultures depends on in vitro passaging, we set up cell...

  12. Use of piracetam improves sickle cell deformability in vitro and in vivo.

    Science.gov (United States)

    Gini, E K; Sonnet, J

    1987-01-01

    Microsieving diluted suspensions of oxygenated sickle cell anaemia (HbSS) cells on polycarbonate filters shows that piracetam improves the red cell deformability in vitro. In vivo an oral intake of 160 mg/kg/day divided in four doses enhances the HbSS cell deformability as actively as it does in in vitro experiments. The drug is also able partially to restore the impaired deformability of physiologically deoxygenated HbSS cells. These findings are consistent with the results of clinical trials, which show that continuous treatment with piracetam reduces the incidence of vaso-occlusive crises in patients with sickle cell disease. PMID:3818978

  13. Oxcarbazepine-loaded polymeric nanoparticles: development and permeability studies across in vitro models of the blood–brain barrier and human placental trophoblast

    Directory of Open Access Journals (Sweden)

    Lopalco A

    2015-03-01

    Full Text Available Antonio Lopalco,1–3,* Hazem Ali,1,* Nunzio Denora,3 Erik Rytting1,4,5 1Department of Obstretrics and Gynecology, University of Texas Medical Branch, Galveston, TX, USA; 2Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, KS, USA; 3Department of Pharmacy – Drug Sciences, University of Bari Aldo Moro, Bari, Italy; 4Center for Biomedical Engineering, University of Texas Medical Branch, Galveston, TX, USA; 5Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, TX, USA *These authors contributed equally to this work Abstract: Encapsulation of antiepileptic drugs (AEDs into nanoparticles may offer promise for treating pregnant women with epilepsy by improving brain delivery and limiting the transplacental permeability of AEDs to avoid fetal exposure and its consequent undesirable adverse effects. Oxcarbazepine-loaded nanoparticles were prepared by a modified solvent displacement method from biocompatible polymers (poly(lactic-co-glycolic acid [PLGA] with or without surfactant and PEGylated PLGA [Resomer® RGPd5055]. The physical properties of the developed nanoparticles were determined with subsequent evaluation of their permeability across in vitro models of the blood–brain barrier (hCMEC/D3 cells and human placental trophoblast cells (BeWo b30 cells. Oxcarbazepine-loaded nanoparticles with encapsulation efficiency above 69% were prepared with sizes ranging from 140–170 nm, polydispersity indices below 0.3, and zeta potential values below −34 mV. Differential scanning calorimetry and X-ray diffraction studies confirmed the amorphous state of the nanoencapsulated drug. The apparent permeability (Pe values of the free and nanoencapsulated oxcarbazepine were comparable across both cell types, likely due to rapid drug release kinetics. Transport studies using fluorescently-labeled nanoparticles (loaded with coumarin-6 demonstrated increased permeability of surfactant-coated nanoparticles

  14. Oxcarbazepine-loaded polymeric nanoparticles: development and permeability studies across in vitro models of the blood-brain barrier and human placental trophoblast.

    Science.gov (United States)

    Lopalco, Antonio; Ali, Hazem; Denora, Nunzio; Rytting, Erik

    2015-01-01

    Encapsulation of antiepileptic drugs (AEDs) into nanoparticles may offer promise for treating pregnant women with epilepsy by improving brain delivery and limiting the transplacental permeability of AEDs to avoid fetal exposure and its consequent undesirable adverse effects. Oxcarbazepine-loaded nanoparticles were prepared by a modified solvent displacement method from biocompatible polymers (poly(lactic-co-glycolic acid) [PLGA] with or without surfactant and PEGylated PLGA [Resomer(®) RGPd5055]). The physical properties of the developed nanoparticles were determined with subsequent evaluation of their permeability across in vitro models of the blood-brain barrier (hCMEC/D3 cells) and human placental trophoblast cells (BeWo b30 cells). Oxcarbazepine-loaded nanoparticles with encapsulation efficiency above 69% were prepared with sizes ranging from 140-170 nm, polydispersity indices below 0.3, and zeta potential values below -34 mV. Differential scanning calorimetry and X-ray diffraction studies confirmed the amorphous state of the nanoencapsulated drug. The apparent permeability (Pe ) values of the free and nanoencapsulated oxcarbazepine were comparable across both cell types, likely due to rapid drug release kinetics. Transport studies using fluorescently-labeled nanoparticles (loaded with coumarin-6) demonstrated increased permeability of surfactant-coated nanoparticles. Future developments in enzyme-prodrug therapy and targeted delivery are expected to provide improved options for pregnant patients with epilepsy.

  15. Oxcarbazepine-loaded polymeric nanoparticles: development and permeability studies across in vitro models of the blood–brain barrier and human placental trophoblast

    Science.gov (United States)

    Lopalco, Antonio; Ali, Hazem; Denora, Nunzio; Rytting, Erik

    2015-01-01

    Encapsulation of antiepileptic drugs (AEDs) into nanoparticles may offer promise for treating pregnant women with epilepsy by improving brain delivery and limiting the transplacental permeability of AEDs to avoid fetal exposure and its consequent undesirable adverse effects. Oxcarbazepine-loaded nanoparticles were prepared by a modified solvent displacement method from biocompatible polymers (poly(lactic-co-glycolic acid) [PLGA] with or without surfactant and PEGylated PLGA [Resomer® RGPd5055]). The physical properties of the developed nanoparticles were determined with subsequent evaluation of their permeability across in vitro models of the blood–brain barrier (hCMEC/D3 cells) and human placental trophoblast cells (BeWo b30 cells). Oxcarbazepine-loaded nanoparticles with encapsulation efficiency above 69% were prepared with sizes ranging from 140–170 nm, polydispersity indices below 0.3, and zeta potential values below -34 mV. Differential scanning calorimetry and X-ray diffraction studies confirmed the amorphous state of the nanoencapsulated drug. The apparent permeability (Pe) values of the free and nanoencapsulated oxcarbazepine were comparable across both cell types, likely due to rapid drug release kinetics. Transport studies using fluorescently-labeled nanoparticles (loaded with coumarin-6) demonstrated increased permeability of surfactant-coated nanoparticles. Future developments in enzyme-prodrug therapy and targeted delivery are expected to provide improved options for pregnant patients with epilepsy. PMID:25792832

  16. Trophoblast cell fusion and differentiation are mediated by both the protein kinase C and a pathways.

    Directory of Open Access Journals (Sweden)

    Waka Omata

    Full Text Available The syncytiotrophoblast of the human placenta is an epithelial barrier that interacts with maternal blood and is a key for the transfer of nutrients and other solutes to the developing fetus. The syncytiotrophoblast is a true syncytium and fusion of progenitor cytotrophoblasts is the cardinal event leading to the formation of this layer. BeWo cells are often used as a surrogate for cytotrophoblasts, since they can be induced to fuse, and then express certain differentiation markers associated with trophoblast syncytialization. Dysferlin, a syncytiotrophoblast membrane repair protein, is up-regulated in BeWo cells induced to fuse by treatment with forskolin; this fusion is thought to occur through cAMP/protein kinase A-dependent mechanisms. We hypothesized that dysferlin may also be up-regulated in response to fusion through other pathways. Here, we show that BeWo cells can also be induced to fuse by treatment with an activator of protein kinase C, and that this fusion is accompanied by increased expression of dysferlin. Moreover, a dramatic synergistic increase in dysferlin expression is observed when both the protein kinase A and protein kinase C pathways are activated in BeWo cells. This synergy in fusion is also accompanied by dramatic increases in mRNA for the placental fusion proteins syncytin 1, syncytin 2, as well as dysferlin. Dysferlin, however, was shown to be dispensable for stimulus-induced BeWo cell syncytialization, since dysferlin knockdown lines fused to the same extent as control cells. The classical trophoblast differentiation marker human chorionic gonadotropin was also monitored and changes in the expression closely parallel that of dysferlin in all of the experimental conditions employed. Thus different biochemical markers of trophoblast fusion behave in concert supporting the hypothesis that activation of both protein kinase C and A pathways lead to trophoblastic differentiation.

  17. Follicle stimulating hormone increases spermatogonial stem cell colonization during in vitro co-culture.

    Science.gov (United States)

    Narenji Sani, Reza; Tajik, Parviz; Yousefi, Mohammad Hassan; Movahedin, Mansoureh; Qasemi-Panahi, Babak; Shafiei, Shiva; Ahmadi Hamedani, Mahmood

    2013-01-01

    The complex process of spermatogenesis is regulated by various factors. Studies on spermatogonial stem cells (SCCs) have provided very important tool to improve herd genetic and different field. 0.2 to 0.3 percent of total cells of seminiferous tubules is consist of spermatogonial stem cells. To investigate and biomanipulation of these cells, proliferation and viability rate of cells should be increased in vitro, at first. Follicle stimulating hormone (FSH) has been suggested to play a determinant role in the survival of germ cells in addition to increasing spermatogonial proliferation. In this study, the in vitro effects of FSH on spermatogonial cell colony formation were investigated. Sertoli and spermatogonial cells were isolated from 3-5 months old calves. The identity of the Sertoli cells and spermatogonial stem cells were confirmed through immunocytochemistry and colony morphology, respectively. Co-cultured Sertoli and spermatogonial cells were treated with FSH in different dose of 10, 20 and 40 IU mL(-1) FSH, before colony assay. Results indicated that, FSH increased in vitro colonization of spermatogonial cells in comparison with control group. In conclusion, using FSH provided proper bovine spermatogonial stem cell culture medium for in vitro study of these cells.

  18. Spontaneous transformation of adult mesenchymal stem cells from cynomolgus macaques in vitro

    International Nuclear Information System (INIS)

    Ren, Zhenhua; Wang, Jiayin; Zhu, Wanwan; Guan, Yunqian; Zou, Chunlin; Chen, Zhiguo; Zhang, Y. Alex

    2011-01-01

    Mesenchymal stem cells (MSCs) have shown potential clinical utility in cell therapy and tissue engineering, due to their ability to proliferate as well as to differentiate into multiple lineages, including osteogenic, adipogenic, and chondrogenic specifications. Therefore, it is crucial to assess the safety of MSCs while extensive expansion ex vivo is a prerequisite to obtain the cell numbers for cell transplantation. Here we show that MSCs derived from adult cynomolgus monkey can undergo spontaneous transformation following in vitro culture. In comparison with MSCs, the spontaneously transformed mesenchymal cells (TMCs) display significantly different growth pattern and morphology, reminiscent of the characteristics of tumor cells. Importantly, TMCs are highly tumorigenic, causing subcutaneous tumors when injected into NOD/SCID mice. Moreover, no multiple differentiation potential of TMCs is observed in vitro or in vivo, suggesting that spontaneously transformed adult stem cells may not necessarily turn into cancer stem cells. These data indicate a direct transformation of cynomolgus monkey MSCs into tumor cells following long-term expansion in vitro. The spontaneous transformation of the cultured cynomolgus monkey MSCs may have important implications for ongoing clinical trials and for models of oncogenesis, thus warranting a more strict assessment of MSCs prior to cell therapy. -- Highlights: ► Spontaneous transformation of cynomolgus monkey MSCs in vitro. ► Transformed mesenchymal cells lack multipotency. ► Transformed mesenchymal cells are highly tumorigenic. ► Transformed mesenchymal cells do not have the characteristics of cancer stem cells.

  19. Gamma-ray mutagenesis of cultured mammalian cells in vitro and in vivo

    International Nuclear Information System (INIS)

    Suzuki, Norio; Okada, Shigefumi

    1977-01-01

    The in vitro assay system used to study the reversion of L5178Y-Ala32 cells from an alanine requiring state to a non-requiring state, has been modified in order to be of use in selected in vivo systems. Gamma-ray induced mutations were compared between cells cultured in vitro and those grown in vivo in the intraperitoneal cavity of mice. The expression time was chosen to be 2 days for cells grown in vitro and 5 days for those grown in vivo. The dose-response curve can be described as cumulative for cells grown in vitro and linear for those grown in vivo. A doserate effect was observed in both systems. The cells grown in vivo were less sensitive to γ-rays with respect to both mutation rate per rad and cell killing as compared to cells grown in vitro. The delayed expression and reduced sensitivity of cells in vivo with respect to induced mutation may be due to factors such as hypoxia and/or reduced availability of essential nutrients. Sensitization in vitro by BUdR was detectable at a concentration as low as 10 -6 M, using an exposure time of 15 h. Under these conditions, BUdR alone did not induce any observable mutations

  20. Study on the cytotoxicity of natural killer cells induced by endothelial cells in vitro in the model of xenotransplantation

    International Nuclear Information System (INIS)

    Huang Haoyue; Shen Zhenya; Liu Hongcheng; Meng Zili; Teng Xiaomei

    2004-01-01

    Objective: To explore the change of the cytotoxicity of natural killer cells induced by vascular endothelial cells in vitro and the relationship between this change and the variety of cytokine level. Methods: After fixed by paraformaldehyde, vascular endothelial cells from pigs were co-cultured in vitro with natural killer cells from Chinese monkeys at different ratios. The change of the cytotoxicity of natural killer cells occurring after this contact and the content of IFN-γ and TNF-α in the supernatants were detected. Results: The cytotoxicity of natural killer cells improved gradually in accordance with the co-culture ratio after co-cultured with fixed vascular endothelial cells. The secretion of INF-γ and TNF-α also improved gradually. Conclusion: After contact with xeno-target cells, the cytotoxicity of natural killer cells and the secretion of cytokines are related to the ratio of effective cells and target cells

  1. In vitro differentiation and maturation of mouse embryonic stem cells into hepatocytes

    International Nuclear Information System (INIS)

    Ishii, Takamichi; Yasuchika, Kentaro; Fujii, Hideaki; Hoppo, Toshitaka; Baba, Shinji; Naito, Masato; Machimoto, Takafumi; Kamo, Naoko; Suemori, Hirofumi; Nakatsuji, Norio; Ikai, Iwao

    2005-01-01

    It is difficult to induce the maturation of embryonic stem (ES) cells into hepatocytes in vitro. We previously reported that Thy1-positive mesenchymal cells derived from the mouse fetal liver promote the maturation of hepatic progenitor cells. Here, we isolated alpha-fetoprotein (AFP)-producing cells from mouse ES cells for subsequent differentiation into hepatocytes in vitro by coculture with Thy1-positive cells. ES cells expressing green fluorescent protein (GFP) under the control of an AFP promoter were cultured under serum- and feeder layer-free culture conditions. The proportion of GFP-positive cells plateaued at 41.6 ± 12.2% (means ± SD) by day 7. GFP-positive cells, isolated by flow cytometry, were cultured in the presence or absence of Thy1-positive cells as a feeder layer. Isolated GFP-positive cells were stained for AFP, Foxa2, and albumin. The expression of mRNAs encoding tyrosine amino transferase, tryptophan 2,3-dioxygenase, and glucose-6-phosphatase were only detected following coculture with Thy1-positive cells. Following coculture with Thy1-positive cells, the isolated cells produced and stored glycogen. Ammonia clearance activity was also enhanced following coculture. Electron microscopic analysis indicated that the cocultured cells exhibited the morphologic features of mature hepatocytes. In conclusion, coculture with Thy1-positive cells in vitro induced the maturation of AFP-producing cells isolated from ES cell cultures into hepatocytes

  2. In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells.

    Science.gov (United States)

    Szaraz, Peter; Gratch, Yarden S; Iqbal, Farwah; Librach, Clifford L

    2017-08-09

    Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading cause of mortality worldwide. Mesenchymal stem cells (MSCs) are a promising option for cell-based therapies to replace current, invasive techniques. MSCs can differentiate into mesenchymal lineages, including cardiac cell types, but complete differentiation into functional cells has not yet been achieved. Previous methods of differentiation were based on pharmacological agents or growth factors. However, more physiologically relevant strategies can also enable MSCs to undergo cardiomyogenic transformation. Here, we present a differentiation method using MSC aggregates on cardiomyocyte feeder layers to produce cardiomyocyte-like contracting cells. Human umbilical cord perivascular cells (HUCPVCs) have been shown to have a greater differentiation potential than commonly investigated MSC types, such as bone marrow MSCs (BMSCs). As an ontogenetically younger source, we investigated the cardiomyogenic potential of first-trimester (FTM) HUCPVCs compared to older sources. FTM HUCPVCs are a novel, rich source of MSCs that retain their in utero immunoprivileged properties when cultured in vitro. Using this differentiation protocol, FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to BMSCs, as indicated by the increased expression of cardiomyocyte markers (i.e., myocyte enhancer factor 2C, cardiac troponin T, heavy chain cardiac myosin, signal regulatory protein α, and connexin 43). They also maintained significantly lower immunogenicity, as demonstrated by their lower HLA-A expression and higher HLA-G expression. Applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells clusters within 1 week of co-culture on cardiac feeder layers, becoming the first MSC type to do so. Our results demonstrate that this

  3. Introduction of in vitro transcribed ENO1 mRNA into neuroblastoma cells induces cell death

    International Nuclear Information System (INIS)

    Ejeskär, Katarina; Krona, Cecilia; Carén, Helena; Zaibak, Faten; Li, Lingli; Martinsson, Tommy; Ioannou, Panayiotis A

    2005-01-01

    Neuroblastoma is a solid tumour of childhood often with an unfavourable outcome. One common genetic feature in aggressive tumours is 1p-deletion. The α-enolase (ENO1) gene is located in chromosome region 1p36.2, within the common region of deletion in neuroblastoma. One alternative translated product of the ENO1 gene, known as MBP-1, acts as a negative regulator of the c-myc oncogene, making the ENO1 gene a candidate as a tumour suppressor gene. Methods used in this study are transfection of cDNA-vectors and in vitro transcribed mRNA, cell growth assay, TUNEL-assay, real-time RT-PCR (TaqMan) for expression studies, genomic sequencing and DHPLC for mutation detection. Here we demonstrate that transfection of ENO1 cDNA into 1p-deleted neuroblastoma cell lines causes' reduced number of viable cells over time compared to a negative control and that it induces apoptosis. Interestingly, a similar but much stronger dose-dependent reduction of cell growth was observed by transfection of in vitro transcribed ENO1 mRNA into neuroblastoma cells. These effects could also be shown in non-neuroblastoma cells (293-cells), indicating ENO1 to have general tumour suppressor activity. Expression of ENO1 is detectable in primary neuroblastomas of all different stages and no difference in the level of expression can be detected between 1p-deleted and 1p-intact tumour samples. Although small numbers (11 primary neuroblastomas), there is some evidence that Stage 4 tumours has a lower level of ENO1-mRNA than Stage 2 tumours (p = 0.01). However, mutation screening of 44 primary neuroblastomas of all different stages, failed to detect any mutations. Our studies indicate that ENO1 has tumour suppressor activity and that high level of ENO1 expression has growth inhibitory effects

  4. Isolation and culture exploration of Anas platyrhynchos amniotic fluid stem cells in vitro

    Directory of Open Access Journals (Sweden)

    Mingming Ning

    2017-06-01

    Conclusion: DAFSCs can be isolated from matrix that have strong self-renewal capacity in vitro. DAFSCs can be induced into adipocyte in vitro. These testify that DAFSCs can be an ideal seeded cells having potentials for preservation and utilization of rare genetic resources. [J Adv Vet Anim Res 2017; 4(2.000: 140-146

  5. Anti·red cell activity of lymphocytotoxic antibodies: and in vitro and in ...

    African Journals Online (AJOL)

    The need to obtain non-toxic antilymphocyre sera (ALS) led to the in vitro and in vivo evaluarion of its crossreactivity for red cells. The findings showed thar the antibodies coating the erythrocytes in vitro are idenrical with the antibodies that sensitize lymphocytes by the cytotoxicity rest. It would appear that rhe observed ...

  6. Differentiation of Human Dental Pulp Stem Cells into Dopaminergic Neuron-like Cells in Vitro.

    Science.gov (United States)

    Chun, So Young; Soker, Shay; Jang, Yu-Jin; Kwon, Tae Gyun; Yoo, Eun Sang

    2016-02-01

    We investigated the potential of human dental pulp stem cells (hDPSCs) to differentiate into dopaminergic neurons in vitro as an autologous stem cell source for Parkinson's disease treatment. The hDPSCs were expanded in knockout-embryonic stem cell (KO-ES) medium containing leukemia inhibitory factor (LIF) on gelatin-coated plates for 3-4 days. Then, the medium was replaced with KO-ES medium without LIF to allow the formation of the neurosphere for 4 days. The neurosphere was transferred into ITS medium, containing ITS (human insulin-transferrin-sodium) and fibronectin, to select for Nestin-positive cells for 6-8 days. The cells were then cultured in N-2 medium containing basic fibroblast growth factor (FGF), FGF-8b, sonic hedgehog-N, and ascorbic acid on poly-l-ornithine/fibronectin-coated plates to expand the Nestin-positive cells for up to 2 weeks. Finally, the cells were transferred into N-2/ascorbic acid medium to allow for their differentiation into dopaminergic neurons for 10-15 days. The differentiation stages were confirmed by morphological, immunocytochemical, flow cytometric, real-time PCR, and ELISA analyses. The expressions of mesenchymal stem cell markers were observed at the early stages. The expressions of early neuronal markers were maintained throughout the differentiation stages. The mature neural markers showed increased expression from stage 3 onwards. The percentage of cells positive for tyrosine hydroxylase was 14.49%, and the amount was 0.526 ± 0.033 ng/mL at the last stage. hDPSCs can differentiate into dopaminergic neural cells under experimental cell differentiation conditions, showing potential as an autologous cell source for the treatment of Parkinson's disease.

  7. Neoplastic transformation of hamster embryo cells irradiated in utero and assayed in vitro

    International Nuclear Information System (INIS)

    Borek, C.; Pain, C.; Mason, H.

    1977-01-01

    It is stated that induction of neoplastic transformation in vitro by x-rays and neutrons has been reported, and the authors had previously found that transformation by x-rays could be detected at doses as low as 1 R and the rate of transformation increased with dose, reaching a peak of 1% between 150 and 300 R. This frequency of neoplastic transformation in vitro is much higher than the frequency of radiation induced tumors observed after exposing animals to similar doses of radiation. Studies are here reported showing that malignant transformed cells can be obtained from embryos irradiated in utero and assayed in vitro, and that the frequency of transformation is at least tenfold lower than when the irradiations are performed in vitro, and thus closer to the incidence in animals. Hamster embryo cells were used for the studies. Questions that arise are as follows: does the host mediate in modulating transformation by radiation; is there a repair of transforming events before they can be expressed; and how significant is the state of cells during irradiation in determining the rate of transformation. It is known from in vitro studies that cell replication is required for fixation of the transformation. With the in vitro technique cells are seeded as single cells with ample opportunity to divide. In addition they are not in contact with one another, and constitute a mixture of cell types from many tissues. In utero the situation is quite different; the embryonic cells are irradiated as tissues where there is cell to cell contact in tissue-specific arrangements, and where the rate of cell replication varies with the tissue. It remains to be seen which of these factors, if any, is responsible for the lowered yield of transformed cells characteristic of in utero as opposed to in vitro irradiation. (U.K.)

  8. Autologous bone marrow Th cells can support multiple myeloma cell proliferation in vitro and in xenografted mice.

    Science.gov (United States)

    Wang, D; Fløisand, Y; Myklebust, C V; Bürgler, S; Parente-Ribes, A; Hofgaard, P O; Bogen, B; Taskén, K; Tjønnfjord, G E; Schjesvold, F; Dalgaard, J; Tveita, A; Munthe, L A

    2017-10-01

    Multiple myeloma (MM) is a plasma cell malignancy where MM cell growth is supported by the bone marrow (BM) microenvironment with poorly defined cellular and molecular mechanisms. MM cells express CD40, a receptor known to activate autocrine secretion of cytokines and elicit proliferation. Activated T helper (Th) cells express CD40 ligand (CD40L) and BM Th cells are significantly increased in MM patients. We hypothesized that activated BM Th cells could support MM cell growth. We here found that activated autologous BM Th cells supported MM cell growth in a contact- and CD40L-dependent manner in vitro. MM cells had retained the ability to activate Th cells that reciprocated and stimulated MM cell proliferation. Autologous BM Th cells supported MM cell growth in xenografted mice and were found in close contact with MM cells. MM cells secreted chemokines that attracted Th cells, secretion was augmented by CD40-stimulation. Within 14 days of culture of whole BM aspirates in autologous serum, MM cells and Th cells mutually stimulated each other, and MM cells required Th cells for further expansion in vitro and in mice. The results suggest that Th cells may support the expansion of MM cells in patients.

  9. In vitro Cell Viability by CellProfiler® Software as Equivalent to MTT Assay.

    Science.gov (United States)

    Gasparini, Luciana S; Macedo, Nayana D; Pimentel, Elisângela F; Fronza, Marcio; Junior, Valdemar L; Borges, Warley S; Cole, Eduardo R; Andrade, Tadeu U; Endringer, Denise C; Lenz, Dominik

    2017-07-01

    This study evaluated in vitro cell viability by the colorimetric MTT stands for 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay compared to image analysis by CellProfiler ® software. Hepatoma (Hepa-1c1c7) and fibroblast (L929) cells were exposed to isolated substances, camptothecin, lycorine, tazettine, albomaculine, 3-epimacronine, trispheridine, galanthine and Padina gymnospora , Sargassum sp. methanolic extract, and Habranthus itaobinus Ravenna ethyl acetate in different concentrations. After MTT assay, cells were stained with Panotic dye kit. Cell images were obtained with an inverted microscope equipped with a digital camera. The images were analyzed by CellProfiler ® . No cytotoxicity at the highest concentration analyzed for 3-epimacronine, albomaculine, galanthine, trispheridine, P. gymnospora extract and Sargassum sp. extract where detected. Tazettine offered cytotoxicity only against the Hepa1c1c7 cell line. Lycorine, camptothecin, and H. itaobinus extract exhibited cytotoxic effects in both cell lines. The viability methods tested were correlated demonstrated by Bland-Atman test with normal distribution with mean difference between the two methods close to zero, bias value 3.0263. The error was within the limits of the confidence intervals and these values had a narrow difference. The correlation between the two methods was demonstrated by the linear regression plotted as R 2 . CellProfiler ® image analysis presented similar results to the MTT assay in the identification of viable cells, and image analysis may assist part of biological analysis procedures. The presented methodology is inexpensive and reproducible. In vitro cell viability assessment with MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay may be replaced by image analysis by CellProfiler ® . The viability methods

  10. The proteomic dataset for bone marrow derived human mesenchymal stromal cells: Effect of in vitro passaging

    Directory of Open Access Journals (Sweden)

    Samuel T. Mindaye

    2015-12-01

    Full Text Available Bone-marrow derived mesenchymal stromal cells (BMSCs have been in clinical trials for therapy. One major bottleneck in the advancement of BMSC-based products is the challenge associated with cell isolation, characterization, and ensuring cell fitness over the course of in vitro cell propagation steps. The data in this report is part of publications that explored the proteomic changes following in vitro passaging of BMSCs [4] and the molecular heterogeneity in cultures obtained from different human donors [5,6].The methodological details involving cell manufacturing, proteome harvesting, protein identification and quantification as well as the bioinformatic analyses were described to ensure reproducibility of the results.

  11. Review of microfluidic cell culture devices for the control of gaseous microenvironments in vitro

    Science.gov (United States)

    Wu, H.-M.; Lee, T.-A.; Ko, P.-L.; Chiang, H.-J.; Peng, C.-C.; Tung, Y.-C.

    2018-04-01

    Gaseous microenvironments play important roles in various biological activities in vivo. However, it is challenging to precisely control gaseous microenvironments in vitro for cell culture due to the high diffusivity nature of gases. In recent years, microfluidics has paved the way for the development of new types of cell culture devices capable of manipulating cellular microenvironments, and provides a powerful tool for in vitro cell studies. This paper reviews recent developments of microfluidic cell culture devices for the control of gaseous microenvironments, and discusses the advantages and limitations of current devices. We conclude with suggestions for the future development of microfluidic cell culture devices for the control of gaseous microenvironments.

  12. Fra-1 induces morphological transformation and increases in vitro invasiveness and motility of epithelioid adenocarcinoma cells

    DEFF Research Database (Denmark)

    Kustikova, O.; Kramerov, D.; Grigorian, M.

    1998-01-01

    in vitro nor in vivo. CSML100 possesses all characteristics of a highly progressive carcinoma. These cells do not form tight contacts, are highly invasive in vitro, and are metastatic in vivo. AP-1 activity was considerably higher in CSML100 cells than in CSML0 cells. There was a common predominant Jun...... from tumors of epithelial origin revealed a correlation of fra-1 expression with mesenchymal characteristics of carcinoma cells. Moreover, we show here for the first time that the expression of exogenous Fra-1 in epithelioid cells results in morphological changes that resemble fibroblastoid conversion...

  13. Internalisation of engineered nanoparticles into mammalian cells in vitro: influence of cell type and particle properties

    International Nuclear Information System (INIS)

    Busch, Wibke; Bastian, Susanne; Trahorsch, Ulrike; Iwe, Maria; Kühnel, Dana; Meißner, Tobias; Springer, Armin; Gelinsky, Michael; Richter, Volkmar; Ikonomidou, Chrysanthy; Potthoff, Annegret; Lehmann, Irina; Schirmer, Kristin

    2011-01-01

    Cellular internalisation of industrial engineered nanoparticles is undesired and a reason for concern. Here we investigated and compared the ability of seven different mammalian cell cultures in vitro to incorporate six kinds of engineered nanoparticles, focussing on the role of cell type and particle properties in particle uptake. Uptake was examined using light and electron microscopy coupled with energy dispersive X-ray spectroscopy (EDX) for particle element identification. Flow cytometry was applied for semi-quantitative analyses of particle uptake and for exploring the influence on uptake by the phagocytosis inhibitor Cytochalasin D (CytoD). All particles studied were found to enter each kind of cultured cells. Yet, particles were never found within cell nuclei. The presence of the respective particles within the cells was confirmed by EDX. Live-cell imaging revealed the time-dependent process of internalisation of technical nanoparticles, which was exemplified by tungsten carbide particle uptake into the human skin cells, HaCaT. Particles were found to co-localise with lysosomal structures within the cells. The incorporated nanoparticles changed the cellular granularity, as measured by flow cytometry, already after 3 h of exposure in a particle specific manner. By correlating particle properties with flow cytometry data, only the primary particle size was found to be a weakly influential property for particle uptake. CytoD, an inhibitor of actin filaments and therewith of phagocytosis, significantly inhibited the internalisation of particle uptake in only two of the seven investigated cell cultures. Our study, therefore, supports the notion that nanoparticles can enter mammalian cells quickly and easily, irrespective of the phagocytic ability of the cells.

  14. Microfabricated Electrochemical Cell-Based Biosensors for Analysis of Living Cells In Vitro

    Directory of Open Access Journals (Sweden)

    Jun Wang

    2012-04-01

    Full Text Available Cellular biochemical parameters can be used to reveal the physiological and functional information of various cells. Due to demonstrated high accuracy and non-invasiveness, electrochemical detection methods have been used for cell-based investigation. When combined with improved biosensor design and advanced measurement systems, the on-line biochemical analysis of living cells in vitro has been applied for biological mechanism study, drug screening and even environmental monitoring. In recent decades, new types of miniaturized electrochemical biosensor are emerging with the development of microfabrication technology. This review aims to give an overview of the microfabricated electrochemical cell-based biosensors, such as microelectrode arrays (MEA, the electric cell-substrate impedance sensing (ECIS technique, and the light addressable potentiometric sensor (LAPS. The details in their working principles, measurement systems, and applications in cell monitoring are covered. Driven by the need for high throughput and multi-parameter detection proposed by biomedicine, the development trends of electrochemical cell-based biosensors are also introduced, including newly developed integrated biosensors, and the application of nanotechnology and microfluidic technology.

  15. In vitro cell-mediated immunity assay using 125I-iododeoxyuridine

    International Nuclear Information System (INIS)

    Morris, J.E.; Graham, T.M.

    1979-01-01

    We investigated an in vitro cell-mediated immunity assay using incorporation of 125 I-iododeoxyuridine as an indicator of lymphocyte responsiveness to mitogen stimulation. The system permits the use of whole-blood cultures in rats and dogs

  16. In vitro cytotoxicity of Indonesian stingless bee products against human cancer cell lines

    Directory of Open Access Journals (Sweden)

    Paula M. Kustiawan

    2014-07-01

    Conclusions: Propolis from T. incisa and Trigona fusco-balteata contain an in vitro cytotoxic activity against human cancer cell lines. Further study is required, including the isolation and characterization of the active antiproliferative agent(s.

  17. ENDOGENOUS PYROGEN RELEASE FROM RABBIT BLOOD CELLS INCUBATED IN VITRO WITH PARAINFLUENZA VIRUS.

    Science.gov (United States)

    ATKINS, E; CRONIN, M; ISACSON, P

    1964-12-11

    Rabbit blood cells incubated in vitro with purified parainfluenza-5 virus (DA strain) released a rapidly acting pyrogen. Spleen and lymph node cells were inactive. The pyrogen resembled in behavior a pyrogen extracted from granulocytic exudates. Similar cells in the blood are believed to be activated by virus in vivo to produce the circulating endogenous pyrogen that mediates virus-induced fever.

  18. Quantitation, in vitro propagation, and characterization of preleukemic cells induced by radiation leukemia virus

    International Nuclear Information System (INIS)

    Yefenof, E.; Epszteyn, S.; Kotler, M.

    1991-01-01

    Intrathymic (i.t.) inoculation of radiation leukemia virus into C57BL/6 mice induces a population of preleukemic (PL) cells that can progress into mature thymic lymphomas upon transfer into syngeneic recipients. A minimum of 10(3) PL thymic cells are required to induce lymphomas in the recipient. Most of the individual lymphomas developed in mice which were inoculated with cells of a single PL thymus, derived from different T-cell precursors. PL thymic cells could be grown in vitro on a feeder layer consisting of splenic stromal cells. Growth medium was supplemented with supernatant harvested from an established radiation leukemia virus-induced lymphoma cell line (SR4). The in vitro-grown PL cells were characterized as Thy-1+, CD4+, CD8- T-cells, most of which expressed radiation leukemia virus antigens. Cultured PL cells were found to be nontumorigenic, based on their inability to form s.c. tumors. However, these cells could develop into thymic lymphomas if inoculated i.t. into syngeneic recipients. A culture of PL cells, maintained for 2 mo, showed clonal T-cell receptor arrangement. Lymphomas which developed in several recipient mice upon injection with these PL cells were found to possess the same T-cell receptor arrangement. These results indicate that PL cells can be adapted for in vitro growth while maintaining their preleukemic character

  19. In vitro differentiation of mouse embryonic stem cells into functional ...

    African Journals Online (AJOL)

    Studies have shown that embryonic stem (ES) cells can be successfully differentiated into liver cells, which offer the potential unlimited cell source for a variety of end-stage liver disease. In our study, in order to induce mouse ES cells to differentiate into hepatocyte-like cells under chemically defined conditions, ES cells ...

  20. Trends in the development of microfluidic cell biochips for in vitro hepatotoxicity.

    Science.gov (United States)

    Baudoin, Régis; Corlu, Anne; Griscom, Laurent; Legallais, Cécile; Leclerc, Eric

    2007-06-01

    Current developments in the technological fields of liver tissue engineering, bioengineering, biomechanics, microfabrication and microfluidics have lead to highly complex and pertinent new tools called "cell biochips" for in vitro toxicology. The purpose of "cell biochips" is to mimic organ tissues in vitro in order to partially reduce the amount of in vivo testing. These "cell biochips" consist of microchambers containing engineered tissue and living cell cultures interconnected by a microfluidic network, which allows the control of microfluidic flows for dynamic cultures, by continuous feeding of nutrients to cultured cells and waste removal. Cell biochips also allow the control of physiological contact times of diluted molecules with the tissues and cells, for rapid testing of sample preparations or specific addressing. Cell biochips can be situated between in vitro and in vivo testing. These types of systems can enhance functionality of cells by mimicking the tissue architecture complexities when compared to in vitro analysis but at the same time present a more rapid and simple process when compared to in vivo testing procedures. In this paper, we first introduce the concepts of microfluidic and biochip systems based on recent progress in microfabrication techniques used to mimic liver tissue in vitro. This includes progress and understanding in biomaterials science (cell culture substrate), biomechanics (dynamic cultures conditions) and biology (tissue engineering). The development of new "cell biochips" for chronic toxicology analysis of engineered tissues can be achieved through the combination of these research domains. Combining these advanced research domains, we then present "cell biochips" that allow liver chronic toxicity analysis in vitro on engineered tissues. An extension of the "cell biochip" idea has also allowed "organ interactions on chip", which can be considered as a first step towards the replacement of animal testing using a combined liver

  1. Expression of apoptotic genes in immature and in vitro matured equine oocytes and cumulus cells.

    Science.gov (United States)

    Leon, P M M; Campos, V F; Kaefer, C; Begnini, K R; McBride, A J A; Dellagostin, O A; Seixas, F K; Deschamps, J C; Collares, T

    2013-08-01

    The gene expression of Bax, Bcl-2, survivin and p53, following in vitro maturation of equine oocytes, was compared in morphologically distinct oocytes and cumulus cells. Cumulus-oocyte complexes (COC) were harvested and divided into two groups: G1 - morphologically healthy cells; and G2 - less viable cells or cells with some degree of atresia. Total RNA was isolated from both immature and in vitro matured COC and real-time reverse transcription polymerase chain reaction (qRT-PCR) was used to quantify gene expression. Our results showed there was significantly higher expression of survivin (P < 0.05) and lower expression of p53 (P < 0.01) in oocytes compared with cumulus cells in G1. No significant difference in gene expression was observed following in vitro maturation or in COC derived from G1 and G2. However, expression of the Bax gene was significantly higher in cumulus cells from G1 (P < 0.02).

  2. Investigation of in vitro bone cell adhesion and proliferation on Ti using direct current stimulation

    International Nuclear Information System (INIS)

    Bodhak, Subhadip; Bose, Susmita; Kinsel, William C.; Bandyopadhyay, Amit

    2012-01-01

    Our objective was to establish an in vitro cell culture protocol to improve bone cell attachment and proliferation on Ti substrate using direct current stimulation. For this purpose, a custom made electrical stimulator was developed and a varying range of direct currents, from 5 to 25 μA, was used to study the current stimulation effect on bone cells cultured on conducting Ti samples in vitro. Cell–material interaction was studied for a maximum of 5 days by culturing with human fetal osteoblast cells (hFOB). The direct current was applied in every 8 h time interval and the duration of electrical stimulation was kept constant at 15 min for all cases. In vitro results showed that direct current stimulation significantly favored bone cell attachment and proliferation in comparison to nonstimulated Ti surface. Immunochemistry and confocal microscopy results confirmed that the cell adhesion was most pronounced on 25 μA direct current stimulated Ti surfaces as hFOB cells expressed higher vinculin protein with increasing amount of direct current. Furthermore, MTT assay results established that cells grew 30% higher in number under 25 μA electrical stimulation as compared to nonstimulated Ti surface after 5 days of culture period. In this work we have successfully established a simple and cost effective in vitro protocol offering easy and rapid analysis of bone cell–material interaction which can be used in promotion of bone cell attachment and growth on Ti substrate using direct current electrical stimulation in an in vitro model. - Highlights: ► D.C. stimulation was used to enhance in vitro bone cell adhesion and proliferation. ► Cells cultured on Ti were stimulated by using a custom made electrical stimulator. ► Optimization was performed by using a varying range of direct currents ∼ 5 to 25 μA. ► 25 μA stimulation was found most beneficial for promotion of cell adhesion/growth.

  3. Immunosuppressive Mesenchymal Stromal Cells Derived from Human-Induced Pluripotent Stem Cells Induce Human Regulatory T Cells In Vitro and In Vivo

    OpenAIRE

    Clémence Roux; Clémence Roux; Clémence Roux; Gaëlle Saviane; Gaëlle Saviane; Jonathan Pini; Jonathan Pini; Nourhène Belaïd; Nourhène Belaïd; Gihen Dhib; Gihen Dhib; Christine Voha; Christine Voha; Christine Voha; Lidia Ibáñez

    2018-01-01

    Despite mesenchymal stromal cells (MSCs) are considered as a promising source of cells to modulate immune functions on cells from innate and adaptive immune systems, their clinical use remains restricted (few number, limited in vitro expansion, absence of a full phenotypic characterization, few insights on their in vivo fate). Standardized MSCs derived in vitro from human-induced pluripotent stem (huIPS) cells, remediating part of these issues, are considered as well as a valuable tool for th...

  4. Novel Cell Preservation Technique to Extend Bovine In Vitro White Blood Cell Viability.

    Directory of Open Access Journals (Sweden)

    Emilie L Laurin

    Full Text Available Although cell-mediated immunity based diagnostics can be integral assays for early detection of various diseases of dairy cows, processing of blood samples for these tests is time-sensitive, often within 24 hours of collection, to maintain white blood cell viability. Therefore, to improve utility and practicality of such assays, the objective of this study was to assess the use of a novel white blood cell preservation technology in whole bovine blood. Blood samples from ten healthy cows were each divided into an unpreserved control sample and a test sample preserved with commercially-available cell transport medium. Samples were maintained at room temperature and stimulated with the mitogens pokeweed and concanavalinA, as well as with interleukin-12 p40. Stimulation was completed on days 1, 5, and 8 post-sampling. Viability of white blood cells was assessed through interferon gamma production determined with a commercial enzyme linked immunosorbent assay. In addition, mononuclear cell viability was assessed with propidium iodide flow cytometry. Greater interferon gamma production was observed on days 5 and 8 post-collection in preserved samples, with both pokeweed and concanavalinA stimulating positive interferon gamma production on day 5 post-collection. A greater proportion of the amount of interferon gamma produced on day 1 continued to be produced on days 5 and 8 post-collection with concanavalinA stimulation (with or without interleukin 12 as compared to pokeweed stimulation. Additionally, viable mononuclear cells were still present at eight days post-collection, with a higher mean proportion detected at days 5 and 8 in all stimulated preserved samples. This practical and simple method to extend in vitro white blood cell viability could benefit the efficient utilization of cell-based blood tests in ruminants.

  5. Active suppression of in vitro reactivity of spleen cells after BCG treatment

    International Nuclear Information System (INIS)

    Orbach-Arbouys, S.; Poupon, M.F.

    1978-01-01

    It was found that spleen cells from mice injected i.v. with large doses of BCG responded to PHA stimulation less intensely than did normal spleen cells. It was shown that nylon wool column purified BCG treated T cells also had a low PHA reactivity. Unfractionated spleen cells, adherent cells or T-enriched populations from BCG treated mice, when added to normal T cells lowered their PHA reactivity. When the same BCG treated cell populations were added to tumor cells in vitro, they inhibited their growth. (author)

  6. In vitro expansion of the mammary stem/progenitor cell population by xanthosine treatment

    Directory of Open Access Journals (Sweden)

    Choudhary Ratan K

    2012-06-01

    Full Text Available Abstract Background Mammary stem cells are critical for growth and maintenance of the mammary gland and therefore are of considerable interest for improving productivity and efficiency of dairy animals. Xanthosine treatment has been demonstrated to promote expansion of putative mammary stem cells in vivo, and hepatic and hair follicle stem cells in vitro. In the latter, xanthosine promoted the symmetrical division of hepatic and hair follicle stem cells. The objective of this study was to determine if treating primary cultures of bovine mammary epithelial cells (MEC with xanthosine increases the stem/progenitor cell population by promoting symmetrical division of mammary stem cells. Results In vitro treatment with xanthosine increased the population of MEC during the exponential phase of cell growth, reducing the doubling time from 86 h in control cultures to 60 h in xanthosine-treated cultures. The bromodeoxyuridine (BrdU labeling index and the proportion of MEC in S-phase both were increased by xanthosine treatment, indicating that increased cell accretion was due to increased cell proliferation. Analysis of daughter-pairs indicated that xanthosine promoted a shift from asymmetric to symmetric cell division. Moreover, the 30 % increase in symmetric cell division was concomitant with an increase in the proportion of MEC that were positive for a putative stem cell marker (FNDC3B and a trend toward increased telomerase activity. These results suggest that xanthosine treatment in vitro can increase cell proliferation, promote symmetric cell division and enhance stem/progenitor cell activity. Conclusions Xanthosine treatment increased the proliferation rate of bovine MEC in vitro. This was likely to be mediated by an increase in the proportion of stem/progenitor cells in the MEC population due to promotion of symmetrical stem cell division by xanthosine.

  7. In vitro differentiation of primordial germ cells and oocyte-like cells from stem cells.

    Science.gov (United States)

    Costa, José J N; Souza, Glaucinete B; Soares, Maria A A; Ribeiro, Regislane P; van den Hurk, Robert; Silva, José R V

    2018-02-01

    Infertility is the result of failure due to an organic disorder of the reproductive organs, especially their gametes. Recently, much progress has been made on generating germ cells, including oocytes, from various types of stem cells. This review focuses on advances in female germ cell differentiation from different kinds of stem cells, with emphasis on embryonic stem cells, adult stem cells, and induced pluripotent stem cells. The advantages and disadvantages of the derivation of female germ cells from several types of stem cells are also highlighted, as well as the ability of stem cells to generate mature and functional female gametes. This review shows that stem cell therapies have opened new frontiers in medicine, especially in the reproductive area, with the possibility of regenerating fertility.

  8. Effect of in vitro irradiation and cell cycle-inhibitory drugs on the spontaneous human IgE synthesis in vitro

    International Nuclear Information System (INIS)

    Del Prete, G.F.; Vercelli, D.; Tiri, A.; Maggi, E.; Rossi, O.; Romagnani, S.; Ricci, M.

    1987-01-01

    The in vitro effects of radiation, diterpine forskolin (FK), and hydrocortisone (HC) on the in vitro spontaneous IgE synthesis by peripheral blood B-lymphocytes from atopic patients were investigated. Without affecting cell viability, in vitro irradiation inhibited in a dose-dependent fashion de novo IgE synthesis in vitro by B cells from all patients examined with a mean 40% reduction of in vitro IgE product after treatment with 100 rads. In contrast, the in vitro IgE production by the U266 myeloma cell line was unaffected, even by irradiation with 1600 rads. The addition to B cell cultures from atopic patients of FK consistently resulted in a dose-dependent inhibition of the spontaneous IgE production in vitro. The addition to cultures of 10(-5) and 10(-6) molar concentrations of HC was also usually inhibitory, whereas lower HC concentrations were uneffective or even enhanced the spontaneous in vitro IgE synthesis. When 10(-6) molar concentrations of both HC and FK were combined in culture, a summation inhibitory effect on the spontaneous IgE synthesis was observed. In contrast, neither FK nor HC had inhibitory effect on the in vitro spontaneous IgE synthesis by the U266 myeloma cell line. The spontaneous in vitro IgE synthesis by B cells from patients with Hodgkin's disease, demonstrating high levels of serum IgE, was strongly reduced or virtually abolished after patients underwent total nodal irradiation to prevent the spread of the disease. In addition, the in vitro spontaneous IgE synthesis by B cells from atopic patients was markedly decreased or abolished by in vivo administration of betamethasone

  9. Generation of Dopamine-Secreting Cells from Human Adipose Tissue-Derived Stem Cells In Vitro.

    Science.gov (United States)

    Soheilifar, Mohammad Hasan; Javeri, Arash; Amini, Hossein; Taha, Masoumeh Fakhr

    2018-03-12

    Several studies have demonstrated the differentiation of human adipose tissue-derived stem cells (hADSCs) to neuronal and glial phenotypes, but directing the fate of these cells toward dopaminergic neurons has not been frequently reported. The aim of this study was to investigate dopaminergic specification of hADSCs in vitro. ADSCs were isolated from subcutaneous abdominal adipose tissue and were characterized. For dopaminergic differentiation, a cocktail of sonic hedgehog, fibroblast growth factor 8, basic fibroblast growth factor, and brain-derived neurotrophic factor were used under a low serum condition. As the control group, the ADSCs were cultured under the same low serum condition without the dopaminergic cocktail. At the end of differentiation period, the cells expressed neuron-specific markers, NES, NSE, and NEFL, and dopaminergic markers, EN1, NURR1, PITX3, VMAT2, TH, and GIRK2 genes. TH, NURR1, and EN1 mRNAs were upregulated in the dopaminergic group compared with the control group. NEFL and TH proteins were also expressed in the differentiated cells. A total of 27.9% of the cells differentiated in dopaminergic induction medium showed positive staining for TH protein. Based on reversed-phase high-performance liquid chromatography analysis, the differentiated cells released a significant amount of dopamine in response to KCl-induced depolarization. In conclusion, results of this study indicate that hADSCs can be induced by a growth factor cocktail to produce dopamine secreting cells with possible applications for future cell replacement therapy of Parkinson's disease.

  10. Expression of a fms-related oncogene in carcinogen-induced neoplastic epithelial cells

    International Nuclear Information System (INIS)

    Walker, C.; Nettesheim, P.; Barrett, J.C.; Gilmer, T.M.

    1987-01-01

    Following carcinogen exposure in vitro, normal rat tracheal epithelial cells are transformed in a multistage process in which the cultured cells become immortal and ultimately, neoplastic. Five cell lines derived from tumors produced by neoplastically transformed rat tracheal epithelial cells were examined for the expression of 11 cellular oncogenes previously implicated in pulmonary or epithelial carcinogenesis. RNA homologous to fms was expressed at a level 5-19 times higher than normal tracheal epithelial cells in three of five of the tumor-derived lines. All three lines expressing high levels of fms-related RNA gave rise to invasive tumors of epithelial origin when injected into nude mice. Increased expression of the fms-related mRNA was not due to gene amplification, and no gene rearrangement was detected by Southern analyses. RNA blot analysis using a 3' v-fms probe detected a 9.5-kilobase message in the three tumor-derived lines, whereas both normal rat aveolar macrophages and the human choriocarcinoma line BeWo expressed a fms transcript of ≅ 4 kilobases. The authors conclude from these data that the gene expressed as a 9.5-kilobase transcript in these neoplastic epithelial cells is a member of a fms-related gene family but may be distinct from the gene that encodes the macrophage colony-stimulating factor (CSF-1) receptor

  11. iNKT cell cytotoxic responses control T-lymphoma growth in vitro and in vivo

    Science.gov (United States)

    Bassiri, Hamid; Das, Rupali; Guan, Peng; Barrett, David M.; Brennan, Patrick J.; Banerjee, Pinaki P.; Wiener, Susan J.; Orange, Jordan S.; Brenner, Michael B.; Grupp, Stephan A.; Nichols, Kim E.

    2013-01-01

    Invariant natural killer T (iNKT) cells comprise a lineage of CD1d-restricted glycolipid-reactive T lymphocytes with important roles in host immunity to cancer. iNKT cells indirectly participate in antitumor responses by inducing dendritic cell maturation and producing cytokines that promote tumor clearance by CD8+ T and NK cells. Although iNKT cells thereby act as potent cellular adjuvants, it is less clear whether they directly control the growth of tumors. To gain insights into the direct contribution of iNKT cells to tumor immune surveillance, we developed in vitro and in vivo systems to selectively examine the antitumor activity of iNKT cells in the absence of other cytolytic effectors. Using the EL4 T-lymphoma cell line as a model, we find that iNKT cells exert robust and specific lysis of tumor cells in vitro in a manner that is differentially-induced by iNKT cell agonists of varying TCR affinities, such as OCH, α-galactosyl ceramide and PBS44. In vitro blockade of CD1d-mediated lipid antigen presentation, disruption of T cell receptor (TCR) signaling, or loss of perforin expression significantly reduce iNKT cell killing. Consistent with these findings, iNKT cell reconstitution of T, B, and NK cell-deficient mice slows EL4 growth in vivo via TCR-CD1d and perforin-dependent mechanisms. Together, these observations establish that iNKT cells are sufficient to control the growth of T-lymphoma in vitro and in vivo. They also suggest that the induction of iNKT cell cytotoxic responses in situ might serve as a more effective strategy to prevent and/or treat CD1d+ cancers, such as T-lymphoma. PMID:24563871

  12. An Abbreviated Protocol for In Vitro Generation of Functional Human Embryonic Stem Cell-Derived Beta-Like Cells

    DEFF Research Database (Denmark)

    Massumi, Mohammad; Pourasgari, Farzaneh; Nalla, Amarnadh

    2016-01-01

    developed an abbreviated five-stage protocol (25-30 days) to generate human Embryonic Stem Cell-Derived Beta-like Cells (ES-DBCs). We showed that Geltrex, as an extracellular matrix, could support the generation of ES-DBCs more efficiently than that of the previously described culture systems......The ability to yield glucose-responsive pancreatic beta-cells from human pluripotent stem cells in vitro will facilitate the development of the cell replacement therapies for the treatment of Type 1 Diabetes. Here, through the sequential in vitro targeting of selected signaling pathways, we have...... positive cells, 1% insulin and glucagon positive cells and 30% insulin and NKX6.1 co-expressing cells. Functionally, ES-DBCs were responsive to high glucose in static incubation and perifusion studies, and could secrete insulin in response to successive glucose stimulations. Mitochondrial metabolic flux...

  13. Comparative mutagenesis of human cells in vivo and in vitro

    International Nuclear Information System (INIS)

    Thilly, W.G.

    1992-05-01

    This report discusses measuring methods of point mutations; high density cell cultures for low dose studies; measurement and sequence determination of mutations in DNA; the mutational spectra of styrene oxide and ethlyene oxide in TK-6 cells; mutational spectrum of Cr in human lymphoblast cells; mutational spectra of radon in TK-6 cells; and the mutational spectra of smokeless tobacco

  14. IN VITRO TRANSPLANTATION OF GENETICALLY MODIFIED CELLS TO THE TENDON SURFACE

    OpenAIRE

    Couvreur, Paulus J. J.; Zhao, Chunfeng; Murphy, Stephen; Amadio, Peter C.

    2008-01-01

    The objective of this paper was to study in vitro transfection of tendon cells and adherence of transfected cells to different tendon surfaces. Achilles tendon fibroblasts from 2-month-old New Zealand white rabbits were cultured to confluence, after which the cells were transfected by an adenovirus carrying either the β-galactosidase reporter gene or the green fluorescent protein (GFP) gene at multiplicities of infection (MOIs) of 50, 100, or 500. Two days later, the cells were transplanted o...

  15. iNKT cell cytotoxic responses control T-lymphoma growth in vitro and in vivo .

    Science.gov (United States)

    Bassiri, Hamid; Das, Rupali; Guan, Peng; Barrett, David M; Brennan, Patrick J; Banerjee, Pinaki P; Wiener, Susan J; Orange, Jordan S; Brenner, Michael B; Grupp, Stephan A; Nichols, Kim E

    2014-01-01

    Invariant natural killer T (iNKT) cells comprise a lineage of CD1d-restricted glycolipid-reactive T lymphocytes with important roles in host immunity to cancer. iNKT cells indirectly participate in antitumor responses by inducing dendritic cell maturation and producing cytokines that promote tumor clearance by CD8+ T and NK cells. Although iNKT cells thereby act as potent cellular adjuvants, it is less clear whether they directly control the growth of tumors. To gain insights into the direct contribution of iNKT cells to tumor immune surveillance, we developed in vitro and in vivo systems to selectively examine the antitumor activity of iNKT cells in the absence of other cytolytic effectors. Using the EL4 T-lymphoma cell line as a model, we found that iNKT cells exert robust and specific lysis of tumor cells in vitro in a manner that is differentially induced by iNKT cell agonists of varying T-cell receptor (TCR) affinities, such as OCH, α-galactosyl ceramide, and PBS44. In vitro blockade of CD1d-mediated lipid antigen presentation, disruption of TCR signaling, or loss of perforin expression significantly reduce iNKT cell killing. Consistent with these findings, iNKT cell reconstitution of T, B, and NK cell–deficient mice slows EL4 growth in vivo via TCR-CD1d and perforin-dependent mechanisms. Together, these observations establish that iNKT cells are sufficient to control the growth of T lymphoma in vitro and in vivo. They also suggest that the induction of iNKT cell cytotoxic responses in situ might serve as a more effective strategy to prevent and/or treat CD1d+ cancers, such as T lymphoma. ©2013 AACR.

  16. Endothelial cell subpopulations in vitro: cell volume, cell cycle, and radiosensitivity

    International Nuclear Information System (INIS)

    Rubin, D.B.; Drab, E.A.; Bauer, K.D.

    1989-01-01

    Vascular endothelial cells (EC) are important clinical targets of radiation and other forms of free radical/oxidant stresses. In this study, we found that the extent of endothelial damage may be determined by the different cytotoxic responses of EC subpopulations. The following characteristics of EC subpopulations were examined: (1) cell volume; (2) cell cycle position; and (3) cytotoxic indexes for both acute cell survival and proliferative capacity after irradiation (137Cs, gamma, 0-10 Gy). EC cultured from bovine aortas were separated by centrifugal elutriation into subpopulations of different cell volumes. Through flow cytometry, we found that cell volume was related to the cell cycle phase distribution. The smallest EC were distributed in G1 phase and the larger cells were distributed in either early S, middle S, or late S + G2M phases. Cell cycle phase at the time of irradiation was not associated with acute cell loss. However, distribution in the cell cycle did relate to cell survival based on proliferative capacity (P less than 0.01). The order of increasing radioresistance was cells in G1 (D0 = 110 cGy), early S (135 cGy), middle S (145 cGy), and late S + G2M phases (180 cGy). These findings (1) suggest an age-related response to radiation in a nonmalignant differentiated cell type and (2) demonstrate EC subpopulations in culture

  17. Human spermatogonial stem cells display limited proliferation in vitro under mouse spermatogonial stem cell culture conditions.

    Science.gov (United States)

    Medrano, Jose V; Rombaut, Charlotte; Simon, Carlos; Pellicer, Antonio; Goossens, Ellen

    2016-11-01

    To study the ability of human spermatogonial stem cells (hSSCs) to proliferate in vitro under mouse spermatogonial stem cell (mSSC) culture conditions. Experimental basic science study. Reproductive biology laboratory. Cryopreserved testicular tissue with normal spermatogenesis obtained from three donors subjected to orchiectomy due to a prostate cancer treatment. Testicular cells used to create in vitro cell cultures corresponding to the following groups: [1] unsorted human testicular cells, [2] differentially plated human testicular cells, and [3] cells enriched with major histocompatibility complex class 1 (HLA - )/epithelial cell surface antigen (EPCAM + ) in coculture with inactivated testicular feeders from the same patient. Analyses and characterization including immunocytochemistry and quantitative reverse-transcription polymerase chain reaction for somatic and germ cell markers, testosterone and inhibin B quantification, and TUNEL assay. Putative hSSCs appeared in singlets, doublets, or small groups of up to four cells in vitro only when testicular cells were cultured in StemPro-34 medium supplemented with glial cell line-derived neurotrophic factor (GDNF), leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF). Fluorescence-activated cell sorting with HLA - /EPCAM + resulted in an enrichment of 27% VASA + /UTF1 + hSSCs, compared to 13% in unsorted controls. Coculture of sorted cells with inactivated testicular feeders gave rise to an average density of 112 hSSCs/cm 2 after 2 weeks in vitro compared with unsorted cells (61 hSSCs/cm 2 ) and differentially plated cells (49 hSSCS/cm 2 ). However, putative hSSCs rarely stained positive for the proliferation marker Ki67, and their presence was reduced to the point of almost disappearing after 4 weeks in vitro. We found that hSSCs show limited proliferation in vitro under mSSC culture conditions. Coculture of HLA - /EPCAM + sorted cells with testicular

  18. In Vitro T-Cell Generation From Adult, Embryonic, and Induced Pluripotent Stem Cells: Many Roads to One Destination.

    Science.gov (United States)

    Smith, Michelle J; Webber, Beau R; Mohtashami, Mahmood; Stefanski, Heather E; Zúñiga-Pflücker, Juan Carlos; Blazar, Bruce R

    2015-11-01

    T lymphocytes are critical mediators of the adaptive immune system and have the capacity to serve as therapeutic agents in the areas of transplant and cancer immunotherapy. While T cells can be isolated and expanded from patients, T cells derived in vitro from both hematopoietic stem/progenitor cells (HSPCs) and human pluripotent stem cells (hPSCs) offer great potential advantages in generating a self-renewing source of T cells that can be readily genetically modified. T-cell differentiation in vivo is a complex process requiring tightly regulated signals; providing the correct signals in vitro to induce T-cell lineage commitment followed by their development into mature, functional, single positive T cells, is similarly complex. In this review, we discuss current methods for the in vitro derivation of T cells from murine and human HSPCs and hPSCs that use feeder-cell and feeder-cell-free systems. Furthermore, we explore their potential for adoption for use in T-cell-based therapies. © 2015 AlphaMed Press.

  19. Gene delivery to pancreatic exocrine cells in vivo and in vitro

    Directory of Open Access Journals (Sweden)

    Houbracken Isabelle

    2012-10-01

    Full Text Available Abstract Background Effective gene transfer to the pancreas or to pancreatic cells has remained elusive although it is essential for studies of genetic lineage tracing and modulation of gene expression. Different transduction methods and viral vectors were tested in vitro and in vivo, in rat and mouse pancreas. Results For in vitro transfection/transduction of rat exocrine cells lipofection reagents, adenoviral vectors, and Mokola- and VSV-G pseudotyped lentiviral vectors were used. For in vivo transduction of mouse and rat pancreas adenoviral vectors and VSV-G lentiviral vectors were injected into the parenchymal tissue. Both lipofection of rat exocrine cell cultures and transduction with Mokola pseudotyped lentiviral vectors were inefficient and resulted in less than 4% EGFP expressing cells. Adenoviral transduction was highly efficient but its usefulness for gene delivery to rat exocrine cells in vitro was hampered by a drastic increase in cell death. In vitro transduction of rat exocrine cells was most optimal with VSV-G pseudotyped lentiviral vectors, with stable transgene expression, no significant effect on cell survival and about 40% transduced cells. In vivo, pancreatic cells could not be transduced by intra-parenchymal administration of lentiviral vectors in mouse and rat pancreas. However, a high efficiency could be obtained by adenoviral vectors, resulting in transient transduction of mainly exocrine acinar cells. Injection in immune-deficient animals diminished leukocyte infiltration and prolonged transgene expression. Conclusions In summary, our study remarkably demonstrates that transduction of pancreatic exocrine cells requires lentiviral vectors in vitro but adenoviral vectors in vivo.

  20. The alkylphospholipid, perifosine, radiosensitizes prostate cancer cells both in vitro and in vivo

    International Nuclear Information System (INIS)

    Gao, Yuanhong; Ittmann, Michael; Thompson, Timothy C; Butler, E Brian; Xu, Bo; Teh, Bin S; Ishiyama, Hiromichi; Sun, Mianen; Brinkman, Kathryn L; Wang, Xiaozhen; Zhu, Julie; Mai, Weiyuan; Huang, Ying; Floryk, Daniel

    2011-01-01

    Perifosine is a membrane-targeted alkylphospholipid developed to inhibit the PI3K/Akt pathway and has been suggested as a favorable candidate for combined use with radiotherapy. In this study, we investigated the effect of the combined treatment of perifosine and radiation (CTPR) on prostate cancer cells in vitro and on prostate cancer xenografts in vivo. Human prostate cancer cell line, CWR22RV1, was treated with perifosine, radiation, or CTPR. Clonogenic survival assays, sulforhodamine B cytotoxity assays and cell density assays were used to assess the effectiveness of each therapy in vitro. Measurements of apoptosis, cell cycle analysis by flow cytometry and Western blots were used to evaluate mechanisms of action in vitro. Tumor growth delay assays were used to evaluate radiation induced tumor responses in vivo. In vitro, CTPR had greater inhibitory effects on prostate cancer cell viability and clonogenic survival than either perifosine or radiation treatment alone. A marked increase in prostate cancer cell apoptosis was noted in CTPR. Phosphorylation of AKT-T308 AKT and S473 were decreased when using perifosine treatment or CTPR. Cleaved caspase 3 was significantly increased in the CTPR group. In vivo, CTPR had greater inhibitory effects on the growth of xenografts when compared with perifosine or radiation treatment alone groups. Perifosine enhances prostate cancer radiosensitivity in vitro and in vivo. These data provide strong support for further development of this combination therapy in clinical studies

  1. In Vitro Generation of Functional Liver Organoid-Like Structures Using Adult Human Cells.

    Science.gov (United States)

    Ramachandran, Sarada Devi; Schirmer, Katharina; Münst, Bernhard; Heinz, Stefan; Ghafoory, Shahrouz; Wölfl, Stefan; Simon-Keller, Katja; Marx, Alexander; Øie, Cristina Ionica; Ebert, Matthias P; Walles, Heike; Braspenning, Joris; Breitkopf-Heinlein, Katja

    2015-01-01

    In this study we used differentiated adult human upcyte® cells for the in vitro generation of liver organoids. Upcyte® cells are genetically engineered cell strains derived from primary human cells by lenti-viral transduction of genes or gene combinations inducing transient proliferation capacity (upcyte® process). Proliferating upcyte® cells undergo a finite number of cell divisions, i.e., 20 to 40 population doublings, but upon withdrawal of proliferation stimulating factors, they regain most of the cell specific characteristics of primary cells. When a defined mixture of differentiated human upcyte® cells (hepatocytes, liver sinusoidal endothelial cells (LSECs) and mesenchymal stem cells (MSCs)) was cultured in vitro on a thick layer of Matrigel™, they self-organized to form liver organoid-like structures within 24 hours. When further cultured for 10 days in a bioreactor, these liver organoids show typical functional characteristics of liver parenchyma including activity of cytochromes P450, CYP3A4, CYP2B6 and CYP2C9 as well as mRNA expression of several marker genes and other enzymes. In summary, we hereby describe that 3D functional hepatic structures composed of primary human cell strains can be generated in vitro. They can be cultured for a prolonged period of time and are potentially useful ex vivo models to study liver functions.

  2. Chemical Characterization and in Vitro Cytotoxicity on Squamous Cell Carcinoma Cells of Carica Papaya Leaf Extracts

    Directory of Open Access Journals (Sweden)

    Thao T. Nguyen

    2015-12-01

    Full Text Available In traditional medicine, Carica papaya leaf has been used for a wide range of therapeutic applications including skin diseases and cancer. In this study, we investigated the in vitro cytotoxicity of aqueous and ethanolic extracts of Carica papaya leaves on the human oral squamous cell carcinoma SCC25 cell line in parallel with non-cancerous human keratinocyte HaCaT cells. Two out of four extracts showed a significantly selective effect towards the cancer cells and were found to contain high levels of phenolic and flavonoid compounds. The chromatographic and mass spectrometric profiles of the extracts obtained with Ultra High Performance Liquid Chromatography-Quadrupole Time of Flight-Mass Spectrometry were used to tentatively identify the bioactive compounds using comparative analysis. The principal compounds identified were flavonoids or flavonoid glycosides, particularly compounds from the kaempferol and quercetin families, of which several have previously been reported to possess anticancer activities. These results confirm that papaya leaf is a potential source of anticancer compounds and warrant further scientific investigation to validate the traditional use of papaya leaf to treat cancer.

  3. Enhanced Radiosensitivity of Tumor Cells Treated with Vanadate in Vitro

    International Nuclear Information System (INIS)

    Lee, Myung Za; Lee, Won Young

    1994-01-01

    Intracellular ions which have a major role in cellular function have been reported to affect repair of radiation damage. Recently it has been reported that ouabain sensitizes A549 tumor cells hut not CCL-120 normal cells to radiation. Ouabain inhibits the Na+-K+-pump rapidly thus it increases intracellular Na concentration. Vanadate which is distributed extensively in almost all living organisms in known to be a Na+-K+-ATPase inhibitors. This study was performed to see any change in radiosensitivity of tumor cell by vanadate and any role of Na+-K+-ATPase in radiosensitization. Experiments have been carried out by pretreatment with vanadate in human cell line(A549, JMG) and mouse cell line(L1210, spleen). For the cell survival MTT assay was performed for A549 and JMG cell and trypan blue dye exclusion test for L120, and spleen cells. Measurements of Na+-K+-ATPase activity in control, vanadate treated cell, radiation treated cell (9 Gy for A549 and JMG, 2 Gy for L1201, spleen), and combined 10-6 M vanadate and radiation treated cells were done. The results were summarized as follows. 1. L1210 cell was most radiosensitive, and spleen cell and JMG cell were intermediate, and A549 cell was least radiosensitive. 2. Minimum or cytotoxicity was seen with vanadate below concentration of 10-6 M. 3. In A549 cells there was a little change in radiosensitivity with treatment of vanadate. However radiation sensitization was shown in low dose level of radiation i. E. 2-Gy. In JMG cells no change in radiosensitivity was noted. Both L1210 and spleen cell had radiosensitization but change was greater in tumor cell. 4. Na+-K+-ATPase activity was inhibited significantly in tumor cell by treatment of vanadate. 5. Radiation itself inhibited Na+-K+-ATPase activity of tumor cell with high Na+- K+-ATPase concention. Increase in radiosensitivity by vanadate was closely associated with original Na+-K+-ATPase contents. From the above results vanadate had little cytotoxicity and it sensitized

  4. A three-dimensional approach to in vitro culture of immune-related cells

    DEFF Research Database (Denmark)

    Hartmann, Sofie Bruun

    setups for improved activation/differentiation of immune cells. Conclusively, this work highlights the importance of acknowledging the effect from external factors when analyzing data generated from in vitro cultures. This being even more important when working with immune cells since these cells adopt......T lymphocytes are key players during the initiation of an adaptive immune response. The activation of these cells in vivo requires migration within the lymph nodes until they encounter antigen presenting cells (APCs) that can activate them to secrete IFN-γ which mediates downstream effector...... functions. The in vitro reactivation of antigen-experienced T lymphocytes and detection of IFN-γ from cell cultures can be used in a diagnostic assay to test for disease or vaccine efficacy. Practical procedures of the IFN-γ release assay (IGRA) was investigated using bovine cells and whole blood cultures...

  5. Intestinal Stem Cell Niche Insights Gathered from Both In Vivo and Novel In Vitro Models

    Directory of Open Access Journals (Sweden)

    Nikolce Gjorevski

    2017-01-01

    Full Text Available Intestinal stem cells are located at the base of the crypts and are surrounded by a complex structure called niche. This environment is composed mainly of epithelial cells and stroma which provides signals that govern cell maintenance, proliferation, and differentiation. Understanding how the niche regulates stem cell fate by controlling developmental signaling pathways will help us to define how stem cells choose between self-renewal and differentiation and how they maintain their undifferentiated state. Tractable in vitro assay systems, which reflect the complexity of the in vivo situation but provide higher level of control, would likely be crucial in identifying new players and mechanisms controlling stem cell function. Knowledge of the intestinal stem cell niche gathered from both in vivo and novel in vitro models may help us improve therapies for tumorigenesis and intestinal damage and make autologous intestinal transplants a feasible clinical practice.

  6. In vitro evaluation of cardiomyogenic differentiation of bone marrow derived mesenchymal stem cells (MSCs)

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Min Hwan; Lee, Yong Jin; Kang, Joo Hyun [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

    2010-10-15

    Bone marrow derived mesenchymal stem cells (MSCs) are excellent candidate as therapeutic agent for cell therapy. MSCs can be expanded in vitro rapidly (more than 3-5 fold in a weeks), and maintained their stem cell properties for a long culture period. Recently, many investigators have suggested that MSCs have ability to differentiate into cardiomyocytes by given appropriate condition in vitro or in vivo. Although, MSCs may be useful cell therapeutic agents in heart disease, there are still exist major barriers to track their capacity to differentiate into functional cardiomyocytes. In our previous study, the transgenic mouse model expressing sodium iodide symporter (NIS) driven by {alpha}-myosin heavy chain ({alpha}-MHC) promoter was developed to image cardiomyocyte with {gamma}-camera and microPET in vivo. In this study, we investigate the monitoring availability of {alpha}-MHC driven NIS gene of MSCs from the transgenic mouse during cardiomyogenic differentiation in vitro

  7. 21 CFR 864.9225 - Cell-freezing apparatus and reagents for in vitro diagnostic use.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Cell-freezing apparatus and reagents for in vitro diagnostic use. 864.9225 Section 864.9225 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH... Establishments That Manufacture Blood and Blood Products § 864.9225 Cell-freezing apparatus and reagents for in...

  8. The odontogenic potential of STRO-1 sorted rat dental pulp stem cells in vitro.

    NARCIS (Netherlands)

    Yang, X.; Dolder, J. van den; Walboomers, X.F.; Zhang, W.; Bian, Z.; Fan, M.; Jansen, J.A.

    2007-01-01

    The presence of heterogeneous cell populations in dental pulp may count for the considerable variation in the outcome of in vitro and in vivo experiments. Here, we intended to determine whether a minor cell sub-population of high proliferation and odontogenic potential existed among a larger

  9. In vitro assays for cobblestone area-forming cells, LTC-IC, and CFU-C

    NARCIS (Netherlands)

    van Os, Ronald P; Dethmers-Ausema, Bertien; de Haan, Gerald; Bunting, Kevin

    2008-01-01

    Various assays exist that measure the function of hematopoietic stemcells (HSCs). In this chapter, in vitro assays are described that measure the frequency of progenitors (colony-forming unit in culture; CFU-C), stem cells (long-term culture-initiating cell; LTC-IC), or both (cobblestone

  10. Propranolol inhibits the in vitro conversion of thyroxine into triiodothyronine by isolated rat liver parenchymal cells

    NARCIS (Netherlands)

    van Noorden, C. J.; Wiersinga, W. M.; Touber, J. L.

    1979-01-01

    A model for the in vitro study of the conversion of thyroxine into triiodothyronine using isolated rat liver parenchymal cells is described. Isolated liver cells (mean protein content 18 mg/ml) convert approximately 0.8% of 1.3 microM exogenously added T4 into T3 during thirty minutes incubation.

  11. Nocardia brasiliensis induces formation of foamy macrophages and dendritic cells in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Irene Meester

    Full Text Available Foamy cells have been described in various infectious diseases, for example in actinomycetoma induced by Nocardia brasiliensis. These cells are generally considered to be macrophages, although they present dendritic cell (DC-specific surface markers. In this study, we determined and confirmed the lineage of possible precursors of foamy cells in vitro and in vivo using an experimental actinomycetoma model in BALB/c mice. Bone marrow-derived macrophages (BMDM or DC (BMDC were infected in vitro with N. brasiliensis or labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE. Both, macrophages and DC, differentiated into foamy cells after in vitro infection. CFSE-labeled BMDM or BMDC were tested for phagocytosis and CD11c/CD11b receptors markers expression before being transferred into the actinomycetoma lesion site of infected mice. In vivo studies showed that BMDM and BMDC were traced at the site where foamy cells are present in the experimental actinomycetoma. Interestingly, many of the transferred BMDM and BMDC were stained with the lipid-droplet fluorophore Nile Red. In conclusion, macrophages and DC cells can be differentiated into foamy cells in vitro and in vivo during N. brasiliensis infection.

  12. Nocardia brasiliensis induces formation of foamy macrophages and dendritic cells in vitro and in vivo.

    Science.gov (United States)

    Meester, Irene; Rosas-Taraco, Adrian Geovanni; Salinas-Carmona, Mario Cesar

    2014-01-01

    Foamy cells have been described in various infectious diseases, for example in actinomycetoma induced by Nocardia brasiliensis. These cells are generally considered to be macrophages, although they present dendritic cell (DC)-specific surface markers. In this study, we determined and confirmed the lineage of possible precursors of foamy cells in vitro and in vivo using an experimental actinomycetoma model in BALB/c mice. Bone marrow-derived macrophages (BMDM) or DC (BMDC) were infected in vitro with N. brasiliensis or labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE). Both, macrophages and DC, differentiated into foamy cells after in vitro infection. CFSE-labeled BMDM or BMDC were tested for phagocytosis and CD11c/CD11b receptors markers expression before being transferred into the actinomycetoma lesion site of infected mice. In vivo studies showed that BMDM and BMDC were traced at the site where foamy cells are present in the experimental actinomycetoma. Interestingly, many of the transferred BMDM and BMDC were stained with the lipid-droplet fluorophore Nile Red. In conclusion, macrophages and DC cells can be differentiated into foamy cells in vitro and in vivo during N. brasiliensis infection.

  13. Novel diffusion cell for in vitro transdermal permeation, compatible with automated dynamic sampling

    NARCIS (Netherlands)

    Bosman, I.J; Lawant, A.L; Avegaart, S.R.; Ensing, K; de Zeeuw, R.A

    The development of a new diffusion cell for in vitro transdermal permeation is described. The so-called Kelder cells were used in combination with the ASPEC system (Automatic Sample Preparation with Extraction Columns), which is designed for the automation of solid-phase extractions (SPE). Instead

  14. Chemosensitivity and radiosensitivity testing of freshly explanted human tumour cells in vitro

    International Nuclear Information System (INIS)

    Wells, J.

    1977-10-01

    In this thesis, in vitro testing for the chemosensitivity and radiosensitivity of freshly explanted human tumour cells is described. The cells were incubated with anti-tumour drugs and either a 6-day growth test performed or a clonal growth test as a measure of survival of cell reproductive capacity. It was shown that if one aims to develop a suitable in vitro method for predicting the subsequent response of human tumour cells in situ to cytotoxic chemotherapy, the test procedure must be initiated before the explanted cells have undergone significant growth in vitro. The survival of the reproductive capacity of tumour cell explants following X-radiation was also studied. Using a 'feeder' layer technique, values for the survival curve parameter Dsub(q) were in the range 400-610 rad and the values for D 0 were in the range 120-160 rad. The shape of the X-ray survival curves did not change when cells were retested after repeated subculturing in vitro. Therefore, unlike chemosensitivity measured by the same biological end-point, radiosensitivity apparently does not change once cells have reached their maximum growth potential. (UK)

  15. In Vitro Derivation and Propagation of Spermatogonial Stem Cell Activity from Mouse Pluripotent Stem Cells.

    Science.gov (United States)

    Ishikura, Yukiko; Yabuta, Yukihiro; Ohta, Hiroshi; Hayashi, Katsuhiko; Nakamura, Tomonori; Okamoto, Ikuhiro; Yamamoto, Takuya; Kurimoto, Kazuki; Shirane, Kenjiro; Sasaki, Hiroyuki; Saitou, Mitinori

    2016-12-06

    The in vitro derivation and propagation of spermatogonial stem cells (SSCs) from pluripotent stem cells (PSCs) is a key goal in reproductive science. We show here that when aggregated with embryonic testicular somatic cells (reconstituted testes), primordial germ cell-like cells (PGCLCs) induced from mouse embryonic stem cells differentiate into spermatogonia-like cells in vitro and are expandable as cells that resemble germline stem cells (GSCs), a primary cell line with SSC activity. Remarkably, GSC-like cells (GSCLCs), but not PGCLCs, colonize adult testes and, albeit less effectively than GSCs, contribute to spermatogenesis and fertile offspring. Whole-genome analyses reveal that GSCLCs exhibit aberrant methylation at vulnerable regulatory elements, including those critical for spermatogenesis, which may restrain their spermatogenic potential. Our study establishes a strategy for the in vitro derivation of SSC activity from PSCs, which, we propose, relies on faithful epigenomic regulation. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  16. Eimeria tenella: in vitro development in irradiated bovine kidney cells

    Energy Technology Data Exchange (ETDEWEB)

    Crane, M.St.J.; Schmatz, D.M.; Stevens, S.; Habbersett, M.C.; Murray, P.K. (Merck Sharp and Dohme Research Labs., Rahway, NJ (USA))

    1984-06-01

    The initial infection and first-generation development of Eimeria tenella was quantified using a cloned MDBK (Madin-Darby Bovine Kidney) cell line, irradiated with gamma radiation prior to infection, as the host cell. Irradiated cell cultures were found to be more susceptible to infection and had a greater capacity to support parasite development than non-irradiated cultures. It was suggested that the larger proportion of cells in the G/sub 2/ phase of the cell cycle, the larger individual cell size and the inhibition of cell division in the irradiated cultures were all factors contributing to the increased susceptibility to infection and capacity to support parasite growth and development. The application of this technique (host cell irradiation) to the cultivation of other intracellular, protozoan parasites is discussed.

  17. Eimeria tenella: in vitro development in irradiated bovine kidney cells

    International Nuclear Information System (INIS)

    Crane, M. St.J.; Schmatz, D.M.; Stevens, S.; Habbersett, M.C.; Murray, P.K.

    1984-01-01

    The initial infection and first-generation development of Eimeria tenella was quantified using a cloned MDBK (Madin-Darby Bovine Kidney) cell line, irradiated with gamma radiation prior to infection, as the host cell. Irradiated cell cultures were found to be more susceptible to infection and had a greater capacity to support parasite development than non-irradiated cultures. It was suggested that the larger proportion of cells in the G 2 phase of the cell cycle, the larger individual cell size and the inhibition of cell division in the irradiated cultures were all factors contributing to the increased susceptibility to infection and capacity to support parasite growth and development. The application of this technique (host cell irradiation) to the cultivation of other intracellular, protozoan parasites is discussed. (author)

  18. [EXPERIMENTAL RESEARCH OF DIFFERENTIATION OF HUMAN AMNIOTIC MESENCHYMAL STEM CELLS INTO LIGAMENT CELLS IN VITRO].

    Science.gov (United States)

    Jin, Ying; Li, Yuwan; Zhang, Chenghao; Wu, Shuhong; Cheng, Daixiong; Liu, Yi

    2016-02-01

    To discuss whether human amniotic mesenchymal stem cells (hAMSCs) possesses the characteristic of mesenchymal stem cells, and could differentiate into ligament cells in vitro after induction. The hAMSCs were separated through enzyme digestion, and the phenotypic characteristics of hAMSCs were tested through flow cytometry. The cells at passage 3 were cultured with L-DMEM/F12 medium containing transforming growth factor beta1 (TGF-beta1) + basic fibroblast growth factor (bFGF) (group A), containing hyaluronic acid (HA) (group B), containing TGF-beta1+bFGF+HA (group C), and simple L-DMEM/F12 medium (group D) as control group. The morphology changes of cells in each group were observed by inverted phase contrast microscope at 21 days after induction; the cellular activities and proliferation were examined by sulforhodamine (SRB) colorimetric method; and specific mRNA and protein expressions of ligament including collagen type I, collagen type III, and tenascin C (TNC) were measured by real-time fluorescence quantitative PCR and immunohistochemical staining. The flow cytometry result indicated that hAMSCs expressed mesenchymal stem cell phenotype. After 21 days of induction, the cells in groups A, B, and C grew like spindle-shaped fibroblasts under inverted phase contrast microscope, and cells showed single shape, obvious directivity, and compact arrangement in group C. The SRB result indicated that the cells in each group reached the peak of growth curve at 6 days; the cellular activities of groups A, B, and C were significantly higher than that of group D at 6 days after induction. Also, the immunohistochemical staining results showed that no expressions of TNC were detected in 4 groups at 7 days; expressions of collagen type I in groups A, B, and C were significantly higher than that in group D at 7, 14, and 21 days (Pligament specific genes can be up-regulated and the synthesis of ligament specific proteins can be also strengthened. As a result, it can be used as

  19. Differentiation ability of rat postnatal dental pulp cells in vitro.

    NARCIS (Netherlands)

    Zhang, W.; Walboomers, X.F.; Wolke, J.G.C.; Bian, Z.; Fan, M.W.; Jansen, J.A.

    2005-01-01

    The current rapid progression in stem cell research has enhanced our knowledge of dental tissue regeneration. In this study, rat dental pulp cells were isolated and their differentiation ability was evaluated. First, dental pulp cells were obtained from maxillary incisors of male Wistar rats.

  20. Isolation and in vitro expansion of human colonic stem cells

    NARCIS (Netherlands)

    Jung, P.; Sato, T.; Merlos-Suarez, A.; Barriga, F.M.; Iglesias, M.; Rossell, D.; Auer, H.; Gallardo, M.; Blasco, M.A.; Sancho, E.; Clevers, H.; Batlle, E.

    2011-01-01

    Here we describe the isolation of stem cells of the human colonic epithelium. Differential cell surface abundance of ephrin type-B receptor 2 (EPHB2) allows the purification of different cell types from human colon mucosa biopsies. The highest EPHB2 surface levels correspond to epithelial colonic

  1. Study of the cetuximab-ionizing radiation association on a model of in vitro endothelial cells

    International Nuclear Information System (INIS)

    Vautravers-Dewas, C.; Rebucci, M.; Lansiaux, A.; Peixoto, P.; Lartigau, E.

    2009-01-01

    These data support the existence of functional signalling tracks downstream epidermal growth factor receptor (E.G.F.R.) in endothelial cells. But our in vitro model, in two dimensional calculations, did not allow to show a radiosensitivity by cetuximab. The study of functional consequences of the association cetuximab-irradiation treatment will be relevant in an in vitro three dimensional model and on xenografts. (N.C.)

  2. Multiple mesodermal lineage differentiation of Apodemus sylvaticus embryonic stem cells in vitro

    Directory of Open Access Journals (Sweden)

    Yu Weihua

    2010-06-01

    Full Text Available Abstract Background Embryonic stem (ES cells have attracted significant attention from researchers around the world because of their ability to undergo indefinite self-renewal and produce derivatives from the three cell lineages, which has enormous value in research and clinical applications. Until now, many ES cell lines of different mammals have been established and studied. In addition, recently, AS-ES1 cells derived from Apodemus sylvaticus were established and identified by our laboratory as a new mammalian ES cell line. Hence further research, in the application of AS-ES1 cells, is warranted. Results Herein we report the generation of multiple mesodermal AS-ES1 lineages via embryoid body (EB formation by the hanging drop method and the addition of particular reagents and factors for induction at the stage of EB attachment. The AS-ES1 cells generated separately in vitro included: adipocytes, osteoblasts, chondrocytes and cardiomyocytes. Histochemical staining, immunofluorescent staining and RT-PCR were carried out to confirm the formation of multiple mesodermal lineage cells. Conclusions The appropriate reagents and culture milieu used in mesodermal differentiation of mouse ES cells also guide the differentiation of in vitro AS-ES1 cells into distinct mesoderm-derived cells. This study provides a better understanding of the characteristics of AS-ES1 cells, a new species ES cell line and promotes the use of Apodemus ES cells as a complement to mouse ES cells in future studies.

  3. Allometric scaling and cell ratios in multi-organ in vitro models of human metabolism

    Directory of Open Access Journals (Sweden)

    Nadia eUcciferri

    2014-12-01

    Full Text Available Intelligent in vitro models able to recapitulate the physiological interactions between tissues in the body have enormous potential as they enable detailed studies on specific two-way or higher order tissue communication. These models are the first step towards building an integrated picture of systemic metabolism and signalling in physiological or pathological conditions. However the rational design of in vitro models of cell-cell or cell-tissue interaction is difficult as quite often cell culture experiments are driven by the device used, rather than by design considerations. Indeed very little research has been carried out on in vitro models of metabolism connecting different cell or tissue types in a physiologically and metabolically relevant manner. Here we analyse the physiologic relationship between cells, cell metabolism and exchange in the human body using allometric rules, downscaling them to an organ-on-a plate device. In particular, in order to establish appropriate cell ratios in the system in a rational manner, two different allometric scaling models (Cell Number Scaling Model, CNSM, and Metabolic and Surface Scaling model, MSSM are proposed and applied to a two compartment model of hepatic-vascular metabolic cross-talk. The theoretical scaling studies illustrate that the design and hence relevance of multi-organ models is principally determined by experimental constraints. Two experimentally feasible model configurations are then implemented in a multi-compartment organ-on-a plate device. An analysis of the metabolic response of the two configurations demonstrates that their glucose and lipid balance is quite different, with only one of the two models recapitulating physiological-like homeostasis. In conclusion, not only do cross-talk and physical stimuli play an important role in in vitro models, but the numeric relationship between cells is also crucial to recreate in vitro interactions which can be extrapolated to the in vivo

  4. Allometric Scaling and Cell Ratios in Multi-Organ in vitro Models of Human Metabolism.

    Science.gov (United States)

    Ucciferri, Nadia; Sbrana, Tommaso; Ahluwalia, Arti

    2014-01-01

    Intelligent in vitro models able to recapitulate the physiological interactions between tissues in the body have enormous potential as they enable detailed studies on specific two-way or higher order tissue communication. These models are the first step toward building an integrated picture of systemic metabolism and signaling in physiological or pathological conditions. However, the rational design of in vitro models of cell-cell or cell-tissue interaction is difficult as quite often cell culture experiments are driven by the device used, rather than by design considerations. Indeed, very little research has been carried out on in vitro models of metabolism connecting different cell or tissue types in a physiologically and metabolically relevant manner. Here, we analyze the physiological relationship between cells, cell metabolism, and exchange in the human body using allometric rules, downscaling them to an organ-on-a-plate device. In particular, in order to establish appropriate cell ratios in the system in a rational manner, two different allometric scaling models (cell number scaling model and metabolic and surface scaling model) are proposed and applied to a two compartment model of hepatic-vascular metabolic cross-talk. The theoretical scaling studies illustrate that the design and hence relevance of multi-organ models is principally determined by experimental constraints. Two experimentally feasible model configurations are then implemented in a multi-compartment organ-on-a-plate device. An analysis of the metabolic response of the two configurations demonstrates that their glucose and lipid balance is quite different, with only one of the two models recapitulating physiological-like homeostasis. In conclusion, not only do cross-talk and physical stimuli play an important role in in vitro models, but the numeric relationship between cells is also crucial to recreate in vitro interactions, which can be extrapolated to the in vivo reality.

  5. Radiation response characteristics of human cell in vitro

    International Nuclear Information System (INIS)

    Hall, E.J.

    1987-01-01

    Improvements in tissue culture techniques and growth media have made it possible to culture a range of cells of human origin, both normal and malignant. The most recent addition to the list are endothelial cells. Interesting results have been obtained, some of which may have implications in Radiation Therapy. (i) Repair of Potentially Lethal Damage (PLDR) has been observed in all cell lines investigated; cells of normal origin repair PLD at least as well as malignant cells, which makes clinical trials of PLDR inhibitors of doubtful usefulness. (ii) PLD in fibroblasts of human origin appears to have a component that is repaired rapidly, in a matter of minutes, as well as a slower component that takes hours to repair. (iii) Sublethal damage repair, manifest by a dose-rate effect, has also been observed in all human cell lines tested. Cells of normal tissue origin, including fibroblasts and endothelial cells, exhibit a dose-rate effect that is intermediate between that for cells from traditionally resistant tumors (melanoma and osteosarcoma) and cells from more sensitive tumors (neuroblastoma and breast). (iv) Fibroblasts from patients with Ataxia Telangectasia (AT) are much more sensitive to x-rays, with a D/sub o/ about half that for normal human fibroblasts. Nevertheless repair of both PLD and SLD can be demonstrated in these cells

  6. Assessing cell fusion and cytokinesis failure as mechanisms of clone 9 hepatocyte multinucleation in vitro.

    Science.gov (United States)

    Simic, Damir; Euler, Catherine; Thurby, Christina; Peden, Mike; Tannehill-Gregg, Sarah; Bunch, Todd; Sanderson, Thomas; Van Vleet, Terry

    2012-08-01

    In this in vitro model of hepatocyte multinucleation, separate cultures of rat Clone 9 cells are labeled with either red or green cell tracker dyes (Red Cell Tracker CMPTX or Vybrant CFDA SE Cell Tracer), plated together in mixed-color colonies, and treated with positive or negative control agents for 4 days. The fluorescent dyes become cell-impermeant after entering cells and are not transferred to adjacent cells in a population, but are inherited by daughter cells after fusion. The mixed-color cultures are then evaluated microscopically for multinucleation and analysis of the underlying mechanism (cell fusion/cytokinesis). Multinucleated cells containing only one dye have undergone cytokinesis failure, whereas dual-labeled multinucleated cells have resulted from fusion. © 2012 by John Wiley & Sons, Inc.

  7. Impact of host cell variation on the neutralization of HIV-1 in vitro.

    Science.gov (United States)

    Polonis, Victoria R; Schuitemaker, Hanneke; Bunnik, Evelien M; Brown, Bruce K; Scarlatti, Gabriella

    2009-09-01

    In this review we present current advances in our understanding of HIV-1 neutralization assays that employ primary cell types, as compared with those that utilize cell lines and the newer, more standardized pseudovirus assays. A commentary on the challenges of standardizing in-vitro neutralization assays using primary cells is included. The data from reporter cell line neutralization assays may agree with results observed in primary cells; however, exceptions have recently been reported. Multiple variables exist in primary cell assays using peripheral blood mononuclear cells from HIV-seronegative donors; in-vitro neutralization titers can vary significantly based on the donor cells used for assay targets and for virus propagation. Thus, more research is required to achieve validated primary cell neutralization assays. HIV-vaccine-induced antibody performance in the current neutralization assays may function as a 'gatekeeper' for HIV-1 subunit vaccine advancement. Development of standardized platforms for reproducible measurement of in-vitro neutralization is therefore a high priority. Given the considerable variation in results obtained from some widely applied HIV neutralization platforms, parallel evaluation of new antibodies using different host cells for assay targets, as well as virus propagation, is recommended until immune correlates of protection are identified.

  8. Tendon synovial cells secrete fibronectin in vivo and in vitro

    International Nuclear Information System (INIS)

    Banes, A.J.; Link, G.W.; Bevin, A.G.; Peterson, H.D.; Gillespie, Y.; Bynum, D.; Watts, S.; Dahners, L.

    1988-01-01

    The chemistry and cell biology of the tendon have been largely overlooked due to the emphasis on collagen, the principle structural component of the tendon. The tendon must not only transmit the force of muscle contraction to bone to effect movement, but it must also glide simultaneously over extratendonous tissues. Fibronectin is classified as a cell attachment molecule that induces cell spreading and adhesion to substratum. The external surface of intact avian flexor tendon stained positively with antibody to cellular fibronectin. However, if the surface synovial cells were first removed with collagenase, no positive reaction with antifibronectin antibody was detected. Analysis of immunologically stained frozen sections of tendon also revealed fibronectin at the tendon synovium, but little was associated with cells internal in tendon. The staining pattern with isolated, cultured synovial cells and fibroblasts from the tendon interior substantiated the histological observations. Analysis of polyacrylamide gel profiles of 35 S-methionine-labeled proteins synthesized by synovial cells and internal fibroblasts indicated that fibronectin was synthesized principally by synovial cells. Fibronectin at the tendon surface may play a role in cell attachment to prevent cell removal by the friction of gliding. Alternatively, fibronectin, with its binding sites for hyaluronic acid and collagen, may act as a complex for boundary lubrication

  9. Morphological evaluation during in vitro chondrogenesis of dental pulp stromal cells

    Directory of Open Access Journals (Sweden)

    Choo-Ryung Chung

    2012-02-01

    Full Text Available Objectives The aim was to confirm the stem cell-like properties of the dental pulp stromal cells and to evaluate the morphologic changes during in vitro chondrogenesis. Materials and Methods Stromal cells were outgrown from the dental pulp tissue of the premolars. Surface markers were investigated and cell proliferation rate was compared to other mesenchymal stem cells. Multipotency of the pulp cells was confirmed by inducing osteogenesis, adipogenesis and chondrogenesis. The morphologic changes in the chondrogenic pellet during the 21 day of induction were evaluated under light microscope and transmission electron microscope. TUNEL assay was used to evaluate apoptosis within the chondrogenic pellets. Results Pulp cells were CD90, 105 positive and CD31, 34 negative. They showed similar proliferation rate to other stem cells. Pulp cells differentiated to osteogenic, adipogenic and chondrogenic tissues. During chondrogenesis, 3-dimensional pellet was created with multi-layers, hypertrophic chondrocyte-like cells and cartilage-like extracellular matrix. However, cell morphology became irregular and apoptotic cells were increased after 7 day of chondrogenic induction. Conclusions Pulp cells indicated mesenchymal stem cell-like characteristics. During the in vitro chondrogenesis, cellular activity was superior during the earlier phase (within 7 day of differentiation.

  10. Stem Cells from Cryopreserved Human Dental Pulp Tissues Sequentially Differentiate into Definitive Endoderm and Hepatocyte-Like Cells in vitro.

    Science.gov (United States)

    Han, Young-Jin; Kang, Young-Hoon; Shivakumar, Sarath Belame; Bharti, Dinesh; Son, Young-Bum; Choi, Yong-Ho; Park, Won-Uk; Byun, June-Ho; Rho, Gyu-Jin; Park, Bong-Wook

    2017-01-01

    We previously described a novel tissue cryopreservation protocol to enable the safe preservation of various autologous stem cell sources. The present study characterized the stem cells derived from long-term cryopreserved dental pulp tissues (hDPSCs-cryo) and analyzed their differentiation into definitive endoderm (DE) and hepatocyte-like cells (HLCs) in vitro . Human dental pulp tissues from extracted wisdom teeth were cryopreserved as per a slow freezing tissue cryopreservation protocol for at least a year. Characteristics of hDPSCs-cryo were compared to those of stem cells from fresh dental pulps (hDPSCs-fresh). hDPSCs-cryo were differentiated into DE cells in vitro with Activin A as per the Wnt3a protocol for 6 days. These cells were further differentiated into HLCs in the presence of growth factors until day 30. hDPSCs-fresh and hDPSCs-cryo displayed similar cell growth morphology, cell proliferation rates, and mesenchymal stem cell character. During differentiation into DE and HLCs in vitro , the cells flattened and became polygonal in shape, and finally adopted a hepatocyte-like shape. The differentiated DE cells at day 6 and HLCs at day 30 displayed significantly increased DE- and hepatocyte-specific markers at the mRNA and protein level, respectively. In addition, the differentiated HLCs showed detoxification and glycogen storage capacities, indicating they could share multiple functions with real hepatocytes. These data conclusively show that hPDSCs-cryo derived from long-term cryopreserved dental pulp tissues can be successfully differentiated into DE and functional hepatocytes in vitro . Thus, preservation of dental tissues could provide a valuable source of autologous stem cells for tissue engineering.

  11. [Relationship between sensitivity of tumor cells to chemotherapeutic agent in vivo and in vitro: experiment with mouse lymphoma cells].

    Science.gov (United States)

    Li, Chuan-gang; Li, Mo-lin; Shu, Xiao-hong; Jia, Yu-jie; Liu, Yong-ji; Li, Ming

    2007-06-12

    To study the relationship of the sensitivity of tumor cells to chemotherapeutic agent between in vivo and in vitro. Mouse lymphoma cells of the line E14 were cultured and melphalan resistant EL4 cell line (EL4/melphalan) was established by culturing EL4 cells with continuous low-concentration and intermittent gradually-increasing-concentration of melphalan in vitro. MTT assay was used to evaluate the drug sensitivity and the resistance index of the EL4/melphalan cells to melphalan was calculated. EL4/melphalan and EL4 cells of the concentration of 5 x 10(8)/L were inoculated separately into 20 C57BL/6 mice subcutaneously. 12 days later, the EL4 and EL4/melphalan tumor-bearing mice were randomly divided into 2 groups respectively, 5 mice in each group. Treatment groups were given 7.5 mg/kg melphalan intraperitoneally, and control groups were given the same volume of normal saline. The tumor size was observed every other day. Compared with the EL4 cells, the EL4/melphalan cells had no obvious changes morphologically. They could grow in RPMI 1640 medium containing 5 mg/ml melphalan. The resistance index was 2.87 against melphalan. After the treatment of melphalan of the dose 7.5 mg/kg, the tumor sizes of the treatment groups and control groups inoculated with both EL4 cells and the EL4/melphalan cells gradually decreased at the similar speed, and about one week later all tumors disappeared. However, the tumors of the control groups grew progressively and all the mice died at last. The chemotherapeutic effects of tumors in vivo have nothing to do with the effects of the chemotherapeutic agents on tumor cells in vitro. The tumor cells resistant to melphalan in vitro remain sensitive to the drug in vivo.

  12. In vitro evaluation of cell-seeded chitosan films for peripheral nerve tissue engineering

    OpenAIRE

    Wrobel, Sandra; Serra, Sofia Cristina; Samy, S. M.; Sousa, Nuno; Heimann, Claudia; Barwig, Christina; Grothe, Claudia; Salgado, A. J.; Talini, Kirsten Haastert

    2014-01-01

    Natural biomaterials have attracted an increasing interest in the field of tissue-engineered nerve grafts, representing a possible alternative to autologous nerve transplantation. With the prospect of developing a novel entubulation strategy for transected nerves with cell-seeded chitosan films, we examined the biocompatibility of such films in vitro. Different types of rat Schwann cells (SCs)-immortalized, neonatal, and adult-as well as rat bone-marrow-derived mesenchymal stromal cells (BMSC...

  13. In vitro Rb-1 gene transfer to retinoblastoma cell lines

    International Nuclear Information System (INIS)

    Choi, Sang Wook; Ham, Yong Hoh; Kim, Mee Heui

    1994-04-01

    After transfection of Rb-vector to packaging cell line (CRIP) by Ca-P precipitation method, we could select nineteen colonies of G-418 resistant clone by ring cloning. Each colony was transduced to NIH3T3 cells to select the one which produces high titer virus. After NIH3T3 cells transduction, we could get 28 colony counts for the high, 127 for the middle, and 6 for the low viral titer. With the supernatant of the high viral titer colony (CRIPRb 2-5). We transduct retinoblastoma cell lines. 5 figs, 11 refs. (Author)

  14. Mechanism of mast cell adhesion to human tenocytes in vitro.

    Science.gov (United States)

    Behzad, Hayedeh; Tsai, Shu-Huei; Nassab, Paulina; Mousavizadeh, Rouhollah; McCormack, Robert G; Scott, Alex

    2015-01-01

    Mast cells and fibroblasts are two key players involved in many fibrotic and degenerative disorders. In the present study we examined the nature of binding interactions between human mast cells and tendon fibroblasts (tenocytes). In the mast cell-fibroblast co-culture model, mast cells were shown to spontaneously bind to tenocytes, in a process that was partially mediated by α5β1 integrin receptors. The same receptors on mast cells significantly mediated binding of these cells to tissue culture plates in the presence of tenocyte-conditioned media; the tenocyte-derived fibronectin in the media was shown to also play a major role in these binding activities. Upon binding to tenocytes or tissue culture plates, mast cells acquired an elongated phenotype, which was dependent on α5β1 integrin and tenocyte fibronectin. Additionally, tenocyte-derived fibronectin significantly enhanced mRNA expression of the adhesion molecule, THY1, by mast cells. Our data suggests that α5β1 integrin mediates binding of mast cells to human tenocyte and to tenocyte-derived ECM proteins, in particular fibronectin. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  15. Interspecific in vitro assay for the chimera-forming ability of human pluripotent stem cells.

    Science.gov (United States)

    Masaki, Hideki; Kato-Itoh, Megumi; Umino, Ayumi; Sato, Hideyuki; Hamanaka, Sanae; Kobayashi, Toshihiro; Yamaguchi, Tomoyuki; Nishimura, Ken; Ohtaka, Manami; Nakanishi, Mahito; Nakauchi, Hiromitsu

    2015-09-15

    Functional assay limitations are an emerging issue in characterizing human pluripotent stem cells (PSCs). With rodent PSCs, chimera formation using pre-implantation embryos is the gold-standard assay of pluripotency (competence of progeny to differentiate into all three germ layers). In human PSCs (hPSCs), however, this can only be monitored via teratoma formation or in vitro differentiation, as ethical concerns preclude generation of human-human or human-animal chimeras. To circumvent this issue, we developed a functional assay utilizing interspecific blastocyst injection and in vitro culture (interspecies in vitro chimera assay) that enables the development and observation of embryos up to headfold stage. The assay uses mouse pre-implantation embryos and rat, monkey and human PSCs to create interspecies chimeras cultured in vitro to the early egg-cylinder stage. Intra- and interspecific chimera assays with rodent PSC lines were performed to confirm the consistency of results in vitro and in vivo. The behavior of chimeras developed in vitro appeared to recapitulate that of chimeras developed in vivo; that is, PSC-derived cells survived and were integrated into the epiblast of egg-cylinder-stage embryos. This indicates that the interspecific in vitro chimera assay is useful in evaluating the chimera-forming ability of rodent PSCs. However, when human induced PSCs (both conventional and naïve-like types) were injected into mouse embryos and cultured, some human cells survived but were segregated; unlike epiblast-stage rodent PSCs, they never integrated into the epiblast of egg-cylinder-stage embryos. These data suggest that the mouse-human interspecies in vitro chimera assay does not accurately reflect the early developmental potential/process of hPSCs. The use of evolutionarily more closely related species as host embryos might be necessary to evaluate the developmental potency of hPSCs. © 2015. Published by The Company of Biologists Ltd.

  16. Patterns of proliferation and differentiation of irradiated haemopoietic stem cells cultured on normal 'stromal' cell colonies in vitro

    International Nuclear Information System (INIS)

    Mori, K.J.

    1981-01-01

    Experiments were designed to elucidate whether or not the irradiated bone marrow cells receive any stimulation for the self-replication and differentiation from normal 'stromal' cell colonies in the bone marrow cell culture in vitro. When irradiated or unirradiated bone marrow cells were overlaid on the normal adherent cell colonies, the proliferation of haemopoietic stem cells was supported, the degree of the stimulation depending on the starting cellular concentration. There was, however, no significant changes in the concentration of either CFUs or CFUc regardless of the dose of irradiation on the bone marrow cells overlaid. This was a great contrast to the dose-dependent decrease of CFUs or CFUc within the culture in which both the stem cells and stromal cells were simultaneously irradiated. These results suggest that the balance of self-replication and differentiation of the haemopoietic stem cells is affected only when haemopoietic microenvironment is perturbed. (author)

  17. Stochastic cellular automata model of cell migration, proliferation and differentiation: validation with in vitro cultures of muscle satellite cells.

    Science.gov (United States)

    Garijo, N; Manzano, R; Osta, R; Perez, M A

    2012-12-07

    Cell migration and proliferation has been modelled in the literature as a process similar to diffusion. However, using diffusion models to simulate the proliferation and migration of cells tends to create a homogeneous distribution in the cell density that does not correlate to empirical observations. In fact, the mechanism of cell dispersal is not diffusion. Cells disperse by crawling or proliferation, or are transported in a moving fluid. The use of cellular automata, particle models or cell-based models can overcome this limitation. This paper presents a stochastic cellular automata model to simulate the proliferation, migration and differentiation of cells. These processes are considered as completely stochastic as well as discrete. The model developed was applied to predict the behaviour of in vitro cell cultures performed with adult muscle satellite cells. Moreover, non homogeneous distribution of cells has been observed inside the culture well and, using the above mentioned stochastic cellular automata model, we have been able to predict this heterogeneous cell distribution and compute accurate quantitative results. Differentiation was also incorporated into the computational simulation. The results predicted the myotube formation that typically occurs with adult muscle satellite cells. In conclusion, we have shown how a stochastic cellular automata model can be implemented and is capable of reproducing the in vitro behaviour of adult muscle satellite cells. Copyright © 2012 Elsevier Ltd. All rights reserved.

  18. Melanoma Cells Can Adopt the Phenotype of Stromal Fibroblasts and Macrophages by Spontaneous Cell Fusion in Vitro.

    Science.gov (United States)

    Kemény, Lajos V; Kurgyis, Zsuzsanna; Buknicz, Tünde; Groma, Gergely; Jakab, Ádám; Zänker, Kurt; Dittmar, Thomas; Kemény, Lajos; Németh, István B

    2016-06-02

    After the removal of primary cutaneous melanoma some patients develop local recurrences, even after having histologically tumor-free re-excision. A potential explanation behind this phenomenon is that tumor cells switch their phenotype, making their recognition via standard histopathological assessments extremely difficult. Tumor-stromal cell fusion has been proposed as a potential mechanism for tumor cells to acquire mesenchymal traits; therefore, we hypothesized that melanoma cells could acquire fibroblast- and macrophage-like phenotypes via cell fusion. We show that melanoma cells spontaneously fuse with human dermal fibroblasts and human peripheral blood monocytes in vitro. The hybrid cells' nuclei contain chromosomes from both parental cells and are indistinguishable from the parental fibroblasts or macrophages based on their morphology and immunophenotype, as they could lose the melanoma specific MART1 marker, but express the fibroblast marker smooth muscle actin or the macrophage marker CD68. Our results suggest that, by spontaneous cell fusion in vitro, tumor cells can adopt the morphology and immunophenotype of stromal cells while still carrying oncogenic, tumor-derived genetic information. Therefore, melanoma-stromal cell fusion might play a role in missing tumor cells by routine histopathological assessments.

  19. Human adipose tissue-derived mesenchymal stem cells inhibit T-cell lymphoma growth in vitro and in vivo.

    Science.gov (United States)

    Ahn, Jin-Ok; Chae, Ji-Sang; Coh, Ye-Rin; Jung, Woo-Sung; Lee, Hee-Woo; Shin, Il-Seob; Kang, Sung-Keun; Youn, Hwa-Young

    2014-09-01

    Human mesenchymal stem cells (hMSCs) are thought to be one of the most reliable stem cell sources for a variety of cell therapies. This study investigated the anti-tumor effect of human adipose tissue-derived mesenchymal stem cells (hAT-MSCs) on EL4 murine T-cell lymphoma in vitro and in vivo. The growth-inhibitory effect of hAT-MSCs on EL4 tumor cells was evaluated using a WST-1 cell proliferation assay. Cell-cycle arrest and apoptosis were investigated by flow cytometry and western blot. To evaluate an anti-tumor effect of hAT-MSCs on T-cell lymphoma in vivo, CM-DiI-labeled hAT-MSCs were circumtumorally injected in tumor-bearing nude mice, and tumor size was measured. hAT-MSCs inhibited T-cell lymphoma growth by altering cell-cycle progression and inducing apoptosis in vitro. hAT-MSCs inhibited tumor growth in tumor-bearing nude mice and prolonged survival time. Immunofluorescence analysis showed that hAT-MSCs migrated to tumor sites. hAT-MSCs suppress the growth of T-cell lymphoma, suggesting a therapeutic option for T-cell lymphoma. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  20. Human serum promotes osteogenic differentiation of human dental pulp stem cells in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Alessandra Pisciotta

    Full Text Available Human dental pulp is a promising alternative source of stem cells for cell-based tissue engineering in regenerative medicine, for the easily recruitment with low invasivity for the patient and for the self-renewal and differentiation potential of cells. So far, in vitro culture of mesenchymal stem cells is usually based on supplementing culture and differentiation media with foetal calf serum (FCS. FCS is known to contain a great quantity of growth factors, and thus to promote cell attachment on plastic surface as well as expansion and differentiation. Nevertheless, FCS as an animal origin supplement may represent a potential means for disease transmission besides leading to a xenogenic immune response. Therefore, a significant interest is focused on investigating alternative supplements, in order to obtain a sufficient cell number for clinical application, avoiding the inconvenients of FCS use. In our study we have demonstrated that human serum (HS is a suitable alternative to FCS, indeed its addition to culture medium induces a high hDPSCs proliferation rate and improves the in vitro osteogenic differentiation. Furthermore, hDPSCs-collagen constructs, pre-differentiated with HS-medium in vitro for 10 days, when implanted in immunocompromised rats, are able to restore critical size parietal bone defects. Therefore these data indicate that HS is a valid substitute for FCS to culture and differentiate in vitro hDPSCs in order to obtain a successful bone regeneration in vivo.

  1. Videomicroscopic extraction of specific information on cell proliferation and migration in vitro

    International Nuclear Information System (INIS)

    Debeir, Olivier; Megalizzi, Veronique; Warzee, Nadine; Kiss, Robert; Decaestecker, Christine

    2008-01-01

    In vitro cell imaging is a useful exploratory tool for cell behavior monitoring with a wide range of applications in cell biology and pharmacology. Combined with appropriate image analysis techniques, this approach has been shown to provide useful information on the detection and dynamic analysis of cell events. In this context, numerous efforts have been focused on cell migration analysis. In contrast, the cell division process has been the subject of fewer investigations. The present work focuses on this latter aspect and shows that, in complement to cell migration data, interesting information related to cell division can be extracted from phase-contrast time-lapse image series, in particular cell division duration, which is not provided by standard cell assays using endpoint analyses. We illustrate our approach by analyzing the effects induced by two sigma-1 receptor ligands (haloperidol and 4-IBP) on the behavior of two glioma cell lines using two in vitro cell models, i.e., the low-density individual cell model and the high-density scratch wound model. This illustration also shows that the data provided by our approach are suggestive as to the mechanism of action of compounds, and are thus capable of informing the appropriate selection of further time-consuming and more expensive biological evaluations required to elucidate a mechanism

  2. Stem cell factor stimulates chicken osteoclast activity in vitro

    NARCIS (Netherlands)

    van't Hof, R. J.; von Lindern, M.; Nijweide, P. J.; Beug, H.

    1997-01-01

    Stem cell factor (SCF) is a polypeptide growth factor active on multiple cell types, mainly of hematopoietic origin. We studied the effects of avian SCF on the differentiation of chicken osteoclasts from their putative progenitors as well as on the bone-resorbing activity of terminally

  3. Biomaterials Influence Macrophage-Mesenchymal Stem Cell Interaction In Vitro

    NARCIS (Netherlands)

    N. Grotenhuis (Nienke); S.F. De Witte (Samantha Fh); G.J.V.M. van Osch (Gerjo); Y. Bayon (Yves); J.F. Lange (Johan); Y.M. Bastiaansen-Jenniskens (Yvonne)

    2016-01-01

    textabstractBackground: Macrophages and mesenchymal stem cells (MSCs) are important cells in wound healing. We hypothesized that the cross-talk between macrophages and adipose tissue-derived MSCs (ASCs) is biomaterial dependent, thereby influencing processes involved in wound healing. Materials and

  4. Cell-Based Microarrays for In Vitro Toxicology

    Science.gov (United States)

    Wegener, Joachim

    2015-07-01

    DNA/RNA and protein microarrays have proven their outstanding bioanalytical performance throughout the past decades, given the unprecedented level of parallelization by which molecular recognition assays can be performed and analyzed. Cell microarrays (CMAs) make use of similar construction principles. They are applied to profile a given cell population with respect to the expression of specific molecular markers and also to measure functional cell responses to drugs and chemicals. This review focuses on the use of cell-based microarrays for assessing the cytotoxicity of drugs, toxins, or chemicals in general. It also summarizes CMA construction principles with respect to the cell types that are used for such microarrays, the readout parameters to assess toxicity, and the various formats that have been established and applied. The review ends with a critical comparison of CMAs and well-established microtiter plate (MTP) approaches.

  5. Physiological oxygen concentration alters glioma cell malignancy and responsiveness to photodynamic therapy in vitro.

    Science.gov (United States)

    Albert, Ina; Hefti, Martin; Luginbuehl, Vera

    2014-11-01

    The partial pressure of oxygen (pO2) in brain tumors ranges from 5 to 15%. Nevertheless, the majority of in vitro experiments with glioblastoma multiforme (GBM) cell lines are carried out under an atmospheric pO2 of 19 to 21%. Recently, 5-aminolevulinic acid (5-ALA), a precursor of protoporphyrin IX (PpIX), has been introduced to neurosurgery to allow for photodynamic diagnosis and photodynamic therapy (PDT) in high-grade gliomas. Here, we investigate whether low pO2 affects GBM cell physiology, PpIX accumulation, or PDT efficacy. GBM cell lines (U-87 MG and U-251 MG) were cultured under atmospheric (pO2  =  19%) and physiological (pO2  =  9%) oxygen concentrations. PpIX accumulation and localization were investigated, and cell survival and cell death were observed following in vitro PDT. A physiological pO2 of 9% stimulated GBM cell migration, increased hypoxia-inducible factor (HIF)-1 alpha levels, and elevated resistance to camptothecin in U-87 MG cells compared to cultivation at a pO2 of 19%. This oxygen reduction did not alter 5-ALA-induced intracellular PpIX accumulation. However, physiological pO2 changed the responsiveness of U-87 MG but not of U-251 MG cells to in vitro PDT. Around 20% more irradiation light was required to kill U-87 MG cells at physiological pO2, resulting in reduced lactate dehydrogenase (LDH) release (one- to two-fold) and inhibition of caspase 3 activation. Reduction of oxygen concentration from atmospheric to a more physiological level can influence the malignant behavior and survival of GBM cell lines after in vitro PDT. Therefore, precise oxygen concentration control should be considered when designing and performing experiments with GBM cells.

  6. Allometric Scaling and Cell Ratios in Multi-Organ in vitro Models of Human Metabolism

    International Nuclear Information System (INIS)

    Ucciferri, Nadia; Sbrana, Tommaso; Ahluwalia, Arti

    2014-01-01

    Intelligent in vitro models able to recapitulate the physiological interactions between tissues in the body have enormous potential as they enable detailed studies on specific two-way or higher order tissue communication. These models are the first step toward building an integrated picture of systemic metabolism and signaling in physiological or pathological conditions. However, the rational design of in vitro models of cell–cell or cell–tissue interaction is difficult as quite often cell culture experiments are driven by the device used, rather than by design considerations. Indeed, very little research has been carried out on in vitro models of metabolism connecting different cell or tissue types in a physiologically and metabolically relevant manner. Here, we analyze the physiological relationship between cells, cell metabolism, and exchange in the human body using allometric rules, downscaling them to an organ-on-a-plate device. In particular, in order to establish appropriate cell ratios in the system in a rational manner, two different allometric scaling models (cell number scaling model and metabolic and surface scaling model) are proposed and applied to a two compartment model of hepatic-vascular metabolic cross-talk. The theoretical scaling studies illustrate that the design and hence relevance of multi-organ models is principally determined by experimental constraints. Two experimentally feasible model configurations are then implemented in a multi-compartment organ-on-a-plate device. An analysis of the metabolic response of the two configurations demonstrates that their glucose and lipid balance is quite different, with only one of the two models recapitulating physiological-like homeostasis. In conclusion, not only do cross-talk and physical stimuli play an important role in in vitro models, but the numeric relationship between cells is also crucial to recreate in vitro interactions, which can be extrapolated to the in vivo reality.

  7. Allometric Scaling and Cell Ratios in Multi-Organ in vitro Models of Human Metabolism

    Energy Technology Data Exchange (ETDEWEB)

    Ucciferri, Nadia [CNR Institute of Clinical Physiology, Pisa (Italy); Interdepartmental Research Center “E. Piaggio”, University of Pisa, Pisa (Italy); Sbrana, Tommaso [Interdepartmental Research Center “E. Piaggio”, University of Pisa, Pisa (Italy); Ahluwalia, Arti, E-mail: arti.ahluwalia@unipi.it [CNR Institute of Clinical Physiology, Pisa (Italy); Interdepartmental Research Center “E. Piaggio”, University of Pisa, Pisa (Italy)

    2014-12-17

    Intelligent in vitro models able to recapitulate the physiological interactions between tissues in the body have enormous potential as they enable detailed studies on specific two-way or higher order tissue communication. These models are the first step toward building an integrated picture of systemic metabolism and signaling in physiological or pathological conditions. However, the rational design of in vitro models of cell–cell or cell–tissue interaction is difficult as quite often cell culture experiments are driven by the device used, rather than by design considerations. Indeed, very little research has been carried out on in vitro models of metabolism connecting different cell or tissue types in a physiologically and metabolically relevant manner. Here, we analyze the physiological relationship between cells, cell metabolism, and exchange in the human body using allometric rules, downscaling them to an organ-on-a-plate device. In particular, in order to establish appropriate cell ratios in the system in a rational manner, two different allometric scaling models (cell number scaling model and metabolic and surface scaling model) are proposed and applied to a two compartment model of hepatic-vascular metabolic cross-talk. The theoretical scaling studies illustrate that the design and hence relevance of multi-organ models is principally determined by experimental constraints. Two experimentally feasible model configurations are then implemented in a multi-compartment organ-on-a-plate device. An analysis of the metabolic response of the two configurations demonstrates that their glucose and lipid balance is quite different, with only one of the two models recapitulating physiological-like homeostasis. In conclusion, not only do cross-talk and physical stimuli play an important role in in vitro models, but the numeric relationship between cells is also crucial to recreate in vitro interactions, which can be extrapolated to the in vivo reality.

  8. Spatial organization of mesenchymal stem cells in vitro--results from a new individual cell-based model with podia.

    Directory of Open Access Journals (Sweden)

    Martin Hoffmann

    Full Text Available Therapeutic application of mesenchymal stem cells (MSC requires their extensive in vitro expansion. MSC in culture typically grow to confluence within a few weeks. They show spindle-shaped fibroblastoid morphology and align to each other in characteristic spatial patterns at high cell density. We present an individual cell-based model (IBM that is able to quantitatively describe the spatio-temporal organization of MSC in culture. Our model substantially improves on previous models by explicitly representing cell podia and their dynamics. It employs podia-generated forces for cell movement and adjusts cell behavior in response to cell density. At the same time, it is simple enough to simulate thousands of cells with reasonable computational effort. Experimental sheep MSC cultures were monitored under standard conditions. Automated image analysis was used to determine the location and orientation of individual cells. Our simulations quantitatively reproduced the observed growth dynamics and cell-cell alignment assuming cell density-dependent proliferation, migration, and morphology. In addition to cell growth on plain substrates our model captured cell alignment on micro-structured surfaces. We propose a specific surface micro-structure that according to our simulations can substantially enlarge cell culture harvest. The 'tool box' of cell migratory behavior newly introduced in this study significantly enhances the bandwidth of IBM. Our approach is capable of accommodating individual cell behavior and collective cell dynamics of a variety of cell types and tissues in computational systems biology.

  9. Efficient Genome Editing in Induced Pluripotent Stem Cells with Engineered Nucleases In Vitro.

    Science.gov (United States)

    Termglinchan, Vittavat; Seeger, Timon; Chen, Caressa; Wu, Joseph C; Karakikes, Ioannis

    2017-01-01

    Precision genome engineering is rapidly advancing the application of the induced pluripotent stem cells (iPSCs) technology for in vitro disease modeling of cardiovascular diseases. Targeted genome editing using engineered nucleases is a powerful tool that allows for reverse genetics, genome engineering, and targeted transgene integration experiments to be performed in a precise and predictable manner. However, nuclease-mediated homologous recombination is an inefficient process. Herein, we describe the development of an optimized method combining site-specific nucleases and the piggyBac transposon system for "seamless" genome editing in pluripotent stem cells with high efficiency and fidelity in vitro.

  10. HLA-DR-specific suppressor cells after repeated allogeneic sensitizations of human lymphocytes in vitro

    International Nuclear Information System (INIS)

    Sasportes, M.; Fradelizi, D.; Dausset, J.

    1978-01-01

    In conclusion, DR-specific suppressor cells can be induced by repeated in vitro sensitizations. They were able to decrease a secondary proliferation, to suppress consistently, in a primary proliferative assay, when added as third cells (primed twice against a DR antigen [PLT II] and γ-irradiated), the response of unprimed cells towards stimulating cells, which share a DR specificity with the priming cell of the PLT II. The suppression follows the D part of the recombinant haplotype within an HLA-B/D recombinant family and is specific for the DR antigen used twice as stimulator for production of the PLT II

  11. Photodynamic action of methylene blue in osteosarcoma cells in vitro.

    Science.gov (United States)

    Guan, Jiemin; Lai, Xiaoping; Wang, Xinna; Leung, Albert Wingnang; Zhang, Hongwei; Xu, Chuanshan

    2014-03-01

    Osteosarcoma is a common malignant bone tumor which threatens the life of young people worldwide. To explore alternative strategy for combating osteosarcoma, a light-emitting diode (LED) that activates methylene blue (MB) was used in the present study to investigate cell death of osteosarcoma-derived UMR106 cells. Photocytotoxicity in UMR106 cells was investigated 24h after photodynamic activation of MB using sulforhodamine B (SRB) assay and light microscopy. Apoptosis induction was observed 24h after photodynamic treatment using a confocal laser scanning microscopy (CLSM) with Hoechst 33342 staining. The change in mitochondrial membrane potential (MMP) was analyzed using a flow cytometry with rhodamine 123 staining. MB under red light irradiation caused a drug-concentration (0-100μM) and light-dose (0-32J/cm(2)) dependent cytotoxicity in UMR106 cells. The SRB assay and light microscopy observed a significant decrease in the number of UMR106 cells attached to the bottom of culture well after LED light-activated MB (100μM, 32J/cm(2)). Nuclear shrinkage, chromatin condensation and fragmentation were found in the treated cells by nuclear staining. In addition, flow cytometry showed that the MMP in UMR106 cells was rapidly reduced by photo-activated MB (100μM, 32J/cm(2)). Photodynamic action of MB under LED irradiation could remarkably kill osteosarcoma cells and induce cell apoptosis as well as MMP collapse. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

  12. Nesfatin-1 inhibits ovarian epithelial carcinoma cell proliferation in vitro

    International Nuclear Information System (INIS)

    Xu, Yang; Pang, Xiaoyan; Dong, Mei; Wen, Fang; Zhang, Yi

    2013-01-01

    Highlights: •Nesfatin-1 inhibits the proliferation and growth of HO-8910 cells by G1 phase arrest. •Nesfatin-1 enhances HO-8910 cell apoptosis. •Nesfatin-1 inhibits HO-8910 cell proliferation via mTOR and RhoA/ROCK signaling pathway. •The first report of nesfatin-1-mediated proliferation in ovarian epithelial carcinoma. -- Abstract: Nesfatin-1, an 82-amino-acid peptide derived from a 396-amino-acid precursor protein nucleobindin 2 (NUCB2), was originally identified in hypothalamic nuclei involved in the regulation of food intake. It was recently reported that nesfatin-1 is a novel depot specific adipokine preferentially produced by subcutaneous tissue, with obesity- and food deprivation-regulated expression. Although a relation between ovarian cancer mortality and obesity has been previously established, a role of nesfatin-1 in ovarian epithelial carcinoma remains unknown. The aim of the present study is to examine the effect of nesfatin-1 on ovary carcinoma cells proliferation. We found that nesfatin-1 inhibits the proliferation and growth of HO-8910 cells by G1 phase arrest, this inhibition could be abolished by nesfatin-1 neutralizing antibody. Nesfatin-1 enhances HO-8910 cell apoptosis, activation of mammalian target of rapamycin (mTOR) and RhoA/ROCK signaling pathway block the effects of nesfatin-1-induced apoptosis, therefore reverses the inhibition of HO-8910 cell proliferation by nesfatin-1. In conclusion, the present study demonstrated that nesfatin-1 can inhibit the proliferation in human ovarian epithelial carcinoma cell line HO-8910 cells through inducing apoptosis via mTOR and RhoA/ROCK signaling pathway. This study provides a novel regulatory signaling pathway of nesfatin-1-regulated ovarian epithelial carcinoma growth and may contribute to ovarian cancer prevention and therapy, especially in obese patients

  13. Nesfatin-1 inhibits ovarian epithelial carcinoma cell proliferation in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Yang; Pang, Xiaoyan; Dong, Mei; Wen, Fang, E-mail: wenfang64@hotmail.com; Zhang, Yi, E-mail: syzi960@yahoo.com

    2013-11-01

    Highlights: •Nesfatin-1 inhibits the proliferation and growth of HO-8910 cells by G1 phase arrest. •Nesfatin-1 enhances HO-8910 cell apoptosis. •Nesfatin-1 inhibits HO-8910 cell proliferation via mTOR and RhoA/ROCK signaling pathway. •The first report of nesfatin-1-mediated proliferation in ovarian epithelial carcinoma. -- Abstract: Nesfatin-1, an 82-amino-acid peptide derived from a 396-amino-acid precursor protein nucleobindin 2 (NUCB2), was originally identified in hypothalamic nuclei involved in the regulation of food intake. It was recently reported that nesfatin-1 is a novel depot specific adipokine preferentially produced by subcutaneous tissue, with obesity- and food deprivation-regulated expression. Although a relation between ovarian cancer mortality and obesity has been previously established, a role of nesfatin-1 in ovarian epithelial carcinoma remains unknown. The aim of the present study is to examine the effect of nesfatin-1 on ovary carcinoma cells proliferation. We found that nesfatin-1 inhibits the proliferation and growth of HO-8910 cells by G1 phase arrest, this inhibition could be abolished by nesfatin-1 neutralizing antibody. Nesfatin-1 enhances HO-8910 cell apoptosis, activation of mammalian target of rapamycin (mTOR) and RhoA/ROCK signaling pathway block the effects of nesfatin-1-induced apoptosis, therefore reverses the inhibition of HO-8910 cell proliferation by nesfatin-1. In conclusion, the present study demonstrated that nesfatin-1 can inhibit the proliferation in human ovarian epithelial carcinoma cell line HO-8910 cells through inducing apoptosis via mTOR and RhoA/ROCK signaling pathway. This study provides a novel regulatory signaling pathway of nesfatin-1-regulated ovarian epithelial carcinoma growth and may contribute to ovarian cancer prevention and therapy, especially in obese patients.

  14. Comparative mutagenesis of human cells in vivo and in vitro

    International Nuclear Information System (INIS)

    Thilly, W.G.

    1990-01-01

    Our goal is to develop the tools of mutational spectrometry in order to discover the cause(s) of genetic change in somatic and germinal cells in humans. Our study of the spectrum of point mutations in human mitochrondrial DNA sequences has revealed that there are multiple point mutation hotspots in each of four separate sequences in the mitochrondrial genome. These spectra were revealed by a combination of high fidelity PCR (modified T 7 polymerase) and denaturing gradient gel electrophoresis which has a limit of detection of about 10 -3 . There appear to be identical hotspot mutations in both cultured B cell and fresh human blood T cell samples

  15. Live cell imaging techniques to study T cell trafficking across the blood-brain barrier in vitro and in vivo

    Directory of Open Access Journals (Sweden)

    Coisne Caroline

    2013-01-01

    Full Text Available Abstract Background The central nervous system (CNS is an immunologically privileged site to which access for circulating immune cells is tightly controlled by the endothelial blood–brain barrier (BBB located in CNS microvessels. Under physiological conditions immune cell migration across the BBB is low. However, in neuroinflammatory diseases such as multiple sclerosis, many immune cells can cross the BBB and cause neurological symptoms. Extravasation of circulating immune cells is a multi-step process that is regulated by the sequential interaction of different adhesion and signaling molecules on the immune cells and on the endothelium. The specialized barrier characteristics of the BBB, therefore, imply the existence of unique mechanisms for immune cell migration across the BBB. Methods and design An in vitro mouse BBB model maintaining physiological barrier characteristics in a flow chamber and combined with high magnification live cell imaging, has been established. This model enables the molecular mechanisms involved in the multi-step extravasation of T cells across the in vitro BBB, to be defined with high-throughput analyses. Subsequently these mechanisms have been verified in vivo using a limited number of experimental animals and a spinal cord window surgical technique. The window enables live observation of the dynamic interaction between T cells and spinal cord microvessels under physiological and pathological conditions using real time epifluorescence intravital imaging. These in vitro and in vivo live cell imaging methods have shown that the BBB endothelium possesses unique and specialized mechanisms involved in the multi-step T cell migration across this endothelial barrier under physiological flow. The initial T cell interaction with the endothelium is either mediated by T cell capture or by T cell rolling. Arrest follows, and then T cells polarize and especially CD4+ T cells crawl over long distances against the direction of

  16. Three-dimensional printing of Hela cells for cervical tumor model in vitro

    International Nuclear Information System (INIS)

    Zhao, Yu; Yao, Rui; Ouyang, Liliang; Ding, Hongxu; Zhang, Ting; Sun, Wei; Zhang, Kaitai; Cheng, Shujun

    2014-01-01

    Advances in three-dimensional (3D) printing have enabled the direct assembly of cells and extracellular matrix materials to form in vitro cellular models for 3D biology, the study of disease pathogenesis and new drug discovery. In this study, we report a method of 3D printing for Hela cells and gelatin/alginate/fibrinogen hydrogels to construct in vitro cervical tumor models. Cell proliferation, matrix metalloproteinase (MMP) protein expression and chemoresistance were measured in the printed 3D cervical tumor models and compared with conventional 2D planar culture models. Over 90% cell viability was observed using the defined printing process. Comparisons of 3D and 2D results revealed that Hela cells showed a higher proliferation rate in the printed 3D environment and tended to form cellular spheroids, but formed monolayer cell sheets in 2D culture. Hela cells in 3D printed models also showed higher MMP protein expression and higher chemoresistance than those in 2D culture. These new biological characteristics from the printed 3D tumor models in vitro as well as the novel 3D cell printing technology may help the evolution of 3D cancer study. (paper)

  17. Exogenous regucalcin suppresses the growth of human liver cancer HepG2 cells in vitro.

    Science.gov (United States)

    Yamaguchi, Masayoshi; Murata, Tomiyasu

    2018-04-05

    Regucalcin, which its gene is localized on the X chromosome, plays a pivotal role as a suppressor protein in signal transduction in various types of cells and tissues. Regucalcin gene expression has been demonstrated to be suppressed in various tumor tissues of animal and human subjects, suggesting a potential role of regucalcin in carcinogenesis. Regucalcin, which is produced from the tissues including liver, is found to be present in the serum of human subjects and animals. This study was undertaken to determine the effects of exogenous regucalcin on the proliferation in cloned human hepatoma HepG2 cells in vitro. Proliferation of HepG2 cells was suppressed after culture with addition of regucalcin (0.01 – 10 nM) into culture medium. Exogenous regucalcin did not reveal apoptotic cell death in HepG2 cells in vitro. Suppressive effects of regucalcin on cell proliferation were not enhanced in the presence of various signaling inhibitors including tumor necrosis factor-α (TNF-α), Bay K 8644, PD98059, staurosporine, worthomannin, 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) or gemcitabine, which were found to suppress the proliferation. In addition, exogenous regucalcin suppressed the formation of colonies of cultured hepatoma cells in vitro. These findings demonstrated that exogenous regucalcin exhibits a suppressive effect on the growth of human hepatoma HepG2 cells, proposing a strategy with the gene therapy for cancer treatment.

  18. Neural differentiation of mouse embryonic stem cells as a tool to assess developmental neurotoxicity in vitro.

    Science.gov (United States)

    Visan, Anke; Hayess, Katrin; Sittner, Dana; Pohl, Elena E; Riebeling, Christian; Slawik, Birgitta; Gulich, Konrad; Oelgeschläger, Michael; Luch, Andreas; Seiler, Andrea E M

    2012-10-01

    Mouse embryonic stem cells (mESCs) represent an attractive cellular system for in vitro studies in developmental biology as well as toxicology because of their potential to differentiate into all fetal cell lineages. The present study aims to establish an in vitro system for developmental neurotoxicity testing employing mESCs. We developed a robust and reproducible protocol for fast and efficient differentiation of the mESC line D3 into neural cells, optimized with regard to chemical testing. Morphological examination and immunocytochemical staining confirmed the presence of different neural cell types, including neural progenitors, neurons, astrocytes, oligodendrocytes, and radial glial cells. Neurons derived from D3 cells expressed the synaptic proteins PSD95 and synaptophysin, and the neurotransmitters serotonin and γ-aminobutyric acid. Calcium ion imaging revealed the presence of functionally active glutamate and dopamine receptors. In addition, flow cytometry analysis of the neuron-specific marker protein MAP2 on day 12 after induction of differentiation demonstrated a concentration dependent effect of the neurodevelopmental toxicants methylmercury chloride, chlorpyrifos, and lead acetate on neuronal differentiation. The current study shows that D3 mESCs differentiate efficiently into neural cells involving a neurosphere-like state and that this system is suitable to detect adverse effects of neurodevelopmental toxicants. Therefore, we propose that the protocol for differentiation of mESCs into neural cells described here could constitute one component of an in vitro testing strategy for developmental neurotoxicity. Copyright © 2012 Elsevier Inc. All rights reserved.

  19. In vitro proliferation of haemopoietic cells in the presence of adherent cell layers. II. Differential effect of adherent cell layers derived from various organs

    NARCIS (Netherlands)

    Reimann, J.; Burger, H.

    1979-01-01

    Mouse bone marrow-derived adherent cell populations promoted proliferation of haemopoietic cells in vitro in a liquid culture system for at least 4 weeks. Adherent cell layers derived from other haemopoietic organs (foetal liver, adult spleen) and fibroblasts from embryonic tissues did not maintain

  20. Insights into female germ cell biology: from in vivo development to in vitro derivations.

    Science.gov (United States)

    Jung, Dajung; Kee, Kehkooi

    2015-01-01

    Understanding the mechanisms of human germ cell biology is important for developing infertility treatments. However, little is known about the mechanisms that regulate human gametogenesis due to the difficulties in collecting samples, especially germ cells during fetal development. In contrast to the mitotic arrest of spermatogonia stem cells in the fetal testis, female germ cells proceed into meiosis and began folliculogenesis in fetal ovaries. Regulations of these developmental events, including the initiation of meiosis and the endowment of primordial follicles, remain an enigma. Studying the molecular mechanisms of female germ cell biology in the human ovary has been mostly limited to spatiotemporal characterizations of genes or proteins. Recent efforts in utilizing in vitro differentiation system of stem cells to derive germ cells have allowed researchers to begin studying molecular mechanisms during human germ cell development. Meanwhile, the possibility of isolating female germline stem cells in adult ovaries also excites researchers and generates many debates. This review will mainly focus on presenting and discussing recent in vivo and in vitro studies on female germ cell biology in human. The topics will highlight the progress made in understanding the three main stages of germ cell developments: namely, primordial germ cell formation, meiotic initiation, and folliculogenesis.

  1. Differential response to gamma radiation of human stomach cancer cells in vitro

    International Nuclear Information System (INIS)

    Jenkins, V.K.; Barranco, S.C.; Townsend, C.M. Jr.; Perry, R.R.; Ives, K.L.

    1986-01-01

    In vitro effects of radiation were studied in two permanent cell lines (AGS and SII) from two patients with stomach adenocarcinoma and three permanent sublines from each cell line. Radiation survival parameters for AGS and SII parent cell lines and sublines were determined after in vitro irradiation with 0.5 to 10 Gy of 60 Co gamma rays. AGS and SII cell lines had different growth properties. DNA contents and radiation survival curves. Surviving fractions of SII parent cells (76 chromosomes) after 2.0 and 10 Gy were 1.22 and 17.8 times greater, respectively, than values for AGS parent cells (47 chromosomes). Sensitivities (D 0 ) were 1.08 and 1.45 Gy for AGS and SII parent lines, respectively. D 0 values for AGS parent cells and sublines were similar (1.01 to 1.08 Gy), but SII parent cells and sublines had D 0 values of 1.45, 1.36, 1.37 and 1.12 Gy (for SII-A). The SII parent cells had survival fractions after 2.0 and 10 Gy that were 1.3 and 11.3 times greater, respectively, than values for the SII-A cells. These data show differences in radiation responses among stomach cancer cell lines and sublines that may relate to DNA content, but there was no consistent correlation between radiation response and a particular cell characteristic. (author)

  2. The selective role of ECM components on cell adhesion, morphology, proliferation and communication in vitro

    International Nuclear Information System (INIS)

    Schlie-Wolter, Sabrina; Ngezahayo, Anaclet; Chichkov, Boris N.

    2013-01-01

    Cell binding to the extracellular matrix (ECM) is essential for cell and tissue functions. In this context, each tissue consists of a unique ECM composition, which may be responsible for tissue-specific cell responses. Due to the complexity of ECM-cell interactions—which depend on the interplay of inside-out and outside-in signaling cascades, cell and tissue specificity of ECM-guidance is poorly understood. In this paper, we investigate the role of different ECM components like laminin, fibronectin, and collagen type I with respect to the essential cell behaviour patterns: attachment dynamics such as adhesion kinetic and force, formation of focal adhesion complexes, morphology, proliferation, and intercellular communication. A detailed in vitro comparison of fibroblasts, endothelial cells, osteoblasts, smooth muscle cells, and chondrocytes reveals significant differences in their cell responses to the ECM: cell behaviour follows a cell specific ligand priority ranking, which was independent of the cell type origin. Fibroblasts responded best to fibronectin, chondrocytes best to collagen I, the other cell types best to laminin. This knowledge is essential for optimization of tissue-biomaterial interfaces in all tissue engineering applications and gives insight into tissue-specific cell guidance. -- Highlights: • We analyse the impact of ECM components on cell behaviour in vitro. • We compare five different cell types, using the same culture conditions. • The ECM significantly guides all cell responses. • Cell behaviour follows a cell specific ligand-priority ranking. • This gives insight in tissue formation and is essential for biomedical applications

  3. The selective role of ECM components on cell adhesion, morphology, proliferation and communication in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Schlie-Wolter, Sabrina, E-mail: s.schlie@lzh.de [Laser Zentrum Hannover e.V., Hollerithallee 8, 30419 Hannover (Germany); Ngezahayo, Anaclet, E-mail: ngezahayo@biophysik.uni-hannover.de [Institute of Biophysics, Leibniz University Hannover, Herrenhäuser Str. 2, Hannover 30419 (Germany); Chichkov, Boris N., E-mail: b.chichkov@lzh.de [Laser Zentrum Hannover e.V., Hollerithallee 8, 30419 Hannover (Germany)

    2013-06-10

    Cell binding to the extracellular matrix (ECM) is essential for cell and tissue functions. In this context, each tissue consists of a unique ECM composition, which may be responsible for tissue-specific cell responses. Due to the complexity of ECM-cell interactions—which depend on the interplay of inside-out and outside-in signaling cascades, cell and tissue specificity of ECM-guidance is poorly understood. In this paper, we investigate the role of different ECM components like laminin, fibronectin, and collagen type I with respect to the essential cell behaviour patterns: attachment dynamics such as adhesion kinetic and force, formation of focal adhesion complexes, morphology, proliferation, and intercellular communication. A detailed in vitro comparison of fibroblasts, endothelial cells, osteoblasts, smooth muscle cells, and chondrocytes reveals significant differences in their cell responses to the ECM: cell behaviour follows a cell specific ligand priority ranking, which was independent of the cell type origin. Fibroblasts responded best to fibronectin, chondrocytes best to collagen I, the other cell types best to laminin. This knowledge is essential for optimization of tissue-biomaterial interfaces in all tissue engineering applications and gives insight into tissue-specific cell guidance. -- Highlights: • We analyse the impact of ECM components on cell behaviour in vitro. • We compare five different cell types, using the same culture conditions. • The ECM significantly guides all cell responses. • Cell behaviour follows a cell specific ligand-priority ranking. • This gives insight in tissue formation and is essential for biomedical applications.

  4. In vitro proliferative capacity of vascular cells irradiated in vivo

    International Nuclear Information System (INIS)

    Fischer-Dzoga, K.; Dimitrievich, G.S.; Griem, M.L.

    1985-01-01

    Explants were prepared from rabbit vascular aortic layers and irradiated with x-ray doses ranging from 100 cGy-5 cGy. This resulted in a 50% reduction in number of outgrowing cells with doses of 100-125 gy. Doses of 250, 500 and 750 gy resulted in a reduction of 70, 90, and 95% respectively. However, when the rabbit was irradiated in vivo to a narrow mediastinal port immediately before the explantation of vascular tissue, the number of outgrowing cells was comparable to that of the irradiated control for doses up to 250 cGy, while doses of 500 and 750 cGy reduced outgrowth by 60 and 93% respectively. To test for in situ repair, the time interval between irradiation and explantation was prolonged from 1-4 hours in one hour increments. The results were scored as average number of cells/explant and average number of cells/growing culture

  5. Cell-free synthetic biology for in vitro prototype engineering.

    Science.gov (United States)

    Moore, Simon J; MacDonald, James T; Freemont, Paul S

    2017-06-15

    Cell-free transcription-translation is an expanding field in synthetic biology as a rapid prototyping platform for blueprinting the design of synthetic biological devices. Exemplar efforts include translation of prototype designs into medical test kits for on-site identification of viruses (Zika and Ebola), while gene circuit cascades can be tested, debugged and re-designed within rapid turnover times. Coupled with mathematical modelling, this discipline lends itself towards the precision engineering of new synthetic life. The next stages of cell-free look set to unlock new microbial hosts that remain slow to engineer and unsuited to rapid iterative design cycles. It is hoped that the development of such systems will provide new tools to aid the transition from cell-free prototype designs to functioning synthetic genetic circuits and engineered natural product pathways in living cells. © 2017 The Author(s).

  6. Propagation of Human Spermatogonial Stem Cells In Vitro

    NARCIS (Netherlands)

    Sadri-Ardekani, Hooman; Mizrak, Sefika C.; van Daalen, Saskia K. M.; Korver, Cindy M.; Roepers-Gajadien, Hermien L.; Koruji, Morteza; Hovingh, Suzanne; de Reijke, Theo M.; de La Rosette, Jean J. M. C. H.; van der Veen, Fulco; de Rooij, Dirk G.; Repping, Sjoerd; van Pelt, Ans M. M.

    2009-01-01

    Context Young boys treated with high-dose chemotherapy are often confronted with infertility once they reach adulthood. Cryopreserving testicular tissue before chemotherapy and autotransplantation of spermatogonial stem cells at a later stage could theoretically allow for restoration of fertility.

  7. In vitro production of azadirachtin from cell suspension cultures of ...

    Indian Academy of Sciences (India)

    PRAKASH KUMAR G

    proven effective in the control of agricultural pests in an environmentally ..... Prakash G and Srivastava A K 2005 Statistical media optimization for cell growth and ... Juss. suspension cultures; Process Biochemistry 40 3795–3800. Prakash G ...

  8. Invasion of vascular cells in vitro by Porphyromonas endodontalis.

    Science.gov (United States)

    Dorn, B R; Harris, L J; Wujick, C T; Vertucci, F J; Progulske-Fox, A

    2002-04-01

    The objective of this study was to determine whether laboratory strains and clinical isolates of microorganisms associated with root canal infections can invade primary cultures of cardiovascular cells. Quantitative levels of bacterial invasion of human coronary artery endothelial cells (HCAEC) and coronary artery smooth muscle cells (CASMC) were measured using a standard antibiotic protection assay. Transmission electron microscopy was used to confirm and visualize internalization within the vascular cells. Of the laboratory and clinical strains tested, only P. endodontalis ATCC 35406 was invasive in an antibiotic protection assay using HCAEC and CASMC. Invasion of P. endodontalis ATCC 35406 was confirmed by transmission electron microscopy. Certain microorganisms associated with endodontic infections are invasive. If bacterial invasion of the vasculature contributes to the pathogenesis of cardiovascular disease, then microorganisms in the pulp chamber represent potential pathogens.

  9. Tumor necrosis factor alpha production in irradiated cells in vitro

    International Nuclear Information System (INIS)

    Koeteles, G.J.; Bognar, G.; Kubasova, T.

    1994-01-01

    Normal and tumor cell lines were used to investigate tumor necrosis factor (TNFα) production and its radiation sensitivity. The cells were irradiated with gamma rays using different doses from 0.25 Gy up to 5 Gy. The number of plated cells, changes of proliferation and TNFα production were determined during the following four post-irradiation days. For TNFα quantity measurement immuno-radiometric assay (IRMA) and enzyme amplified sensitivity assay (EASIA) was used. The results suggest that though gamma irradiation decreased cell proliferation in a dose dependent manner, the quantity produced in the post-irradiation period increased considerably in each irradiated sample. (N.T.) 3 refs.; 2 figs.; 1 tab

  10. In Vitro Cytotoxicity of Nanoparticles in Mammalian Germline Stem Cells

    OpenAIRE

    Braydich-Stolle, Laura; Hussain, Saber; Schlager, John J.; Hofmann, Marie-Claude

    2005-01-01

    Gametogenesis is a complex biological process that is particularly sensitive to environmental insults such as chemicals. Many chemicals have a negative impact on the germline, either by directly affecting the germ cells, or indirectly through their action on the somatic nursing cells. Ultimately, these effects can inhibit fertility, and they may have negative consequences for the development of the offspring. Recently, nanomaterials such as nanotubes, nanowires, fullerene derivatives (buckyba...

  11. Intracameral voriconazole: In vitro safety for human ocular cells

    International Nuclear Information System (INIS)

    Kernt, M.; Kampik, A.

    2009-01-01

    Fungal keratitis is a sight-threatening infection of the cornea. It sometimes leads to loss of the eye. Despite an expanding range of fungal pathogens, there are only few therapeutic agents for its treatment available. Voriconazole is a second-generation synthetic triazole with a broad action against yeasts and molds. The current study investigates the safety of voriconazole for intracameral application in a cell culture model. Endothelial toxicity of voriconazole was evaluated in cultured human corneas. Possible toxic effects of voriconazole (10 μg/mL-10 mg/mL) in corneal endothelial cells (CEC), primary human trabecular meshwork cells (TMC), and primary human retinal pigment epithelium (RPE) cells were evaluated after 24 h and under conditions of inflammatory stress by treatment with tumor-necrosis-factor alpha (TNF-α), lipopolysaccharides (LPS), or interleukin-6 (IL-6) and hydrogen peroxide. Toxicity was evaluated by tetrazolium dye-reduction assay, and cell viability was quantified by a microscopic live-dead assay. No corneal endothelial toxicity could be detected after 30 days of treatment with 250 μg/mL of voriconazole. Concentrations up to 1 mg/mL had no influence on CEC, TMC, or RPE cell proliferation, or on cell viability when administered for 24 h. Hydrogen peroxide exposure did not increase cellular toxicity of voriconazole at concentrations from 10 to 250 μg/mL. After preincubation with TNF-α, LPS, or IL-6 for 24 h and subsequent voriconazole treatment for 24 h, no significant decrease in proliferation or viability was observed. This study showed no significant toxicity for voriconazole on CEC, TMC, RPE cells, or human corneal endothelium when administered in therapeutic concentrations up to 250 μg/mL

  12. Response-predictive gene expression profiling of glioma progenitor cells in vitro.

    Directory of Open Access Journals (Sweden)

    Sylvia Moeckel

    Full Text Available High-grade gliomas are amongst the most deadly human tumors. Treatment results are disappointing. Still, in several trials around 20% of patients respond to therapy. To date, diagnostic strategies to identify patients that will profit from a specific therapy do not exist.In this study, we used serum-free short-term treated in vitro cell cultures to predict treatment response in vitro. This approach allowed us (a to enrich specimens for brain tumor initiating cells and (b to confront cells with a therapeutic agent before expression profiling.As a proof of principle we analyzed gene expression in 18 short-term serum-free cultures of high-grade gliomas enhanced for brain tumor initiating cells (BTIC before and after in vitro treatment with the tyrosine kinase inhibitor Sunitinib. Profiles from treated progenitor cells allowed to predict therapy-induced impairment of proliferation in vitro.For the tyrosine kinase inhibitor Sunitinib used in this dataset, the approach revealed additional predictive information in comparison to the evaluation of classical signaling analysis.

  13. Differences in pyrimidine dimer removal between rat skin cells in vitro and in vivo

    International Nuclear Information System (INIS)

    Mullaart, E.; Lohman, P.H.; Vijg, J.

    1988-01-01

    Pyrimidine dimers, the most abundant type of DNA lesions induced by ultraviolet light (UV), are rapidly repaired in human skin fibroblasts in vitro. In the same cell type from rats, however, there is hardly any removal of such dimers. To investigate whether this low capacity of rat skin cells to repair lesions in their DNA is an inherent characteristic of this species or an artifact due to cell culturing, we measured the removal of UV-induced pyrimidine dimers from rat epidermal keratinocytes both in vitro and in vivo. Epidermal keratinocytes in vitro were unable to remove any dimers over the first 3 h after UV-irradiation, while only about 20% was removed during a repair period of 24 h. In this respect, these cells were not different from cultured rat fibroblasts. In contrast to the results obtained with keratinocytes in vitro, we observed a rapid repair of pyrimidine dimers in UV-irradiated keratinocytes in vivo over the first 3 h; this rapid repair phase was followed by a much slower repair phase between 3 and 24 h. These results are discussed in terms of the possibility that mammalian cells are able to switch from one DNA repair pathway to another

  14. A cell culture technique for human epiretinal membranes to describe cell behavior and membrane contraction in vitro.

    Science.gov (United States)

    Wertheimer, Christian; Eibl-Lindner, Kirsten H; Compera, Denise; Kueres, Alexander; Wolf, Armin; Docheva, Denitsa; Priglinger, Siegfried G; Priglinger, Claudia; Schumann, Ricarda G

    2017-11-01

    To introduce a human cell culture technique for investigating in-vitro behavior of primary epiretinal cells and membrane contraction of fibrocellular tissue surgically removed from eyes with idiopathic macular pucker. Human epiretinal membranes were harvested from ten eyes with idiopathic macular pucker during standard vitrectomy. Specimens were fixed on cell culture plastic using small entomological pins to apply horizontal stress to the tissue, and then transferred to standard cell culture conditions. Cell behavior of 400 epiretinal cells from 10 epiretinal membranes was observed in time-lapse microscopy and analyzed in terms of cell migration, cell velocity, and membrane contraction. Immunocytochemistry was performed for cell type-specific antigens. Cell specific differences in migration behavior were observed comprising two phenotypes: (PT1) epiretinal cells moving fast, less directly, with small round phenotype and (PT2) epiretinal cells moving slowly, directly, with elongated large phenotype. No mitosis, no outgrowth and no migration onto the plastic were seen. Horizontal contraction measurements showed variation between specimens. Masses of epiretinal cells with a myofibroblast-like phenotype expressed cytoplasmatic α-SMA stress fibers and correlated with cell behavior characteristics (PT2). Fast moving epiretinal cells (PT1) were identified as microglia by immunostaining. This in-vitro technique using traction application allows for culturing surgically removed epiretinal membranes from eyes with idiopathic macular pucker, demonstrating cell behavior and membrane contraction of primary human epiretinal cells. Our findings emphasize the abundance of myofibroblasts, the presence of microglia and specific differences of cell behavior in these membranes. This technique has the potential to improve the understanding of pathologies at the vitreomacular interface and might be helpful in establishing anti-fibrotic treatment strategies.

  15. Reassembly of adult human testicular cells: can testis cord-like structures be created in vitro?

    Science.gov (United States)

    Mincheva, M; Sandhowe-Klaverkamp, R; Wistuba, J; Redmann, K; Stukenborg, J-B; Kliesch, S; Schlatt, S

    2018-02-01

    Can enzymatically dispersed testicular cells from adult men reassemble into seminiferous cord-like structures in vitro? Adult human testicular somatic cells reassembled into testicular cord-like structures via dynamic interactions of Sertoli and peritubular cells. In vitro approaches using dispersed single cell suspensions of human testes to generate seminiferous tubule structures and to initiate their functionality have as yet shown only limited success. Testes from 15 adult gender dysphoria patients (mean ± standard deviation age 35 ± 9.3 years) showing spermatogonial arrest became available for this study after sex-reassignment surgery. In vitro primary testicular somatic cell cultures were generated to explore the self-organizing ability of testicular somatic cells to form testis cords over a 2-week period. Morphological phenotype, protein marker expression and temporal dynamics of cell reassembly were analyzed. Cell suspensions obtained by two-step enzymatic digestion were plated onto glass coverslips in 24-well plates. To obtain adherent somatic cells, the supernatant was discarded on Day 2. The culture of the attached cell population was continued. Reassembly into cord-like structures was analyzed daily by microscopic observations. Endpoints were qualitative changes in morphology. Cell types were characterized by phase-contrast microscopy and immunohistochemistry. Dynamics of cord formation were recorded by time-lapse microscopy. Primary adult human testicular cells underwent sequential morphological changes including compaction and reaggregation resulting in round or elongated cord-like structures. Time-lapse video recordings within the first 4 days of culture revealed highly dynamic processes of migration and coalescence of reaggregated cells. The cellular movements were mediated by peritubular cells. Immunohistochemical analysis showed that both SRY-related high mobility box 9-positive Sertoli and α-smooth muscle actin-positive peritubular myoid cells

  16. Inhibition of Autophagy Potentiates Atorvastatin-Induced Apoptotic Cell Death in Human Bladder Cancer Cells in Vitro

    Science.gov (United States)

    Kang, Minyong; Jeong, Chang Wook; Ku, Ja Hyeon; Kwak, Cheol; Kim, Hyeon Hoe

    2014-01-01

    Statins are cholesterol reduction agents that exhibit anti-cancer activity in several human cancers. Because autophagy is a crucial survival mechanism for cancer cells under stress conditions, cooperative inhibition of autophagy acts synergistically with other anti-cancer drugs. Thus, this study investigates whether combined treatment of atorvastatin and autophagy inhibitors results in enhancing the cytotoxic effects of atorvastatin, upon human bladder cancer cells, T24 and J82, in vitro. To measure cell viability, we performed the EZ-Cytox cell viability assay. We examined apoptosis by flow cytometry using annexin-V/propidium iodide (PI and western blot using procaspase-3 and poly (ADP-ribose) polymerase (PARP) antibodies. To examine autophagy activation, we evaluated the co-localization of LC3 and LysoTracker by immunocytochemistry, as well as the expression of LC3 and p62/sequestosome-1 (SQSTM1) by western blot. In addition, we assessed the survival and proliferation of T24 and J82 cells by a clonogenic assay. We found that atorvastatin reduced the cell viability of T24 and J82 cells via apoptotic cell death and induced autophagy activation, shown by the co-localization of LC3 and LysoTracker. Moreover, pharmacologic inhibition of autophagy significantly enhanced atorvastatin-induced apoptosis in T24 and J82 cells. In sum, inhibition of autophagy potentiates atorvastatin-induced apoptotic cell death in human bladder cancer cells in vitro, providing a potential therapeutic approach to treat bladder cancer. PMID:24815071

  17. Cytotoxic effects of betaxolol on healthy corneal endothelial cells both in vitro and in vivo

    Directory of Open Access Journals (Sweden)

    Ying Miao

    2014-02-01

    Full Text Available AIM: To demonstrate the cytotoxic effect of betaxolol and its underlying mechanism on human corneal endothelial cells(HCE cells in vitro and cat corneal endothelial cells(CCE cells in vivo, providing experimental basis for safety anti-glaucoma drug usage in clinic of ophthalmology.METHODS: In vivo and in vitro experiments were conducted to explore whether and how betaxolol participates in corneal endothelial cell injury. The in vitro morphology, growth status, plasma membrane permeability, DNA fragmentation, and ultrastructure of HCE cells treated with 0.021875-0.28g/L betaxolol were examined by light microscope, 3-(4,5-dimethylthiahiazo (-z-y1-3,5-di-phenytetrazoliumromide (MTT assay, acridine orange (AO/ethidium bromide (EB double-fluorescent staining, DNA agarose gel electrophoresis, and transmission electron microscope (TEM. The in vivo density, morphology, and ultrastructure of CCE cells, corneal thickness, and eye pressure of cat eyes treated with 0.28g/L betaxolol were investigated by specular microscopy, applanation tonometer, alizarin red staining, scanning electron microscope (SEM, and TEM.RESULTS: Exposure to betaxolol at doses from 0.0875g/L to 2.8g/L induced morphological and ultrastructural changes of in vitro cultured HCE cells such as cytoplasmic vacuolation, cellular shrinkage, structural disorganization, chromatin condensation, and apoptotic body appearance. Simultaneously, betaxolol elevated plasma membrane permeability and induced DNA fragmentation of these cells in a dose-dependent manner in AO/EB staining. Furthermore, betaxolol at a dose of 2.8g/L also induced decrease of density of CCE cells in vivo, and non-hexagonal and shrunk apoptotic cells were also found in betaxolol-treated cat corneal endothelia.CONCLUSION: Betaxolol has significant cytotoxicity on HCE cells in vitro by inducing apoptosis of these cells, and induced apoptosis of CCE cells in vivo as well. The findings help provide new insight into the apoptosis

  18. An Abbreviated Protocol for In Vitro Generation of Functional Human Embryonic Stem Cell-Derived Beta-Like Cells.

    Directory of Open Access Journals (Sweden)

    Mohammad Massumi

    Full Text Available The ability to yield glucose-responsive pancreatic beta-cells from human pluripotent stem cells in vitro will facilitate the development of the cell replacement therapies for the treatment of Type 1 Diabetes. Here, through the sequential in vitro targeting of selected signaling pathways, we have developed an abbreviated five-stage protocol (25-30 days to generate human Embryonic Stem Cell-Derived Beta-like Cells (ES-DBCs. We showed that Geltrex, as an extracellular matrix, could support the generation of ES-DBCs more efficiently than that of the previously described culture systems. The activation of FGF and Retinoic Acid along with the inhibition of BMP, SHH and TGF-beta led to the generation of 75% NKX6.1+/NGN3+ Endocrine Progenitors. The inhibition of Notch and tyrosine kinase receptor AXL, and the treatment with Exendin-4 and T3 in the final stage resulted in 35% mono-hormonal insulin positive cells, 1% insulin and glucagon positive cells and 30% insulin and NKX6.1 co-expressing cells. Functionally, ES-DBCs were responsive to high glucose in static incubation and perifusion studies, and could secrete insulin in response to successive glucose stimulations. Mitochondrial metabolic flux analyses using Seahorse demonstrated that the ES-DBCs could efficiently metabolize glucose and generate intracellular signals to trigger insulin secretion. In conclusion, targeting selected signaling pathways for 25-30 days was sufficient to generate ES-DBCs in vitro. The ability of ES-DBCs to secrete insulin in response to glucose renders them a promising model for the in vitro screening of drugs, small molecules or genes that may have potential to influence beta-cell function.

  19. [Inhibitory effect of 17-AAG combined with paclitaxel on proliferation of esophageal squamous cell carcinoma Eca-109 cells in vitro].

    Science.gov (United States)

    Chen, Size; Chen, Xuemei; Li, Yuqi; Yang, Shu; Mo, Xianyi; Zhang, Fan; Mo, Kailan; Ding, Ying

    2015-06-01

    To investigate the effect of 17-AAG combined with paclitaxel (PTX) on the proliferation and apoptosis of esophageal squamous cell carcinoma cell line Eca-109 in vitro. Eca-109 cells were treated with 17-AAG and PTX either alone or in combination. The proliferation of Eca-109 cells was detected by MTT assay, and the cell cycle changes and cell apoptosis were determined by flow cytometry. Compared with the control group, both 17-AAG and PTX significantly inhibited the proliferation of Eca-109 cells. A combined treatment of the cells with 0.5 µmol/L PTX and 0.625 µmol/L 17-AAG produced an obviously stronger inhibitory effect on the cell proliferation than either of the agents used alone (PAAG and PTX used alone caused Eca-109 cell cycle arrest in G2/M phase and S phase, respectively, and their combined use caused cell cycle arrest in both G2/M and S phases. The cell apoptosis rates of Eca-109 cells treated with 17-AAG, PTX and their combination were 4.52%, 10.91%, and 29.88%, respectively, all significantly higher than that in the control group (1.32%); the combined treatment resulted in a distinct apoptotic peak that was significantly higher than that caused by either of the agents alone. 17-AAG and PTX can inhibit cell proliferation and promote apoptosis of Eca-109 cells, and their combination produces stronger effects in inhibiting cell proliferation and increasing cell apoptosis.

  20. Proteome analysis of human Wharton's jelly cells during in vitro expansion

    Directory of Open Access Journals (Sweden)

    Sulpizio Marilisa

    2010-03-01

    Full Text Available Abstract Background The human umbilical cord contains mucoid connective tissue and fibroblast-like cells. These cells named Wharton's jelly cells, (WJCs display properties similar to mesenchymal stem cells therefore representing a rich source of primitive cells to be potentially used in regenerative medicine. Results To better understand their self-renewal and potential in vitro expansion capacity, a reference 2D map was constructed as a proteomic data set. 158 unique proteins were identified. More than 30% of these proteins belong to cytoskeleton compartment. We also found that several proteins including Shootin1, Adenylate kinase 5 isoenzyme and Plasminogen activator-inhibitor 2 are no longer expressed after the 2nd passage of in vitro replication. This indicates that the proliferative potency of these cells is reduced after the initial stage of in vitro growing. At the end of cellular culturing, new synthesized proteins, including, ERO1-like protein alpha, Aspartyl-tRNA synthetase and Prolyl-4-hydroxylase were identified. It is suggested that these new synthesized proteins are involved in the impairment of cellular surviving during replication and differentiation time. Conclusions Our work represents an essential step towards gaining knowledge of the molecular properties of WJCs so as to better understand their possible use in the field of cell therapy and regenerative medicine.

  1. Alginate hydrogel as a promising scaffold for dental-derived stem cells: an in vitro study.

    Science.gov (United States)

    Moshaverinia, Alireza; Chen, Chider; Akiyama, Kentaro; Ansari, Sahar; Xu, Xingtian; Chee, Winston W; Schricker, Scott R; Shi, Songtao

    2012-12-01

    The objectives of this study were to: (1) develop an injectable and biodegradable scaffold based on oxidized alginate microbeads encapsulating periodontal ligament (PDLSCs) and gingival mesenchymal stem cells (GMSCs); and (2) investigate the stem cell viability, and osteogenic differentiation of the stem cells in vitro. Stem cells were encapsulated using alginate hydrogel. The stem cell viability, proliferation and differentiation to adipogenic and osteogenic tissues were studied. To investigate the expression of both adipogenesis and ontogenesis related genes, the RNA was extracted and RT-PCR was performed. The degradation behavior of hydrogel based on oxidized sodium alginate with different degrees of oxidation was studied in PBS at 37 °C as a function of time by monitoring the changes in weight loss. The swelling kinetics of alginate hydrogel was also investigated. The results showed that alginate is a promising candidate as a non-toxic scaffold for PDLSCs and GMSCs. It also has the ability to direct the differentiation of these stem cells to osteogenic and adipogenic tissues as compared to the control group in vitro. The encapsulated stem cells remained viable in vitro and both osteo-differentiated and adipo-differentiated after 4 weeks of culturing in the induction media. It was found that the degradation profile and swelling kinetics of alginate hydrogel strongly depends on the degree of oxidation showing its tunable chemistry and degradation rate. These findings demonstrate for the first time that immobilization of PDLSCs and GMSCs in the alginate microspheres provides a promising strategy for bone tissue engineering.

  2. Cell-based in vitro models in environmental toxicology: a review

    Directory of Open Access Journals (Sweden)

    Poteser Michael

    2017-10-01

    Full Text Available An analysis of biological effects induced by environmental toxins and exposure-related evaluation of potential risks for health and environment represent central tasks in classical biomonitoring. While epidemiological data and population surveys are clearly the methodological frontline of this scientific field, cellbased in vitro assays provide information on toxin-affected cellular pathways and mechanisms, and are important sources for the identification of relevant biomarkers. This review provides an overview on currently available in vitro methods based on cultured cells, as well as some limitations and considerations that are of specific interest in the context of environmental toxicology. Today, a large number of different endpoints can be determined to pinpoint basal and specific toxicological cellular effects. Technological progress and increasingly refined protocols are extending the possibilities of cell-based in vitro assays in environmental toxicology and promoting their increasingly important role in biomonitoring.

  3. Sup(99m) Technetium - labeled red blood cells 'in vitro'

    International Nuclear Information System (INIS)

    Bernardo Filho, M.; Souza Moura, I.N. de; Boasquevisque, E.M.

    1983-01-01

    A simple technique for the preparation of sup(99m) Tc labeled red blood cells using a comercial kit is described. To each 3ml of plain blood with anti-coagulant was added 1ml of solution of commercial kit with 6.8 μg of stannous chloride. This mixture was incubated in water bath, at 37 0 C, for 60 minutes. Then technetium-99m was added and the mixture was left for another ten minutes, in water bath, at 37 0 C. Under these conditions there was the best labeling of the red blood cells. Similar results were obtained with a solution of stannous chloride prepared freshly. The labeling is strong for 6.8 μg stannous chloride because the labeling was not removed by the several washes of the red blood cells or by the left in water bath. (Author) [pt

  4. In vitro transdifferentiation of umbilical cord stem cells into cardiac myocytes: Role of growth factors

    Directory of Open Access Journals (Sweden)

    Rasha A.M. Khattab

    2013-04-01

    Full Text Available Recently, stem cell based cell therapy has become a realistic option to replace damaged cardiomyocytes. Most studies on stem cell transplantation therapy have focused on the use of undifferentiated stem cells. There is a strong possibility that some cardiogenic differentiation of the stem cell in vitro prior to transplantation would result in higher engraftment efficiency, as well as enhanced myocardial regeneration and recovery of heart function. In this study we aimed to define the conditions for ex-vivo differentiation of cord blood stem cells to cardiomyocytes and endothelial cells. These conditions include the combination of vascular endothelial growth factor (VEGF; basic fibroblast growth factor (FGF-2 and platelet derived growth factor AB (PDGF-AB. Forty cord blood samples were included in this work. In this work, the percentage of CD34+ cells, CD31+ cells and CD34/31+ cells in mononuclear cells (MNC suspension was counted prior to culture (day zero, and day 10 in the different growth factor cocktails used as well as the control tube, from which the fold increase of CD34+ cells, CD31+ cells and CD34/31+ cells was calculated. Detection of cardiac troponin I in the cultured cells to confirm cardiac differentiation was done at day 10 using Mouse anti-troponin I monoclonal antibody. From the present study, it was concluded that the growth factor cocktail in protocol 2 (FGF2+VEGF+PDGF-AB gives better in vitro trans-differentiation of stem/progenitor cells in umbilical cord blood into cardiomyocytes and endothelial cells than the cytokines cocktail in protocol 1 (FGF2+VEGF alone.

  5. In vitro activation of retinal cells: estimating location of stimulated cell by using a mathematical model

    Science.gov (United States)

    Ziv, Ofer R.; Rizzo, Joseph F., III; Jensen, Ralph J.

    2005-03-01

    Activation of neurons at different depths within the retina and at various eccentricities from the stimulating electrode will presumably influence the visual percepts created by a retinal prosthesis. With an electrical prosthesis, neurons will be activated in relation to the stimulating charge that impacts their cell membranes. The common model used to predict charge density is Coulomb's law, also known as the square law. We propose a modified model that can be used to predict neuronal depth that takes into account: (1) finite dimensions related to the position and size of the stimulating and return electrodes and (2) two-dimensional displacements of neurons with respect to the electrodes, two factors that are not considered in the square law model. We tested our model by using in vitro physiological threshold data that we had obtained previously for eight OFF-center brisk-transient rabbit retinal ganglion cells. For our most spatially dense threshold data (25 µm increments up to 100 µm from the cell body), our model estimated the depth of one RGC to be 76 ± 76 µm versus 87 ± 62 µm (median: SD) for the square law model, respectively. This difference was not statistically significant. For the seven other RGCs for which we had obtained threshold data up to 800 µm from the cell body, the estimate of the RGC depth (using data obtained along the X axis) was 96 ± 74 versus 20 ± 20 µm for the square law and our modified model, respectively. Although this difference was not statistically significant (Student t-test: p = 0.12), our model provided median values much closer to the estimated depth of these RGCs (Gt25 µm). This more realistic estimate of cell depth predicted by our model is not unexpected in this latter data set because of the more spatially distributed threshold data points that were evaluated. Our model has theoretical advantages over the traditional square law model under certain conditions, especially when considering neurons that are

  6. [Anti-tumor effects of DDP-PLLA-CNTs on human cholangiocarcinoma cell line in vitro].

    Science.gov (United States)

    Li, Maolan; Lu, Wei; Zhang, Fei; Ding, Qichen; Wu, Xiangsong; Tan, Zhujun; Wu, Wenguang; Weng, Hao; Wang, Xuefeng; Shi, Weibin; Dong, Ping; Gu, Jun; Liu, Yingbin

    2014-11-04

    To explore the antitumor effects of DDP-PLLA-CNTs on human cholangiocarcinoma cell line. DDP-PLLA-CNTs were prepared with the method of ultrasound emulsification. The morphology of DDP-PLLA-CNTs was determined by scanning electron microscope (SEM). And its drug loading and drug release curve in vitro was detected by UV-Vis-NIR spectrophotometer. CCK8 was used to test the cytotoxic effects of DDP-PLLA-CNTs at different concentrations on QBC939 cell proliferation.Flow cytometry was employed to measure the changes of apoptotic rate. With excellent controlled-release characteristic of in vitro drug release, DDP-PLLA-CNTs inhibited the proliferation and significantly increased the apoptotic rate of QBC939 cell line. DDP-PLLA-CNTs have drug sustained-release characteristics and can significantly inhibit the proliferation of QBC939 cell line.

  7. Targeted femtosecond laser driven drug delivery within HIV-1 infected cells: In-vitro studies [conference paper

    CSIR Research Space (South Africa)

    Maphanga, Charles

    2017-01-01

    Full Text Available of SPIE 10062, Optical Interactions with Tissue and Cells XXVIIISan Francisco, California, USA, 26 January - 03 February 2017 Targeted femtosecond laser driven drug delivery within HIV-1 infected cells: In-vitro studies Charles Maphanga 1, 2...

  8. Critical Evaluation of Air-Liquid Interface Cell Exposure Systems for in Vitro Assessment of Atmospheric Pollutants

    Science.gov (United States)

    We compared various in vitro exposure systems for their ability to expose cells to particles and gases. The systems tested use different mechanisms to deliver multi-pollutants to the cells: diffusion, sedimentation, thermophoresis (THP) and electrostatic precipitation (ESP). Vari...

  9. Rapid and non-enzymatic in vitro retrieval of tumour cells from surgical specimens.

    Directory of Open Access Journals (Sweden)

    Brigitte Mack

    Full Text Available The study of tumourigenesis commonly involves the use of established cell lines or single cell suspensions of primary tumours. Standard methods for the generation of short-term tumour cell cultures include the disintegration of tissue based on enzymatic and mechanical stress. Here, we describe a simple and rapid method for the preparation of single cells from primary carcinomas, which is independent of enzymatic treatment and feeder cells. Tumour biopsies are processed to 1 mm(3 cubes termed explants, which are cultured 1-3 days on agarose-coated well plates in specified medium. Through incisions generated in the explants, single cells are retrieved and collected from the culture supernatant and can be used for further analysis including in vitro and in vivo studies. Collected cells retain tumour-forming capacity in xenotransplantation assays, mimic the phenotype of the primary tumour, and facilitate the generation of cell lines.

  10. Senescent changes in the ribosomes of animal cells in vivo and in vitro

    Science.gov (United States)

    Miquel, J.; Johnson, J. E., Jr.

    1979-01-01

    The paper examines RNA-ribosomal changes observed in protozoa and fixed postmitotic cells, as well as the characteristics of intermitotic cells. Attention is given to a discussion of the implications of the reported ribosomal changes as to the senescent deterioration of protein synthesis and physiological functions. A survey of the literature suggests that, while the data on ribosomal change in dividing cells both in vivo and in vitro are inconclusive, there is strong histological and biochemical evidence in favor of some degree of quantitative ribosomal loss in fixed postmitotic cells. Since these decreases in ribosomes are demonstrated in differential cells from nematodes, insects and mammals, they may represent a universal manifestation of cytoplasmic senescence in certain types of fixed postmitotic animal cells. The observed variability in ribosomal loss for cells belonging to the same type suggests that this involution phenomenon is rather related to the wear and tear suffered by a particular cell.

  11. Cytotoxic properties of a 4-nitroimidazole (NSC 38087): a radiosensitizer of hypoxic cells in vitro

    International Nuclear Information System (INIS)

    Stratford, I.J.; Williamson, C.; Hardy, C.

    1981-01-01

    5-Phenoxysulphonyl-1-methyl-4-nitroimidazole (NSC 38087) can act as a sensitizer of hypoxic mammalian cells to radiation in vitro. The degree of sensitization achieved is greater than would be predicted from the drug's electron affinity. Cytotoxicity studies have shown that 5sub(μM) NSC 38087 can reduce the surviving fraction of log-phase V79 cells in air at 37 0 C to 10 -2 after 2 h exposure. This toxicity is considerably increased by small rises in temperature. In contrast to other nitroheterocyclic radiosensitizers, NSC 38087 and related 5-substituted 4-nitroimidazoles show greater toxicity towards aerobic than to hypoxic cells. Log-phase cells show the highest sensitivity to the toxic action of NSC 38087, with plateau-phase cells, cells with a history of chronic hypoxia, and thermotolerant cells showing greater resistance. These toxicity data are compared to those for the 2-nitroimidazole hypoxic-cell sensitizer misonidazole. (author)

  12. Reversible gelling culture media for in-vitro cell culture in three-dimensional matrices

    Science.gov (United States)

    An, Yuehuei H.; Mironov, Vladimir A.; Gutowska, Anna

    2000-01-01

    A gelling cell culture medium useful for forming a three dimensional matrix for cell culture in vitro is prepared by copolymerizing an acrylamide derivative with a hydrophilic comonomer to form a reversible (preferably thermally reversible) gelling linear random copolymer in the form of a plurality of linear chains having a plurality of molecular weights greater than or equal to a minimum gelling molecular weight cutoff, mixing the copolymer with an aqueous solvent to form a reversible gelling solution and adding a cell culture medium to the gelling solution to form the gelling cell culture medium. Cells such as chondrocytes or hepatocytes are added to the culture medium to form a seeded culture medium, and temperature of the medium is raised to gel the seeded culture medium and form a three dimensional matrix containing the cells. After propagating the cells in the matrix, the cells may be recovered by lowering the temperature to dissolve the matrix and centrifuging.

  13. Transfection of bovine spermatogonial stem cells in vitro.

    Science.gov (United States)

    Tajik, P; Hoseini Pajooh, Kh; Fazle Elahi, Z; Javdani Shahedin, G; Ghasemzadeh-Nava, H

    2017-01-01

    Spermatogonial stem cells (SSCs) are the only stem cells in adults that can transfer genetic information to the future generations. Considering the fact that a single SSC gives rise to a vast number of spermatozoa, genetic manipulation of these cells is a potential novel technology with feasible application to various animal species. The aim of this study was to evaluate enhanced green fluorescent protein (EGFP) gene transfection into bovine SSCs via liposome carrier and assess the best incubation day in uptake exogenous gene by SSCs. Transfection efficiency of EGFP gene with lipofectamine 2000 was determined in days following each three day of transfection (day 4, 6 and 8 of the culture) by fluorescent microscope. Results showed that the transfected cells through lipofection increased significantly (Ptransfection in comparison with those of the control groups. The transfected SSCs were higher in comparison with those of the free exogenous gene carrier groups (Ptransfection proceeds at day four. It was concluded that lipofectamine can be used safely for direct loading exogenous DNA to SSCs particularly during the fourth day of culture.

  14. Sensitization of in vitro mammalian cells by nitrous oxide

    International Nuclear Information System (INIS)

    Ewing, D.

    1984-01-01

    Powers and his colleagues showed almost ten years ago that sensitization by nitrous oxide required two radiolytic products: OH radicals and hydrogen peroxide. That observation with bacterial spores has been confirmed and extended with spores and several strains of bacteria. OH must be present to form hydrogen peroxide, but, in addition, OH must also be present with the hydrogen peroxide for damage to occur. (Reagent hydrogen peroxide, except at very high concentrations, will not sensitize unless OH radicals are present.) The authors have now tested nitrous oxide with two Chinese hamster cell lines, V79 and CHO. The responses in nitrogen and nitrous oxide are the same for each. The authors have tentatively concluded that insufficient hydrogen peroxide is formed in the cells' suspending fluid for damage from nitrous oxide to occur. Several results support this conclusion: reagent hydrogen peroxide is a potent sensitizer of either cell line tested in nitrogen or nitrous oxide and an assay for radiolytic hydrogen peroxide confirms that only minimal levels are formed at the doses used in these survival curves. The authors also present results of other tests to further complement work with procaryotic cells

  15. In vitro developmental toxicity test detects inhibition of stem cell differentiation by silica nanoparticles.

    NARCIS (Netherlands)

    Park, M.V.; Annema, W.; Salvati, A.; Lesniak, A.; Elsaesser, A.; Barnes, C.; McKerr, G.; Howard, C.; Lynch, I.; Dawson, K.; Piersma, A.H.; de Jong, W.H.

    2009-01-01

    While research into the potential toxic properties of nanomaterials is now increasing, the area of developmental toxicity has remained relatively uninvestigated. The embryonic stem cell test is an in vitro screening assay used to investigate the embryotoxic potential of chemicals by determining

  16. Toxicity of formaldehyde and acrolein mixtures : in vitro studies using nasal epithelial cells

    NARCIS (Netherlands)

    Cassee, F.R.; Stenhuis, W.S.; Groten, J.P.; Feron, V.J.

    1996-01-01

    In vitro studies with human and rat nasal epithelial cells were carried out to investigate the combined toxicity of formaldehyde and acrolein and the role of aldehyde dehydrogenases in this process. These studies showed that the toxic effect of mixtures of aldehydes was additive. In addition,

  17. Characterization of calcium responses and electrical activity in differentiating mouse neural progenitor cells in vitro

    NARCIS (Netherlands)

    de Groot, Martje W G D M; Dingemans, Milou M L; Rus, Katinka H; de Groot, Aart; Westerink, Remco H S

    In vitro methods for developmental neurotoxicity (DNT) testing have the potential to reduce animal use and increase insight into cellular and molecular mechanisms underlying chemical-induced alterations in the development of functional neuronal networks. Mouse neural progenitor cells (mNPCs)

  18. UP-REGULATION OF ANTITHROMBOTIC ECTONUCLEOTIDASES BY ASPIRIN IN HUMAN ENDOTHELIAL-CELLS IN-VITRO

    NARCIS (Netherlands)

    CHEUNG, PK; VISSER, J; BAKKER, WW

    1994-01-01

    Ecto ATP-diphosphohydrolase (apyrase) activity of human endothelial cells following aspirin treatment has been studied in-vitro. It was shown by HPLC analysis of supernatant samples that pre-incubation of the cultures with aspirin resulted in a significantly increased turnover of supplemented ATP

  19. Effect of Acrylamide on Oocyte Nuclear Maturation and Cumulus Cells Apoptosis in Mouse In Vitro.

    Directory of Open Access Journals (Sweden)

    Shuzhen Liu

    Full Text Available Acrylamide (ACR is a chemical compound with severe neurotoxicity, genotoxicity, carcinogenicity and reproductive toxicity. Recent studies showed that ACR impairs the function of reproductive organs, e.g., epididymis and testes. In vitro maturation of mouse oocyte is a sensitive assay to identify potential chemical hazard to female fertility. The aim of this study was to evaluate the adverse effects of ACR on the nuclear maturation and cumulus cells apoptosis of mouse oocytes in vitro. Cumulus-oocyte complexes were incubated in a maturation medium containing 0, 5, 10 and 20 μM of ACR. Chromosome alignment and spindle morphology of oocytes was determined by immunofluorescence and confocal microscopy. Our results showed that oocytes exposed to different doses of ACR in vitro were associated with a significant decrease of oocyte maturation, significant increase of chromosome misalignment rate, occurrence of abnormal spindle configurations, and the inhibition of oocyte parthenogenetic activation. Furthermore, apoptosis of cumulus cells was determined by TUNEL and CASPASE-3 assay. Results showed that apoptosis in cumulus cells was enhanced and the expression of CASPASE-3 was increased after cumulus-oocyte complexes were exposed to ACR. Therefore, ACR may affect the nuclear maturation of oocytes via the apoptosis of cumulus cells in vitro.

  20. Imatinib prevents beta cell death in vitro but does not improve islet transplantation outcome.

    Science.gov (United States)

    King, Aileen J F; Griffiths, Lisa A; Persaud, Shanta J; Jones, Peter M; Howell, Simon L; Welsh, Nils

    2016-05-01

    Introduction Improving islet transplantation outcome could not only bring benefits to individual patients but also widen the patient pool to which this life-changing treatment is available. Imatinib has previously been shown to protect beta cells from apoptosis in a variety of in vitro and in vivo models. The aim of this study was to investigate whether imatinib could be used to improve islet transplantation outcome. Methods Islets were isolated from C57Bl/6 mice and pre-cultured with imatinib prior to exposure to streptozotocin and cytokines in vitro. Cell viability and glucose-induced insulin secretion were measured. For transplantation experiments, islets were pre-cultured with imatinib for either 72 h or 24 h prior to transplantation into streptozotocin-diabetic C57Bl/6 mice. In one experimental series mice were also administered imatinib after islet transplantation. Results Imatinib partially protected islets from beta cell death in vitro. However, pre-culturing islets in imatinib or administering the drug to the mice in the days following islet transplantation did not improve blood glucose concentrations more than control-cultured islets. Conclusion Although imatinib protected against beta cell death from cytokines and streptozotocin in vitro, it did not significantly improve syngeneic islet transplantation outcome.

  1. Identification of potential biomarkers in donor cows for in vitro embryo production by granulosa cell transcriptomics

    DEFF Research Database (Denmark)

    Mazzoni, Gianluca; Salleh, Suraya M; Freude, Kristine

    2017-01-01

    The Ovum Pick Up-In vitro Production (OPU-IVP) of embryos is an advanced reproductive technology used in cattle production but the complex biological mechanisms behind IVP outcomes are not fully understood. In this study we sequenced RNA of granulosa cells collected from Holstein cows at oocyte...

  2. Effect of in vitro exposure to cadmium and copper on sea bass blood cells

    Directory of Open Access Journals (Sweden)

    Vincenzo Arizza

    2010-01-01

    Full Text Available Blood cells freshly collected from sea bass (Dicentrarchus labrax were exposed in vitro to different concentrations of cadmium (Cd and copper (Cu at 10-7 M, 10-5 M, 10-3 M, and exam- ined for neutral red retention capacity and for cell vitality with MTT assay. A relationship between heavy metal exposure and alteration in responses of blood cells in a dose-time-dependent was found. Our results showed that fish blood cells may constitute an interesting biological model for experimen- tal and applied toxicology, especially in the case of environmental pollution.

  3. Effects of cell concentrations on the survival and repopulation of haemopoietic stem cells in irradiated bone marrow cell culture in vitro

    International Nuclear Information System (INIS)

    Fujitake, Hideki; Okamoto, Yuruko; Okubo, Hiroshi; Miyanomae, Takeshi; Kumagai, Keiko; Mori, K.J.

    1981-01-01

    Effects of cell concentrations on the survival and repopulation of haemopoietic stem cells after irradiation were studied in the long-term culture of mouse bone marrow cells in vitro. No difference was observed in the survival of the stem cells among cultures in which 0 - 10 7 cells were re-inoculated on the adherent cell colonies in the culture flask. Stem cells showed a significant proliferation within 1 week and the number of the stem cells exceeded the control in 3 weeks after irradiation in the cultures with less than 10 6 re-inoculated cells per flask. In contrast, there was a considerable delay in the onset of stem cell proliferation after irradiation in the culture with 10 7 cells per flask. Based on these results, a possibility that a stimulator of stem cell proliferation, released from irradiated stromal cells, is cancelled by an inhibitory factor produced by irradiated or unirradiated haemopoietic cells is postulated. (author)

  4. The in vitro biokinetics of chlorpromazine and diazepam in aggregating rat brain cell cultures after repeated exposure

    NARCIS (Netherlands)

    Broeders, Jessica J W; Hermens, Joop L M; Blaauboer, Bas J; Zurich, Marie-Gabrielle

    2015-01-01

    Neurotoxic effects of compounds can be tested in vitro using cell systems. One example is aggregating rat brain cell cultures. For the extrapolation of in vitro data to the in vivo situation, it is important to take the biokinetics of the test compound into account. In addition, the exposure in vivo

  5. Detection and characterization of side population in Ewing's sarcoma SK-ES-1 cells in vitro

    International Nuclear Information System (INIS)

    Yang, Min; Zhang, Rui; Yan, Ming; Ye, Zhengxu; Liang, Wei; Luo, Zhuojing

    2010-01-01

    Dye exclusion is a valuable technique to isolate cancer stem cells (CSCs) based on an ability of stem cell to efflux fluorescent DNA-binding dye, especially for tumors without unique surface markers. It has been proven that side population (SP) cells that exclude Hoechst 33342 dye are enriched with stem-like cells in several cancer cell lines. In this study, we isolated and characterized SP cells from human Ewing's sarcoma cell line SK-ES-1 in vitro. SP cells were detected in SK-ES-1 and comprised 1.2% of total cell population. Only SP cells had the capacity to regenerate both SP and non-SP cells. The proliferation rates were similar between SP and non-SP cells. However, the clonogenicity and invasiveness of SP cells were significantly higher than that of non-SP cells. Further characterization of this SP phenotype presented other properties. SP cells exhibited increased multi-drug resistance and the ATP binding cassette protein (ABC) transporters were up-regulated in SP population. These findings suggest that SP cells derived from Ewing's sarcoma play the critical role in tumor metastasis and recurrence and might be an ideal target for clinical therapy.

  6. Mechanisms of cell death induced by infusion sets leachables in in vitro experimental settings.

    Science.gov (United States)

    Kozlovskaya, Luba; Stepensky, David

    2015-01-30

    Leachable materials that are released from infusion sets during their use can induce local and systemic toxic effects. We studied the mechanisms and kinetics of cell death induced by infusion sets leachates in vitro using L-929 and bEnd. 3 cells. Changes in cell morphology and metabolic activity were determined using light microscopy and the MTT test, respectively. Detailed analysis of the mechanisms of cell death was performed using membrane integrity and caspases 3 and 7 activity tests, annexin V-FITC/7-AAD analysis by FACS, and DAPI nuclear staining followed by confocal microscopy. Infusion sets released toxic leachables and induced toxic effects. Latex flashball was the most toxic part of the studied infusion sets, and it potently induced cell oncosis via increased permeability of the cell membrane. Latex-induced decrease in cells metabolic activity and cell death were not accompanied by activation of caspases 3 and 7, changes in nuclear morphology, or substantial annexin V-FITC cell staining. Leachables from the tube part of the infusion sets were less toxic, and induced some biochemical changes without altering the cells morphology. Further studies are needed to reveal the in vivo toxicity of infusion sets and its correlation with the results of in vitro toxicity studies. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. Epithelial cells as active player in fibrosis: findings from an in vitro model.

    Directory of Open Access Journals (Sweden)

    Solange Moll

    Full Text Available Kidney fibrosis, a scarring of the tubulo-interstitial space, is due to activation of interstitial myofibroblasts recruited locally or systemically with consecutive extracellular matrix deposition. Newly published clinical studies correlating acute kidney injury (AKI to chronic kidney disease (CKD challenge this pathological concept putting tubular epithelial cells into the spotlight. In this work we investigated the role of epithelial cells in fibrosis using a simple controlled in vitro system. An epithelial/mesenchymal 3D cell culture model composed of human proximal renal tubular cells and fibroblasts was challenged with toxic doses of Cisplatin, thus injuring epithelial cells. RT-PCR for classical fibrotic markers was performed on fibroblasts to assess their modulation toward an activated myofibroblast phenotype in presence or absence of that stimulus. Epithelial cell lesion triggered a phenotypical modulation of fibroblasts toward activated myofibroblasts as assessed by main fibrotic marker analysis. Uninjured 3D cell culture as well as fibroblasts alone treated with toxic stimulus in the absence of epithelial cells were used as control. Our results, with the caveats due to the limited, but highly controllable and reproducible in vitro approach, suggest that epithelial cells can control and regulate fibroblast phenotype. Therefore they emerge as relevant target cells for the development of new preventive anti-fibrotic therapeutic approaches.

  8. Chromium cytotoxic effects on mammalian cells in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Levis, A G; Buttignol, M; Vettorato, L

    1975-01-01

    Chromium compounds, which have several industrial uses, are reported to be carcinogenic. The author, have, therefore, undertaken the study of K/sub 2/Cr/sub 2/O/sub 7/ effects on a cell line of Hamster fibroblasts (BHK), using specific radioactive precursors and determining the acid soluble pool, the RNA, DNA, and protein contents and specific activities. The K/sub 2/Cr/sub 2/O/sub 7/ induced changes in nucleic acid and protein specific activities are related to two different, dissociable effects: (1) a sudden inhibition of macromolecular syntheses, followed by a recovery period, and (2) an immediate, drastic stimulation of nucleoside transport into the cell, whereas amino acid transport is reduced. The effects on precursor permeability are not related to non specific changes of the plasma membrane, but they seem to be due to specific simulations and inhibitions of nucleoside and amino acid transport mechanisms. A human cell line (HEp) has been also tested, which is more sensitive to K/sup 2/Cr/sub 2/O/sub 7/ action than the BHK line. DNA synthesis as well as survival in single cell plating conditions show the same difference in sensitivity to K/sub 2/Cr/sub 2/O/sub 7/. Thus, the loss of indefinite cell division ability could be due to the blockage of DNA replication. It is suggested that the main chromium cytotoxic effect lies in a multiple attack to the DNA molecule, which gradually alters the DNA tertiary structure resulting in the blockage of replication capacity. This blockage may be reversible owing to the breakage of chromium-DNA bonds and to the induced instability of phosphodiester internucleotide bonds.

  9. Lethal Effect of Thermal Neutrons on Hypoxic Elirlich Ascites Tumour Cells in vitro

    OpenAIRE

    MITSUHIKO, AKABOSHI; KENICHI, KAWAI; HIROTOSHI, MAKI; Research Reactor Institute, Kyoto University; Research Reactor Institute, Kyoto University; Research Reactor Institute, Kyoto University

    1985-01-01

    Ehrlich ascites tumour cells were irradiated in vitro with thermal neutrons under aerobic and hypoxic conditions, and the survival of their reproductive capacity was assayed in vivo. Only a slight hypoxic protection was observed for thermal neutron irradiation with an oxygen enhancement ratio (OER) of 1.2, as compared with OER of 3.3 for ^Co-γ-rays. Absorbed dose of thermal neutrons was calculated by assuming that the energies of recoiled nuclei were completely absorbed within a cell nucleus....

  10. Nano- and microstructured materials for in vitro studies of the physiology of vascular cells

    OpenAIRE

    Alexandra M. Greiner; Adria Sales; Hao Chen; Sarah A. Biela; Dieter Kaufmann; Ralf Kemkemer

    2016-01-01

    The extracellular environment of vascular cells in vivo is complex in its chemical composition, physical properties, and architecture. Consequently, it has been a great challenge to study vascular cell responses in vitro, either to understand their interaction with their native environment or to investigate their interaction with artificial structures such as implant surfaces. New procedures and techniques from materials science to fabricate bio-scaffolds and surfaces have enabled novel studi...

  11. Paracetamol (acetaminophen) attenuates in vitro mast cell and peripheral blood mononucleocyte cell histamine release induced by N-acetylcysteine.

    Science.gov (United States)

    Coulson, James; Thompson, John Paul

    2010-02-01

    The treatment of acute paracetamol (acetaminophen) poisoning with N-acetylcysteine (NAC) is frequently complicated by an anaphylactoid reaction to the antidote. The mechanism that underlies this reaction is unclear. We used the human mast cell line 1 (HMC-1) and human peripheral blood mononucleocytes (PBMCs) to investigate the effects of NAC and paracetamol on histamine secretion in vitro. HMC-1 and human PBMCs were incubated in the presence of increasing concentrations of NAC +/- paracetamol. Cell viability was determined by the Trypan Blue Assay, and histamine secretion was measured by ELISA. NAC was toxic to HMC-1 cells at 100 mg/mL and to PBMCs at 67 mg/mL. NAC increased HMC-1 and PBMC histamine secretion at concentrations of NAC from 20 to 50 mg/mL and 2.5 to 100 mg/mL, respectively. NAC-induced histamine secretion by both cell types was reduced by co-incubation with 2.5 mg/mL of paracetamol. Paracetamol (acetaminophen) is capable of modifying histamine secretion in vitro. This may explain the clinical observation of a lower incidence of adverse reactions to NAC in vivo when higher concentrations of paracetamol are present than when paracetamol concentrations are low. Paracetamol (acetaminophen) attenuates in vitro mast cell and PBMC cell histamine release induced by NAC.

  12. Surface chemical functionalities affect the behavior of human adipose-derived stem cells in vitro

    International Nuclear Information System (INIS)

    Liu, Xujie; Feng, Qingling; Bachhuka, Akash; Vasilev, Krasimir

    2013-01-01

    This study examines the effect of surface chemical functionalities on the behavior of human adipose-derived stem cells (hASCs) in vitro. Plasma polymerized films rich in amine (-NH 2 ), carboxyl (-COOH) and methyl (-CH 3 ), were generated on hydroxyapatite (HAp) substrates. The surface chemical functionalities were characterized by X-ray photoelectron spectroscopy (XPS). The ability of different substrates to absorb proteins was evaluated. The results showed that substrates modified with hydrophilic functional group (-COOH and -NH 2 ) can absorb more proteins than these modified with more hydrophobic functional group (-CH 3 ). The behavior of human adipose-derived stem cells (hASCs) cultured on different substrates was investigated in vitro: cell counting kit-8 (CCK-8) analysis was used to characterize cell proliferation, scanning electronic microscopy (SEM) analysis was used to characterize cell morphology and alkaline phosphatase (ALP) activity analysis was used to account for differentiation. The results of this study demonstrated that the -NH 2 modified surfaces encourage osteogenic differentiation; the -COOH modified surfaces promote cell adhesion and spreading and the -CH 3 modified surfaces have the lowest ability to induce osteogenic differentiation. These findings confirmed that the surface chemical states of biomaterials can affect the behavior of hASCs in vitro.

  13. Surface chemical functionalities affect the behavior of human adipose-derived stem cells in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Xujie [State key laboratory of new ceramics and fine processing, School of Materials Science and Engineering, Tsinghua University, Beijing 100084 (China); Feng, Qingling, E-mail: biomater@mail.tsinghua.edu.cn [State key laboratory of new ceramics and fine processing, School of Materials Science and Engineering, Tsinghua University, Beijing 100084 (China); Bachhuka, Akash [Mawson Institute, University of South Australia, Mawson Lakes 5095 (Australia); Vasilev, Krasimir [Mawson Institute, University of South Australia, Mawson Lakes 5095 (Australia); School of Advanced Manufacturing, University of South Australia, Mawson Lakes 5095 (Australia)

    2013-04-01

    This study examines the effect of surface chemical functionalities on the behavior of human adipose-derived stem cells (hASCs) in vitro. Plasma polymerized films rich in amine (-NH{sub 2}), carboxyl (-COOH) and methyl (-CH{sub 3}), were generated on hydroxyapatite (HAp) substrates. The surface chemical functionalities were characterized by X-ray photoelectron spectroscopy (XPS). The ability of different substrates to absorb proteins was evaluated. The results showed that substrates modified with hydrophilic functional group (-COOH and -NH{sub 2}) can absorb more proteins than these modified with more hydrophobic functional group (-CH{sub 3}). The behavior of human adipose-derived stem cells (hASCs) cultured on different substrates was investigated in vitro: cell counting kit-8 (CCK-8) analysis was used to characterize cell proliferation, scanning electronic microscopy (SEM) analysis was used to characterize cell morphology and alkaline phosphatase (ALP) activity analysis was used to account for differentiation. The results of this study demonstrated that the -NH{sub 2} modified surfaces encourage osteogenic differentiation; the -COOH modified surfaces promote cell adhesion and spreading and the -CH{sub 3} modified surfaces have the lowest ability to induce osteogenic differentiation. These findings confirmed that the surface chemical states of biomaterials can affect the behavior of hASCs in vitro.

  14. Evidence of In Vitro Preservation of Human Nephrogenesis at the Single-Cell Level

    Directory of Open Access Journals (Sweden)

    Naomi Pode-Shakked

    2017-07-01

    Full Text Available During nephrogenesis, stem/progenitor cells differentiate and give rise to early nephron structures that segment to proximal and distal nephron cell types. Previously, we prospectively isolated progenitors from human fetal kidney (hFK utilizing a combination of surface markers. However, upon culture nephron progenitors differentiated and could not be robustly maintained in vitro. Here, by culturing hFK in a modified medium used for in vitro growth of mouse nephron progenitors, and by dissection of NCAM+/CD133− progenitor cells according to EpCAM expression (NCAM+/CD133−/EpCAM−, NCAM+/CD133−/EpCAMdim, NCAM+/CD133−/EpCAMbright, we show at single-cell resolution a preservation of uninduced and induced cap mesenchyme as well as a transitioning mesenchymal-epithelial state. Concomitantly, differentiating and differentiated epithelial lineages are also maintained. In vitro expansion of discrete stages of early human nephrogenesis in nephron stem cell cultures may be used for drug screening on a full repertoire of developing kidney cells and for prospective isolation of mesenchymal or epithelial renal lineages for regenerative medicine.

  15. Live Cell in Vitro and in Vivo Imaging Applications: Accelerating Drug Discovery

    Directory of Open Access Journals (Sweden)

    Neil O Carragher

    2011-04-01

    Full Text Available Dynamic regulation of specific molecular processes and cellular phenotypes in live cell systems reveal unique insights into cell fate and drug pharmacology that are not gained from traditional fixed endpoint assays. Recent advances in microscopic imaging platform technology combined with the development of novel optical biosensors and sophisticated image analysis solutions have increased the scope of live cell imaging applications in drug discovery. We highlight recent literature examples where live cell imaging has uncovered novel insight into biological mechanism or drug mode-of-action. We survey distinct types of optical biosensors and associated analytical methods for monitoring molecular dynamics, in vitro and in vivo. We describe the recent expansion of live cell imaging into automated target validation and drug screening activities through the development of dedicated brightfield and fluorescence kinetic imaging platforms. We provide specific examples of how temporal profiling of phenotypic response signatures using such kinetic imaging platforms can increase the value of in vitro high-content screening. Finally, we offer a prospective view of how further application and development of live cell imaging technology and reagents can accelerate preclinical lead optimization cycles and enhance the in vitro to in vivo translation of drug candidates.

  16. Effects of Pollutants on Vertebrate Cells in Vitro.

    Science.gov (United States)

    1981-01-01

    ex- Barkla and Tutton, 1978). More specifically, perimental setups, the cells were enzymatically re- some of these effects are produced in moved from...upon re- biological membranes (Balduini et al., 1977, exposure to fresh hydrazine, they appeared Barkla and Tutton, 1977; Braun and Wolfe, to attain a...Schneideberg’s Phartna- Chient. 358. 1143-1148. col. 298, 75-77. BARKLA . D. H., AN D TUTTON. P. J. M. (1977). Surface JAIN, S. K.. AND SUBRAHMANVAm. D. (1978

  17. Cellular automata model for human articular chondrocytes migration, proliferation and cell death: An in vitro validation.

    Science.gov (United States)

    Vaca-González, J J; Gutiérrez, M L; Guevara, J M; Garzón-Alvarado, D A

    2017-01-01

    Articular cartilage is characterized by low cell density of only one cell type, chondrocytes, and has limited self-healing properties. When articular cartilage is affected by traumatic injuries, a therapeutic strategy such as autologous chondrocyte implantation is usually proposed for its treatment. This approach requires in vitro chondrocyte expansion to yield high cell number for cell transplantation. To improve the efficiency of this procedure, it is necessary to assess cell dynamics such as migration, proliferation and cell death during culture. Computational models such as cellular automata can be used to simulate cell dynamics in order to enhance the result of cell culture procedures. This methodology has been implemented for several cell types; however, an experimental validation is required for each one. For this reason, in this research a cellular automata model, based on random-walk theory, was devised in order to predict articular chondrocyte behavior in monolayer culture during cell expansion. Results demonstrated that the cellular automata model corresponded to cell dynamics and computed-accurate quantitative results. Moreover, it was possible to observe that cell dynamics depend on weighted probabilities derived from experimental data and cell behavior varies according to the cell culture period. Thus, depending on whether cells were just seeded or proliferated exponentially, culture time probabilities differed in percentages in the CA model. Furthermore, in the experimental assessment a decreased chondrocyte proliferation was observed along with increased passage number. This approach is expected to having other uses as in enhancing articular cartilage therapies based on tissue engineering and regenerative medicine.

  18. Ultrastructural analysis of different human mesenchymal stem cells after in vitro expansion: a technical review

    Directory of Open Access Journals (Sweden)

    M. Miko

    2015-10-01

    Full Text Available Transmission electron microscopy reveals ultrastructural details of cells, and it is a valuable method for studying cell organelles. That is why we used this method for detailed morphological description of different adult tissuederived stem cells, focusing on the morphological signs of their functions (proteosynthetic activity, exchange with external environment, etc. and their comparison. Preparing a specimen from the cell culture suitable for transmission electron microscopy is, however, much more challenging than routine tissue processing for normal histological examination. There are several issues that need to be solved while working with cell pellets instead of solid tissue. Here we describe a simple protocol for the isolation and culture of mesenchymal stem cells from different adult tissues, with applications to stem cell biology and regenerative medicine. Since we are working with population of cells that was obtained after many days of passaging, very efficient and gentle procedures are highly necessary. We demonstrated that our semi-conservative approach regarding to histological techniques and processing of cells for transmission electron microscopy is a well reproducible procedure which results in quality pictures and images of cell populations with minimum distortions and artifacts. We also commented about riskiest steps and histochemical issues (e.g., precise pH, temperature while preparing the specimen. We bring full and detailed procedures of fixation, post-fixation, infiltration, embedding, polymerization and contrasting of cell obtained from in vitro cell and tissue cultures, with modifications according to our experience. All this steps are essential for us to know more about adult stem cells derived from different sources or about other random cell populations. The knowledge about detailed ultra-structure of adult stem cells cultured in vitro are also essential for their using in regenerative medicine and tissue engineering.

  19. Ultrastructural Analysis of Different Human Mesenchymal Stem Cells After in Vitro Expansion: A Technical Review

    Science.gov (United States)

    Danišovič, Ľ.; Majidi, A.; Varga, I.

    2015-01-01

    Transmission electron microscopy reveals ultrastructural details of cells, and it is a valuable method for studying cell organelles. That is why we used this method for detailed morphological description of different adult tissue-derived stem cells, focusing on the morphological signs of their functions (proteosynthetic activity, exchange with external environment, etc.) and their comparison. Preparing a specimen from the cell culture suitable for transmission electron microscopy is, however, much more challenging than routine tissue processing for normal histological examination. There are several issues that need to be solved while working with cell pellets instead of solid tissue. Here we describe a simple protocol for the isolation and culture of mesenchymal stem cells from different adult tissues, with applications to stem cell biology and regenerative medicine. Since we are working with population of cells that was obtained after many days of passaging, very efficient and gentle procedures are highly necessary. We demonstrated that our semi-conservative approach regarding to histological techniques and processing of cells for transmission electron microscopy is a well reproducible procedure which results in quality pictures and images of cell populations with minimum distortions and artifacts. We also commented about riskiest steps and histochemical issues (e.g., precise pH, temperature) while preparing the specimen. We bring full and detailed procedures of fixation, post-fixation, infiltration, embedding, polymerization and contrasting of cell obtained from in vitro cell and tissue cultures, with modifications according to our experience. All this steps are essential for us to know more about adult stem cells derived from different sources or about other random cell populations. The knowledge about detailed ultra-structure of adult stem cells cultured in vitro are also essential for their using in regenerative medicine and tissue engineering. PMID:26708176

  20. Non-multipotent stroma inhibit the proliferation and differentiation of mesenchymal stromal cells in vitro.

    Science.gov (United States)

    Rosu-Myles, Michael; Fair, Joel; Pearce, Nelson; Mehic, Jelica

    2010-10-01

    The ability to expand and maintain bone marrow (BM)-derived mesenchymal stem cells (MSC) in vitro is an important aspect of their therapeutic potential. Despite this, the exact composition of stromal cell types within these cultures and the potential effects of non-stem cells on the maintenance of MSC are poorly understood. C57BL/6J BM stroma was investigated as a model to determine the relationship between MSC and non-multipotent cells in vitro. Whole BM and single-cell derived cultures were characterized using flow cytometry and cell sorting combined with multipotent differentiation. Proliferation of individual stromal populations was evaluated using BrdU. At a single-cell level, MSC were distinguished from committed progenitors, and cells lacking differentiation ability, by the expression of CD105 (CD105+). A 3-fold reduction in the percentage of CD105+ cells was detected after prolonged culture and correlated with loss of MSC. Depletion of CD105+ cells coincided with a 10-20% increase in the frequency of proliferating CD105(-) cells. Removal of CD105(-) stroma caused increased proliferation in CD105+ cells, which could be diminished by conditioned media from parent cultures. Comparison of the multipotent differentiation potential in purified and non-purified CD105+ cells determined that MSC were detectable for at least 3 weeks longer when cultured in the absence of CD105(-) cells. This work identifies a simple model for characterizing the different cellular components present in BM stromal cultures and demonstrates that stromal cells lacking multipotent differentiating capacity greatly reduce the longevity of MSC.

  1. Fibroblasts derived from human pluripotent stem cells activate angiogenic responses in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Yulia Shamis

    Full Text Available Human embryonic and induced pluripotent stem cells (hESC/hiPSC are promising cell sources for the derivation of large numbers of specific cell types for tissue engineering and cell therapy applications. We have describe a directed differentiation protocol that generates fibroblasts from both hESC and hiPSC (EDK/iPDK that support the repair and regeneration of epithelial tissue in engineered, 3D skin equivalents. In the current study, we analyzed the secretory profiles of EDK and iPDK cells to investigate the production of factors that activate and promote angiogenesis. Analysis of in vitro secretion profiles from EDK and iPDK cells demonstrated the elevated secretion of pro-angiogenic soluble mediators, including VEGF, HGF, IL-8, PDGF-AA, and Ang-1, that stimulated endothelial cell sprouting in a 3D model of angiogenesis in vitro. Phenotypic analysis of EDK and iPDK cells during the course of differentiation from hESCs and iPSCs revealed that both cell types progressively acquired pericyte lineage markers NG2, PDGFRβ, CD105, and CD73 and demonstrated transient induction of pericyte progenitor markers CD31, CD34, and Flk1/VEGFR2. Furthermore, when co-cultured with endothelial cells in 3D fibrin-based constructs, EDK and iPDK cells promoted self-assembly of vascular networks and vascular basement membrane deposition. Finally, transplantation of EDK cells into mice with hindlimb ischemia significantly reduced tissue necrosis and improved blood perfusion, demonstrating the potential of these cells to stimulate angiogenic responses in vivo. These findings demonstrate that stable populations of pericyte-like angiogenic cells can be generated with high efficiency from hESC and hiPSC using a directed differentiation approach. This provides new cell sources and opportunities for vascular tissue engineering and for the development of novel strategies in regenerative medicine.

  2. Paramagnetic particles carried by cell-penetrating peptide tracking of bone marrow mesenchymal stem cells, a research in vitro

    International Nuclear Information System (INIS)

    Liu Min; Guo Youmin; Wu Qifei; Yang Junle; Wang Peng; Wang Sicen; Guo Xiaojuan; Qiang Yongqian; Duan Xiaoyi

    2006-01-01

    The ability to track the distribution and differentiation of stem cells by high-resolution imaging techniques would have significant clinical and research implications. In this study, a model cell-penetrating peptide was used to carry gadolinium particles for magnetic resonance imaging of the mesenchymal stem cells. The mesenchymal stem cells were isolated from rat bone marrow by Percoll and identified by osteogenic differentiation in vitro. The cell-penetrating peptides labeled with fluorescein-5-isothiocyanate and gadolinium were synthesized by a solid-phase peptide synthesis method and the relaxivity of cell-penetrating peptide-gadolinium paramagnetic conjugate on 400 MHz nuclear magnetic resonance was 5.7311 ± 0.0122 mmol -1 s -1 , higher than that of diethylenetriamine pentaacetic acid gadolinium (p < 0.05). Fluorescein imaging confirmed that this new peptide could internalize into the cytoplasm and nucleus. Gadolinium was efficiently internalized into mesenchymal stem cells by the peptide in a time- or concentration-dependent fashion, resulting in intercellular T1 relaxation enhancement, which was obviously detected by 1.5 T magnetic resonance imaging. Cytotoxicity assay and flow cytometric analysis showed the intercellular contrast medium incorporation did not affect cell viability and membrane potential gradient. The research in vitro suggests that the newly constructed peptides could be a vector for tracking mesenchymal stem cells

  3. Examination of tetrachlorosalicylanilide (TCSA) photoallergy using in vitro photohapten-modified Langerhans cell-enriched epidermal cells

    International Nuclear Information System (INIS)

    Gerberick, G.F.; Ryan, C.A.; Von Bargen, E.C.; Stuard, S.B.; Ridder, G.M.

    1991-01-01

    Lymphocytes from BALB/c mice photosensitized in vivo to tetrachlorosalicylanilide (TCSA) were investigated to determine whether they could be stimulated to proliferate when cultured with Langerhans cell-enriched cultured epidermal cells (LC-EC) photohapten-modified in vitro with TCSA + UVA radiation. Cultured LC-EC were photohapten-modified in vitro by irradiation in TCSA-containing medium using a 1000-watt solar simulator equipped with filters to deliver primarily UVA radiation (320-400 nm). Lymphocytes from TCSA-photosensitized mice were incubated with LC-EC that had been treated in vitro with 0.1 mM TCSA and 2 J/cm2 UVA radiation (TCSA + UVA). Responder lymphocytes demonstrated a significant increase in their blastogenesis response compared to lymphocytes that were incubated with LC-EC irradiated with UVA prior to treatment with TCSA (UVA/TCSA) or with LC-EC that had received no treatment. Lymphocytes from naive mice or mice photosensitized with musk ambrette (MA) demonstrated a significantly lower response to LC-EC modified with TCSA + UVA, indicating the specificity of the response. Maximum blastogenesis response was achieved when LC-EC were treated with 0.1 mM TCSA and a UVA radiation dose of at least 0.5 J/cm2. Epidermal cells depleted of LC by treatment with anti-Ia antibody plus complement or by an adherence procedure were unable to stimulate this blastogenesis response. Epidermal cells treated in vitro with TCSA + UVA demonstrated enhanced fluorescence compared to control cells. The fluorescence observed was not restricted to any specific epidermal cell type; however, fluorescence microscopy studies revealed that dendritic Ia-positive cells, presumably LC, were also TCSA fluorescent

  4. Dual effects exerted in vitro by micromolar concentrations of deoxynivalenol on undifferentiated caco-2 cells.

    Science.gov (United States)

    Manda, Gina; Mocanu, Mihaela Andreea; Marin, Daniela Eliza; Taranu, Ionelia

    2015-02-16

    Contamination of crops used for food and feed production with Fusarium mycotoxins, such as deoxynivalenol (DON), raise important health and economic issues all along the food chain. Acute exposure to high DON concentrations can alter the intestinal barrier, while chronic exposure to lower doses may exert more subtle effects on signal transduction pathways, leading to disturbances in cellular homeostasis. Using real-time cellular impedance measurements, we studied the effects exerted in vitro by low concentrations of DON (0.37-1.50 μM), relevant for mycotoxin-contaminated food, on the proliferation of undifferentiated Caco-2 cells presenting a tumorigenic phenotype. A 1.5 μM concentration of DON maintained cell adherence of non-proliferating Caco-2 cells, whilst arresting the growth of actively proliferating cells compared with control Caco-2 cells in vitro. At 0.37 μM, DON enhanced Caco-2 cell metabolism, thereby triggering a moderate increase in cell proliferation. The results of the current study suggested that low concentrations of DON commonly detected in food may either limit or sustain the proliferation of colon cancer cells, depending on their proliferation status and on DON concentration. Soluble factors released by Lactobacillus strains can partially counteract the inhibitory action of DON on actively proliferating colon cancer cells. The study also emphasized that real-time cellular impedance measurements were a valuable tool for investigating the dynamics of cellular responses to xenobiotics.

  5. Dual Effects Exerted in Vitro by Micromolar Concentrations of Deoxynivalenol on Undifferentiated Caco-2 Cells

    Directory of Open Access Journals (Sweden)

    Gina Manda

    2015-02-01

    Full Text Available Contamination of crops used for food and feed production with Fusarium mycotoxins, such as deoxynivalenol (DON, raise important health and economic issues all along the food chain. Acute exposure to high DON concentrations can alter the intestinal barrier, while chronic exposure to lower doses may exert more subtle effects on signal transduction pathways, leading to disturbances in cellular homeostasis. Using real-time cellular impedance measurements, we studied the effects exerted in vitro by low concentrations of DON (0.37–1.50 μM, relevant for mycotoxin-contaminated food, on the proliferation of undifferentiated Caco-2 cells presenting a tumorigenic phenotype. A 1.5 μM concentration of DON maintained cell adherence of non-proliferating Caco-2 cells, whilst arresting the growth of actively proliferating cells compared with control Caco-2 cells in vitro. At 0.37 μM, DON enhanced Caco-2 cell metabolism, thereby triggering a moderate increase in cell proliferation. The results of the current study suggested that low concentrations of DON commonly detected in food may either limit or sustain the proliferation of colon cancer cells, depending on their proliferation status and on DON concentration. Soluble factors released by Lactobacillus strains can partially counteract the inhibitory action of DON on actively proliferating colon cancer cells. The study also emphasized that real-time cellular impedance measurements were a valuable tool for investigating the dynamics of cellular responses to xenobiotics.

  6. 5-Aminolevulinic acid-mediated sonosensitization of rat RG2 glioma cells in vitro

    Directory of Open Access Journals (Sweden)

    Krzysztof Bilmin

    2016-10-01

    Full Text Available Sonodynamic therapy (SDT is a promising technique based on the ability of certain substances, called sonosensitizers, to sensitize cancer cells to non-thermal effects of low-energy ultrasound waves, allowing their destruction. Sonosensitization is thought to induce cell death by direct physical effects such as cavitation and acoustical streaming as well as by complementary chemical reactions generating oxygen free radicals. One of the promising sonosensitizers is 5-aminolevulinic acid (ALA which upon selective uptake by cancer cells is metabolized and accumulated as protoporphyrin IX. The objective of the study was to describe ALA-mediated sonodynamic effects in vitro on a rat RG2 glioma cell line. Glioma cells, seeded at the bottom of 96-well plates and incubated with ALA (10 µg/ml for 6 h, were exposed to the sinusoidal US pulses with a resonance frequency of 1 MHz, 1000 µs duration, 0.4 duty-cycle, and average acoustic power varying from 2 W to 6 W. Ultrasound waves were generated by a flat circular piezoelectric transducer with a diameter of 25 mm. Cell viability was determined by MTT assay. Structural cellular changes were visualized with a fluorescence microscope. Signs of cytotoxicity such as a decrease in cell viability, chromatin condensation and apoptosis were found. ALA-mediated SDT evokes cytotoxic effects of low intensity US on rat RG2 glioma cells in vitro . This cell line is indicated for further preclinical assessment of SDT in in vivo conditions.

  7. In vitro extracellular matrix model to evaluate stroma cell response to transvaginal mesh.

    Science.gov (United States)

    Wu, Ming-Ping; Huang, Kuan-Hui; Long, Cheng-Yu; Yang, Chau-Chen; Tong, Yat-Ching

    2014-04-01

    The use of surgical mesh for female pelvic floor reconstruction has increased in recent years. However, there is paucity of information about the biological responses of host stroma cells to different meshes. This study was aimed to establish an in vitro experimental model to study the micro-environment of extracellular matrix (ECM) with embedded mesh and the stroma cell behaviors to different synthetic meshes. Matrigel multi-cellular co-culture system with embedded mesh was used to evaluate the interaction of stroma cells and synthetic mesh in a simulated ECM environment. Human umbilical vein endothelial cells (HUVEC) and NIH3T3 fibroblasts were inoculated in the system. The established multi-cellular Matrigel co-culture system was used to detect stroma cell recruitment and tube formation ability for different synthetic meshes. HUVEC and NIH3T3 cells were recruited into the mesh interstices and organized into tube-like structures in type I mesh material from Perigee, Marlex and Prolift 24 hr after cell inoculation. On the contrary, there was little recruitment of HUVEC and NIH3T3 cells into the type III mesh of intra-vaginal sling (IVS). The Matrigel multi-cellular co-culture system with embedded mesh offers a useful in vitro model to study the biological behaviors of stroma cells in response to different types of synthetic meshes. The system can help to select ideal mesh candidates before actual implantation into the human body. © 2013 Wiley Periodicals, Inc.

  8. Repetitive cryotherapy attenuates the in vitro and in vivo mononuclear cell activation response.

    Science.gov (United States)

    Lindsay, Angus; Othman, Mohd Izani; Prebble, Hannah; Davies, Sian; Gieseg, Steven P

    2016-07-01

    What is the central question of this study? Acute and repetitive cryotherapy are routinely used to accelerate postexercise recovery, although the effect on resident immune cells and repetitive exposure has largely been unexplored and neglected. What is the main finding and its importance? Using blood-derived mononuclear cells and semi-professional mixed martial artists, we show that acute and repetitive cryotherapy reduces the in vitro and in vivo T-cell and monocyte activation response whilst remaining independent of the physical performance of elite athletes. We investigated the effect of repetitive cryotherapy on the in vitro (cold exposure) and in vivo (cold water immersion) activation of blood-derived mononuclear cells following high-intensity exercise. Single and repeated cold exposure (5°C) of a mixed cell culture (T cells and monocytes) was investigated using in vitro tissue culture experimentation for total neopterin production (neopterin plus 7,8-dihydroneopterin). Fourteen elite mixed martial art fighters were also randomly assigned to either a cold water immersion (15 min at 10°C) or passive recovery protocol, which they completed three times per week during a 6 week training camp. Urine was collected and analysed for neopterin and total neopterin three times per week, and perceived soreness, fatigue, physical performance (broad jump, push-ups and pull-ups) and training performance were also assessed. Single and repetitive cold exposure significantly (P cryotherapy attenuates in vitro T-cell and monocyte activation. This may explain the disparity in in vivo neopterin and total neopterin between cold water immersion and passive recovery following repetitive exposure during a high-intensity physical impact sport that remains independent of physical performance. © 2016 The Authors. Experimental Physiology © 2016 The Physiological Society.

  9. Sodium hyaluronate enhances colorectal tumour cell metastatic potential in vitro and in vivo.

    LENUS (Irish Health Repository)

    Tan, B

    2012-02-03

    BACKGROUND: Sodium hyaluronate has been used intraperitoneally to prevent postoperative adhesions. However, the effect of sodium hyaluronate on tumour growth and metastasis in vitro and in vivo is still unknown. METHODS: Human colorectal tumour cell lines SW480, SW620 and SW707 were treated with sodium hyaluronate (10-500 microg\\/ml) and carboxymethylcellulose (0.125-1 per cent), and tumour cell proliferation and motility were determined in vitro. For the in vivo experiments male BD IX rats were randomized to a sodium hyaluronate group (n = 11; intraperitoneal administration of 0.5 x 10(6) DHD\\/K12 tumour cells and 5 ml 0.4 per cent sodium hyaluronate) or a phosphate-buffered saline group (n = 11; 0.5 x 10(6) DHD\\/K12 tumour cells and 5 ml phosphate-buffered saline intraperitoneally). Four weeks later the intraperitoneal tumour load was visualized directly. RESULTS: In vitro sodium hyaluronate increased tumour cell proliferation and motility significantly. Sodium hyaluronate-induced tumour cell motility appeared to be CD44 receptor dependent, whereas sodium hyaluronate-induced tumour cell proliferation was CD44 receptor independent. In vivo there was a significantly higher total tumour nodule count in the peritoneal cavity of the sodium hyaluronate-treated group compared with the control (P = 0.016). CONCLUSION: Sodium hyaluronate enhances tumour metastatic potential in vitro and in vivo, which suggests that use of sodium hyaluronate to prevent adhesions in colorectal cancer surgery may also potentiate intraperitoneal tumour growth. Presented to the Patey Prize Session of the Surgical Research Society and the annual scientific meeting of the Association of Surgeons of Great Britain and Ireland, Brighton, UK, 4-7 May 1999

  10. Release of an endogenous pyrogen in vitro from rabbit mononuclear cells.

    Science.gov (United States)

    Atkins, E; Bodel, P; Francis, L

    1967-08-01

    The capacity of rabbit mononuclear cells to release an endogenous pyrogen (EP) in vitro has been studied. After incubation with tuberculin, preparations of predominantly monocytic cells, derived from the respiratory passages of the lungs of rabbits sensitized with BCG, were activated to release EP. Pyrogen production occurred more slowly with lung monocytes than with blood leukocytes of similarly sensitized rabbits and 9 to 10 hr incubation in a fully supportive medium was required to produce clear-cut results. As previously reported with blood leukocytes, mononuclear cells from the lungs of normal animals were also activated by tuberculin but to a lesser degree than were those from specifically sensitized rabbits. Under a variety of conditions, mononuclear cells from either spleen or lymph nodes of the same sensitized rabbits failed to release detectable amounts of pyrogen when incubated with tuberculin in vitro but were activated in a majority of instances when phagocytosis of heat-killed staphylococci was used as the stimulus. Release of pyrogen from lung monocytes appears to be an active process that is both temperature-dependent and requires protein synthesis. Neither serum antibody nor complement appears to play a role in this process. Evidence is presented that the granulocyte is the main source of pyrogen evolved by blood leukocytes incubated in vitro with OT or heat-killed staphylococci, whereas the lung macrophage and/or monocyte is responsible for most of the pyrogen released from the lung cell preparations. From these studies, it is concluded that mononuclear cells can be activated in vitro by several microbial stimuli and must be considered an additional cellular source of EP. The clinical implications of these findings for the pathogenesis of fever in granulomatous diseases where the monocyte is the predominant cell are discussed.

  11. Effects of sciatic-conditioned medium on neonatal rat retinal cells in vitro

    Directory of Open Access Journals (Sweden)

    Torres P.M.M.

    1998-01-01

    Full Text Available Schwann cells produce and release trophic factors that induce the regeneration and survival of neurons following lesions in the peripheral nerves. In the present study we examined the in vitro ability of developing rat retinal cells to respond to factors released from fragments of sciatic nerve. Treatment of neonatal rat retinal cells with sciatic-conditioned medium (SCM for 48 h induced an increase of 92.5 ± 8.8% (N = 7 for each group in the amount of total protein. SCM increased cell adhesion, neuronal survival and glial cell proliferation as evaluated by morphological criteria. This effect was completely blocked by 2.5 µM chelerythrine chloride, an inhibitor of protein kinase C (PKC. These data indicate that PKC activation is involved in the effect of SCM on retinal cells and demonstrate that fragments of sciatic nerve release trophic factors having a remarkable effect on neonatal rat retinal cells in culture.

  12. In vitro interactions of lymphocytes and cultured cells from beagles with plutonium-induced bone tumors

    International Nuclear Information System (INIS)

    Frazier, M.E.; Lund, J.E.; Busch, R.H.

    1976-01-01

    Cell cultures have been prepared from lung and bone tumors arising in beagle dogs following exposure to inhaled plutonium. Evaluation of the cultured cells by commonly applied criteria (i.e., cell morphology, lack of contact inhibitory mechanisms, cloning efficiency, growth in soft agar, and tumor production in vivo) indicated that tumor cells were being grown in culture. Blood leukocytes and peripheral lymphocytes from beagle dogs were tested for cytotoxic effects against several cell cultures. Lymphocytes from normal dogs or dogs with unrelated tumors would not kill the bone tumor cells unless monocytes (macrophage) were present, in which case the leukocyte preparation was capable of mounting de novo cytotoxic immune reactions after 3 to 5 days in culture. In contrast, the dogs with plutonium-induced bone tumors had circulating lymphocytes that appeared to have undergone presensitization to bone-tumor-distinctive antigens in vivo. Consequently these lymphocytes interacted with cultured cells promptly after encounter in vitro

  13. Combined action of radiation, salts of copper and nickel on cell viability in vitro

    Directory of Open Access Journals (Sweden)

    D. D. Gapeenko

    2014-09-01

    Full Text Available Experimental study of the combined action of heavy metals and ionizing radiation on the viability of cells in culture was made. We established a significant toxic effect of copper and nickel in the proliferative and mitotic activity of cells in vitro. Under the combined effects of radiation and copper ions on cells we observed the mor-phological changes in morphologically-functional properties of cells that were determined by or radiation dose or by concentration of copper ions. While incubation of irradiated cells with nickel ions we observed sensitiza-tion of cells by nickel ions under the irradiation dose of 0.5 and 5.0 Gy, and the resistance of cells to exposure to sublethal dose of 10.0 Gy.

  14. In vitro stimulation of rabbit T lymphocytes by cells expressing herpes simplex antigens.

    Science.gov (United States)

    Kapoor, A K; Ling, N R; Nash, A A; Bachan, A; Wildy, P

    1982-04-01

    Lymphocyte stimulation responses to herpes antigens were studied using virus-infected X-irradiated cells. Rabbits were immunized with herpes simplex virus type 1 (strain HFEM) grown in RK 13 cells. For in vitro stimulation assay BHK21 cells were X-irradiated (15 000 rad) and infected with a high m.o.i. of a temperature-sensitive (ts) mutant (N102) of HFEM strain at the non-permissive temperature (38.5 degrees C) of virus. Virus antigens were expressed on the infected cells and there was no leakage of infectious virus into the medium at 38.5 degrees C. T lymphocytes from rabbits immunized with herpes simplex virus were specifically activated by herpesvirus-infected X-irradiated cells; lymph node cells from rabbits immunized with RK13 cells and from non-immune rabbits showed no proliferative response.

  15. In vitro study of stem cell communication via gap junctions for fibrocartilage regeneration at entheses.

    Science.gov (United States)

    Nayak, Bibhukalyan Prasad; Goh, James Cho Hong; Toh, Siew Lok; Satpathy, Gyan Ranjan

    2010-03-01

    Entheses are fibrocartilaginous organs that bridge ligament with bone at their interface and add significant insertional strength. To replace a severely damaged ligament, a tissue-engineered graft preinstalled with interfacial fibrocartilage, which is being regenerated from stem cells, appears to be more promising than ligament-alone graft. Such a concept can be realized by a biomimetic approach of establishing a dynamic communication of stem cells with bone cells and/or ligament fibroblasts in vitro. The current study has two objectives. The first objective is to demonstrate functional coculture of bone marrow-derived stem cells (BMSCs) with mature bone cells/ligament fibroblasts as evidenced by gap-junctional communication in vitro. The second objective is to investigate the role of BMSCs in the regeneration of fibrocartilage within the coculture. Rabbit bone/ligament fibroblasts were dual-stained with DiI-Red and calcein (gap-junction permeable dye), and cocultured with unlabeled BMSCs at fixed ratio (1:10). The functional gap junction was demonstrated by the transfer of calcein from donor to recipient cells that was confirmed and quantified by flow cytometry. Type 2 collagen (cartilage extracellular matrix-specific protein) expressed by the mixed cell lines in the cocultures were estimated by real-time reverse transcription PCR and compared with that of the ligament-bone coculture (control). Significant transfer of calcein into BMSCs was observed and flow cytometry analyses showed a gradual increase in the percentage of BMSCs acquiring calcein with time. Cocultures that included BMSCs expressed significantly more type 2 collagen compared with the control. The current study, for the first time, reported the expression of gap-junctional communication of BMSCs with two adherent cell lines of musculoskeletal system in vitro and also confirmed that incorporation of stem cells augments fibrocartilage regeneration. The results open up a path to envisage a composite

  16. In vitro toxicity of polycyclic aromatic hydrocarbons and halogenated aromatic hydrocarbons to cetacean cells and tissues

    Energy Technology Data Exchange (ETDEWEB)

    Carvan, M.J. III.

    1993-01-01

    Cetaceans bioaccumulate high aromatic hydrocarbon tissue residues, and elevated levels of PCB residues in tissues are proposed to have occurred concurrently with recent epizootic deaths of dolphins. The objectives of this study were: (1) to develop and characterize an epithelial cell line derived from dolphin tissues, (2) to investigate the effects of hydrocarbon pollutants on those cells, and (3) to analyze the toxicity of hydrocarbon pollutants on cetacean tissues in vitro. An epithelial cell line, Carvan dolphin kidney (CDK), isolated from a spontaneously aborted female bottlenose dolphin, Tursiops truncatus, grew rapidly. These cells were neither transformed nor immortal. Velocity sedimentation analysis showed CDK cells contained nuclear aryl hydrocarbon receptor, suggestive of cytochrome P450 inducibility. BaP inhibited mitosis in CDK cells in a dose-dependent manner. Data indicate that CDK cells metabolize BaP, that BaP metabolites bind to cellular DNA initiating unscheduled DNA synthesis, and that the inhibition of cytochrome P450 metabolism decrease the BaP-associated inhibition of mitosis in dolphin cells. The data also suggest that TCDD acts synergistically to increase the levels of DNA damage by the procarcinogen BaP. Cetacean liver microsomes was isolated and evaluated for the presence of cytochrome P450 proteins by SDS-PAGE, apparent minimum molecular weight determination, and immunoblot analysis. P450 activity was induced in cetacean tissue samples and CDK cells by exposure in vitro to one of several cytochrome P450-inducing chemicals. The data suggest that cetacean tissues and cells can be utilized to study the in vitro induction of cytochrome P450, resultant metabolism of xenobiotic contaminants, and the subsequent cellular and molecular responses. However, the identity of specific P450 isozymes involved in this process will remain undetermined until monoclonal antibodies that recognize cetacean P450s can be generated.

  17. Brassinosteroids inhibit in vitro angiogenesis in human endothelial cells

    Czech Academy of Sciences Publication Activity Database

    Rárová, L.; Zahler, S.; Liebl, J.; Kryštof, Vladimír; Sedlák, David; Bartůněk, Petr; Kohout, Ladislav; Strnad, Miroslav

    2012-01-01

    Roč. 77, č. 13 (2012), s. 1502-1509 ISSN 0039-128X R&D Projects: GA MŠk(CZ) LC06077 Grant - others:GA MŠk(CZ) ED0007/01/01 Program:ED Institutional research plan: CEZ:AV0Z50380511; CEZ:AV0Z50520514; CEZ:AV0Z40550506 Keywords : Angiogenesis * Human umbilical vein endothelial cells * Migration Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.803, year: 2012

  18. Automated autoradiographic analysis of tumor cell colonies in vitro

    International Nuclear Information System (INIS)

    Kallman, R.F.

    1984-01-01

    The studies reported are basic to the design of therapeutic strategies against cancer and to the understanding of therapeutic effects. It is unlikely that clinical therapy could employ the same methodology, however, so this approach could not be incorporated into 'kinetics-directe' therapy. As defined and implemented successfully by Barranco et al. therapy is kinetics-directed when the kinetic properties of cell populations under treatment are actually determined and used to schedule the timing of multiple treatments. Thus, the substance of this paper is of potential importance to kinetic-based cancer therapy, i.e. using historical or empirical data to formulate models. (orig./AJ)

  19. Chemical Carcinogen (Hydrazine et al.) Induced Carcinogenesis of Human Diploid Cells in Vitro

    Science.gov (United States)

    1982-09-07

    untreated cell popula- pared from the stock solutions in complete growth tions were seeded onto CES in vitro. The CES medium (CIM). Thee solutions were added...time from induction pared to metastases IFig. IBl. We want to imply to neoplasia of 6 to 10 wk instead of 1 to 1.5 yr. that these chemical carcinogen...This definition was used because the pathologist was ds:b celular invasion Into CE,, an organ culture, In vitro. Thw two Interpretations am not

  20. Long-term in vitro, cell-type-specific genome-wide reprogramming of gene expression

    International Nuclear Information System (INIS)

    Hakelien, Anne-Mari; Gaustad, Kristine G.; Taranger, Christel K.; Skalhegg, Bjorn S.; Kuentziger, Thomas; Collas, Philippe

    2005-01-01

    We demonstrate a cell extract-based, genome-wide and heritable reprogramming of gene expression in vitro. Kidney epithelial 293T cells have previously been shown to take on T cell properties following a brief treatment with an extract of Jurkat T cells. We show here that 293T cells exposed for 1 h to a Jurkat cell extract undergo genome-wide, target cell-type-specific and long-lasting transcriptional changes. Microarray analyses indicate that on any given week after extract treatment, ∼2500 genes are upregulated >3-fold, of which ∼900 are also expressed in Jurkat cells. Concomitantly, ∼1500 genes are downregulated or repressed, of which ∼500 are also downregulated in Jurkat cells. Gene expression changes persist for over 30 passages (∼80 population doublings) in culture. Target cell-type specificity of these changes is shown by the lack of activation or repression of Jurkat-specific genes by extracts of 293T cells or carcinoma cells. Quantitative RT-PCR analysis confirms the long-term transcriptional activation of genes involved in key T cell functions. Additionally, growth of cells in suspended aggregates, expression of CD3 and CD28 T cell surface markers, and interleukin-2 secretion by 293T cells treated with extract of adult peripheral blood T cells illustrate a functional nuclear reprogramming. Therefore, target cell-type-specific and heritable changes in gene expression, and alterations in cell function, can be promoted by extracts derived from transformed cells as well as from adult primary cells

  1. Effect of oviduct epithelial cells on the fertilization and development of sheep oocytes in vitro

    DEFF Research Database (Denmark)

    Holm, Peter; Irvine, Brendon J.; Armstrong, David T.

    1994-01-01

    The study examined whether co-culture with oviductal epithelial cells was of benefit to ovine in vitro fertilization ( IVF) and embryo culture procedures utilizing ·a well charac- terized culture system based on a synthetic oviductal fluid medium (SOFM) supple- mented with serum in a 90% N2, 5% 0 2......, 5% C02, atmosphere at 38.6°C. Two experiments were carried out. In Experiment 1, comparison was made between the frequency of fertil- ization and development of in vitro matured ( IVM) oocytes cultured in the absence (Group 1) or presence of oviductal cells for a 24 h (Group 2), 48 h (Group 3) or 96...... h (Group 4) period post insemination. In Experiment 2, comparison was made between the develop- ment of IVM oocytes fertilized and cultured in vitro for 7. 5 days in the absence or presence of oviductal cells with IVM oocytes which had been fertilized in vitro for 20 h in the pres- ence of oviductal...

  2. Juglans mandshurica Maxim extracts exhibit antitumor activity on HeLa cells in vitro.

    Science.gov (United States)

    Xin, Nian; Hasan, Murtaza; Li, Wei; Li, Yan

    2014-04-01

    The present study examined the potential application of Juglans mandshurica Maxim extracts (HT) for cancer therapy by assessing their anti‑proliferative activity, reduction of telomerase activity, induction of apoptosis and cell cycle arrest in S phase in HeLa cells. From the perspective of using HT as a herbal medicine, photomicroscopy and florescent microscopy techniques were utilized to characterize the effect of the extracts on telomerase activity and cell morphology. Flow cytometry was employed to study apoptosis and cell cycle of HeLa cells, and DNA laddering was performed. The results showed that HT inhibited cell proliferation and telomerase activity, induced apoptosis and caused S phase arrest of HeLa cells in vitro. HT inhibited HeLa cell proliferation significantly, and the highest inhibition rate was 83.7%. A trap‑silver staining assay showed that HT was capable of markedly decreasing telomerase activity of HeLa cells and this inhibition was enhanced in a time‑ and dose‑dependent manner. Results of a Hoechst 33258 staining assay showed that HeLa cells treated by HT induced cell death. Through DNA agarose gel electrophoresis, DNA ladders of HeLa cells treated with HT were observed, indicating apoptosis. In conclusion, the present study demonstrated that HT exhibited anti‑tumor effects comprising the inhibition of growth and telomerase activity as well as apoptosis and cell cycle arrest in HeLa cells.

  3. Studies of rhodamine-123: effect on rat prostate cancer and human prostate cancer cells in vitro.

    Science.gov (United States)

    Arcadi, J A; Narayan, K S; Techy, G; Ng, C P; Saroufeem, R M; Jones, L W

    1995-06-01

    The effect of the lipophilic, cationic dye, Rhodamine-123 (Rh-123), on prostate cancer in rats, and on three tumor cell lines in vitro is reported here. The general toxicity of Rh-123 in mice has been found to be minimal. Lobund-Wistar (L-W) rats with the autochthonous prostate cancer of Pollard were treated for six doses with Rh-123 at a dose of 15 mg/kg subcutaneously every other day. Microscopic examination of the tumors revealed cellular and acinar destruction. The effectiveness of Rh-123 as a cytotoxic agent was tested by clonogenic and viability assays in vitro with three human prostate cancer cell lines. Severe (60-95%) growth inhibition was observed following Rh-123 exposure for 2-5 days at doses as low as 1.6 micrograms/ml in all three prostate cancer cell lines.

  4. Influence of in vitro irradiation upon LIF production by ConA stimulated mononuclear cells

    International Nuclear Information System (INIS)

    Sandru, G.; Veraguth, P.

    1981-01-01

    Leukocyte migration inhibitory factor (LIF) activity of culture supernatants of in vitro irradiated Concanavalin A (ConA) stimulated lymphocytes was tested by measuring granulocyte migration from clotted plasma droplets placed in flat bottom microplates. The specificity of inhibition was assured by pretreating the assay supernatants with anti-LIF antibodies which abrogated granulocyte migration inhibition but did not impair guinea pig Peritoneal Exudate Cells (PEC) migration inhibition. In vitro irradiation (150-1200 rads) of MNC cultures either before or after ConA stimulation did not impair lymphokine production and sometimes significantly improved the supernatants' LIF activity as compared with that of unirradiated cultures. The existence of radiosensitive suppressor cells regulating LIF production by ConA stimulated mononuclear cells is suggested

  5. In vitro expansion of Lin+ and Lin− mononuclear cells from human peripheral blood

    International Nuclear Information System (INIS)

    Norhaiza, H. Siti; Zarina, Z. A. Intan; Hisham, Z. A. Shahrul; Rohaya, M. A. W.

    2013-01-01

    Haematopoietic stem cells (HSCs) are used in the therapy of blood disorders due to the ability of these cells to reconstitute haematopoietic lineage cells when transplanted into myeloablative recipients. However, substantial number of cells is required in order for the reconstitution to take place. Since HSCs present in low frequency, larger number of donor is required to accommodate the demand of transplantable HSCs. Therefore, in vitro expansion of HSCs will have profound impact on clinical purposes. The aim of this study was to expand lineage negative (Lin − ) stem cells from human peripheral blood. Total peripheral blood mononuclear cells (PBMNCs) were fractionated from human blood by density gradient centrifugation. Subsequently, PBMNCs were subjected to magnetic assisted cell sorter (MACS) which depletes lineage positive (Lin + ) mononuclear cells expressing lineage positive markers such as CD2, CD3, CD11b, CD14, CD15, CD16, CD19, CD56, CD123, and CD235a to obtained Lin − cell population. The ability of Lin + and Lin − to survive in vitro was explored by culturing both cell populations in complete medium consisting of Alpha-Minimal Essential Medium (AMEM) +10% (v/v) Newborn Calf Serum (NBCS)+ 2% (v/v) pen/strep. In another experiment, Lin + and Lin − were cultured with complete medium supplemented with 10ng/mL of the following growth factors: stem cell factor (SCF), interleukin (IL)-3, granulocyte-macrophage colony stimulating factor (GM-CSF), 2IU/mL of Erythropoietin (Epo) and 20ng/mL of IL-6. Three samples were monitored in static culture for 22 days. The expansion potential was assessed by the number of total viable cells, counted by trypan blue exclusion assay. It was found that Lin + mononuclear cells were not able to survive either in normal proliferation medium or proliferation medium supplemented with cytokines. Similarly, Lin − stem cells were not able to survive in proliferation medium however, addition of cytokines into the proliferation

  6. In vitro expansion of Lin+ and Lin- mononuclear cells from human peripheral blood

    Science.gov (United States)

    Norhaiza, H. Siti; Rohaya, M. A. W.; Zarina, Z. A. Intan; Hisham, Z. A. Shahrul

    2013-11-01

    Haematopoietic stem cells (HSCs) are used in the therapy of blood disorders due to the ability of these cells to reconstitute haematopoietic lineage cells when transplanted into myeloablative recipients. However, substantial number of cells is required in order for the reconstitution to take place. Since HSCs present in low frequency, larger number of donor is required to accommodate the demand of transplantable HSCs. Therefore, in vitro expansion of HSCs will have profound impact on clinical purposes. The aim of this study was to expand lineage negative (Lin-) stem cells from human peripheral blood. Total peripheral blood mononuclear cells (PBMNCs) were fractionated from human blood by density gradient centrifugation. Subsequently, PBMNCs were subjected to magnetic assisted cell sorter (MACS) which depletes lineage positive (Lin+) mononuclear cells expressing lineage positive markers such as CD2, CD3, CD11b, CD14, CD15, CD16, CD19, CD56, CD123, and CD235a to obtained Lin- cell population. The ability of Lin+ and Lin- to survive in vitro was explored by culturing both cell populations in complete medium consisting of Alpha-Minimal Essential Medium (AMEM) +10% (v/v) Newborn Calf Serum (NBCS)+ 2% (v/v) pen/strep. In another experiment, Lin+ and Lin- were cultured with complete medium supplemented with 10ng/mL of the following growth factors: stem cell factor (SCF), interleukin (IL)-3, granulocyte-macrophage colony stimulating factor (GM-CSF), 2IU/mL of Erythropoietin (Epo) and 20ng/mL of IL-6. Three samples were monitored in static culture for 22 days. The expansion potential was assessed by the number of total viable cells, counted by trypan blue exclusion assay. It was found that Lin+ mononuclear cells were not able to survive either in normal proliferation medium or proliferation medium supplemented with cytokines. Similarly, Lin- stem cells were not able to survive in proliferation medium however, addition of cytokines into the proliferation medium support Lin

  7. Insulin-Producing Endocrine Cells Differentiated In Vitro From Human Embryonic Stem Cells Function in Macroencapsulation Devices In Vivo.

    Science.gov (United States)

    Agulnick, Alan D; Ambruzs, Dana M; Moorman, Mark A; Bhoumik, Anindita; Cesario, Rosemary M; Payne, Janice K; Kelly, Jonathan R; Haakmeester, Carl; Srijemac, Robert; Wilson, Alistair Z; Kerr, Justin; Frazier, Mauro A; Kroon, Evert J; D'Amour, Kevin A

    2015-10-01

    The PEC-01 cell population, differentiated from human embryonic stem cells (hESCs), contains pancreatic progenitors (PPs) that, when loaded into macroencapsulation devices (to produce the VC-01 candidate product) and transplanted into mice, can mature into glucose-responsive insulin-secreting cells and other pancreatic endocrine cells involved in glucose metabolism. We modified the protocol for making PEC-01 cells such that 73%-80% of the cell population consisted of PDX1-positive (PDX1+) and NKX6.1+ PPs. The PPs were further differentiated to islet-like cells (ICs) that reproducibly contained 73%-89% endocrine cells, of which approximately 40%-50% expressed insulin. A large fraction of these insulin-positive cells were single hormone-positive and expressed the transcription factors PDX1 and NKX6.1. To preclude a significant contribution of progenitors to the in vivo function of ICs, we used a simple enrichment process to remove remaining PPs, yielding aggregates that contained 93%-98% endocrine cells and 1%-3% progenitors. Enriched ICs, when encapsulated and implanted into mice, functioned similarly to the VC-01 candidate product, demonstrating conclusively that in vitro-produced hESC-derived insulin-producing cells can mature and function in vivo in devices. A scaled version of our suspension culture was used, and the endocrine aggregates could be cryopreserved and retain functionality. Although ICs expressed multiple important β cell genes, the cells contained relatively low levels of several maturity-associated markers. Correlating with this, the time to function of ICs was similar to PEC-01 cells, indicating that ICs required cell-autonomous maturation after delivery in vivo, which would occur concurrently with graft integration into the host. Type 1 diabetes (T1D) affects approximately 1.25 million people in the U.S. alone and is deadly if not managed with insulin injections. This paper describes the production of insulin-producing cells in vitro and a new

  8. Fucoidan from Sargassum sp. and Fucus vesiculosus reduces cell viability of lung carcinoma and melanoma cells in vitro and activates natural killer cells in mice in vivo

    DEFF Research Database (Denmark)

    Ale, Marcel Tutor; Maruyama, Hiroko; Tamauchi, Hidekazu

    2011-01-01

    Fucoidan is known to exhibit crucial biological activities, including anti-tumor activity. In this study, we examined the influence of crude fucoidan extracted from Sargassum sp. (MTA) and Fucus vesiculosus (SIG) on Lewis lung carcinoma cells (LCC) and melanoma B16 cells (MC). In vitro studies we...

  9. The production of fluorescent transgenic trout to study in vitro myogenic cell differentiation

    Directory of Open Access Journals (Sweden)

    Rescan Pierre-Yves

    2010-05-01

    Full Text Available Abstract Background Fish skeletal muscle growth involves the activation of a resident myogenic stem cell population, referred to as satellite cells, that can fuse with pre-existing muscle fibers or among themselves to generate a new fiber. In order to monitor the regulation of myogenic cell differentiation and fusion by various extrinsic factors, we generated transgenic trout (Oncorhynchus mykiss carrying a construct containing the green fluorescent protein reporter gene driven by a fast myosin light chain 2 (MlC2f promoter, and cultivated genetically modified myogenic cells derived from these fish. Results In transgenic trout, green fluorescence appeared in fast muscle fibers as early as the somitogenesis stage and persisted throughout life. Using an in vitro myogenesis system we observed that satellite cells isolated from the myotomal muscle of transgenic trout expressed GFP about 5 days post-plating as they started to fuse. GFP fluorescence persisted subsequently in myosatellite cell-derived myotubes. Using this in vitro myogenesis system, we showed that the rate of muscle cell differentiation was strongly dependent on temperature, one of the most important environmental factors in the muscle growth of poikilotherms. Conclusions We produced MLC2f-gfp transgenic trout that exhibited fluorescence in their fast muscle fibers. The culture of muscle cells extracted from these trout enabled the real-time monitoring of myogenic differentiation. This in vitro myogenesis system could have numerous applications in fish physiology to evaluate the myogenic activity of circulating growth factors, to test interfering RNA and to assess the myogenic potential of fish mesenchymal stem cells. In ecotoxicology, this system could be useful to assess the impact of environmental factors and marine pollutants on fish muscle growth.

  10. Functional analysis of HPV-like particle-activated Langerhans cells in vitro.

    Science.gov (United States)

    Yan, Lisa; Woodham, Andrew W; Da Silva, Diane M; Kast, W Martin

    2015-01-01

    Langerhans cells (LCs) are antigen-presenting cells responsible for initiating an immune response against human papillomaviruses (HPVs) entering the epithelial layer in vivo as they are the first immune cell that HPV comes into contact with. LCs become activated in response to foreign antigens, which causes internal signaling resulting in the increased expression of co-stimulatory molecules and the secretion of inflammatory cytokines. Functionally activated LCs are then capable of migrating to the lymph nodes where they interact with antigen-specific T cells and initiate an adaptive T-cell response in vivo. However, HPV has evolved in a manner that suppresses LC function, and thus the induction of antigen-specific T cells is hindered. While many methods exist to monitor the activity of LCs in vitro, the migration and induction of cytotoxic T cells is ultimately indicative of a functional immune response. Here, methods in analyzing functional migration and induction of antigen-specific T cells after stimulation of LCs with HPV virus-like particles in vitro are described.

  11. Diethyldithiocarbamate enhancement of radiation and hyperthermic effects on Chinese hamster cells in vitro

    International Nuclear Information System (INIS)

    Lin, P.S.; Kwock, L.; Butterfield, C.E.

    1979-01-01

    To test the possibility of enhancing O 2 - toxicity during radiation therapy and/or hyperthermia, Chinese hamster cells (Don) were treated with the drug diethyldithiocarbamate (DDC) in vitro. This drug has been shown to inhibit, both in vitro and in vivo, the enzyme superoxide dismutase (SOD) which is responsible for eliminating the 0 2 - radical in cells. We observed that the cytocidal effect of DDC on Don cells is dependent on both DDC concentration and exposure time. After 8 to 10 days of incubation with 10 -9 M DDC, no change in cell survival was noted; however, incubation with 10 -4 M DDC produced a marked loss in colony formation. When DDC-treated cells were γ-irradiated, they did not survive as well as cells that were treated with either radiation or DDC alone. The combined effects of hyperthermia and DDC were dramatic. Cells treated 5 min at 43 0 C with 10 -4 M DDC or 10 min at 43 0 C with 10 -5 M DDC showed significant decrease in survival. These results suggest that DDC may be a potentially powerful sensitizing agent in tumor therapy, since there is increasing evidence that tumor cells have a lower level of superoxide dismutase

  12. Spontaneous pyrogen production by mouse histiocytic and myelomonocytic tumor cell lines in vitro.

    Science.gov (United States)

    Bodel, P

    1978-05-01

    Tumor-associated fever occurs commonly in acute leukemias and lymphomas. We investigated the capacity for in vitro production of pyrogen by three mouse histiocytic lymphoma cell lines (J-774, PU5-1.8, p 388 D1), one myelomonoyctic line (WEHI-3), and tow lymphoma-derived lines, RAW-8 and R-8. Pyrogen was released spontaneously into the culture medium during growth by all cell lines with macrophage or myeloid characteristics including lysozyme production; R-8 cells, of presumed B-lymphocyte origin, did not produce pyrogen. When injected into mice, the pyrogens gave fever curves typical of endogenous pyrogen, were inactived by heating to 56 degrees C and by pronase digestion, and appeared to be secreted continuously by viable cells. Two pyrogenic molecular species produced by H-774 cells were identified by Sephadex filtration, one of mol wt approximately equal to 30,000, and the other greater than or equal to 60,000. By contrast, three carcinoma cell lines of human origin and SV-40 3T3 mouse fibroblasts did not produce pyrogen in vitro. These results suggest that some malignant cells derived from phagocytic cells of bone marrow origin retain their capacity for pyrogen production, and may spontaneously secrete pyrogen during growth.

  13. Generation of autologous tumor-specific T cells for adoptive transfer based on vaccination, in vitro restimulation and CD3/CD28 dynabead-induced T cell expansion

    DEFF Research Database (Denmark)

    Brimnes, Marie Klinge; Gang, Anne Ortved; Donia, Marco

    2012-01-01

    Adoptive cell transfer (ACT) of in vitro expanded autologous tumor-infiltrating lymphocytes (TIL) has been shown to exert therapeutic efficacy in melanoma patients. We aimed to develop an ACT protocol based on tumor-specific T cells isolated from peripheral blood and in vitro expanded by Dynabeads...

  14. In vitro recapitulation of the urea cycle using murine embryonic stem cell-derived in vitro liver model.

    Science.gov (United States)

    Tamai, Miho; Aoki, Mami; Nishimura, Akihito; Morishita, Koji; Tagawa, Yoh-ichi

    2013-12-01

    Ammonia, a toxic metabolite, is converted to urea in hepatocytes via the urea cycle, a process necessary for cell/organismal survival. In liver, hepatocytes, polygonal and multipolar structures, have a few sides which face hepatic sinusoids and adjacent hepatocytes to form intercellular bile canaliculi connecting to the ductules. The critical nature of this three-dimensional environment should be related to the maintenance of hepatocyte function such as urea synthesis. Recently, we established an in vitro liver model derived from murine embryonic stem cells, IVL(mES), which included the hepatocyte layer and a surrounding sinusoid vascular-like network. The IVL(mES) culture, where the hepatocyte is polarized in a similar fashion to its in vivo counterpart, could successfully recapitulate in vivo results. L-Ornithine is an intermediate of the urea cycle, but supplemental L-ornithine does not activate the urea cycle in the apolar primary hepatocyte of monolayer culture. In the IVL(mES), supplemental L-ornithine could activate the urea cycle, and also protect against ammonium/alcohol-induced hepatocyte death. While the IVL(mES) displays architectural and functional properties similar to the liver, primary hepatocyte of monolayer culture fail to model critical functional aspects of liver physiology. We propose that the IVL(mES) will represent a useful, humane alternative to animal studies for drug toxicity and mechanistic studies of liver injury.

  15. Interaction of osteoblast-like cells with serum and fibronectin: effects on cell motility and proliferation in vitro

    International Nuclear Information System (INIS)

    Zuk, A.

    1986-01-01

    Osteoblast migration and proliferation are believed to occur during bone remodelling, in particular after osteoclastic bone resorption and prior to osteoblastic bone formation. In order to study migration and proliferation in vitro, the model of Alessandri et al. (1983) was modified. The model entailed seeding osteoblast-like cells into wells cut in agar and quantifying migration and proliferation peripheral to the well. Cell morphology also was described. The data indicated that on growth surfaces enriched with varying concentrations of fetal calf serum (FSC), the quantification of migration and proliferation was related both to percent cell attachment and to FCS-concentration. Because few osteoblast-like cells incorporated ( 3 H-TdR), it was concluded that the appearance of cells peripheral to the well was due to migration, and not to proliferation. Cell morphology and myosin distribution and organization indicated that osteoblast-like cells at the periphery of the cell culture (i.e. leading edge) may have been directionally migrating whereas cells behind the leading edge may have been engaged in non-directional migration. The migration, proliferation, and morphology of osteoblast-like cells cultured on fibronectin (FN) enriched growth surfaces also was examined. The quantification of migration and proliferation was related to the FN-concentration applied to the growth surface. Because few osteoblast-like cells incorporated 3 H-TdR and cell morphology indicated migration, it was concluded that osteoblast-like cells on FN-enriched growth surfaces are specialized, in part, for migration

  16. Induced synthesis of metallothionein by pig kidney cells in vitro in response to cadmium

    Energy Technology Data Exchange (ETDEWEB)

    Webb, M; Daniel, M

    1975-01-01

    Cells of a line (K7), derived from the cortex of the adult pig kidney, synthesize and accumulate high levels of metallothionein when grown in vitro in the presence of low concentrations (0.5 ..mu..g/ml) of Cd/sup 2 +/. This indicates that the accumulation of this protein in the kidneys of animals exposed to cadmium is due at least partly to synthesis in situ, and not solely to uptake by the renal cells of metallothionein produced by the liver. It is suggested that the ability to synthesize large amounts of metallothionein indicates the tubular origin of the cells of this line.

  17. The SGBS cell strain as a model for the in vitro study of obesity and cancer.

    LENUS (Irish Health Repository)

    Allott, Emma H

    2012-10-01

    The murine adipocyte cell line 3T3-L1 is well characterised and used widely, while the human pre-adipocyte cell strain, Simpson-Golabi-Behmel Syndrome (SGBS), requires validation for use in human studies. Obesity is currently estimated to account for up to 41 % of the worldwide cancer burden. A human in vitro model system is required to elucidate the molecular mechanisms for this poorly understood association. This work investigates the relevance of the SGBS cell strain for obesity and cancer research in humans.

  18. Uncovering cancer cell behavioral phenotype in 3-D in vitro metastatic landscapes

    Science.gov (United States)

    Liu, Liyu; Sun, Bo; Duclos, Guillaume; Kam, Yoonseok; Gatenby, Robert; Stone, Howard; Austin, Robert

    2012-02-01

    One well-known fact is that cancer cell genetics determines cell metastatic potentials. However, from a physics point of view, genetics as cell properties cannot directly act on metastasis. An agent is needed to unscramble the genetics first before generating dynamics for metastasis. Exactly this agent is cell behavioral phenotype, which is rarely studied due to the difficulties of real-time cell tracking in in vivo tissue. Here we have successfully constructed a micro in vitro environment with collagen based Extracellular Matrix (ECM) structures for cell 3-D metastasis. With stable nutrition (glucose) gradient inside, breast cancer cell MDA-MB-231 is able to invade inside the collagen from the nutrition poor site towards the nutrition rich site. Continuous confocal microscopy captures images of the cells every 12 hours and tracks their positions in 3-D space. The micro fluorescent beads pre-mixed inside the ECM demonstrate that invasive cells have altered the structures through mechanics. With the observation and the analysis of cell collective behaviors, we argue that game theory may exist between the pioneering cells and their followers in the metastatic cell group. The cell collaboration may explain the high efficiency of metastasis.

  19. Follicle stimulating hormone increases spermatogonial stem cell colonization during in vitro co - culture

    Directory of Open Access Journals (Sweden)

    Reza Narenji Sani

    2013-03-01

    Full Text Available The complex process of spermatogenesis is regulated by various factors. Studies onspermatogonial stem cells(SCCshave provided very important tool to improve herd geneticand different field. 0.2 to 0.3 percent of total cells of seminiferous tubules is consist ofspermatogonial stem cells. To investigate and biomanipulation of these cells, proliferationand viability rate of cells should be increasedin vitro, at first. Follicle stimulating hormone(FSH has been suggested to play a determinant role in the survival of germ cells in additionto increasing spermatogonial proliferation. In this study, thein vitroeffects ofFSHonspermatogonial cell colony formation were investigated. Sertoli and spermatogonial cellswere isolated from 3-5 months old calves. The identity of theSertoli cells and spermatogonialstem cells were confirmed through immunocytochemistry and colony morphology,respectively. Co-cultured Sertoli and spermatogonial cells were treatedwithFSHin differentdose of10, 20 and 40 IU mL-1FSH, before colony assay.Results indicated that,FSHincreasedin vitrocolonization of spermatogonial cells in comparison with control group. In conclusion,usingFSHprovided proper bovine spermatogonial stem cell culture medium forin vitrostudy of these cells.

  20. In vitro generation of motor neuron precursors from mouse embryonic stem cells using mesoporous nanoparticles

    DEFF Research Database (Denmark)

    Garcia-Bennett, Alfonso E; König, Niclas; Abrahamsson, Ninnie

    2014-01-01

    nanoparticles could be effective for stem cell differentiation in vitro. Materials & methods: We used a mouse embryonic stem cell line expressing green fluorescent protein under the promoter for the MN-specific gene Hb9 to visualize the level of MN differentiation. The differentiation of stem cells......Aim: Stem cell-derived motor neurons (MNs) are utilized to develop replacement strategies for spinal cord disorders. Differentiation of embryonic stem cells into MN precursors involves factors and their repeated administration. We investigated if delivery of factors loaded into mesoporous...... was evaluated by expression of MN-specific transcription factors monitored by quantitative real-time PCR reactions and immunocytochemistry. Results: Mesoporous nanoparticles have strong affiliation to the embryoid bodies, penetrate inside the embryoid bodies and come in contact with differentiating cells...

  1. Tuning differentiation signals for efficient propagation and in vitro validation of rat embryonic stem cell cultures.

    Science.gov (United States)

    Meek, Stephen; Sutherland, Linda; Burdon, Tom

    2015-01-01

    The rat is one of the most commonly used laboratory animals in biomedical research and the recent isolation of genuine pluripotent rat embryonic stem (ES) cell lines has provided new opportunities for applying contemporary genetic engineering techniques to the rat and enhancing the use of this rodent in scientific research. Technical refinements that improve the stability of the rat ES cell cultures will undoubtedly further strengthen and broaden the use of these stem cells in biomedical research. Here, we describe a relatively simple and robust protocol that supports the propagation of germ line competent rat ES cells, and outline how tuning stem cell signaling using small molecule inhibitors can be used to both stabilize self-renewal of rat ES cell cultures and aid evaluation of their differentiation potential in vitro.

  2. In vitro atrazine-exposure inhibits human natural killer cell lytic granule release

    International Nuclear Information System (INIS)

    Rowe, Alexander M.; Brundage, Kathleen M.; Barnett, John B.

    2007-01-01

    The herbicide atrazine is a known immunotoxicant and an inhibitor of human natural killer (NK) cell lytic function. The precise changes in NK cell lytic function following atrazine exposure have not been fully elucidated. The current study identifies the point at which atrazine exerts its affect on the stepwise process of human NK cell-mediated lyses of the K562 target cell line. Using intracellular staining of human peripheral blood lymphocytes, it was determined that a 24-h in vitro exposure to atrazine did not decrease the level of NK cell lytic proteins granzyme A, granzyme B or perforin. Thus, it was hypothesized that atrazine exposure was inhibiting the ability of the NK cells to bind to the target cell and subsequently inhibit the release of lytic protein from the NK cell. To test this hypothesis, flow cytometry and fluorescent microscopy were employed to analyze NK cell-target cell co-cultures following atrazine exposure. These assays demonstrated no significant decrease in the level of target cell binding. However, the levels of NK intracellular lytic protein retained and the amount of lytic protein released were assessed following a 4-h incubation with K562 target cells. The relative level of intracellular lytic protein was 25-50% higher, and the amount of lytic protein released was 55-65% less in atrazine-treated cells than vehicle-treated cells following incubation with the target cells. These results indicate that ATR exposure inhibits the ability of NK cells to lyse target cells by blocking lytic granule release without affecting the ability of the NK cell to form stable conjugates with target cells

  3. Differentiation potential of human CD133 positive hematopoietic stem cells into motor neuron- like cells, in vitro.

    Science.gov (United States)

    Moghaddam, Sepideh Alavi; Yousefi, Behnam; Sanooghi, Davood; Faghihi, Faezeh; Hayati Roodbari, Nasim; Bana, Nikoo; Joghataei, Mohammad Taghi; Pooyan, Paria; Arjmand, Babak

    2017-12-01

    Spinal cord injuries and motor neuron-related disorders impact on life of many patients around the world. Since pharmacotherapy and surgical approaches were not efficient to regenerate these types of defects; stem cell therapy as a good strategy to restore the lost cells has become the focus of interest among the scientists. Umbilical cord blood CD133 + hematopoietic stem cells (UCB- CD133 + HSCs) with self- renewal property and neural lineage differentiation capacity are ethically approved cell candidate for use in regenerative medicine. In this regard the aim of this study was to quantitatively evaluate the capability of these cells to differentiate into motor neuron-like cells (MNL), in vitro. CD133 + HSCs were isolated from human UCB using MACS system. After cell characterization using flow cytometry, the cells were treated with a combination of Retinoic acid, Sonic hedgehog, Brain derived neurotrophic factor, and B27 through a 2- step procedure for two weeks. The expression of MN-specific markers was examined using qRT- PCR, flow cytometry and immunocytochemistry. By the end of the two-week differentiation protocol, CD133 + cells acquired unipolar MNL morphology with thin and long neurites. The expression of Isl-1(62.15%), AChE (41.83%), SMI-32 (21.55%) and Nestin (17.46%) was detected using flow cytometry and immunocytochemistry. The analysis of the expression of PAX6, ISL-1, ACHE, CHAT and SMI-32 revealed that MNLs present these neural markers at levels comparable with undifferentiated cells. In Conclusion Human UCB- CD133 + HSCs are remarkably potent cell candidates to transdifferentiate into motor neuron-like cells, in vitro. Copyright © 2017. Published by Elsevier B.V.

  4. In vitro and in vivo motility studies of radiolabelled sperm cells

    International Nuclear Information System (INIS)

    Balogh, L.; Szasz, F.; Janoki, Gy.A.; Toth, L.; Zoldag, L.; Huszenicza, Gy.

    1994-01-01

    A new method for radiolabelling of sperm cells with 99m Tc HM-PAO (hexamethyl-propylene-amine-oxide) - LEUCO-SCINT kit, is investigated. The labelling technique for fresh rabbit, bull, sheep and horse as well as frozen-thawed bull sperm was optimized. The optimum conditions for sperm cell labelling (incubation volume, incubation time, initial activity of 99m Tc HM-PAO, cell number) yielded a high labelling efficiency (70-80%) and survival rate (50-60%). The labelled sperm cells were used to study their motility in vitro. The migrating at 37 o C cells incubated capillary tubes containing bovine cervical mucus. The tubes were cut and the activity of the parts measured and valued. We compared the results of living and killed sperm cells and the label alone by the change of species and running time. Ten minutes after the labelling procedures the total activity of microtubes was 2-3 times higher and the activity distribution was different from the results obtained 3 hours after the labelling. The sperm migration in vivo in the living female animals using a non invasive technique was also visualized. The sperm flow was clearly demonstrated in 3 different animal model (rabbit, ewe, hen) under gamma camera. The comparison of the in vivo migration of rabbit and bull sperm cells showed that the homologous sperm migrated faster and farther. On study of bull sperm migration in the ewe genital tract the cornu uteri was clearly visualized. In the hen model the whole genital tract was demonstrated with considerable free activity in the cavum abdominal 24 hours after the artificial insemination. The new method is developed and manufactured by NRIRR, Budapest, originally designed for radiolabelling leucocytes. The 99m Tc HM-PAO Labelled sperm cells with their retained migration properties are suitable for in vitro motility assays and in vitro migration studies in both human and veterinary medicine. (author)

  5. Pro-neurogenic effects of andrographolide on RSC96 Schwann cells in vitro

    Science.gov (United States)

    Xu, Fuben; Wu, Huayu; Zhang, Kun; Lv, Peizhen; Zheng, Li; Zhao, Jinmin

    2016-01-01

    Nerve regeneration remains a challenge to the treatment of peripheral nerve injury. Andrographolide (Andro) is the main active constituent of Andrographis paniculata, which has been applied in the treatment of several diseases, including inflammation, in ancient China. Andro has been reported to facilitate the reduction of edema and to exert analgesic effects in the treatment of various diseases. These findings suggest that Andro may be considered a promising anti-inflammatory agent that may suppress destruction and accelerate proliferation of Schwann cells following peripheral nerve injury. In the present study, the effects of Andro on RSC96 cells were investigated in vitro. The RSC96 cell line is a spontaneously immortalized rat Schwann cell line, which was originally derived from a long-term culture of rat primary Schwann cells. RSC96 cells were treated with a range of 0 to 50 µM Andro prior to the MTT assay. Cell proliferation, morphology, synthesis and nerve-specific gene expression were performed to detect the effect of Andro on RSC96 cells. The results of the present study demonstrated that the recommended doses of Andro ranged between 0.78 and 12.5 µM, among which the most obvious response was observed when used at 3.125 µM (P<0.05). DNA content was improved in Andro groups compared with the control group (P<0.05). In addition, Andro was able to promote the gene expression of glial cell line-derived neurotrophic factor, brain-derived neurotrophic factor, ciliary neurotrophic factor, and the specific Schwann cell marker S100β (P<0.05). The results of a viability assay, hematoxylin-eosin staining, and immunohistochemistry were also improved in Andro groups. These results indicated that Andro may accelerate proliferation of RSC96 cells in vitro, whilst maintaining the Schwann cell phenotype; therefore, the present study may provide valuable evidence for the further exploration of the effects of Andro on peripheral nerves. PMID:27599453

  6. In vitro and in vivo characterization of highly purified Human Mesothelioma derived cells

    International Nuclear Information System (INIS)

    Melotti, Alice; Daga, Antonio; Marubbi, Daniela; Zunino, Annalisa; Mutti, Luciano; Corte, Giorgio

    2010-01-01

    Malignant pleural mesothelioma is a rare disease known to be resistant to conventional therapies. A better understanding of mesothelioma biology may provide the rationale for new therapeutic strategies. In this regard, tumor cell lines development has been an important tool to study the biological properties of many tumors. However all the cell lines established so far were grown in medium containing at least 10% serum, and it has been shown that primary cell lines cultured under these conditions lose their ability to differentiate, acquire gene expression profiles that differ from that of tissue specific stem cells or the primary tumor they derive from, and in some cases are neither clonogenic nor tumorigenic. Our work was aimed to establish from fresh human pleural mesothelioma samples cell cultures maintaining tumorigenic properties. The primary cell cultures, obtained from four human pleural mesotheliomas, were expanded in vitro in a low serum proliferation-permissive medium and the expression of different markers as well as the tumorigenicity in immunodeficient mice was evaluated. The established mesothelioma cell cultures are able to engraft, after pseudo orthotopic intraperitoneal transplantation, in immunodeficient mouse and maintain this ability to after serial transplantation. Our cell cultures were strongly positive for CD46, CD47, CD56 and CD63 and were also strongly positive for some markers never described before in mesothelioma cell lines, including CD55, CD90 and CD99. By real time PCR we found that our cell lines expressed high mRNA levels of typical mesothelioma markers as mesothelin (MSLN) and calretinin (CALB2), and of BMI-1, a stemness marker, and DKK1, a potent Wingless [WNT] inhibitor. These cell cultures may provide a valuable in vitro and in vivo model to investigate mesothelioma biology. The identification of new mesothelioma markers may be useful for diagnosis and/or prognosis of this neoplasia as well as for isolation of mesothelioma

  7. In vitro analysis of the invasive phenotype of SUM 149, an inflammatory breast cancer cell line

    Directory of Open Access Journals (Sweden)

    Dharmawardhane Suranganie F

    2005-04-01

    Full Text Available Abstract Background Inflammatory breast cancer (IBC is the most lethal form of locally invasive breast cancer known. However, very little information is available on the cellular mechanisms responsible for manifestation of the IBC phenotype. To understand the unique phenotype of IBC, we compared the motile and adhesive interactions of an IBC cell line, SUM 149, to the non-IBC cell line SUM 102. Results Our results demonstrate that both IBC and non-IBC cell lines exhibit similar adhesive properties to basal lamina, but SUM 149 showed a marked increase in adhesion to collagen I. In vitro haptotaxis assays demonstrate that SUM 149 was less invasive, while wound healing assays show a less in vitro migratory phenotype for SUM 149 cells relative to SUM 102 cells. We also demonstrate a role for Rho and E-cadherin in the unique invasive phenotype of IBC. Immunoblotting reveals higher E-cadherin and RhoA expression in the IBC cell line but similar RhoC expression. Rhodamine phalloidin staining demonstrates increased formation of actin stress fibers and larger focal adhesions in SUM 149 relative to the SUM 102 cell line. Conclusion The observed unique actin and cellular architecture as well as the invasive and adhesive responses to the extracellular matrix of SUM 149 IBC cells suggest that the preference of IBC cells for connective tissue, possibly a mediator important for the vasculogenic mimicry via tubulogenesis seen in IBC pathological specimens. Overexpression of E-cadherin and RhoA may contribute to passive dissemination of IBC by promoting cell-cell adhesion and actin cytoskeletal structures that maintain tissue integrity. Therefore, we believe that these findings indicate a passive metastatic mechanism by which IBC cells invade the circulatory system as tumor emboli rather than by active migratory mechanisms.

  8. Human Dental Pulp Cells Differentiate toward Neuronal Cells and Promote Neuroregeneration in Adult Organotypic Hippocampal Slices In Vitro.

    Science.gov (United States)

    Xiao, Li; Ide, Ryoji; Saiki, Chikako; Kumazawa, Yasuo; Okamura, Hisashi

    2017-08-11

    The adult mammalian central nerve system has fundamental difficulties regarding effective neuroregeneration. The aim of this study is to investigate whether human dental pulp cells (DPCs) can promote neuroregeneration by (i) being differentiated toward neuronal cells and/or (ii) stimulating local neurogenesis in the adult hippocampus. Using immunostaining, we demonstrated that adult human dental pulp contains multipotent DPCs, including STRO-1, CD146 and P75-positive stem cells. DPC-formed spheroids were able to differentiate into neuronal, vascular, osteogenic and cartilaginous lineages under osteogenic induction. However, under neuronal inductive conditions, cells in the DPC-formed spheroids differentiated toward neuronal rather than other lineages. Electrophysiological study showed that these cells consistently exhibit the capacity to produce action potentials, suggesting that they have a functional feature in neuronal cells. We further co-cultivated DPCs with adult mouse hippocampal slices on matrigel in vitro. Immunostaining and presto blue assay showed that DPCs were able to stimulate the growth of neuronal cells (especially neurons) in both the CA1 zone and the edges of the hippocampal slices. Brain-derived neurotrophic factor (BDNF), was expressed in co-cultivated DPCs. In conclusion, our data demonstrated that DPCs are well-suited to differentiate into the neuronal lineage. They are able to stimulate neurogenesis in the adult mouse hippocampus through neurotrophic support in vitro.

  9. [Differentiation of human umbilical cord derived mesenchymal stem cells into low immunogenic and functional hepatocyte-like cells in vitro].

    Science.gov (United States)

    Ren, Hong-ying; Zhao, Qin-jun; Xing, Wen; Yang, Shao-guang; Lu, Shi-hong; Ren, Qian; Zhang, Lei; Han, Zhong-chao

    2010-04-01

    To investigate the biological function of hepatocyte-like cells derived from mesenchymal stem cells that isolated from human umbilical cord UC-MSCs in vitro, and to detect the changes in the immunogenicity of the differentiated hepatocyte-like cells (DHC). Transdifferentiation of UC-MSCs into hepatic lineage in vitro was induced in modified two-step induction medium. The expressions of hepatic specific markers were detected by RT-PCR analysis and immunofluorescence staining at different time points after induction. The levels of albumin and urea in the supernatants of cultures were measured by enzyme-linked immunosorbent assay. Furthermore, the immunosuppressive property of DHC was detected by one-way mixed lymphocyte culture. The mRNA and proteins of alpha fetoprotein (AFP), albumin (ALB),and cytokeratin-19 (CK-19) were expressed in naive UC-MSCs at low levels. DHC highly expressed hepatic markers AFP, ALB, CK-19, and tryptophan 2, 3-dioxygenase 14 and 28 days after hepatic differentiation and were accompanied by an increased production of ALB and urea in supernatant in a time-dependent manner. DHC did not express human leukocyte antigen DR antigen and significantly decreased the lymphocyte proliferation. UC-MSCs are able to differentiate into functional hepatocyte-like cells in vitro, while the immunogenicity of DHC remains low.

  10. In vitro motility evaluation of aggregated cancer cells by means of automatic image processing.

    Science.gov (United States)

    De Hauwer, C; Darro, F; Camby, I; Kiss, R; Van Ham, P; Decaesteker, C

    1999-05-01

    Set up of an automatic image processing based method that enables the motility of in vitro aggregated cells to be evaluated for a number of hours. Our biological model included the PC-3 human prostate cancer cell line growing as a monolayer on the bottom of Falcon plastic dishes containing conventional culture media. Our equipment consisted of an incubator, an inverted phase contrast microscope, a Charge Coupled Device (CCD) video camera, and a computer equipped with an image processing software developed in our laboratory. This computer-assisted microscope analysis of aggregated cells enables global cluster motility to be evaluated. This analysis also enables the trajectory of each cell to be isolated and parametrized within a given cluster or, indeed, the trajectories of individual cells outside a cluster. The results show that motility inside a PC-3 cluster is not restricted to slight motion due to cluster expansion, but rather consists of a marked cell movement within the cluster. The proposed equipment enables in vitro aggregated cell motility to be studied. This method can, therefore, be used in pharmacological studies in order to select anti-motility related compounds. The compounds selected by the equipment described could then be tested in vivo as potential anti-metastatic.

  11. Immunophenotypic characterization of human T cells after in vitro exposure to different silicone breast implant surfaces.

    Directory of Open Access Journals (Sweden)

    Giuseppe Cappellano

    Full Text Available The most common complication of silicone breast implants is capsular contracture (massive scar formation around the implant. We postulate that capsular contracture is always a sequel to inflammatory processes, with both innate and adaptive immune mechanisms participating. In general, fibroblasts and macrophages have been used as cell types to evaluate in vitro the biocompatibility of breast implant surfaces. Moreover, also T cells have been found at the implant site at the initial stage of fibrous capsule formation. However, only few studies have addressed the influence of surfaces with different textures on T-cell responses. The aim of the present study was to investigate the immune response of human peripheral blood mononuclear cells (PBMC to commercially available silicone breast implants in vitro. PBMC from healthy female blood donors were cultured on each silicone surface for 4 days. Proliferation and phenotype of cultured cells were assessed by flow cytometry. Cytokine levels were determined by multiplex and real-time assay. We found that silicone surfaces do not induce T-cell proliferation, nor do they extensively alter the proportion of T cell subsets (CD4, CD8, naïve, effector memory. Interestingly, cytokine profiling identified matrix specific differences, especially for IL-6 and TNF-α on certain surface topographies that could lead to increased fibrosis.

  12. Direct cell writing of 3D microorgan for in vitro pharmacokinetic model.

    Science.gov (United States)

    Chang, Robert; Nam, Jae; Sun, Wei

    2008-06-01

    A novel targeted application of tissue engineering is the development of an in vitro pharmacokinetic model for drug screening and toxicology. An in vitro pharmacokinetic model is needed to realistically and reliably predict in vivo human response to drug administrations and potential toxic exposures. This paper details the fabrication process development and adaptation of microfluidic devices for the creation of such a physiologically relevant pharmacokinetic model. First, an automated syringe-based, layered direct cell writing (DCW) bioprinting process creates a 3D microorgan that biomimics the cell's natural microenvironment with enhanced functionality. Next, soft lithographic micropatterning techniques are used to fabricate a microscale in vitro device to house the 3D microorgan. This paper demonstrates the feasibility of the DCW process for freeform biofabrication of 3D cell-encapsulated hydrogel-based tissue constructs with defined reproducible patterns, direct integration of 3D constructs onto a microfluidic device for continuous perfusion drug flow, and characterization of 3D tissue constructs with predictable cell viability/proliferation outcomes and enhanced functionality over traditional culture methods.

  13. Scutellarin promotes in vitro angiogenesis in human umbilical vein endothelial cells

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    Gao, Zhong-Xiu-Zi [Department of Anatomy, Basic Medical Science College, Harbin Medical University, Harbin (China); Huang, Da-Yong [Department of Oncology, The Second Clinical Hospital, Harbin Medical University, Harbin (China); Li, Hai-Xia; Zhang, Li-Na; Lv, Yan-Hong; Cui, Hai-Dong [Department of Anatomy, Basic Medical Science College, Harbin Medical University, Harbin (China); Zheng, Jin-Hua, E-mail: jhzhenghrbmu@yahoo.cn [Department of Anatomy, Basic Medical Science College, Harbin Medical University, Harbin (China)

    2010-09-10

    Research highlights: {yields} It has been shown that scutellarin exhibits a variety of pharmacological actions, including anti-oxidative, anti-inflammatory, vasodilator as well as cardiovascular and cerebrovascular ischemia protective effects, indicating beneficial vascular effects of scutellarin. Therefore, it is speculated that scutellarin may be able to stimulate angiogenesis, which could be beneficial in the treatment of ischemic disease, wound healing and tissue regeneration. {yields} The purpose of the present study was to elucidate the direct angiogenic actions of scutellarin on human umbilical vein endothelial cells (HUVECs) in vitro. {yields} Our results showed that scutellarin to directly induce in vitro angiogenesis, which is closely correlated with upregulated MMP-2 expression, suggesting a potential for increasing angiogenesis. -- Abstract: Angiogenesis is critical to a wide range of physiological and pathological processes. Scutellarin, a major flavonoid of a Chinese herbal medicine Erigeron breviscapus (Vant.) Hand. Mazz. has been shown to offer beneficial effects on cardiovascular and cerebrovascular functions. However, scutellarin's effects on angiogenesis and underlying mechanisms are not fully elucidated. Here, we studied angiogenic effects of scutellarin on human umbilical vein endothelial cells (HUVECs) in vitro. Scutellarin was found by MTT assay to induce proliferation of HUVECs. In scutellarin-treated HUVECs, a dramatic increase in migration was measured by wound healing assay; Transwell chamber assay found significantly more invading cells in scutellarin-treated groups. Scutellarin also promoted capillary-like tube formation in HUVECs on Matrigel, and significantly upregulated platelet endothelial cell adhesion molecule-1 at both mRNA and protein levels. Scutellarin's angiogenic mechanism was investigated in vitro by measuring expression of angiogenic factors associated with cell migration and invasion. Scutellarin strongly

  14. Scutellarin promotes in vitro angiogenesis in human umbilical vein endothelial cells

    International Nuclear Information System (INIS)

    Gao, Zhong-Xiu-Zi; Huang, Da-Yong; Li, Hai-Xia; Zhang, Li-Na; Lv, Yan-Hong; Cui, Hai-Dong; Zheng, Jin-Hua

    2010-01-01

    Research highlights: → It has been shown that scutellarin exhibits a variety of pharmacological actions, including anti-oxidative, anti-inflammatory, vasodilator as well as cardiovascular and cerebrovascular ischemia protective effects, indicating beneficial vascular effects of scutellarin. Therefore, it is speculated that scutellarin may be able to stimulate angiogenesis, which could be beneficial in the treatment of ischemic disease, wound healing and tissue regeneration. → The purpose of the present study was to elucidate the direct angiogenic actions of scutellarin on human umbilical vein endothelial cells (HUVECs) in vitro. → Our results showed that scutellarin to directly induce in vitro angiogenesis, which is closely correlated with upregulated MMP-2 expression, suggesting a potential for increasing angiogenesis. -- Abstract: Angiogenesis is critical to a wide range of physiological and pathological processes. Scutellarin, a major flavonoid of a Chinese herbal medicine Erigeron breviscapus (Vant.) Hand. Mazz. has been shown to offer beneficial effects on cardiovascular and cerebrovascular functions. However, scutellarin's effects on angiogenesis and underlying mechanisms are not fully elucidated. Here, we studied angiogenic effects of scutellarin on human umbilical vein endothelial cells (HUVECs) in vitro. Scutellarin was found by MTT assay to induce proliferation of HUVECs. In scutellarin-treated HUVECs, a dramatic increase in migration was measured by wound healing assay; Transwell chamber assay found significantly more invading cells in scutellarin-treated groups. Scutellarin also promoted capillary-like tube formation in HUVECs on Matrigel, and significantly upregulated platelet endothelial cell adhesion molecule-1 at both mRNA and protein levels. Scutellarin's angiogenic mechanism was investigated in vitro by measuring expression of angiogenic factors associated with cell migration and invasion. Scutellarin strongly induced MMP-2 activation and m

  15. Effects of Lactobacillus strains on cancer cell proliferation and oxidative stress in vitro.

    Science.gov (United States)

    Choi, S S; Kim, Y; Han, K S; You, S; Oh, S; Kim, S H

    2006-05-01

    The objective of this study was to assess in vitro, whether heat-killed (HK) lactic acid bacteria cells and fractionations of HK cells could suppress the viability of human cancer cells and inhibit the cytotoxicity associated with oxidative stress. Among the strains, the HK cells of Lactobacillus acidophilus 606 and Lactobacillus casei ATCC 393 exhibited the most profound inhibitory activity in all of the tested cell lines. HK cells of L. acidophilus 606 were determined to be less toxic to healthy human embryo fibroblasts (hEF cells) than were HK cells of L. casei ATCC 393. The soluble polysaccharides from L. acidophilus 606 evidenced the most effective anticancer activity, but inhibited hEF cell growth by only 20%. The soluble polysaccharides from L. acidophilus 606 were partly observed to induce apoptosis in the HT-29 cells by DNA fragmentation and propidium iodine staining. Both the HK cells of L. acidophilus 606 and the soluble polysaccharide components of this strain also exhibited potent antioxidative activity. Our findings suggest that the soluble polysaccharide fraction from L. acidophilus 606 may constitute a novel anticancer agent, which manifests a high degree of selectivity for human cancer cells and antioxidative agent in the food industry. These soluble polysaccharide components from Lactobacillus may be applied to various foods, and used as adjuncts for cancer therapy and prevention.

  16. Nano- and microstructured materials for in vitro studies of the physiology of vascular cells

    Directory of Open Access Journals (Sweden)

    Alexandra M. Greiner

    2016-11-01

    Full Text Available The extracellular environment of vascular cells in vivo is complex in its chemical composition, physical properties, and architecture. Consequently, it has been a great challenge to study vascular cell responses in vitro, either to understand their interaction with their native environment or to investigate their interaction with artificial structures such as implant surfaces. New procedures and techniques from materials science to fabricate bio-scaffolds and surfaces have enabled novel studies of vascular cell responses under well-defined, controllable culture conditions. These advancements are paving the way for a deeper understanding of vascular cell biology and materials–cell interaction. Here, we review previous work focusing on the interaction of vascular smooth muscle cells (SMCs and endothelial cells (ECs with materials having micro- and nanostructured surfaces. We summarize fabrication techniques for surface topographies, materials, geometries, biochemical functionalization, and mechanical properties of such materials. Furthermore, various studies on vascular cell behavior and their biological responses to micro- and nanostructured surfaces are reviewed. Emphasis is given to studies of cell morphology and motility, cell proliferation, the cytoskeleton and cell-matrix adhesions, and signal transduction pathways of vascular cells. We finalize with a short outlook on potential interesting future studies.

  17. Deciduous and permanent dental pulp mesenchymal cells acquire hepatic morphologic and functional features in vitro.

    Science.gov (United States)

    Ishkitiev, Nikolay; Yaegaki, Ken; Calenic, Bogdan; Nakahara, Taka; Ishikawa, Hiroshi; Mitiev, Vanyo; Haapasalo, Markus

    2010-03-01

    Mesenchymal stem cells display extensive proliferative capacity of multilineage differentiation. The stromal compartment of mesenchymal tissues is considered to harbor stem cells. We assessed the endodermal differentiation of mesenchymal cells from deciduous and wisdom tooth pulp. Dental mesenchymal cells were isolated and expanded in vitro. After cell cultures had been established, cells were characterized using known stem cell markers. For hepatic differentiation the media was supplemented with hepatic growth factor, dexamethasone, Insulin-Transferrin-Selenium-X, and oncostatin. Both cultures showed a number of cells positive for specific hepatic markers including alpha-fetoprotein, albumin, and hepatic nuclear factor 4alpha after differentiation. Also, small clusters of cells positive for insulin-like growth factor 1 were found. The concentration of urea increased significantly in the media. Moreover, a significant amount of glycogen was found in the cells. Because the cells proved to produce specific hepatic proteins and to start functions specific for hepatocytes, such as storing glycogen and urea production, we may state that the mesenchymal cell cultures from wisdom and deciduous tooth pulp acquired morphologic and functional characteristics of hepatocytes. Copyright (c) 2010 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  18. Functional properties of hepatocytes in vitro are correlated with cell polarity maintenance.

    Science.gov (United States)

    Zeigerer, Anja; Wuttke, Anne; Marsico, Giovanni; Seifert, Sarah; Kalaidzidis, Yannis; Zerial, Marino

    2017-01-01

    Exploring the cell biology of hepatocytes in vitro could be a powerful strategy to dissect the molecular mechanisms underlying the structure and function of the liver in vivo. However, this approach relies on appropriate in vitro cell culture systems that can recapitulate the cell biological and metabolic features of the hepatocytes in the liver whilst being accessible to experimental manipulations. Here, we adapted protocols for high-resolution fluorescence microscopy and quantitative image analysis to compare two primary hepatocyte culture systems, monolayer and collagen sandwich, with respect to the distribution of two distinct populations of early endosomes (APPL1 and EEA1-positive), endocytic capacity, metabolic and signaling activities. In addition to the re-acquisition of hepatocellular polarity, primary hepatocytes grown in collagen sandwich but not in monolayer culture recapitulated the apico-basal distribution of EEA1 endosomes observed in liver tissue. We found that such distribution correlated with the organization of the actin cytoskeleton in vitro and, surprisingly, was dependent on the nutritional state in vivo. Hepatocytes in collagen sandwich also exhibited faster kinetics of low-density lipoprotein (LDL) and epidermal growth factor (EGF) internalization, showed improved insulin sensitivity and preserved their ability for glucose production, compared to hepatocytes in monolayer cultures. Although no in vitro culture system can reproduce the exquisite structural features of liver tissue, our data nevertheless highlight the ability of the collagen sandwich system to recapitulate key structural and functional properties of the hepatocytes in the liver and, therefore, support the usage of this system to study aspects of hepatocellular biology in vitro. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  19. Differentiation of PDX1 gene-modified human umbilical cord mesenchymal stem cells into insulin-producing cells in vitro.

    Science.gov (United States)

    He, Dongmei; Wang, Juan; Gao, Yangjun; Zhang, Yuan

    2011-12-01

    Mesenchymal stem cells (MSCs) have significant advantages over other stem cell types, and greater potential for immediate clinical application. MSCs would be an interesting cellular source for treatment of type 1 diabetes. In this study, MSCs from human umbilical cord were differentiated into functional insulin-producing cells in vitro by introduction of the pancreatic and duodenal homeobox factor 1 (PDX1) and in the presence of induction factors. The expressions of cell surface antigens were detected by flow cytometry. After induction in an adipogenic medium or an osteogenic medium, the cells were observed by Oil Red O staining and alkaline phosphatase staining. Recombinant adenovirus carrying the PDX1 gene was constructed and MSCs were infected by the recombinant adenovirus, then treated with several inducing factors for differentiation into islet β-like cells. The expression of the genes and protein related to islet β-cells was detected by immunocytochemistry, RT-PCR and Western blot analysis. Insulin and C-peptide secretion were assayed. Our results show that the morphology and immunophenotype of MSCs from human umbilical cord were similar to those present in human bone marrow. The MSCs could be induced to differentiate into osteocytes and adipocytes. After induction by recombined adenovirus vector with induction factors, MSCs were aggregated and presented islet-like bodies. Dithizone staining of these cells was positive. The genes' expression related to islet β-cells was found. After induction, insulin and C-peptide secretion in the supernatant were significantly increased. In conclusion, our results demonstrated that PDX1 gene-modified human umbilical cord mesenchymal stem cells could be differentiated into insulin-producing cells in vitro.

  20. Flow Cytometry Assessment of In Vitro Generated CD138+ Human Plasma Cells

    Directory of Open Access Journals (Sweden)

    Rayelle Itoua Maïga

    2014-01-01

    Full Text Available The in vitro CD40-CD154 interaction promotes human B lymphocytes differentiation into plasma cells. Currently, CD138 is the hallmark marker enabling the detection of human plasma cells, both in vitro and in vivo; its presence can be monitored by flow cytometry using a specific antibody. We have developed a culture system allowing for the differentiation of memory B lymphocytes. In order to detect the newly formed plasma cells, we have compared their staining using five anti-CD138 monoclonal antibodies (mAbs. As a reference, we also tested human cell lines, peripheral blood mononuclear cells, and bone marrow samples. The five anti-CD138 mAbs stained RPMI-8226 cells (>98% with variable stain index (SI. The highest SI was obtained with B-A38 mAb while the lowest SI was obtained with DL-101 and 1D4 mAbs. However, the anti-CD138 mAbs were not showing equivalent CD138+ cells frequencies within the generated plasma cells. B-A38, B-B4, and MI-15 were similar (15–25% while DL-101 mAb stained a higher proportion of CD138-positive cells (38–42%. DL-101 and B-A38 mAbs stained similar populations in bone marrow samples but differed in their capacity to bind to CD138high and CD138lo cell lines. In conclusion, such cellular fluctuations suggest heterogeneity in human plasma cell populations and/or in CD138 molecules.

  1. In vitro characterization of cancer cell morphology, chemokinesis, and matrix invasion using a novel microfabricated system

    Science.gov (United States)

    Blaha, Laura

    A diagnosis of metastatic cancer reduces a patient's 5-year survival rate by nearly 80% compared to a primary tumor diagnosed at an early stage. While gene expression arrays have revealed unique gene signatures for metastatic cancer cells, we are lacking an understanding of the tangible physical changes that distinguish metastatic tumor cells from each other and from their related primary tumors. At the fundamental level, this translates into first characterizing the phenotype of metastatic cancer cells in vitro both in 2D - looking at morphology and migration - and in 3D - focusing on matrix invasion. While 2D in vitro studies have provided insight into the effects of specific environmental conditions on specific cancer cell lines, the unique details included in each experimental design make it challenging to compare cell phenotype across different in vitro platforms as well as between laboratories and disciplines that share the goal of understanding cancer. While 3D phenotype studies have employed more standardized and ubiquitous assays, most available tools lack the imaging capability and geometry to effectively characterize all factors driving 3D matrix invasion. In this work, we present protocols and platforms aimed at addressing the problems identified in the tools currently available for studying metastatic cancer in vitro. First, we present a 2D study of morphology and migration using widely accepted protocols. The study is applied to characterizing phenotypes of three breast cancer cell lines with different metastatic organ tropisms. The results show that general populations of cells from each of the 3 lines are unique in shape and motility despite being derived from the same tumor line and that the observed phenotype differences may be related to differences in focal adhesion assembly. More broadly, these studies suggest that standardizing phenotype studies using commonly available techniques may provide a platform by which to compare phenotypic studies

  2. Quantum dot labeling and tracking of cultured limbal epithelial cell transplants in-vitro

    Science.gov (United States)

    Genicio, Nuria; Paramo, Juan Gallo; Shortt, Alex J.

    2015-01-01

    PURPOSE Cultured human limbal epithelial cells (HLEC) have shown promise in the treatment of limbal stem cell deficiency but little is known about their survival, behaviour and long-term fate post transplantation. The aim of this research was to evaluate, in-vitro, quantum dot (QDot) technology as a tool for tracking transplanted HLEC. METHODS In-vitro cultured HLEC were labeled with Qdot nanocrystals. Toxicity was assessed using live-dead assays. The effect on HLEC function was assessed using colony forming efficiency assays and expression of CK3, P63alpha and ABCG2. Sheets of cultured HLEC labeled with Qdot nanocrystals were transplanted onto decellularised human corneo-scleral rims in an organ culture model and observed to investigate the behaviour of transplanted cells. RESULTS Qdot labeling had no detrimental effect on HLEC viability or function in-vitro. Proliferation resulted in a gradual reduction in Qdot signal but sufficient signal was present to allow tracking of cells through multiple generations. Cells labeled with Qdots could be reliably detected and observed using confocal microscopy for at least 2 weeks post transplantation in our organ culture model. In addition it was possible to label and observe epithelial cells in intact human corneas using the Rostock corneal module adapted for use with the Heidelberg HRA. CONCLUSIONS This work demonstrates that Qdots combined with existing clinical equipment could be used to track HLEC for up to 2 weeks post transplantation, however, our model does not permit the assessment of cell labeling beyond 2 weeks. Further characterisation in in-vivo models are required. PMID:26024089

  3. In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells.

    Science.gov (United States)

    Fang, Mingzhu; Kang, Hwan-Goo; Park, Youngil; Estrella, Brian; Zarbl, Helmut

    2017-09-28

    The circadian rhythm is a fundamental physiological process present in all organisms that regulates biological processes ranging from gene expression to sleep behavior. In vertebrates, circadian rhythm is controlled by a molecular oscillator that functions in both the suprachiasmatic nucleus (SCN; central pacemaker) and individual cells comprising most peripheral tissues. More importantly, disruption of circadian rhythm by exposure to light-at-night, environmental stressors and/or toxicants is associated with increased risk of chronic diseases and aging. The ability to identify agents that can disrupt central and/or peripheral biological clocks, and agents that can prevent or mitigate the effects of circadian disruption, has significant implications for prevention of chronic diseases. Although rodent models can be used to identify exposures and agents that induce or prevent/mitigate circadian disruption, these experiments require large numbers of animals. In vivo studies also require significant resources and infrastructure, and require researchers to work all night. Thus, there is an urgent need for a cell-type appropriate in vitro system to screen for environmental circadian disruptors and enhancers in cell types from different organs and disease states. We constructed a vector that drives transcription of the destabilized luciferase in eukaryotic cells under the control of the human PERIOD 2 gene promoter. This circadian reporter construct was stably transfected into human mammary epithelial cells, and circadian responsive reporter cells were selected to develop the in vitro bioluminescence assay. Here, we present a detailed protocol to establish and validate the assay. We further provide details for proof of concept experiments demonstrating the ability of our in vitro assay to recapitulate the in vivo effects of various chemicals on the cellular biological clock. The results indicate that the assay can be adapted to a variety of cell types to screen for both

  4. The interaction between Sertoli cells and luekemia inhibitory factor on the propagation and differentiation of spermatogonial stem cells in vitro.

    Science.gov (United States)

    Rastegar, Tayebeh; Habibi Roudkenar, Mehryar; Parvari, Soraya; Baazm, Maryam

    2015-11-01

    Sertoli cells play a pivotal role in creating microenvironments essential for spermatogonial stem cells (SSCs) self-renewal and commitment to differentiation. Maintenance of SSCs and or induction of in vitro spermiogenesis may provide a therapeutic strategy to treat male infertility. This study investigated the role of luekemia inhibitory factor (LIF) on the propagation of SSCs and both functions of Sertoli cells on the proliferation and differentiation of these cells. SSCs were sorted from the testes of adult male mice by magnetic activated cell sorting and thymus cell antigen 1 antibody. On the other hand, isolated Sertoli cells were enriched using lectin coated plates. SSCs were cultured on Sertoli cells for 7 days in the absence or presence of LIF. The effects of these conditions were evaluated by microscopy and expression of meiotic and post meiotic transcripts by reverse transcriptase polymerase chain reaction. Our data showed that SSCs co-cultured with Sertoli cells in the presence of LIF formed colonies on top of the Sertoli cells. These colonies had alkaline phosphatesase activity and expressed SSCs specific genes. SSCs were enjoyed limited development after the mere removal of LIF, and exhibiting expression of meiotic and postmeiotic transcript and loss of SSCs specific gene expression (p< 0.05). Our findings represent co-culture of SSCs with Sertoli cells provides conditions that may allow efficient proliferation and differentiation of SSCs for male infertility treatment.

  5. In Vitro Effects of Bromoalkyl Phenytoin Derivatives on Regulated Death, Cell Cycle and Ultrastructure of Leukemia Cells.

    Science.gov (United States)

    Śladowska, Katarzyna; Opydo-Chanek, Małgorzata; Król, Teodora; Trybus, Wojciech; Trybus, Ewa; Kopacz-Bednarska, Anna; Handzlik, Jadwiga; Kieć-Kononowicz, Katarzyna; Mazur, Lidia

    2017-11-01

    To search for new antileukemic agents, the chemical structure of phenytoin was modified. A possible cytotoxic activity of three bromoalkyl phenytoin analogs, methyl 2-(1-(3-bromopropyl)-2,4-dioxo-5,5-diphenylimidazolidin-3-yl) propanoate (PH2), 1-(3-bromopropyl)-3-methyl-5,5-diphenylimidazolidine-2,4-dione (PH3) and 1-(4-bromobutyl)-3-methyl-5,5-diphenylimidazolidine-2,4-dione (PH4) on regulated cell death, the cell cycle and cell ultrastructure was assessed. The experiments were performed in vitro on HL-60 and U937 cells, using flow cytometry and electron microscopy methods. Application of PH2, PH3, and PH4 resulted in cell surface exposure of phosphatidylserine and plasma membrane impairment, caspase-8, -9, and -3/7 activation, dissipation of mitochondrial membrane potential, DNA breakage, cell-cycle disturbance and cell ultrastructural changes. In general, PH3 appeared to be the most active against the leukemia cells, and all bromoalkyl hydantoins, PH2-PH4, were more active in HL-60 cells than in U937 cells. The antileukemic activity of the bromoalkyl phenytoin analogs depended on the combination of N-hydantoin substituents and the human cell line used. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  6. The interaction between Sertoli cells and luekemia inhibitory factor on the propagation and differentiation of spermatogonial stem cells in vitro

    Directory of Open Access Journals (Sweden)

    Tayebeh Rastegar

    2015-11-01

    Full Text Available Background: Sertoli cells play a pivotal role in creating microenvironments essential for spermatogonial stem cells (SSCs self-renewal and commitment to differentiation. Maintenance of SSCs and or induction of in vitro spermiogenesis may provide a therapeutic strategy to treat male infertility. Objective: This study investigated the role of luekemia inhibitory factor (LIF on the propagation of SSCs and both functions of Sertoli cells on the proliferation and differentiation of these cells. Materials and Methods: SSCs were sorted from the testes of adult male mice by magnetic activated cell sorting and thymus cell antigen 1 antibody. On the other hand, isolated Sertoli cells were enriched using lectin coated plates. SSCs were cultured on Sertoli cells for 7 days in the absence or presence of LIF. The effects of these conditions were evaluated by microscopy and expression of meiotic and post meiotic transcripts by reverse transcriptase polymerase chain reaction. Results: Our data showed that SSCs co-cultured with Sertoli cells in the presence of LIF formed colonies on top of the Sertoli cells. These colonies had alkaline phosphatesase activity and expressed SSCs specific genes. SSCs were enjoyed limited development after the mere removal of LIF, and exhibiting expression of meiotic and postmeiotic transcript and loss of SSCs specific gene expression (p< 0.05. Conclusion: Our findings represent co-culture of SSCs with Sertoli cells provides conditions that may allow efficient proliferation and differentiation of SSCs for male infertility treatment.

  7. FLAX OIL FROM TRANSGENIC LINUM USITATISSIMUM SELECTIVELY INHIBITS IN VITRO PROLIFERATION OF HUMAN CANCER CELL LINES.

    Science.gov (United States)

    Gebarowski, Tomasz; Gebczak, Katarzyna; Wiatrak, Benita; Kulma, Anna; Pelc, Katarzyna; Czuj, Tadeusz; Szopa, Jan; Gasiorowski, Kazimierz

    2017-03-01

    Emulsions made of oils from transgenic flaxseeds significantly decreased in vitro proliferation of six tested human cancer cell lines in 48-h cultures, as assessed with the standard sulforhodamine assay. However, the emulsions also increased proliferation rate of normal human dermal fibroblasts and, to a lower extend, of keratinocytes. Both inhibition of in vitro proliferation of human cancer cell lines and stimulation of proliferation of normal dermal fibroblasts and keratinocytes were especially strong with the emulsion type B and with emulsion type M. Oils from seeds of transgenic flax type B and M should be considered as valuable adjunct to standard cytostatic therapy of human cancers and also could be applied to improve the treatment of skin lesions in wound healing.

  8. Zoledronic acid inhibits vasculogenic mimicry in murine osteosarcoma cell line in vitro.

    Science.gov (United States)

    Fu, Dehao; He, Xianfeng; Yang, Shuhua; Xu, Weihua; Lin, Tao; Feng, Xiaobo

    2011-06-30

    To study the effects of zoledronic acid (ZA) on the vasculogenic mimicry of osteosarcoma cells in vitro. A Three-dimensional culture of LM8 osteosarcoma cells on a type I collagen matrix was used to investigate whether osteosarcoma cells can develop vasculogenic mimicry, and to determine the effects of ZA on this process. In addition, the cellular ultrastructural changes were observed using scanning electron microscopy and laser confocal microscopy. The effects of ZA on the translocation of RhoA protein from the cytosol to the membrane in LM8 cells were measured via immunoblotting. ZA inhibited the development of vasculogenic mimicry by the LM8 osteosarcoma cells, decreased microvilli formation on the cell surface, and disrupted the F-actin cytoskeleton. ZA prevented translocation of RhoA protein from the cytosol to the membrane in LM8 cells. ZA can impair RhoA membrane localization in LM8 cells, causing obvious changes in the ultrastructure of osteosarcoma cells and induce cell apoptosis, which may be one of the underlying mechanisms by which the agent inhibits the development of vasculogenic mimicry by the LM8 cells.

  9. Zoledronic acid inhibits vasculogenic mimicry in murine osteosarcoma cell line in vitro

    Directory of Open Access Journals (Sweden)

    Lin Tao

    2011-06-01

    Full Text Available Abstract Background To study the effects of zoledronic acid (ZA on the vasculogenic mimicry of osteosarcoma cells in vitro. Methods A Three-dimensional culture of LM8 osteosarcoma cells on a type I collagen matrix was used to investigate whether osteosarcoma cells can develop vasculogenic mimicry, and to determine the effects of ZA on this process. In addition, the cellular ultrastructural changes were observed using scanning electron microscopy and laser confocal microscopy. The effects of ZA on the translocation of RhoA protein from the cytosol to the membrane in LM8 cells were measured via immunoblotting. Results ZA inhibited the development of vasculogenic mimicry by the LM8 osteosarcoma cells, decreased microvilli formation on the cell surface, and disrupted the F-actin cytoskeleton. ZA prevented translocation of RhoA protein from the cytosol to the membrane in LM8 cells. Conclusions ZA can impair RhoA membrane localization in LM8 cells, causing obvious changes in the ultrastructure of osteosarcoma cells and induce cell apoptosis, which may be one of the underlying mechanisms by which the agent inhibits the development of vasculogenic mimicry by the LM8 cells.

  10. Effect of chemotherapy and irradiation on interactions between stromal and hemopoietic cells in vitro

    International Nuclear Information System (INIS)

    Cohen, G.I.; Greenberger, J.S.; Canellos, G.P.

    1982-01-01

    We examined the interactions between stromal and hemopoietic cells in mouse long-term bone marrow cultures. The adherent stroma is formed by several layers of cells consisting of macrophage, fibroblasts, and adventitial cells which accumulate lipid to become adipocytes. Stromal cells become closely apposed to loosely adherent hemopoietic cells but gap junctions occur only among cells in the adherent layer. The hemopoietic cells form tightly packed structures resembling cobblestones which contain granulocytes in all stages of differentiation. Using an in vitro model for bone marrow transplantation (BMT), we treated pure mouse stromal cell cultures with irradiation (1000 R) or chemotherapy (BCNU) prior to engraftment with hemopoietic stem cells. After two weeks, engrafted cultures were indistinguishable from the long-term bone marrow cultures previously described by Dexter. The adipocytes in irradiated cultures developed numerous submembrane pinocytotic vesicles but stromal-hemopoietic cell interactions remained unchanged compared to unirradiated controls. By contrast, granulocytes grafted onto chemotherapy treated stroma showed swelling of endoplasmic reticulum suggesting early toxic injury. These findings are consistent with functional studies of hemopoiesis after engraftment onto treated stroma and confirm an important role for stromal cells in the support of hemopoiesis

  11. Differentiation of Human Malignant Melanoma Cells that Escape Apoptosis After Treatment with 9-Nitrocamptothecin In Vitro

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    Panayotis Pantazis

    1999-08-01

    Full Text Available After in-vitro exposure to 0.05 μmol/L 9-nitrocamptothecin (9NC for periods of time longer than 5 days, 65% to 80% of the human malignant melanoma SB1 B cells die by apoptosis, whereas the remaining cells are arrested at the G2-phase of the cell cycle. Upon discontinuation of exposure to 9NC the G2-arrested cells resume cell cycling or remain arrested depending on the duration of 9NC exposure. In contrast to cycling malignant cells, the cells irreversibly arrested at G2 exhibit features of normal-like cells, the melanocytes, as assessed by the appearance of dendrite-like structures; loss of proliferative activity; synthesis of the characteristic pigment, melanin; and, particularly, loss of tumorigenic ability after xenografting in immunodeficient mice. Further, the expression of the cyclin-dependent kinase inhibitor p16 is upregulated in the 9NC-treated, G1-arrested, but downregulated in density G1-arrested cells, whereas the reverse is observed in the expression of another cyclin-dependent kinase inhibitor, p21. These results suggest that malignant melanoma SB1B cells that escape 9NC-induced death by apoptosis undergo differentiation toward nonmalignant, normal-like cells.

  12. PCL-coated hydroxyapatite scaffold derived from cuttlefish bone: In vitro cell culture studies

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    Milovac, Dajana; Gamboa-Martínez, Tatiana C.; Ivankovic, Marica; Gallego Ferrer, Gloria; Ivankovic, Hrvoje

    2014-01-01

    In the present study, we examined the potential of using highly porous poly(ε-caprolactone) (PCL)-coated hydroxyapatite (HAp) scaffold derived from cuttlefish bone for bone tissue engineering applications. The cell culture studies were performed in vitro with preosteoblastic MC3T3-E1 cells in static culture conditions. Comparisons were made with uncoated HAp scaffold. The attachment and spreading of preosteoblasts on scaffolds were observed by Live/Dead staining Kit. The cells grown on the HAp/PCL composite scaffold exhibited greater spreading than cells grown on the HAp scaffold. DNA quantification and scanning electron microscopy (SEM) confirmed a good proliferation of cells on the scaffolds. DNA content on the HAp/PCL scaffold was significantly higher compared to porous HAp scaffolds. The amount of collagen synthesis was determined using a hydroxyproline assay. The osteoblastic differentiation of the cells was evaluated by determining alkaline phosphatase (ALP) activity and collagen type I secretion. Furthermore, cell spreading and cell proliferation within scaffolds were observed using a fluorescence microscope. - Highlights: • Hydroxyapatite/poly(ε-caprolactone) scaffold with interconnected pores was prepared • Cytotoxicity test showed that the scaffold was not cytotoxic towards MC3T3-E1 cells • The scaffold supported the attachment, proliferation and differentiation of cells • A 3D cell colonization was confirmed using the fluorescence microscopy • The scaffold might be a promising candidate for bone tissue engineering

  13. Recapitulation of Extracellular LAMININ Environment Maintains Stemness of Satellite Cells In Vitro.

    Science.gov (United States)

    Ishii, Kana; Sakurai, Hidetoshi; Suzuki, Nobuharu; Mabuchi, Yo; Sekiya, Ichiro; Sekiguchi, Kiyotoshi; Akazawa, Chihiro

    2018-02-13

    Satellite cells function as precursor cells in mature skeletal muscle homeostasis and regeneration. In healthy tissue, these cells are maintained in a state of quiescence by a microenvironment formed by myofibers and basement membrane in which LAMININs (LMs) form a major component. In the present study, we evaluated the satellite cell microenvironment in vivo and found that these cells are encapsulated by LMα2-5. We sought to recapitulate this satellite cell niche in vitro by culturing satellite cells in the presence of recombinant LM-E8 fragments. We show that treatment with LM-E8 promotes proliferation of satellite cells in an undifferentiated state, through reduced phosphorylation of JNK and p38. On transplantation into injured muscle tissue, satellite cells cultured with LM-E8 promoted the regeneration of skeletal muscle. These findings represent an efficient method of culturing satellite cells for use in transplantation through the recapitulation of the satellite cell niche using recombinant LM-E8 fragments. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  14. [Effects of metformin on human oral cancer KB cell proliferation and apoptosis in vitro].

    Science.gov (United States)

    Wang, Fang; Xu, Jincheng; Xia, Fei; Liu, Zhe; Zhao, Surong; Liu, Hao; Jiang, Zhiwen

    2014-02-01

    To investigate the effects of metformin on the proliferation and apoptosis of human oral cancer cell line KB in vitro. Human oral cancer cell line KB was exposed to different doses of metformin (0, 1.25, 2.5, 5, 10, and 20 mmol/L), and the changes in cell viability were detected using MTT assay. Colony formation of the cells was observed following an 8-day metformin exposure. The changes in mitochondrial membrane potential were measured by JC-1 assay, and PI staining was used to observe the cell apoptosis. Western blotting was employed to detect the changes in the protein expressions of GRP78 and activated caspase-3. Metformin exposure caused time- and dose-dependent suppression of KB cell proliferation, and exposure to 5 mmol/L metformin for 24, 48 and 72 h resulted in cell survival rates of 68.0%, 36.9%, and 14.5%, respectively. Metformin significantly inhibited KB cell colony formation. Exposure of the cells to increased concentrations of metformin gradually increased the apoptotic rate and decreased mitochondrial membrane potential. Metformin caused an initial up-regulation followed by a down-regulation of GRP78 expression in KB cells and increased the expression of activated caspase-3. Metformin can inhibit the proliferation and induce apoptosis of KB cells, the mechanism of which may involve the activation of the mitochondrial apoptotic pathway and endoplasmic reticulum stress.

  15. Selection of genetically modified hematopoietic cells in vitro and in vivo using alkylating agent lysomustine.

    Science.gov (United States)

    Rozov, F N; Grinenko, T S; Levit, G L; Krasnov, V P; Belyavsky, A V

    2010-09-15

    Efficient gene transfer into hematopoietic stem cells is vital for the success of gene therapy of hematopoietic and immune system disorders. An in vivo selection system based on a mutant form of the O(6)-methylguanine-DNA-methyltransferase gene (MGMTm) is considered one of the more promising strategies for expansion of hematopoietic cells transduced with viral vectors. Here we demonstrate that MGMTm-expressing cells can be efficiently selected using lysomustine, a nitrosourea derivative of lysine. K562 and murine bone marrow cells expressing MGMTm are protected from the cytotoxic action of lysomustine in vitro. We also show in a murine model that MGMTm-transduced hematopoietic cells can be expanded in vivo on transplantation into sublethally irradiated recipients followed by lysomustine treatment. These results indicate that lysomustine can be used as a potent novel chemoselection drug applicable for gene therapy of hematopoietic and immune system disorders. 2010 Elsevier Inc. All rights reserved.

  16. In Vitro Efficient Expansion of Tumor Cells Deriving from Different Types of Human Tumor Samples

    Directory of Open Access Journals (Sweden)

    Ilaria Turin

    2014-03-01

    Full Text Available Obtaining human tumor cell lines from fresh tumors is essential to advance our understanding of antitumor immune surveillance mechanisms and to develop new ex vivo strategies to generate an efficient anti-tumor response. The present study delineates a simple and rapid method for efficiently establishing primary cultures starting from tumor samples of different types, while maintaining the immuno-histochemical characteristics of the original tumor. We compared two different strategies to disaggregate tumor specimens. After short or long term in vitro expansion, cells analyzed for the presence of malignant cells demonstrated their neoplastic origin. Considering that tumor cells may be isolated in a closed system with high efficiency, we propose this methodology for the ex vivo expansion of tumor cells to be used to evaluate suitable new drugs or to generate tumor-specific cytotoxic T lymphocytes or vaccines.

  17. Drug-induced in vitro inhibition of neutrophil-endothelial cell adhesion.

    Science.gov (United States)

    Pellegatta, F.; Lu, Y.; Radaelli, A.; Zocchi, M. R.; Ferrero, E.; Chierchia, S.; Gaja, G.; Ferrero, M. E.

    1996-01-01

    1. Leukocyte-endothelial cell interactions play an important role during ischaemia-reperfusion events. Adhesion molecules are specifically implicated in this interaction process. 2. Since defibrotide has been shown to be an efficient drug in reducing damage due to ischaemia-reperfusion in many experimental models, we analysed the effect of defibrotide in vitro on leukocyte adhesion to endothelial cells in basal conditions and after their stimulation. 3. In basal conditions, defibrotide (1000 micrograms ml-1) partially inhibited leukocyte adhesion to endothelial cells by 17.3% +/- 3.6 (P defibrotide. 5. This result was confirmed in NIH/3T3-ICAM-1 transfected cells. 6. We conclude that defibrotide is able to interfere with leukocyte adhesion to endothelial cells mainly in activated conditions and that the ICAM-1/LFA-1 adhesion system is involved in the defibrotide mechanism of action. PMID:8762067

  18. Ascorbate concentrations in vitro and in vivo, and their role in the radiation response of cells

    International Nuclear Information System (INIS)

    Stratford, M.R.L.; Hodgkiss, R.J.

    1985-01-01

    Hydrogen-atom or electron-transfer reactions of ascorbate are often invoked in discussing its potential role in radiobiology and free radical damage by cytotoxins, but detailed information on actual levels in experimental systems is lacking. A range of 0-250 μM ascorbate is present in several commonly used mammalian cell culture media. V79 379A Chinese hamster cells can concentrate ascorbate from medium containing 200 or 500 μM ascorbate but when ascorbate is absent in medium, cells do not appear to contain a significant amount. Tumour concentrations are approximately 1mM, similar to that of glutathione (GSH). There is much current interest in depleting cells of GSH to enhance radiosensitivity, and ascorbate is maintained by a GSH dependent enzyme, glutathione dehydrogenase. Data is presented on the effect of GSH depletion by buthionine sulphoximine on cell and tumour ascorbate levels, and the effect of ascorbate on in vitro radiosensitivity, and misonidazole sensitizing efficiency

  19. Inhibitory effect of calcium channel blockers on proliferation of human glioma cells in vitro

    International Nuclear Information System (INIS)

    Kunert-Radek, J.; Stepien, H.; Lyson, K.; Pawlikowski, M.; Radek, A.

    1989-01-01

    The effects of 2 specific calcium channel blockers, verapamil and nimodipine, on the proliferation of human glioma tumour cells were investigated in vitro. Tumour tissues for primary cell cultures were obtained bioptically from 3 patients with the histopathological diagnosis of glioblastoma. The [ 3 H]-thymidine incorporation into glioma tumour cells DNA was used as a sensitive index of the cell proliferation. It was found that varapamil (10 4 -10 5 M) and nimodipine (10 4 -10 6 M) significantly inhibited the [ 3 H]-thymidine uptake in a dose-related manner. The inhibitory effect of both calcium channel antagonists was reversed by stimultancous addition of calcium chloride (5x10 3 M). These results indicate that verapamil and nimodipine may exert an antiproliferative effect on glioma cells growth acting through a blokade of specific voltage-dependent calcium channels. (author)

  20. Apoptosis induced by cold shock in vitro is dependent on cell growth phase.

    Science.gov (United States)

    Soloff, B L; Nagle, W A; Moss, A J; Henle, K J; Crawford, J T

    1987-06-15

    Chinese hamster V79 fibroblast cells were exposed to brief periods of cold but non-freezing temperatures at different points on the population growth curve. Upon rewarming, cells at the transition from logarithmic to stationary growth exhibited apoptosis (programmed cell death). Cells in other stages of growth, or after reentry into logarithmic growth by refeeding, did not exhibit apoptosis. Apoptosis was expressed by marked cytoplasmic blebbing, by a characteristic non-random fragmentation of DNA into nucleosomal-sized pieces, and by loss of colony-forming ability. The data suggest that cold shock served as a stimulus for susceptible cells to undergo apoptosis. Thus, the experiments describe a new in vitro system for studying the mechanisms of apoptosis.

  1. The Antidiabetic Drug Metformin Inhibits the Proliferation of Bladder Cancer Cells in Vitro and in Vivo

    Directory of Open Access Journals (Sweden)

    Tao Zhang

    2013-12-01

    Full Text Available Recent studies suggest that metformin, a widely used antidiabetic agent, may reduce cancer risk and improve prognosis of certain malignancies. However, the mechanisms for the anti-cancer effects of metformin remain uncertain. In this study, we investigated the effects of metformin on human bladder cancer cells and the underlying mechanisms. Metformin significantly inhibited the proliferation and colony formation of 5637 and T24 cells in vitro; specifically, metformin induced an apparent cell cycle arrest in G0/G1 phases, accompanied by a strong decrease of cyclin D1, cyclin-dependent kinase 4 (CDK4, E2F1 and an increase of p21waf-1. Further experiments revealed that metformin activated AMP-activated protein kinase (AMPK and suppressed mammalian target of rapamycin (mTOR, the central regulator of protein synthesis and cell growth. Moreover, daily treatment of metformin led to a substantial inhibition of tumor growth in a xenograft model with concomitant decrease in the expression of proliferating cell nuclear antigen (PCNA, cyclin D1 and p-mTOR. The in vitro and in vivo results demonstrate that metformin efficiently suppresses the proliferation of bladder cancer cells and suggest that metformin may be a potential therapeutic agent for the treatment of bladder cancer.

  2. Bio-EdIP: An automatic approach for in vitro cell confluence images quantification.

    Science.gov (United States)

    Cardona, Andrés; Ariza-Jiménez, Leandro; Uribe, Diego; Arroyave, Johanna C; Galeano, July; Cortés-Mancera, Fabian M

    2017-07-01

    Cell imaging is a widely-employed technique to analyze multiple biological processes. Therefore, simple, accurate and quantitative tools are needed to understand cellular events. For this purpose, Bio-EdIP was developed as a user-friendly tool to quantify confluence levels using cell culture images. The proposed algorithm combines a pre-processing step with subsequent stages that involve local processing techniques and a morphological reconstruction-based segmentation algorithm. Segmentation performance was assessed in three constructed image sets, comparing F-measure scores and AUC values (ROC analysis) for Bio-EdIP, its previous version and TScratch. Furthermore, segmentation results were compared with published algorithms using eight public benchmarks. Bio-EdIP automatically segmented cell-free regions from images of in vitro cell culture. Based on mean F-measure scores and ROC analysis, Bio-EdIP conserved a high performance regardless of image characteristics of the constructed dataset, when compared with its previous version and TScratch. Although acquisition quality of the public dataset affected Bio-EdIP segmentation, performance was better in two out of eight public sets. Bio-EdIP is a user-friendly interface, which is useful for the automatic analysis of confluence levels and cell growth processes using in vitro cell culture images. Here, we also presented new manually annotated data for algorithms evaluation. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Arachidonate metabolism increases as rat alveolar type II cells differentiate in vitro

    International Nuclear Information System (INIS)

    Lipchik, R.J.; Chauncey, J.B.; Paine, R.; Simon, R.H.; Peters-Golden, M.

    1990-01-01

    Rat type II alveolar epithelial cells are known to undergo morphological and functional changes when maintained in culture for several days. Having previously demonstrated that these cells can deacylate free arachidonic acid (AA) and metabolize it to products of the cyclooxygenase pathway, the present study was undertaken to determine whether in vitro differentiation was accompanied by alterations in the availability and metabolism of AA. We assessed the constitutive and ionophore A23187-induced deacylation and metabolism of endogenous AA, as well as the metabolism of exogenously supplied AA, in primary cultures of rat type II cells at days 2, 4, and 7 after isolation. Levels of free endogenous AA were increased at day 4, whereas eicosanoid synthesis, predominantly prostaglandin E2 and prostacyclin, increased markedly only at day 7. A similar time course of augmentation of prostanoid release was seen in response to exogenous AA. Type II cells cultured on fibronectin, intended to hasten cell flattening and spreading, demonstrated accelerated increases in available free AA in response to A23187; cells cultured on basement membrane derived from Engelbreth-Holm-Swarm mouse sarcoma, known to maintain the type II phenotype, exhibited diminished levels of available free AA. From these findings, we conclude that alterations in arachidonate metabolism are linked to alterations in cellular phenotype. The potentiation of eicosanoid synthesis accompanying in vitro differentiation suggests a possible role for the alveolar epithelium in the modulation of inflammation and fibrosis in the distal lung

  4. Chemopreventive effects of in vitro digested and fermented bread in human colon cells.

    Science.gov (United States)

    Schlörmann, Wiebke; Hiller, Beate; Jahns, Franziska; Zöger, Romy; Hennemeier, Isabell; Wilhelm, Anne; Lindhauer, Meinolf G; Glei, Michael

    2012-10-01

    Bread as a staple food product represents an important source for dietary fibre consumption. Effects of wheat bread, wholemeal wheat bread and wholemeal rye bread on mechanisms which could have impact on chemoprevention were analysed in colon cells after in vitro fermentation. Effects of fermented bread samples on gene expression, glutathione S-transferase activity and glutathione content, differentiation, growth and apoptosis were investigated using the human colon adenoma cell line LT97. Additionally, apoptosis was studied in normal and tumour colon tissue by determination of caspase activities. The expression of 76 genes (biotransformation, differentiation, apoptosis) was significantly upregulated (1.5-fold) in LT97 cells. The fermented bread samples were able to significantly increase glutathione S-transferase activity (1.8-fold) and glutathione content (1.4-fold) of the cells. Alkaline phosphatase activity as a marker of differentiation was also significantly enhanced (1.7-fold). The fermented bread samples significantly inhibited LT97 cell growth and increased the level of apoptotic cells (1.8-fold). Only marginal effects on apoptosis in tumour compared to normal tissue were observed. This is the first study which presents chemopreventive effects of different breads after in vitro fermentation. In spite of differences in composition, the results were comparable between the bread types. Nevertheless, they indicate a potential involvement of this staple food product regarding the prevention of colon cancer.

  5. The influence of immunosuppressive drugs on neural stem/progenitor cell fate in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Skardelly, Marco, E-mail: Marco.Skardelly@med.uni-tuebingen.de [Department of Neurosurgery, University Hospital, Leipzig (Germany); Translational Centre for Regenerative Medicine, University of Leipzig, Leipzig (Germany); Glien, Anja; Groba, Claudia; Schlichting, Nadine [Department of Neurosurgery, University Hospital, Leipzig (Germany); Kamprad, Manja [Institute of Clinical Immunology, Medical Faculty, University of Leipzig, Leipzig (Germany); Meixensberger, Juergen [Department of Neurosurgery, University Hospital, Leipzig (Germany); Milosevic, Javorina [Translational Centre for Regenerative Medicine, University of Leipzig, Leipzig (Germany)

    2013-12-10

    In allogenic and xenogenic transplantation, adequate immunosuppression plays a major role in graft survival, especially over the long term. The effect of immunosuppressive drugs on neural stem/progenitor cell fate has not been sufficiently explored. The focus of this study is to systematically investigate the effects of the following four different immunotherapeutic strategies on human neural progenitor cell survival/death, proliferation, metabolic activity, differentiation and migration in vitro: (1) cyclosporine A (CsA), a calcineurin inhibitor; (2) everolimus (RAD001), an mTOR-inhibitor; (3) mycophenolic acid (MPA, mycophenolate), an inhibitor of inosine monophosphate dehydrogenase and (4) prednisolone, a steroid. At the minimum effective concentration (MEC), we found a prominent decrease in hNPCs' proliferative capacity (BrdU incorporation), especially for CsA and MPA, and an alteration of the NAD(P)H-dependent metabolic activity. Cell death rate, neurogenesis, gliogenesis and cell migration remained mostly unaffected under these conditions for all four immunosuppressants, except for apoptotic cell death, which was significantly increased by MPA treatment. - Highlights: • Four immunosuppresants (ISs) were tested in human neural progenitor cells in vitro. • Cyclosporine A and mycophenolic acid showed a prominent anti-proliferative activity • Mycophenolic acid exhibited a significant pro-apoptotic effect. • NAD(P)H-dependent metabolic activity was occasionally induced by ISs. • Neuronal differentiation and migration potential remained unaffected by ISs treatment.

  6. 3D cell culture to determine in vitro biocompatibility of bioactive glass in association with chitosan.

    Science.gov (United States)

    Bédouin, Y; Pellen Mussi, P; Tricot-Doleux, S; Chauvel-Lebret, D; Auroy, P; Ravalec, X; Oudadesse, H; Perez, F

    2015-01-01

    This study reports the in vitro biocompatibility of a composite biomaterial composed of 46S6 bioactive glass in association with chitosan (CH) by using 3D osteoblast culture of SaOS2. The 46S6 and CH composite (46S6-CH) forms small hydroxyapatite crystals on its surface after only three days immersion in the simulated body fluid. For 2D osteoblast culture, a significant increase in cell proliferation was observed after three days of contact with 46S6 or 46S6-CH-immersed media. After six days, 46S6-CH led to a significant increase in cell proliferation (128%) compared with pure 46S6 (113%) and pure CH (122%). For 3D osteoblast culture, after six days of culture, there was an increase in gene expression of markers of the early osteoblastic differentiation (RUNX2, ALP, COL1A1). Geometric structures corresponding to small apatite clusters were observed by SEM on the surface of the spheroids cultivated with 46S6 or 46S6-CH-immersed media. We showed different cellular responses depending on the 2D and 3D cell culture model. The induction of osteoblast differentiation in the 3D cell culture explained the differences of cell proliferation in contact with 46S6, CH or 46S6-CH-immersed media. This study confirmed that the 3D cell culture model is a very promising tool for in vitro biological evaluation of bone substitutes' properties.

  7. Activation of Wnt Planar Cell Polarity (PCP) signaling promotes growth plate column formation in vitro.

    Science.gov (United States)

    Randall, Rachel M; Shao, Yvonne Y; Wang, Lai; Ballock, R Tracy

    2012-12-01

    Disrupting the Wnt Planar Cell Polarity (PCP) signaling pathway in vivo results in loss of columnar growth plate architecture, but it is unknown whether activation of this pathway in vitro is sufficient to promote column formation. We hypothesized that activation of the Wnt PCP pathway in growth plate chondrocyte cell pellets would promote columnar organization in these cells that are normally oriented randomly in culture. Rat growth plate chondrocytes were transfected with plasmids encoding the Fzd7 cell-surface Wnt receptor, a Fzd7 deletion mutant lacking the Wnt-binding domain, or Wnt receptor-associated proteins Ror2 or Vangl2, and then cultured as three-dimensional cell pellets in the presence of recombinant Wnt5a or Wnt5b for 21 days. Cellular morphology was evaluated using histomorphometric measurements. Activation of Wnt PCP signaling components promoted the initiation of columnar morphogenesis in the chondrocyte pellet culture model, as measured by histomorphometric analysis of the column index (ANOVA p = 0.01). Activation of noncanonical Wnt signaling through overexpression of both the cell-surface Wnt receptor Fzd7 and receptor-associated protein Ror2 with addition of recombinant Wnt5a promotes the initiation of columnar architecture of growth plate chondrocytes in vitro, representing an important step toward growth plate regeneration. Copyright © 2012 Orthopaedic Research Society.

  8. SH-SY5Y human neuroblastoma cell line: in vitro cell model of dopaminergic neurons in Parkinson's disease.

    Science.gov (United States)

    Xie, Hong-rong; Hu, Lin-sen; Li, Guo-yi

    2010-04-20

    To evaluate the human neuroblastoma SH-SY5Y cell line as an in vitro model of dopaminergic (DAergic) neurons for Parkinson's disease (PD) research and to determine the effect of differentiation on this cell model. The data of this review were selected from the original reports and reviews related to SH-SY5Y cells published in Chinese and foreign journals (Pubmed 1973 to 2009). After searching the literature, 60 articles were selected to address this review. The SH-SY5Y cell line has become a popular cell model for PD research because this cell line posses many characteristics of DAergic neurons. For example, these cells express tyrosine hydroxylase and dopamine-beta-hydroxylase, as well as the dopamine transporter. Moreover, this cell line can be differentiated into a functionally mature neuronal phenotype in the presence of various agents. Upon differentiation, SH-SY5Y cells stop proliferating and a constant cell number is subsequently maintained. However, different differentiating agents induce different neuronal phenotypes and biochemical changes. For example, retinoic acid induces differentiation toward a cholinergic neuronal phenotype and increases the susceptibility of SH-SY5Y cells to neurotoxins and neuroprotective agents, whereas treatment with retinoic acid followed by phorbol ester 12-O-tetradecanoylphorbol-13-acetate results in a DAergic neuronal phenotype and decreases the susceptibility of cells to neurotoxins and neuroprotective agents. Some differentiating agents also alter kinetics of 1-methyl-4-phenyl-pyridinium (MPP(+)) uptake, making SH-SY5Y cells more similar to primary mesencephalic neurons. Differentiated and undifferentiated SH-SY5Y cells have been widely used as a cell model of DAergic neurons for PD research. Some differentiating agents afford SH-SY5Y cells with more potential for studying neurotoxicity and neuroprotection and are thus more relevant to experimental PD research.

  9. In vitro characterization of bone marrow stromal cells from osteoarthritic donors

    Directory of Open Access Journals (Sweden)

    Maik Stiehler

    2016-05-01

    Overall, the in vitro characteristics of BMSCs are not markedly influenced by OA. However, increased SOX9 and CD90 as well as reduced CD166 expression levels in OA-BMSCs warrant further investigation. These data will help to further understand the role of BMSC in OA and facilitate the application of autologous cell-based strategies for musculoskeletal tissue regeneration in OA patients.

  10. In vitro culture of various species of microsporidia causing keratitis: Evaluation of three immortalized cell lines

    Directory of Open Access Journals (Sweden)

    Joseph J

    2009-01-01

    Full Text Available Being intracellular parasites, microsporidia can only be propagated in cell culture systems. This study evaluated three cell lines to determine the most suitable host-parasite In vitro system. Confluent monolayers of vero, SIRC, and HeLa cell lines, grown in 24-well tissue culture plates, were inoculated with varying concentrations (1 x 10 4 to 1 x 10 8 spores/mL of Vittaforma corneae, Encephalitozoon hellem, Encephalitozoon cuniculi, and Encephalitozoon intestinalis spores. Growth was compared quantitatively at weekly intervals. Encephalitozoon species showed the highest amount of growth when cultured in vero cell line, while there was no significant difference in their growth in SIRC and HeLa cell lines. In comparison, V. corneae showed the highest growth in SIRC cells, followed by vero cells. The analytical sensitivity was found to be 1 x 10 4 spores/mL for vero cell line compared to 1 x 10 5 spores/mL for SIRC cell line and 1 x 10 7 spores/mL for HeLa cell line. HeLa cells also showed rapid disruption of cells, and the spores could not be easily distinguished from cell debris. This is the first report of the comparison of vero, SIRC, and HeLa for the propagation of microsporidial spores. Vero cell line was found to be more sensitive than SIRC and HeLa cells, and we believe that the inclusion of vero cell line in the routine culture protocols of ocular parasitology laboratories would result in a significant increase in the diagnostic yield.

  11. The effects of levofloxacin on rabbit anterior cruciate ligament cells in vitro

    International Nuclear Information System (INIS)

    Deng, Yu; Chen, Biao; Qi, Yongjian; Magdalou, Jacques; Wang, Hui; Chen, Liaobin

    2011-01-01

    Articular cartilage, epiphyseal growth plate and tendons have been recognized as targets of fluoroquinolone-induced connective tissue toxicity. The effects of fluoroquinolones on ligament tissues are still unknown. The aim of this study was to investigate the effects of levofloxacin, a typical fluoroquinolone antibiotic drug, on rabbit anterior cruciate ligament (ACL) cells in vitro. Rabbit ACL cells were treated with levofloxacin at different concentrations (0, 14, 28, 56, 112 and 224 μM) and were assessed to determine the possible cytotoxic effects of levofloxacin on ACL cells. Levofloxacin, with concentrations ranging from 28 to 224 μM, induced dose-dependent ACL cell apoptosis. Characteristic markers of programmed cell death and degenerative changes were identified by electron microscopy in the ACL cells treated with 28 μM of levofloxacin. Moreover, levofloxacin significantly increased the mRNA expression of matrix metalloproteinase 3 (MMP-3) and MMP-13 and decreased the expression of tissue inhibitors of metalloproteinase 1 (TIMP-1) in a concentration-dependent manner; TIMP-3 and collagen type I alpha 1 (Col1A1) mRNA expression was not affected. Immunocytochemical analysis indicated that levofloxacin markedly increased the expression of active caspase-3 within a concentration range of 28 to 224 μM, whereas a clear-cut decrease in Col1A1 expression was found with levofloxacin treatment concentrations of 112 and 224 μM, compared to controls. Our data suggest that levofloxacin has cytotoxic effects on ACL cells characterized by enhanced apoptosis and decreased extracellular matrix, which suggest a potential adverse effect of fluoroquinolones. -- Highlights: ► Levofloxacin has cytotoxic effect on rabbit ACL cells in vitro. ► Levofloxacin induces apoptosis in ACL cells. ► It decreases extracellular matrix by upregulation of matrix degrading enzymes. ► ACL cells are more susceptible to cytotoxicity by fluoroquinolones. ► Our study suggests a potential

  12. Translation of LINE-1 DNA elements in vitro and in human cells

    International Nuclear Information System (INIS)

    Leibold, D.M.; Swergold, G.D.; Thayer, R.E.; Singer, M.F.; Fanning, T.G.; Dombroski, B.A.

    1990-01-01

    The LINE-1(L1) family of interspread DNA sequences found throughout the human genome (L1 Homo sapiens, L1Hs) includes active transposable elements. Current models for the mechanism of transposition involve reverse transcription of an RNA intermediate and utilization of element-encoded proteins. The authors report that an antiserum against the polypeptide encoded by the L1Hs 5' open reading frame (ORF1) detects, in human cells, an endogenous ORF1 protein as well as the ORG1 product of an appropriate transfecting recombinant vector. The endogenous polypeptide is most abundant in teratocarcinoma and choriocarcinoma cells, among those cell lines tested; it appears to be a single species of ∼38 kDa. In contrast, RNAs synthesized in vitro from cDNAs representing full-length, polyadenylylated cytoplasmic L1Hs RNA yield, upon in vitro translation, ORF1 products of slightly different sizes. This is consistent with the fact that the various cDNAs are different and represent transcription of different genomic L1Hs elements. In vitro studies additionally suggest that translation of ORF1 is initiated at the first AUG codon. Finally, in no case was an ORF1-ORF2 fusion protein detected

  13. In vitro anti-tubulin effects of mebendazole and fenbendazole on canine glioma cells.

    Science.gov (United States)

    Lai, S R; Castello, S A; Robinson, A C; Koehler, J W

    2017-12-01

    Benzimidazole anthelmintics have reported anti-neoplastic effects both in vitro and in vivo. The purpose of this study was to evaluate the in vitro chemosensitivity of three canine glioma cell lines to mebendazole and fenbendazole. The mean inhibitory concentration (IC 50 ) (±SD) obtained from performing the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay after treating J3T, G06-A, and SDT-3G cells for 72 h with mebendazole were 0.030 ± 0.003, 0.080 ± 0.015 and 0.030 ± 0.006 μM respectively, while those for fenbendazole were 0.550  ± 0.015, 1.530 ± 0.159 and 0.690 ± 0.095 μM; treatment of primary canine fibroblasts for 72 h at IC 50 showed no significant effect. Immunofluorescence studies showed disruption of tubulin after treatment. Mebendazole and fenbendazole are cytotoxic in canine glioma cell lines in vitro and may be good candidates for treatment of canine gliomas. Further in vivo studies are required. © 2017 John Wiley & Sons Ltd.

  14. In vitro degradation and cell attachment studies of a new electrospun polymeric tubular graft.

    Science.gov (United States)

    Patel, Harsh N; Thai, Kevin N; Chowdhury, Sami; Singh, Raj; Vohra, Yogesh K; Thomas, Vinoy

    Electrospinning technique was utilized to engineer a small-diameter (id = 4 mm) tubular graft. The tubular graft was made from biocompatible and biodegradable polymers polycaprolactone (PCL) and poliglecaprone with 3:1 (PCL:PGC) ratio. Enzymatic degradation effect on the mechanical properties and fiber morphology in the presence of lipase enzyme were observed. Significant changes in tensile strength (1.86-1.49 MPa) and strain (245-205 %) were noticed after 1 month in vitro degradation. The fiber breakage was clearly evident through scanning electron microscopy (SEM) after 4 weeks in vitro degradation. Then, the graft was coated with a collagenous protein matrix to impart bioactivity. Human umbilical vein endothelial cells (HUVECs) and aortic artery smooth muscle cells (AoSMCs) attachment on the coated graft were observed in static condition. Further, HUVECs were seeded on the lumen surface of the grafts and exposed to laminar shear stress for 12 h to understand the cell attachment. The coated graft was aged in PBS solution (pH 7.3) at 37 °C for 1 month to understand the coating stability. Differential scanning calorimetry (DSC) and Fourier transform infrared spectroscopy (FTIR) suggested the erosion of the protein matrix from the coated graft under in vitro condition.

  15. The In Vitro Anti-Tumor Activity of Phycocyanin against Non-Small Cell Lung Cancer Cells

    Directory of Open Access Journals (Sweden)

    Shuai Hao

    2018-05-01

    Full Text Available Phycocyanin, a type of functional food colorant, is shown to have a potent anti-cancer property. Non-small cell lung cancer (NSCLC is one of the most aggressive form of cancers with few effective therapeutic options. Previous studies have demonstrated that phycocyanin exerts a growth inhibitory effect on NSCLC A549 cells. However, its biological function and underlying regulatory mechanism on other cells still remain unknown. Here, we investigated the in vitro function of phycocyanin on three typical NSCLC cell lines, NCI-H1299, NCI-H460, and LTEP-A2, for the first time. The results showed that phycocyanin could significantly induce apoptosis, cell cycle arrest, as well as suppress cell migration, proliferation, and the colony formation ability of NSCLC cells through regulating multiple key genes. Strikingly, phycocyanin was discovered to affect the cell phenotype through regulating the NF-κB signaling of NSCLC cells. Our findings demonstrated the anti-neoplastic function of phycocyanin and provided valuable information for the regulation of phycocyanin in NSCLC cells.

  16. The in Vitro Assessment of Biochemical Factors in Hepatocyte like Cells Derived from Umbilical Cord Blood Stem Cells

    Directory of Open Access Journals (Sweden)

    A KHoramroodi

    2009-10-01

    Full Text Available Introduction & Objective: Umbilical cord blood (UCB is a source of Hematopoietic Stem Cells (HSC and progenitor cells that can reconstitute the hematopoietic system in patients with malignant and nonmalignant disorders. Mesenchymal stem cell-derived from umbilical cord blood (UCB have been differentiated to some kind of cells, such as osteobblast, adipoblast and chondroblast in Vitro. This study examined the differentiation of Umbilical Cord Blood (UCB derived stem cells to functional hepatocytes. Materials & Methods: The present study was an experimental study which was carried out in the Payam-e-Noor University of Tehran in cooperation with Hamedan University of Medical Sciences in 2008. Umbilical cord blood (UCB was obtained from Fatemieh hospital (Hamadan, Iran. Stem cells were isolated from the cord blood by combining density gradient centrifugation with plastic adherence. When the isolated cells reached 80% confluence, they differentiated to hepatocyte like cells. The medium which was used was consists of DMEM and 10% Fetal Bovine Serum (FBS supplemented with 20 ng/mL Hepatocyte Growth Factor (HGF, 10 ng/mL basic Fibroblast Growth Factor (bFGF and 20 ng/mL Oncostatin M (OSM.The medium was changed every 3 days and stored for Albumin (ALB, Alpha Fetoprotein (AFP, Alkaline Phosphatase (ALP, and urea assay. Finally PAS stain was done to study Glycogen storage in the differentiated cell. Results: Measurement of biochemical factors in different days showed that concentration of albumin (ALB, alpha fetoprotein (AFP, alkaline phosphatase (ALP, and Urea gradually increased. Also, PAS staining showed the storage of glycogen in these cells. Conclusion: Stem cell-derived from human umbilical cord blood (HUCB is a new source of cell types for cell transplantation therapy of hepatic diseases and under certain conditions these cells can differentiate into liver cells.

  17. Fluoxetine Induces Proliferation and Inhibits Differentiation of Hypothalamic Neuroprogenitor Cells In Vitro

    Science.gov (United States)

    Sousa-Ferreira, Lígia; Aveleira, Célia; Botelho, Mariana; Álvaro, Ana Rita; Pereira de Almeida, Luís; Cavadas, Cláudia

    2014-01-01

    A significant number of children undergo maternal exposure to antidepressants and they often present low birth weight. Therefore, it is important to understand how selective serotonin reuptake inhibitors (SSRIs) affect the development of the hypothalamus, the key center for metabolism regulation. In this study we investigated the proliferative actions of fluoxetine in fetal hypothalamic neuroprogenitor cells and demonstrate that fluoxetine induces the proliferation of these cells, as shown by increased neurospheres size and number of proliferative cells (Ki-67+ cells). Moreover, fluoxetine inhibits the differentiation of hypothalamic neuroprogenitor cells, as demonstrated by decreased number of mature neurons (Neu-N+ cells) and increased number of undifferentiated cells (SOX-2+ cells). Additionally, fluoxetine-induced proliferation and maintenance of hypothalamic neuroprogenitor cells leads to changes in the mRNA levels of appetite regulator neuropeptides, including Neuropeptide Y (NPY) and Cocaine-and-Amphetamine-Regulated-Transcript (CART). This study provides the first evidence that SSRIs affect the development of hypothalamic neuroprogenitor cells in vitro with consequent alterations on appetite neuropeptides. PMID:24598761

  18. In vitro culture and characterization of a mammary epithelial cell line from Chinese Holstein dairy cow.

    Directory of Open Access Journals (Sweden)

    Han Hu

    Full Text Available BACKGROUND: The objective of this study was to establish a culture system and elucidate the unique characteristics of a bovine mammary epithelial cell line in vitro. METHODOLOGY: Mammary tissue from a three year old lactating dairy cow (ca. 100 d relative to parturition was used as a source of the epithelial cell line, which was cultured in collagen-coated tissue culture dishes. Fibroblasts and epithelial cells successively grew and extended from the culturing mammary tissue at the third day. Pure epithelial cells were obtained by passages culture. PRINCIPAL FINDINGS: The strong positive immunostaining to cytokeratin 18 suggested that the resulting cell line exhibited the specific character of epithelial cells. Epithelial cells cultured in the presence of 10% FBS, supraphysiologic concentrations of insulin, and hydrocortisone maintained a normal diploid chromosome modal number of 2n=60. Furthermore, they were capable of synthesizing beta-casein (CSN2, acetyl-CoA carboxylase-alpha (ACACA and butyrophilin (BTN1A1. An important finding was that frozen preservation in a mixture of 90% FBS and 10% DMSO did not influence the growth characteristics, chromosome number, or protein secretion of the isolated epithelial cell line. CONCLUSIONS: The obtained mammary epithelial cell line had normal morphology, growth characteristics, cytogenetic and secretory characteristics, thus, it might represent an useful tool for studying the function of Chinese Holstein dairy cows mammary epithelial cell (CMECs.

  19. TFF1 inhibits proliferation and induces apoptosis of gastric cancer cells in vitro

    Directory of Open Access Journals (Sweden)

    Yanli Ge

    2012-05-01

    Full Text Available Trefoil Factor Family (TFF plays an essential role in the intestinal epithelial restitution, but the relationship between TFF1 and gastric cancer (GC is still unclear. The present study aimed to determine the role of TFF1 in repairing gastric mucosa and in the pathogenesis of GC.The TFF1 expression in different gastric mucosas was measured with immunohistochemistry. Then, siRNA targeting TFF1 or plasmids expressing TFF1 gene were transfected into BGC823 cells, SGC7901 cells and GES-1 cells. The cell proliferation was detected with MTT assay and apoptosis and cell cycle measured by flow cytometry.From normal gastric mucosa to mucosa with dysplasia and to gastric cancer, the TFF1 expression had a decreasing trend. Down-regulation of TFF1 expression significantly reduced the apoptosis of three cell lines and markedly facilitated their proliferation but had no significant effect on cell cycle. Over-expression of TFF1 could promote apoptosis of three cell lines and inhibit proliferation but had no pronounced effect on cell cycle. TFF1 can inhibit proliferation and induce apoptosis of GC cells in vitro.

  20. KPNA7, a nuclear transport receptor, promotes malignant properties of pancreatic cancer cells in vitro

    International Nuclear Information System (INIS)

    Laurila, Eeva; Vuorinen, Elisa; Savinainen, Kimmo; Rauhala, Hanna; Kallioniemi, Anne

    2014-01-01

    Pancreatic cancer is an aggressive malignancy and one of the leading causes of cancer deaths. The high mortality rate is mostly due to the lack of appropriate tools for early detection of the disease and a shortage of effective therapies. We have previously shown that karyopherin alpha 7 (KPNA7), the newest member of the alpha karyopherin family of nuclear import receptors, is frequently amplified and overexpressed in pancreatic cancer. Here, we report that KPNA7 expression is absent in practically all normal human adult tissues but elevated in several pancreatic cancer cell lines. Inhibition of KPNA7 expression in AsPC-1 and Hs700T pancreatic cancer cells led to a reduction in cell growth and decreased anchorage independent growth, as well as increased autophagy. The cell growth effects were accompanied by an induction of the cell cycle regulator p21 and a G1 arrest of the cell cycle. Interestingly, the p21 induction was caused by increased mRNA synthesis and not defective nuclear transport. These data strongly demonstrate that KPNA7 silencing inhibits the malignant properties of pancreatic cancer cells in vitro and thereby provide the first evidence on the functional role for KPNA7 in human cancer. - Highlights: • KPNA7 expression is elevated in several pancreatic cancer cell lines. • KPNA7 silencing in high expressing cancer cells leads to growth inhibition. • The cell growth reduction is associated with p21 induction and G1 arrest. • KPNA7 silencing is also accompanied with increased autophagy

  1. Human Primary Intestinal Epithelial Cells as an Improved In Vitro Model for Cryptosporidium parvum Infection

    Science.gov (United States)

    Cabada, Miguel M.; Nichols, Joan; Gomez, Guillermo; White, A. Clinton

    2013-01-01

    The study of human intestinal pathogens has been limited by the lack of methods for the long-term culture of primary human intestinal epithelial cells (PECs). The development of infection models with PECs would allow a better understanding of host-parasite interactions. The objective of this study was to develop a novel method for prolonged in vitro cultivation of PECs that can be used to study Cryptosporidium infection. We isolated intact crypts from human intestines removed during weight loss surgery. The fragments of intestinal layers were cultivated with culture medium supplemented with growth factors and antiapoptotic molecules. After 7 days, the PECs formed self-regenerating cell clusters, forming villi that resemble intestinal epithelium. The PECs proliferated and remained viable for at least 60 days. The cells expressed markers for intestinal stem cells, epithelial cells, and mature enterocytes. The PECs were infected with Cryptosporidium. In contrast to older models in which parasite numbers decay, the burden of parasites increased for >120 h. In summary, we describe here a novel method for the cultivation of self-regenerating human epithelial cells from small intestinal crypts, which contain both intestinal stem cells and mature villus cells. We present data that suggest these cells support Cryptosporidium better than existing cell lines. PECs should provide an improved tool for studying host-parasite interactions involving Cryptosporidium and other intestinal pathogens. PMID:23509153

  2. An electron microscopy study of the diversity of Streptococcus sanguinis cells induced by lysozyme in vitro.

    Science.gov (United States)

    Hao, Yuqing; Li, Li; Li, Wei; Zhou, Xuedong; Lu, Junjun

    2010-01-01

    Bacterial virulence could be altered by the antimicrobial agents of the host. Our aim was to identify the damage and survival of Streptococcus sanguinis induced by lysozymes in vitro and to analyse the potential of oral microorganisms to shirk host defences, which cause infective endocarditis. S. sanguinis ATCC 10556 received lysozyme at concentrations of 12.5, 25, 50 and 100 microg/ml. Cells were examined by electron microscopy. The survival was assessed by colony counting and construction of a growth curve. Challenged by lysozymes, cells mainly exhibited cell wall damage, which seemed to increase with increasing lysozyme concentration and longer incubation period in the presence of ions. Cells with little as well as apparent lesion were observed under the same treatment set, and anomalous stick and huge rotund bodies were occasionally observed. After the removal of the lysozyme, some damaged cells could be reverted to its original form with brain heart infusion (BHI), and their growth curve was similar to the control cells. After further incubation in BHI containing lysozyme, S. sanguinis cell damage stopped progressing, and their growth curve was also similar to the control cells. The results suggested that the S. sanguinis lesions caused by the lysozyme in the oral cavity may be nonhomogeneous and that some damaged cells could self-repair and survive. It also indicated that S. sanguinis with damaged cell walls may survive and be transmitted in the bloodstream.

  3. In vitro developmental toxicity test detects inhibition of stem cell differentiation by silica nanoparticles

    International Nuclear Information System (INIS)

    Park, Margriet V.D.Z.; Annema, Wijtske; Salvati, Anna; Lesniak, Anna; Elsaesser, Andreas; Barnes, Clifford; McKerr, George; Howard, C. Vyvyan; Lynch, Iseult; Dawson, Kenneth A.; Piersma, Aldert H.; Jong, Wim H. de

    2009-01-01

    While research into the potential toxic properties of nanomaterials is now increasing, the area of developmental toxicity has remained relatively uninvestigated. The embryonic stem cell test is an in vitro screening assay used to investigate the embryotoxic potential of chemicals by determining their ability to inhibit differentiation of embryonic stem cells into spontaneously contracting cardiomyocytes. Four well characterized silica nanoparticles of various sizes were used to investigate whether nanomaterials are capable of inhibition of differentiation in the embryonic stem cell test. Nanoparticle size distributions and dispersion characteristics were determined before and during incubation in the stem cell culture medium by means of transmission electron microscopy (TEM) and dynamic light scattering. Mouse embryonic stem cells were exposed to silica nanoparticles at concentrations ranging from 1 to 100 μg/ml. The embryonic stem cell test detected a concentration dependent inhibition of differentiation of stem cells into contracting cardiomyocytes by two silica nanoparticles of primary size 10 (TEM 11) and 30 (TEM 34) nm while two other particles of primary size 80 (TEM 34) and 400 (TEM 248) nm had no effect up to the highest concentration tested. Inhibition of differentiation of stem cells occurred below cytotoxic concentrations, indicating a specific effect of the particles on the differentiation of the embryonic stem cells. The impaired differentiation of stem cells by such widely used particles warrants further investigation into the potential of these nanoparticles to migrate into the uterus, placenta and embryo and their possible effects on embryogenesis.

  4. Responses to hyperthermia (420, 440) and/or radiation in four mammalian cell lines in vitro

    International Nuclear Information System (INIS)

    Miyakoshi, Junji

    1981-01-01

    Survival in response to hyperthermia at 42 and 44 0 C, both alone and in combination with X-irradiation was examined in vitro in Chinese hamster V-79, HeLa-S3, murine Ehrlich ascites tumor cells (EH) and murine L-fibroblasts. L-cells were markedly thermo- and radiosensitive, while the other three cell lines, although not so sensitive did show similar responses. When each cell line was exposed to split dose Hyperthermia in the 42 → 44 0 C sequence, survival after the second treatment was increased for V-79 and HeLa-S3 cells, but was not significantly changed for EH and L-cells. In the case of split dose exposure in the 44 → 42 0 C sequence, survival after the second treatment was markedly decreased for V-79, EH and HeLa-S3 cells, but only slightly for L-cells. When hyperthermia at 42 or 44 0 C was followed by X-irradiation immediately, V-79, EH and HeLa-S3 cells showed decrements in both D sub(q) and D sub(o) values, while L-cells showed a decrement only in D sub(q) but no significant change in D sub(o). From these results, it seems that the hyperthermic damage by exposure to 44 0 C may be different from that by exposure to 42 0 C. (author)

  5. An Epigenetic Modifier Results in Improved In Vitro Blastocyst production after Somatic Cell Nuclear Transfer

    DEFF Research Database (Denmark)

    Zhang, Yunhai; Li, Juan; Villemoes, Klaus

    2007-01-01

    The present study was designed to examine the effect of trichostatin A (TSA), an inhibitor of histone deacetylase, on development of porcine cloned embryos. Our results showed that treatment of cloned embryos derived from sow oocytes with 50 nM TSA for up to 24 h after the onset of activation cou...... were tested, and for all cell lines an enhancement in blastocyst development compared to their corresponding control was observed. Our data demonstrate that TSA treatment after somatic cell nuclear transfer in the pig can significantly improve the in vitro blastocyst production...

  6. Improved modification for in vitro labeling of red blood cells with Technetium-99m

    International Nuclear Information System (INIS)

    Gerson, B.; Ballinger, J.R.; Gulenchyn, K.Y.

    1988-01-01

    The authors have tested a modification of Brookhaven method for in vitro labeling of red blood cells (RBCs) with technetium-99m by adding an initial centrifugation step and performing the labeling on packed RBCs. This results in reproducible, high labeling efficiencies (99.3% +/- 0.4%, n = 50) after 15 min of incubation. The use of packed RBCs also results in a higher concentration of labeled RBCs (smaller bolus for injection) and less radiation exposure to the technologist. This technique has proved useful for radionuclide angiography, venography, gastrointestinal bleeding studies, and red cell mass determinations. It is particularly advantageous for RBC labeling in patients receiving chemotherapy

  7. Arctigenin inhibits prostate tumor cell growth in vitro and in vivo

    OpenAIRE

    Piwen Wang; Walter Solorzano; Tanya Diaz; Clara E. Magyar; Susanne M. Henning; Jaydutt V. Vadgama

    2017-01-01

    The low bioavailability of most phytochemicals limits their translation to humans. We investigated whether arctigenin, a novel anti-inflammatory lignan from the seeds of Arctium lappa, has favorable bioavailability/potency against prostate cancer. The anticarcinogenic activity of arctigenin was investigated both in vitro using the androgen-sensitive LNCaP and LAPC-4 human prostate cancer cells and pre-malignant WPE1-NA22 cells, and in vivo using xenograft mouse models. Arctigenin at lower dos...

  8. Hyperosmolarity potentiates toxic effects of benzalkonium chloride on conjunctival epithelial cells in vitro.

    Science.gov (United States)

    Clouzeau, Chloé; Godefroy, David; Riancho, Luisa; Rostène, William; Baudouin, Christophe; Brignole-Baudouin, Françoise

    2012-01-01

    Benzalkonium chloride (BAK), the most commonly used preservative in eye drops, is known to induce ocular irritation symptoms and dry eye in long-term treated patients and animal models. As tear film hyperosmolarity is diagnostic of some types of dry eye disease, we determined in vitro on conjunctival epithelial cells the cytoxicity of BAK in hyperosmolar conditions through cell viability, apoptosis, and oxidative stress assays. The Wong Kilbourne derivative of Chang conjunctival epithelial cells were cultured for 24 h or 48 h either in NaCl-induced hyperosmolar conditions (400-425-500 mOsM), in low concentrations of BAK (10(-4)%, 3.10(-4)%, and 5.10(-4)%), or in combination of both. We investigated cell viability through lysosomal integrity evaluation, cell death (cell membrane permeability and chromatin condensation), and oxidative stress (reactive oxygen species, superoxide anion) using spectrofluorimetry. Immunohistochemistry was performed for cytoskeleton shrinkage (phalloidin staining), mitochondrial permeability transition pore (cytochrome c release), the apoptosis effector active caspase-3, and the caspase-independent apoptosis factor AIF. We also observed early effects induced by the experimental conditions on the conjunctival cell layers using phase contrast imaging of live cells. As compared to standard culture solutions, hyperosmolar stress potentiated BAK cytotoxicity on conjunctival cells through the induction of oxidative stress; reduction of cell viability; cell membrane permeability increase; cell shrinkage with cell blebbing, as shown in phase contrast imaging of live cells; and chromatin condensation. Like BAK, but to a much lesser extent, hyperosmolarity increased cell death in a concentration-dependent manner through a caspase-dependent apoptosis characterized by a release of cytochrome c in the cytoplasm from mitochondria and the activation of caspase-3. Moreover, the caspase-independent apoptosis factor AIF was found translocated from

  9. Migratory and adhesive properties of Xenopus laevis primordial germ cells in vitro

    Directory of Open Access Journals (Sweden)

    Aliaksandr Dzementsei

    2013-11-01

    The directional migration of primordial germ cells (PGCs to the site of gonad formation is an advantageous model system to study cell motility. The embryonic development of PGCs has been investigated in different animal species, including mice, zebrafish, Xenopus and Drosophila. In this study we focus on the physical properties of Xenopus laevis PGCs during their transition from the passive to the active migratory state. Pre-migratory PGCs from Xenopus laevis embryos at developmental stages 17–19 to be compared with migratory PGCs from stages 28–30 were isolated and characterized in respect to motility and adhesive properties. Using single-cell force spectroscopy, we observed a decline in adhesiveness of PGCs upon reaching the migratory state, as defined by decreased attachment to extracellular matrix components like fibronectin, and a reduced adhesion to somatic endodermal cells. Data obtained from qPCR analysis with isolated PGCs reveal that down-regulation of E-cadherin might contribute to this weakening of cell-cell adhesion. Interestingly, however, using an in vitro migration assay, we found that movement of X. laevis PGCs can also occur independently of specific interactions with their neighboring cells. The reduction of cellular adhesion during PGC development is accompanied by enhanced cellular motility, as reflected in increased formation of bleb-like protrusions and inferred from electric cell-substrate impedance sensing (ECIS as well as time-lapse image analysis. Temporal alterations in cell shape, including contraction and expansion of the cellular body, reveal a higher degree of cellular dynamics for the migratory PGCs in vitro.

  10. Coenzyme Q10 protects retinal cells from apoptosis induced by radiation in vitro and in vivo

    International Nuclear Information System (INIS)

    Lulli, M.; Witort, E.; Papucci, L.; Torre, E.; Schiavone, N.; Capaccioli, S.; Dal Monte, M.

    2012-01-01

    The key pathogenetic event of many retinopathies is apoptosis of retinal cells. Our previous studies have demonstrated that Coenzyme Q10 (CoQ10) prevents apoptosis of corneal keratocytes both in vitro and in vivo, by virtue of its ability to inhibit mitochondrial depolarization, independently of its free radical scavenger role. The aim of this study was to evaluate whether CoQ10 can protect cultured retinal cells and the retinas of rats from radiation-induced apoptosis, if instilled as eye drops in the cornea. In vitro experiments were carried out on cultured ARPE-19 or retinal ganglion cells (RGC)-5 cells pretreated with CoQ10 before eliciting apoptosis by ultraviolet (UV)- and γ-radiation, chemical hypoxia (Antimycin A) and serum starvation. Cell viability was evaluated by light microscopy and fluorescence activated cell sorting analysis. Apoptotic events were scored by time-lapse videomicroscopy. Mitochondrial permeability transition was evaluated by JC-1. The anti-apoptotic effectiveness of CoQ10 in retina was also evaluated by an in situ end-labeling assay in Wistar albino rats treated with CoQ10 eye drops prior to UV irradiation of the eye. CoQ10 substantially increased cell viability and lowered retinal cell apoptosis in response both to UV- and γ-radiation and to chemical hypoxia or serum starvation by inhibiting mitochondrion depolarization. In the rat, CoQ10, even when applied as eye drops on the cornea, protected all retina layers from ultraviolet radiation (UVR)-induced apoptosis. The ability of CoQ10 to protect retinal cells from radiation-induced apoptosis following its instillation on the cornea suggests the possibility for CoQ10 eye drops to become a future therapeutic countermeasure for radiation-induced retinal lesions. (author)

  11. In vitro gamma irradiation Medical Center of leukemic cells in mice, rats, and guinea pigs

    International Nuclear Information System (INIS)

    Gross, L.; Dreyfuss, Y.; Ehrenreich, T.; Feldman, D.; Limbert, L.M.

    1980-01-01

    In vitro gamma irradiation of virus-induced (Gross) mouse leukemia cells at doses of 350 to 1600 rads (1 rad = 0.01 gray) had no effect on their ability to induce leukemia, usually within 2 weeks, after transplantation into syngeneic mice. However, when cells irradiated at doses of 2000-20,000 rads were transplanted, they induced leukemia after a latency period exceeding 2.5 months, similar to the results observed in mice inoculated with filtered mouse leukemia extracts. Similar results were also obtained after irradiation of leukemic cells derived from rats in which leukemia had been induced by rat-adapted mouse leukemia virus. Apparently, gamma irradiation at a dose of, or exceeding, 2000 rads, inhibits the ability of mouse and rat leukemic cells to induce leukemia after transplantation into syngeneic hosts; however, it does not inactivate the virus carried by such cells nor prevent it from inducing leukemia. [In previous experiments, doses of more than 4,500,000 rads were needed to inactivate the passage A (Gross) leukemia virus carried in either mouse or rat leukemic cells.] In vitro gamma irradiation of L2C guinea pig leukemic cells at doses of 750 to 2500 rads had no apparent effect on their ability to induce leukemia after transplantation into strain 2 guinea pigs. However, irradiation at doses of 3250 to 20,000 rads inactivated their ability to do so. The morphology of mouse, rat, and guinea pig leukemic cells and the virus particles present in such cells was not affected by irradiation at doses of 20,000 rads

  12. Optimal Population of Embryonic Stem Cells in "Hanging Drop" Culture for in-vitro Differentiation to Cardiac Myocytes

    OpenAIRE

    MIWA, Keiko; LEE, Jong-Kook; HIDAKA, Kyoko; SHI, Rong-qian; MORISAKI, Takayuki; KODAMA, Itsuo

    2002-01-01

    Pluripotent embryonic stem (ES) cells differentiate to cardiac myocytes in vitro by many other previous reports demonstrated "hanging-drop" method. In this study, the number of ES cells in each hanging-drop plays an important role in the cultivation of cardiac myocytes. We examined the optimal hanging-drop size to obtain embryonic stem cell-derived cardiac cells (ESCMs) in vitro using specific labeled mouse ES cells (hCGP7) which were stably transfected with the enhanced green fluorescent pro...

  13. In vitro generation of renal tubular epithelial cells from fibroblasts: implications for precision and regenerative medicine in nephrology.

    Science.gov (United States)

    Wyatt, Christina M; Dubois, Nicole

    2017-02-01

    Prior efforts to generate renal epithelial cells in vitro have relied on pluripotent or bone marrow-derived mesenchymal stem cells. A recent publication in Nature Cell Biology describes the generation of induced tubular epithelial cells from fibroblasts, potentially offering a novel platform for personalized drug toxicity screening and in vitro disease modeling. This report serves as a promising proof of principle study and opens future research directions, including the optimization of the reprogramming process, efficient translation to adult human fibroblasts, and the generation of highly specific functional renal cell types. Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

  14. Human adipose cells in vitro are either refractory or responsive to insulin, reflecting host metabolic state.

    Directory of Open Access Journals (Sweden)

    Vladimir A Lizunov

    Full Text Available While intercellular communication processes are frequently characterized by switch-like transitions, the endocrine system, including the adipose tissue response to insulin, has been characterized by graded responses. Yet here individual cells from adipose tissue biopsies are best described by a switch-like transition between the basal and insulin-stimulated states for the trafficking of the glucose transporter GLUT4. Two statistically-defined populations best describe the observed cellular heterogeneity, representing the fractions of refractive and responsive adipose cells. Furthermore, subjects exhibiting high systemic insulin sensitivity indices (SI have high fractions of responsive adipose cells in vitro, while subjects exhibiting decreasing SI have increasing fractions of refractory cells in vitro. Thus, a two-component model best describes the relationship between cellular refractory fraction and subject SI. Since isolated cells exhibit these different response characteristics in the presence of constant culture conditions and milieu, we suggest that a physiological switching mechanism at the adipose cellular level ultimately drives systemic SI.

  15. Embryonic stem-like cells derived from in vitro produced bovine blastocysts

    Directory of Open Access Journals (Sweden)

    Erika Regina Leal de Freitas

    2011-06-01

    Full Text Available The aim of this work was to study the derivation of bovine embryonic stem-like (ES-like cells from the inner cell mass (ICM of in vitro produced blastocysts. The ICMs were mechanically isolated and six out of seventeen (35% ICMs could attach to a monolayer of murine embryonic fibroblasts (MEF. Ten days after, primary outgrowths were mechanically dissected into several small clumps and transferred to a new MEF layer. Cells were further propagated and passaged by physical dissociation over a 60 days period. The pluripotency of the bovine ES-like cells was confirmed by RT-PCR of Oct-4 and STAT-3 gene markers. The colonies were weakly stained for alkaline phosphatase and the mesoderm and endoderm differentiation gene markers such as GATA-4 and Flk-1, respectively, were not expressed. Embryoid bodies were spontaneously formed at the seventh passage. Results showed that bovine ES-like cells could be obtained and passaged by mechanical procedures from the fresh in vitro produced blastocysts.

  16. Schiff base-Poloxamer P85 combination demonstrates chemotherapeutic effect on prostate cancer cells in vitro.

    Science.gov (United States)

    Demirci, Selami; Doğan, Ayşegül; Türkmen, Neşe Başak; Telci, Dilek; Rizvanov, Albert A; Şahin, Fikrettin

    2017-02-01

    Prostate cancer is a multistep and complicated cancer type that is regulated by androgens at the cellular level and remains the second commonest cause of death among men. Discovery and development of novel chemotherapeutic agents enabling rapid tumor cell death with minimal toxic effects to healthy tissues might greatly improve the safety of chemotherapy. The present study evaluates the anti-cancer activity of a novel heterodinuclear copper(II)Mn(II) complex (Schiff base) in combination with poly(ethylene oxide) and poly(propylene oxide) block copolymer (Pluronic) P85. We used assays for cell proliferation, apoptosis, cell migration and invasion, DNA binding and cleavage to elucidate the molecular mechanisms of action, in addition to the anti-inflammatory potency of the new combination. The combined treatment of Schiff base and P85 lead to a remarkable anti-cancer effect on prostate cancer cell lines. Cell proliferation was inhibited in Schiff base-P85 treatment. The activity of this formulation is on DNA binding and cleavage and prevents inflammation in in vitro conditions. This is the first study presenting the anti-cancer activity of the present Schiff base derivative and its combination with P85 to treat prostate cancer in vitro. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  17. Furano diterpenes from Pterodon pubescens Benth with selective in vitro anticancer activity for prostate cell line

    International Nuclear Information System (INIS)

    Spindola, Humberto M.; Carvalho, Joao E. de; Ruiz, Ana Lucia T.G.; Rodrigues, Rodney A. F.; Denny, Carina; Sousa, Ilza M. de Oliveira; Foglio, Mary Ann; Tamashiro, Jorge Y.

    2009-01-01

    Activity guided fractionation of Pterodon pubescens Benth. methylene chloride-soluble fraction afforded novel 6α-acetoxi 7β-hydroxy-vouacapan 1 and four known diterpene furans 2, 3, 4, 5. The compounds were evaluated for in vitro cytotoxic activities against human normal cells and tumour cell lines UACC-62 (melanoma), MCF-7 (breast), NCI-H460 (lung, non-small cells), OVCAR-03 (ovarian), PC-3 (prostate), HT-29 (colon), 786-0 (renal), K562 (leukemia) and NCI-ADR/RES (ovarian expressing phenotype multiple drugs resistance). Results were expressed by three concentration dependent parameters GI 50 (concentration that produces 50% growth inhibition), TGI (concentration that produces total growth inhibition or cytostatic effect) and LC 50 (concentration that produces .50% growth, a cytotoxicity parameter). Also, in vitro cytotoxicity was evaluated against 3T3 cell line (mouse embryonic fibroblasts). Antiproliferative properties of compounds 1, 4 and 5 are herein reported for the first time. These compounds showed selectivity in a concentration-dependent way against human PC-3. Compound 1 demonstrated selectivity 26 fold more potent than the positive control, doxorubicin, for PC-3 (prostrate) cell line based on GI 50 values, causing cytostatic effect (TGI value) at a concentration fifteen times less than positive control. Moreover comparison of 50% lethal concentration (LC 50 value) with positive control (doxorubicin) suggested that compound 1 was less toxic. (author)

  18. Toxic effect of C60 fullerene-doxorubicin complex towards tumor and normal cells in vitro

    Directory of Open Access Journals (Sweden)

    Prylutska S. V.

    2014-09-01

    Full Text Available Creation of new nanostructures possessing high antitumor activity is an important problem of modern biotechnology. Aim. To evaluate cytotoxicity of created complex of pristine C60 fullerene with the anthracycline antibiotic doxorubicin (Dox, as well as of free C60 fullerene and Dox, towards different cell types – tumor, normal immunocompetent and hepatocytes. Methods. Measurement of size distribution for particles in C60 + Dox mixture was performed by a dynamic light scattering (DLS technique. Toxic effect of C60 + Dox complex in vitro towards tumor and normal cells was studied using the MTT assay. Results. DLS experiment demonstrated that the main fraction of the particles in C60 + Dox mixture had a diameter in the range of about 132 nm. The toxic effect of C60 + Dox complex towards normal (lymphocytes, macrophages, hepatocytes and tumor (Ehrlich ascites carcinoma, leukemia L1210, Lewis lung carcinoma cells was decreased by ~10–16 % and ~7–9 %, accordingly, compared with the same effect of free Dox. Conclusions. The created C60 + Dox composite may be considered as a new pharmacological agent that kills effectively tumor cells in vitro and simultaneously prevents a toxic effect of the free form of Dox on normal cells.

  19. Ensuring the Quality of Stem Cell-Derived In Vitro Models for Toxicity Testing.

    Science.gov (United States)

    Stacey, Glyn N; Coecke, Sandra; Price, Anna-Bal; Healy, Lyn; Jennings, Paul; Wilmes, Anja; Pinset, Christian; Ingelman-Sundberg, Magnus; Louisse, Jochem; Haupt, Simone; Kidd, Darren; Robitski, Andrea; Jahnke, Heinz-Georg; Lemaitre, Gilles; Myatt, Glenn

    Quality control of cell cultures used in new in vitro toxicology assays is crucial to the provision of reliable, reproducible and accurate toxicity data on new drugs or constituents of new consumer products. This chapter explores the key scientific and ethical criteria that must be addressed at the earliest stages of developing toxicology assays based on human pluripotent stem cell (hPSC) lines. It also identifies key considerations for such assays to be acceptable for regulatory, laboratory safety and commercial purposes. Also addressed is the development of hPSC-based assays for the tissue and cell types of greatest interest in drug toxicology. The chapter draws on a range of expert opinion within the European Commission/Cosmetics Europe-funded alternative testing cluster SEURAT-1 and consensus from international groups delivering this guidance such as the International Stem Cell Banking Initiative. Accordingly, the chapter summarizes the most up-date best practices in the use and quality control of human Pluripotent Stem Cell lines in the development of in vitro toxicity assays from leading experts in the field.

  20. In vitro radiation and chemotherapy sensitivity of established cell lines of human small cell lung cancer and its large cell morphological variants

    International Nuclear Information System (INIS)

    Carney, D.N.; Mitchell, J.B.; Kinsella, T.J.

    1983-01-01

    The in vitro response to radiation and chemotherapeutic drugs of cell lines established from 7 patients with small cell (SC) lung cancer were tested using a soft agarose clonogenic assay. Five cell lines retained the typical morphological and biochemical amine precursor uptake decarboxylation characteristics of SC, while two cell lines had undergone ''transformation'' to large cell (LC) morphological variants with loss of amine precursor uptake decarboxylation cell characteristics of SC. The radiation survival curves for the SC lines were characterized by D0 values ranging from 51 to 140 rads and extrapolation values (n) ranging from 1.0 to 3.3. While the D0 values of the radiation survival curves of the LC variants were similar (91 and 80 rads), the extrapolation values were 5.6 and 11.1 In vitro chemosensitivity testing of the cell lines revealed an excellent correlation between prior treatment status of the patient and in vitro sensitivity or resistance. No correlation was observed between in vitro chemosensitivity and radiation response. These data suggest that transformation of SC to LC with loss of amine precursor uptake and decarboxylation characteristics is associated with a marked increase in radiation resistance (n) in vitro. The observation of a 2- to 5-fold increase in survival of the LC compared to the SC lines following 200 rads suggests that the use of larger daily radiation fractions and/or radiation-sensitizing drugs might lead to a significantly greater clinical response in patients with LC morphology. This clinical approach may have a major impact on patient response and survival

  1. Direct Cell-Cell Contact between Mesenchymal Stem Cells and Endothelial Progenitor Cells Induces a Pericyte-Like Phenotype In Vitro

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    Markus Loibl

    2014-01-01

    Full Text Available Tissue engineering techniques for the regeneration of large bone defects require sufficient vascularisation of the applied constructs to ensure a sufficient supply of oxygen and nutrients. In our previous work, prevascularised 3D scaffolds have been successfully established by coculture of bone marrow derived stem cells (MSCs and endothelial progenitor cells (EPCs. We identified stabilising pericytes (PCs as part of newly formed capillary-like structures. In the present study, we report preliminary data on the interactions between MSCs and EPCs, leading to the differentiation of pericyte-like cells. MSCs and EPCs were seeded in transwell cultures, direct cocultures, and single cultures. Cells were cultured for 10 days in IMDM 10% FCS or IMDM 5% FCS 5% platelet lysate medium. Gene expression of PC markers, CD146, NG2, αSMA, and PDGFR-β, was analysed using RT-PCR at days 0, 3, 7, and 10. The upregulation of CD146, NG2, and αSMA in MSCs in direct coculture with EPCs advocates the MSCs’ differentiation towards a pericyte-like phenotype in vitro. These results suggest that pericyte-like cells derive from MSCs and that cell-cell contact with EPCs is an important factor for this differentiation process. These findings emphasise the concept of coculture strategies to promote angiogenesis for cell-based tissue engineered bone grafts.

  2. Radiosensitizing potential of gemcitabine (2',2'-difluoro-2'-deoxycytidine) within the cell cycle in vitro

    International Nuclear Information System (INIS)

    Latz, Detlev; Fleckenstein, Katharina; Eble, Michael; Blatter, Johannes; Wannenmacher, Michael; Weber, Klaus J.

    1998-01-01

    Purpose: Gemcitabine (2',2'-difluorodeoxycytidine; dFdCyd) is a new deoxycitidine analog which exhibits substantial activity against solid tumors and radiosensitizing properties in vitro. To examine cell cycle-specific effects of a combined treatment with gemcitabine and radiation, the in vitro clonogenic survival of two different cell lines was measured for cells from log-phase culture, G1 and S-phase cells. Methods and Materials: Chinese hamster (V79) and human colon carcinoma (Widr) cells were exposed to different radiation doses and for different points of time relative to gemcitabine treatment (2 h). Experiments were also carried out with different cell-cycle populations obtained after mitotic selection (V79) or after serum stimulation of plateau-phase cells (Widr). The resulting survival curves were analyzed according to the LQ model, and mean inactivation doses (MID) and the cell cycle-specific enhancement ratios (ER) were calculated from the survival curve parameters. Results: Effectiveness of combined treatment of log-phase cells was greatest when cells were irradiated at the end of the gemcitabine exposure [ER: 1.28 (V79), 1.24 (Widr)]. For later times after the removal of the drug, radiosensitization declined, approaching independent toxicity. From the time course of interactive-type damage decay half-life values of 75 min (V79) and 92 min (Widr) were derived. Gemcitabine did not radiosensitize G1 Widr cells or V79 cells from the G1/S border, but substantial radiosensitization was observed for the S-phase cell preparations [ER: 1.45 (V79-lateS), 1.57 (Widr)]. Conclusions: Treatment of cells with gemcitabine immediately before irradiation eliminates, or at least greatly reduces, the variation in radiosensitivity during the cell cycle that is manifested by radioresistance during S phase. This reversal of S-phase radioresistance could imply that gemcitabine interferes with the potentially lethal damage repair/fixation pathway. Other approaches have been

  3. Culture of Dendritic Cells in vitro and Its Anti-tumor Immonotherapy

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    Yanwen ZHOU

    2010-05-01

    Full Text Available Background and objective Immunocompromised patients with malignant tumor always lack of strong anti-tumor immune response, because the antigenicity of tumor cells is weak, and antigen-presenting cell function is low, so that can not be effectively presenting tumor antigens to the lymphocytes. Therefore, how to effectively induce anti-tumor immune response is the key issue. Through the study on establishing a method to culture dendritic cells (DC in vitro and to observe the anti-lung cancer immunological effect induced by DC, we provided definite experiment basis for the clinic application of vaccine based on DC. Methods Through the experiment we get the soluble antigen polypeptide from lung cancer cells GLC-82 by 3 mol/L potassium chloride. DCs are cultured and obtained from peripheral blood mononuclear cell by GM-CSF, IL-4 and TNF-a. DCs are identified by flow cytometer (FCM and immunostaining. DCs modified by lung cancer tumor soluble antigen (TSA and staphylococcal enterotox in A (SEA, DCs modified by TSA or DCs modified by SEA or DCs modified by nothing were cultivated together with T lymphocyte, and the obtained cells are named TSA-SEA-DCL or TSA-DCL or SEA-DCL or DCL as effector cells. The anti-tumor activity of every effector cells against target cells was assayed with MTT method. Shape of DCs and effector cells, and the process of killing target cells were observed in microscope. Results Induced DCs expressed more CD1a, CD80 and HLA-DR, which had typical cell traits such as tree branch. The killing ratio of the TSA-SEA-DCL in vitro to GLC-82 is larger than TSA-DCL, SEA-DCL and DCL, also larger than to K562. When the effector cells cultivate with target cells, we can observe the CTL approach and gather to the cancer cell, induce it necrosis and apoptosis. Conclusion Ripe DCs that have typical characteristic and phenotype could be induced successfully. High potency and relatively specific antilung caner effect can be prepared in virtue of

  4. [Analysis on clone in vitro and tumorigenic capacity in vivo of different subsets cells from the MCF-7 human breast cancer cell line].

    Science.gov (United States)

    Li, Zhi; Liu, Chun-ping; He, Yan-li; Tian, Yuan; Huang, Tao

    2008-07-01

    To investigate whether there are cancer stem cells in the MCF-7 human breast cancer cell line. Flow cytometry was applied to separate different subpopulation cells from MCF-7 cells, and their ability of clone in vitro and reconstruction tumor in vivo were determined. The ability of clone in vitro and reconstruction tumor in vivo were observed in some MCF-7 cells. Contrast with CD44+ CD24+ cells, the proportion of tumorigenic cancer cells in CD44+ CD24- cells is higher. Breast cancer stem cell exists in MCF-7 and it mainly locates the subpopulation of CD44+ CD24- cells, CD44+ CD24+ cell possibly is breast cancer progenitor cell.

  5. Articular cartilage-derived cells hold a strong osteogenic differentiation potential in comparison to mesenchymal stem cells in vitro

    International Nuclear Information System (INIS)

    Salamon, Achim; Jonitz-Heincke, Anika; Adam, Stefanie; Rychly, Joachim; Müller-Hilke, Brigitte; Bader, Rainer; Lochner, Katrin; Peters, Kirsten

    2013-01-01

    Cartilaginous matrix-degenerative diseases like osteoarthritis (OA) are characterized by gradual cartilage erosion, and also by increased presence of cells with mesenchymal stem cell (MSC) character within the affected tissues. Moreover, primary chondrocytes long since are known to de-differentiate in vitro and to be chondrogenically re-differentiable. Since both findings appear to conflict with each other, we quantitatively assessed the mesenchymal differentiation potential of OA patient cartilage-derived cells (CDC) towards the osteogenic and adipogenic lineage in vitro and compared it to that of MSC isolated from adipose tissue (adMSC) of healthy donors. We analyzed expression of MSC markers CD29, CD44, CD105, and CD166, and, following osteogenic and adipogenic induction in vitro, quantified their expression of osteogenic and adipogenic differentiation markers. Furthermore, CDC phenotype and proliferation were monitored. We found that CDC exhibit an MSC CD marker expression pattern similar to adMSC and a similar increase in proliferation rate during osteogenic differentiation. In contrast, the marked reduction of proliferation observed during adipogenic differentiation of adMSC was absent in CDC. Quantification of differentiation markers revealed a strong osteogenic differentiation potential for CDC, however almost no capacity for adipogenic differentiation. Since in the pathogenesis of OA, cartilage degeneration coincides with high bone turnover rates, the high osteogenic differentiation potential of OA patient-derived CDC may affect clinical therapeutic regimens aiming at autologous cartilage regeneration in these patients. - Highlights: • We analyze the mesenchymal differentiation capacity of cartilage-derived cells (CDC). • CDC express mesenchymal stem cell (MSC) markers CD29, CD44, CD105, and CD166. • CDC and MSC proliferation is reduced in adipogenesis and increased in osteogenesis. • Adipogenic differentiation is virtually absent in CDC, but

  6. Articular cartilage-derived cells hold a strong osteogenic differentiation potential in comparison to mesenchymal stem cells in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Salamon, Achim, E-mail: achim.salamon@med.uni-rostock.de [Department of Cell Biology, Rostock University Medical Center, Schillingallee 69, D-18057 Rostock (Germany); Jonitz-Heincke, Anika, E-mail: anika.jonitz@med.uni-rostock.de [Biomechanics and Implant Technology Research Laboratory, Department of Orthopedics, Rostock University Medical Center, Doberaner Straße 142, D-18057 Rostock (Germany); Adam, Stefanie, E-mail: stefanie.adam@med.uni-rostock.de [Department of Cell Biology, Rostock University Medical Center, Schillingallee 69, D-18057 Rostock (Germany); Rychly, Joachim, E-mail: joachim.rychly@med.uni-rostock.de [Department of Cell Biology, Rostock University Medical Center, Schillingallee 69, D-18057 Rostock (Germany); Müller-Hilke, Brigitte, E-mail: brigitte.mueller-hilke@med.uni-rostock.de [Institute of Immunology, Rostock University Medical Center, Schillingallee 68, D-18057 Rostock (Germany); Bader, Rainer, E-mail: rainer.bader@med.uni-rostock.de [Biomechanics and Implant Technology Research Laboratory, Department of Orthopedics, Rostock University Medical Center, Doberaner Straße 142, D-18057 Rostock (Germany); Lochner, Katrin, E-mail: katrin.lochner@med.uni-rostock.de [Biomechanics and Implant Technology Research Laboratory, Department of Orthopedics, Rostock University Medical Center, Doberaner Straße 142, D-18057 Rostock (Germany); Peters, Kirsten, E-mail: kirsten.peters@med.uni-rostock.de [Department of Cell Biology, Rostock University Medical Center, Schillingallee 69, D-18057 Rostock (Germany)

    2013-11-01

    Cartilaginous matrix-degenerative diseases like osteoarthritis (OA) are characterized by gradual cartilage erosion, and also by increased presence of cells with mesenchymal stem cell (MSC) character within the affected tissues. Moreover, primary chondrocytes long since are known to de-differentiate in vitro and to be chondrogenically re-differentiable. Since both findings appear to conflict with each other, we quantitatively assessed the mesenchymal differentiation potential of OA patient cartilage-derived cells (CDC) towards the osteogenic and adipogenic lineage in vitro and compared it to that of MSC isolated from adipose tissue (adMSC) of healthy donors. We analyzed expression of MSC markers CD29, CD44, CD105, and CD166, and, following osteogenic and adipogenic induction in vitro, quantified their expression of osteogenic and adipogenic differentiation markers. Furthermore, CDC phenotype and proliferation were monitored. We found that CDC exhibit an MSC CD marker expression pattern similar to adMSC and a similar increase in proliferation rate during osteogenic differentiation. In contrast, the marked reduction of proliferation observed during adipogenic differentiation of adMSC was absent in CDC. Quantification of differentiation markers revealed a strong osteogenic differentiation potential for CDC, however almost no capacity for adipogenic differentiation. Since in the pathogenesis of OA, cartilage degeneration coincides with high bone turnover rates, the high osteogenic differentiation potential of OA patient-derived CDC may affect clinical therapeutic regimens aiming at autologous cartilage regeneration in these patients. - Highlights: • We analyze the mesenchymal differentiation capacity of cartilage-derived cells (CDC). • CDC express mesenchymal stem cell (MSC) markers CD29, CD44, CD105, and CD166. • CDC and MSC proliferation is reduced in adipogenesis and increased in osteogenesis. • Adipogenic differentiation is virtually absent in CDC, but

  7. Vitamin K2 improves proliferation and migration of bovine skeletal muscle cells in vitro.

    Science.gov (United States)

    Rønning, Sissel Beate; Pedersen, Mona Elisabeth; Berg, Ragnhild Stenberg; Kirkhus, Bente; Rødbotten, Rune

    2018-01-01

    Skeletal muscle function is highly dependent on the ability to regenerate, however, during ageing or disease, the proliferative capacity is reduced, leading to loss of muscle function. We have previously demonstrated the presence of vitamin K2 in bovine skeletal muscles, but whether vitamin K has a role in muscle regulation and function is unknown. In this study, we used primary bovine skeletal muscle cells, cultured in monolayers in vitro, to assess a potential effect of vitamin K2 (MK-4) during myogenesis of muscle cells. Cell viability experiments demonstrate that the amount of ATP produced by the cells was unchanged when MK-4 was added, indicating viable cells. Cytotoxicity analysis show that MK-4 reduced the lactate dehydrogenase (LDH) released into the media, suggesting that MK-4 was beneficial to the muscle cells. Cell migration, proliferation and differentiation was characterised after MK-4 incubation using wound scratch analysis, immunocytochemistry and real-time PCR analysis. Adding MK-4 to the cells led to an increased muscle proliferation, increased gene expression of the myogenic transcription factor myod as well as increased cell migration. In addition, we observed a reduction in the fusion index and relative gene expression of muscle differentiation markers, with fewer complex myotubes formed in MK-4 stimulated cells compared to control cells, indicating that the MK-4 plays a significant role during the early phases of muscle proliferation. Likewise, we see the same pattern for the relative gene expression of collagen 1A, showing increased gene expression in proliferating cells, and reduced expression in differentiating cells. Our results also suggest that MK-4 incubation affect low density lipoprotein receptor-related protein 1 (LRP1) and the low-density lipoprotein receptor (LDLR) with a peak in gene expression after 45 min of MK-4 incubation. Altogether, our experiments show that MK-4 has a positive effect on muscle cell migration and

  8. Mycoplasma agalactiae Induces Cytopathic Effects in Infected Cells Cultured In Vitro.

    Directory of Open Access Journals (Sweden)

    Shrilakshmi Hegde

    Full Text Available Mycoplasma agalactiae is the etiological agent of the contagious agalactia syndrome in sheep and goats and causes significant economic losses worldwide. Yet the mechanism of pathogenesis is largely unknown. Even whole-genome sequence analysis of its pathogenic type strain did not lead to any conclusions regarding its virulence or pathogenicity factors. Although inflammation and tissue destruction at the local site of M. agalactiae infection are largely considered as effects of the host immune response, the direct effect of the agent on host cells is not completely understood. The aim of this study was to investigate the effect of M. agalactiae infection on the quality and viability of host cells in vitro. Changes in cell morphology including cell elongation, cytoplasm shrinkage and membrane blebbing were observed in infected HeLa cells. Chromatin condensation and increased caspase-3 cleavage in infected HeLa cells 48 h after infection suggests an apoptosis-like phenomenon in M. agalactiae-infected cells. In compliance with these results, decreased viability and cell lysis of M. agalactiae-infected HeLa cells was also observed. Measurement of the amount of LDH released after M. agalactiae infection revealed a time- and dose-dependent increase in HeLa cell lysis. A significant decrease in LDH released after gentamicin treatment of infected cells confirmed the major role of cytadherent M. agalactiae in inducing host cell lysis. This is the first study illustrating M. agalactiae's induction of cytopathic effects in infected HeLa cells. Further detailed investigation of infected host tissue for apoptotic markers might demonstrate the association between M. agalactiae-induced host cell lysis and the tissue destruction observed during M. agalactiae natural infection.

  9. An in vitro study of bone cells grown on an electrospun scaffold for bone repair and reconstruction

    CSIR Research Space (South Africa)

    Wepener, I

    2012-10-01

    Full Text Available This presentation focuses on the manufacturing of the electrospun scaffold and the in vitro testing of this scaffold by making use of human cells. This scaffold is a possible candidate for repair and reconstruction of bone tissue....

  10. Human mesenchymal stem cells are resistant to cytotoxic and genotoxic effects of cisplatin in vitro

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    Bruno Corrêa Bellagamba

    2016-03-01

    Full Text Available Abstract Mesenchymal stem cells (MSCs are known for their important properties involving multilineage differentiation potential., trophic factor secretion and localization along various organs and tissues. On the dark side, MSCs play a distinguished role in tumor microenvironments by differentiating into tumor-associated fibroblasts or supporting tumor growth via distinct mechanisms. Cisplatin (CIS is a drug widely applied in the treatment of a large number of cancers and is known for its cytotoxic and genotoxic effects, both in vitro and in vivo. Here we assessed the effects of CIS on MSCs and the ovarian cancer cell line OVCAR-3, by MTT and comet assays. Our results demonstrated the resistance of MSCs to cell death and DNA damage induction by CIS, which was not observed when OVCAR-3 cells were exposed to this drug.

  11. In vitro cytotoxicity of alpha conjugates for human pancreatic cancer cell lines

    International Nuclear Information System (INIS)

    Qu, C.; Li, Y.; Rizvi, M.A.; Allen, B.; Samra, J.; Smith, R.

    2003-01-01

    Targeted Alpha therapy (TAT) can inhibit the growth of micrometastases by selectively killing isolated and preangiogenic clusters of cancer cells. The aim of this study is to demonstrate the cytotoxicity of different alpha conjugates in vitro to human metastatic pancreatic cancer cell lines (CAPAN-1, CFPAN-1 and PANC-1). We are labeling the C595 and J591 (non-specific controls) monoclonal antibodies (Mabs) with 213 Bi were performed according to the standard methods in our laboratory. 213 Bi-C595 is specifically cytotoxic to CAPAN-1, CFPAN-1 and PANC-1cell lines in a concentration-dependent fashion. While non-specific alpha conjugates only killed very small fractions of pancreatic cancer cells. These alpha conjugates might be useful agents for the treatment of micro-metastases in pancreatic cancer patients with over-expression of the targeted receptors

  12. EMMPRIN contributes to the in vitro invasion of human salivary adenoid cystic carcinoma cells

    Science.gov (United States)

    YANG, XINJIE; ZHANG, PU; MA, QIN; KONG, LIANG; LI, YUAN; LIU, BAOLIN; LEI, DELIN

    2012-01-01

    Extracellular matrix metalloproteinase inducer (EMMPRIN) is a transmembrane glycoprotein that is involved in tumor invasion by stimulating matrix metalloproteinase (MMP) expression. Our previous immunohistochemical study found that the expression of EMMPRIN in salivary adenoid cystic carcinoma (SACC) was positively correlated with tumor perineural and perivascular invasion. The present study was designed to further investigate the role of EMMPRIN in the invasion of SACC. Western blot results showed that EMMPRIN was upregulated in the highly metastatic SACC cell line SACC-LM, compared to SACC-83, a SACC cell line with low metastatic ability. Blocking of EMMPRIN by its antibody significantly decreased the adhesion, secretion of MMP-2 and MMP-9, and invasion activity of SACC-LM cells in vitro (PEMMPRIN may play an important role in the invasion of SACC by stimulating the expression of MMP-2 and MMP-9 in tumor and stromal cells. PMID:22200897

  13. Human epithelial cells increase their rigidity with ageing in vitro: direct measurements

    International Nuclear Information System (INIS)

    Berdyyeva, Tamara K; Woodworth, Craig D; Sokolov, Igor

    2005-01-01

    The decrease in elasticity of epithelial tissues with ageing contributes to many human diseases. This change was previously attributed to increased crosslinking of extracellular matrix proteins. Here we show that individual human epithelial cells also become significantly more rigid during ageing in vitro. Using atomic force microscopy (AFM), we found that the Young's modulus of viable cells was consistently increased two- to four-fold in older versus younger cells. Direct visualization of the cytoskeleton using a novel method involving the AFM suggested that increased rigidity of ageing cells was due to a higher density of cytoskeletal fibres. Our results identify a unique mechanism that might contribute to the age-related loss of elasticity in epithelial tissues

  14. In vitro organogenesis in three dimensions: self-organising stem cells.

    Science.gov (United States)

    Sasai, Yoshiki; Eiraku, Mototsugu; Suga, Hidetaka

    2012-11-01

    Organ formation during embryogenesis is a complex process that involves various local cell-cell interactions at the molecular and mechanical levels. Despite this complexity, organogenesis can be modelled in vitro. In this article, we focus on two recent examples in which embryonic stem cells can self-organise into three-dimensional structures - the optic cup and the pituitary epithelium; and one case of self-organising adult stem cells - the gut epithelium. We summarise how these approaches have revealed intrinsic programs that drive locally autonomous modes of organogenesis and homeostasis. We also attempt to interpret the results of previous in vivo studies of retinal development in light of the self-organising nature of the retina.

  15. Implications of silver nanoparticle induced cell apoptosis for in vitro gene therapy

    International Nuclear Information System (INIS)

    Gopinath, P; Ghosh, Siddhartha Sankar; Gogoi, Sonit Kumar; Chattopadhyay, Arun

    2008-01-01

    The impact of manufactured nanomaterials on human health and the environment is a major concern for commercial use of nanotechnology based products. A judicious choice of selective usage, lower nanomaterial concentration and use in combination with conventional therapeutic materials may provide the best solution. For example, silver nanoparticles (Ag NPs) are known to be bactericidal and also cytotoxic to mammalian cells. Herein, we investigate the molecular mechanism of Ag NP mediated cytotoxicity in both cancer and non-cancer cells and find that optimum particle concentration leads to programmed cell death in vitro. Also, the benefit of the cytotoxic effects of Ag NPs was tested for therapeutic use in conjunction with conventional gene therapy. The synergistic effect of Ag NPs on the uracil phosphoribosyltransferase expression system sensitized the cells more towards treatment with the drug 5-fluorouracil. Induction of the apoptotic pathway makes Ag NPs a representative of a new chemosensitization strategy for future application in gene therapy

  16. Molecular analysis of human papillomavirus virus-like particle activated Langerhans cells in vitro.

    Science.gov (United States)

    Woodham, Andrew W; Raff, Adam B; Da Silva, Diane M; Kast, W Martin

    2015-01-01

    Langerhans cells (LC) are the resident antigen-presenting cells in human epithelium, and are therefore responsible for initiating immune responses against human papillomaviruses (HPV) entering the epithelial and mucosal layers in vivo. Upon proper pathogenic stimulation, LC become activated causing an internal signaling cascade that results in the up-regulation of co-stimulatory molecules and the release of inflammatory cytokines. Activated LC then migrate to lymph nodes where they interact with antigen-specific T cells and initiate an adaptive T-cell response. However, HPV manipulates LC in a suppressive manner that alters these normal maturation responses. Here, in vitro LC activation assays for the detection of phosphorylated signaling intermediates, the up-regulation of activation-associated surface markers, and the release of inflammatory cytokines in response to HPV particles are described.

  17. In vitro evaluation of three different biomaterials as scaffolds for canine mesenchymal stem cells

    Directory of Open Access Journals (Sweden)

    Oduvaldo Câmara Marques Pereira-Junior

    2013-05-01

    Full Text Available PURPOSE: To evaluate in vitro ability the of three different biomaterials - purified hydroxyapatite, demineralized bone matrix and castor oil-based polyurethane - as biocompatible 3D scaffolds for canine bone marrow mesenchymal stem cell (MSC intending bone tissue engineering. METHODS: MSCs were isolated from canine bone marrow, characterized and cultivated for seven days with the biomaterials. Cell proliferation and adhesion to the biomaterial surface were evaluated by scanning electron microscopy while differentiation into osteogenic lineage was evaluated by Alizarin Red staining and Sp7/Osterix surface antibody marker. RESULTS: The biomaterials allowed cellular growth, attachment and proliferation. Osteogenic differentiation occurred in the presence of hydroxyapatite, and matrix deposition commenced in the presence of the castor oil-based polyurethane. CONCLUSION: All the tested biomaterials may be used as mesenchymal stem cell scaffolds in cell-based orthopedic reconstructive therapy.

  18. Effects of blood products on inflammatory response in endothelial cells in vitro.

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    Martin Urner

    Full Text Available BACKGROUND: Transfusing blood products may induce inflammatory reactions within the vascular compartment potentially leading to a systemic inflammatory response. Experiments were designed to assess the inflammatory potential of different blood products in an endothelial cell-based in vitro model and to compare baseline levels of potentially activating substances in transfusion products. METHODS: The inflammatory response from pre-activated (endotoxin-stimulated and non-activated endothelial cells as well as neutrophil endothelial transmigration in response to packed red blood cells (PRBC, platelet concentrates (PC and fresh frozen plasma (FFP was determined. Baseline inflammatory mediator and lipid concentrations in blood products were evaluated. RESULTS: Following incubation with all blood products, an increased inflammatory mediator release from endothelial cells was observed. Platelet concentrates, and to a lesser extent also FFP, caused the most pronounced response, which was accentuated in already pre-stimulated endothelial cells. Inflammatory response of endothelial cells as well as blood product-induced migration of neutrophils through the endothelium was in good agreement with the lipid content of the according blood product. CONCLUSION: Within the group of different blood transfusion products both PC and FFP have a high inflammatory potential with regard to activation of endothelial cells. Inflammation upon blood product exposure is strongly accentuated when endothelial cells are pre-injured. High lipid contents in the respective blood products goes along with an accentuated inflammatory reaction from endothelial cells.

  19. Effect of repeated irradiation on biological characteristics of lung adenocarcinoma cell line Anip973 in vitro

    International Nuclear Information System (INIS)

    Xu Qingyong; Xu Xiangying; Yang Zhiwei

    2008-01-01

    Objective: To study the effect of repeated irradiation on biological characteristics of human lung adenocarcinoma cell line Anip973 in vitro. Methods: Anip973 cells were treated with high energy X-ray to a total dose of 60 Gy at 4 Gy fractions. The radiosensitivity of Anip973R and its parental cell were measured by clonogenic assay. The biological parameters were fitted to the single hit multitarget formula. Furthermore, the population double time(PDT) and cell cycle distribution were measured by cell growth curve and flow cytometry, respectively. Results: Comparing with its parental cell, Anip973 R acquired radioresistance showing increased D 0 , D q and SF 2 and a broader shoulder. PDT of Anip973R extended 3 h more than that of Anip973. The Anip973R also showed higher and lower percentage of cells in G 1 and S phase (P 2 /M distribution (P>0.05). Conclusions: A radioresistant lung adenocarcinoma cell line Anip973R is established by repeatedly irradiation. Its radioresistance displays obviously in lower dose area. However, its characteristic of cell cycle is not completely coincident with the classical radiobiological theory. (authors)

  20. Adult Murine Skeletal Muscle Contains Cells That Can Differentiate into Beating Cardiomyocytes In Vitro

    Directory of Open Access Journals (Sweden)

    Winitsky Steve O

    2005-01-01

    Full Text Available It has long been held as scientific fact that soon after birth, cardiomyocytes cease dividing, thus explaining the limited restoration of cardiac function after a heart attack. Recent demonstrations of cardiac myocyte differentiation observed in vitro or after in vivo transplantation of adult stem cells from blood, fat, skeletal muscle, or heart have challenged this view. Analysis of these studies has been complicated by the large disparity in the magnitude of effects seen by different groups and obscured by the recently appreciated process of in vivo stem-cell fusion. We now show a novel population of nonsatellite cells in adult murine skeletal muscle that progress under standard primary cell-culture conditions to autonomously beating cardiomyocytes. Their differentiation into beating cardiomyocytes is characterized here by video microscopy, confocal-detected calcium transients, electron microscopy, immunofluorescent cardiac-specific markers, and single-cell patch recordings of cardiac action potentials. Within 2 d after tail-vein injection of these marked cells into a mouse model of acute infarction, the marked cells are visible in the heart. By 6 d they begin to differentiate without fusing to recipient cardiac cells. Three months later, the tagged cells are visible as striated heart muscle restricted to the region of the cardiac infarct.

  1. Adult murine skeletal muscle contains cells that can differentiate into beating cardiomyocytes in vitro.

    Directory of Open Access Journals (Sweden)

    Steve O Winitsky

    2005-04-01

    Full Text Available It has long been held as scientific fact that soon after birth, cardiomyocytes cease dividing, thus explaining the limited restoration of cardiac function after a heart attack. Recent demonstrations of cardiac myocyte differentiation observed in vitro or after in vivo transplantation of adult stem cells from blood, fat, skeletal muscle, or heart have challenged this view. Analysis of these studies has been complicated by the large disparity in the magnitude of effects seen by different groups and obscured by the recently appreciated process of in vivo stem-cell fusion. We now show a novel population of nonsatellite cells in adult murine skeletal muscle that progress under standard primary cell-culture conditions to autonomously beating cardiomyocytes. Their differentiation into beating cardiomyocytes is characterized here by video microscopy, confocal-detected calcium transients, electron microscopy, immunofluorescent cardiac-specific markers, and single-cell patch recordings of cardiac action potentials. Within 2 d after tail-vein injection of these marked cells into a mouse model of acute infarction, the marked cells are visible in the heart. By 6 d they begin to differentiate without fusing to recipient cardiac cells. Three months later, the tagged cells are visible as striated heart muscle restricted to the region of the cardiac infarct.

  2. Cell Spheroids with Enhanced Aggressiveness to Mimic Human Liver Cancer In Vitro and In Vivo.

    Science.gov (United States)

    Jung, Hong-Ryul; Kang, Hyun Mi; Ryu, Jea-Woon; Kim, Dae-Soo; Noh, Kyung Hee; Kim, Eun-Su; Lee, Ho-Joon; Chung, Kyung-Sook; Cho, Hyun-Soo; Kim, Nam-Soon; Im, Dong-Soo; Lim, Jung Hwa; Jung, Cho-Rok

    2017-09-05

    We fabricated a spheroid-forming unit (SFU) for efficient and economic production of cell spheroids. We optimized the protocol for generating large and homogenous liver cancer cell spheroids using Huh7 hepatocellular carcinoma (HCC) cells. The large Huh7 spheroids showed apoptotic and proliferative signals in the centre and at the surface, respectively. In particular, hypoxia-induced factor-1 alpha (HIF-1α) and ERK signal activation were detected in the cell spheroids. To diminish core necrosis and increase the oncogenic character, we co-cultured spheroids with 2% human umbilical vein endothelial cells (HUVECs). HUVECs promoted proliferation and gene expression of HCC-related genes and cancer stem cell markers in the Huh7 spheroidsby activating cytokine signalling, mimicking gene expression in liver cancer. HUVECs induced angiogenesis and vessel maturation in Huh7 spheroids in vivo by activating epithelial-mesenchymal transition and angiogenic pathways. The large Huh7 cell spheroids containing HUVECs survived at higher concentrations of anti-cancer drugs (doxorubicin and sorafenib) than did monolayer cells. Our large cell spheroid provides a useful in vitro HCC model to enable intuitive observation for anti-cancer drug testing.

  3. Effect of Lipopolysaccharide on Progesterone Production during Luteinization of Granulosa and Theca cells In Vitro.

    Science.gov (United States)

    Shimizu, Takashi; Echizenya, Riku; Miyamoto, Akio

    2016-04-01

    The aim of this study is to examine the effect of lipopolysaccharide (LPS) on progesterone production during luteinization of granulosa and theca cells isolated from bovine large follicles. Granulosa and theca cells isolated from large follicles of bovine ovaries were exposed to LPS under appropriate hormone conditions in vitro. Progesterone (P4) production in theca cells, but not granulosa cells, was decreased by long-term exposure of LPS. Long-term exposure of LPS suppressed the gene expression of luteinizing hormone receptor in theca cells. Although long-term exposure of LPS did not affect the expression of steroidogenic acute regulatory protein (StAR) and 3β-hydroxy-steroid dehydrogenase (3β-HSD) genes, it did inhibit the protein expression of StAR and 3β-HSD in theca cells. These findings suggest that theca cells, rather than granulosa cells, are susceptible to LPS during luteinization and that LPS inhibits P4 production by decreasing protein levels of StAR during luteinization of theca cells. © 2016 Wiley Periodicals, Inc.

  4. In Vitro Propagation and Branching Morphogenesis from Single Ureteric Bud Cells.

    Science.gov (United States)

    Yuri, Shunsuke; Nishikawa, Masaki; Yanagawa, Naomi; Jo, Oak D; Yanagawa, Norimoto

    2017-02-14

    A method to maintain and rebuild ureteric bud (UB)-like structures from UB cells in vitro could provide a useful tool for kidney regeneration. We aimed in our present study to establish a serum-free culture system that enables the expansion of UB progenitor cells, i.e., UB tip cells, and reconstruction of UB-like structures. We found that fibroblast growth factors or retinoic acid (RA) was sufficient for the survival of UB cells in serum-free condition, while the proliferation and maintenance of UB tip cells required glial cell-derived neurotrophic factor together with signaling from either WNT-β-catenin pathway or RA. The activation of WNT-β-catenin signaling in UB cells by endogenous WNT proteins required R-spondins. Together with Rho kinase inhibitor, our culture system facilitated the expansion of UB tip cells to form UB-like structures from dispersed single cells. The UB-like structures thus formed retained the original UB characteristics and integrated into the native embryonic kidneys. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  5. In Vitro Propagation and Branching Morphogenesis from Single Ureteric Bud Cells

    Directory of Open Access Journals (Sweden)

    Shunsuke Yuri

    2017-02-01

    Full Text Available A method to maintain and rebuild ureteric bud (UB-like structures from UB cells in vitro could provide a useful tool for kidney regeneration. We aimed in our present study to establish a serum-free culture system that enables the expansion of UB progenitor cells, i.e., UB tip cells, and reconstruction of UB-like structures. We found that fibroblast growth factors or retinoic acid (RA was sufficient for the survival of UB cells in serum-free condition, while the proliferation and maintenance of UB tip cells required glial cell-derived neurotrophic factor together with signaling from either WNT-β-catenin pathway or RA. The activation of WNT-β-catenin signaling in UB cells by endogenous WNT proteins required R-spondins. Together with Rho kinase inhibitor, our culture system facilitated the expansion of UB tip cells to form UB-like structures from dispersed single cells. The UB-like structures thus formed retained the original UB characteristics and integrated into the native embryonic kidneys.

  6. [Inhibitory effect of diclofenac sodium on the proliferation of rabbit corneal epithelial cells in vitro].

    Science.gov (United States)

    Wu, Ningling; Du, Zhiyu

    2010-11-01

    To investigate the inhibitory effect of diclofenac sodium on rabbit corneal epithelial cells (RCECs) in vitro and explore its pharmacological mechanism. The fresh rabbit cornea was cultured to obtain the primary RCECs, and RCECs of passage 2 were used in this research. The cells were divided into experimental groups, the cells in which were incubated with different concentrations (18.18, 27.27, 36.36, 45.45, 54.55 μg/ml) of diclofenac sodium, and control group. The effect of diclofenac sodium on the proliferation of cells was measured by methyl thiazolyl thiazolium (MTT) assay 24, 48 and 72 h after incubation. While the RCECs were divided into experimental groups, the cells in which were incubated with 9 and 12.5 μg/ml diclofenac sodium, and control group. The cell cycle and apoptotic rate were observed by flow cytometer. MTT assay showed that diclofenac sodium had obvious inhibitory effect on RCECs, and the inhibition rate was increasing along with the increasing concentration of diclofenac sodium and the incubation time(Pdiclofenac sodium, the cells in G0/G1 phase were obviously increased, and the apoptosis cusp and apoptotic rate were increased. Diclofenac sodium exerts significant inhibitory effect on RCECs in a dosage-dependent manner, and it may function by inducing cell apoptosis and ceasing cell cycles.

  7. In vitro evaluation of the interactions between human corneal endothelial cells and extracellular matrix proteins

    International Nuclear Information System (INIS)

    Choi, Jin San; Kim, Eun Young; Kim, Min Jeong; Giegengack, Matthew; Khan, Faraaz A; Soker, Shay; Khang, Gilson

    2013-01-01

    The corneal endothelium is the innermost cell layer of the cornea and rests on Descemet's membrane consisting of various extracellular matrix (ECM) proteins which can directly affect the cellular behaviors such as cell adhesion, proliferation, polarity, morphogenesis and function. The objective of this study was to investigate the interactions between the ECM environment and human corneal endothelial cells (HCECs), with the ultimate goal to improve cell proliferation and function in vitro. To evaluate the interaction of HCECs with ECM proteins, cells were seeded on ECM-coated tissue culture dishes, including collagen type I (COL I), collagen type IV (COL IV), fibronectin (FN), FNC coating mix (FNC) and laminin (LM). Cell adhesion and proliferation of HCECs on each substratum and expression of CEC markers were studied. The results showed that HCECs plated on the COL I, COL IV, FN and FNC-coated plates had enhanced cell adhesion initially; the number for COL I, COL IV, FN and FNC was significantly higher than the control (P < 0.05). In addition, cells grown on ECM protein-coated dishes showed more compact cellular morphology and CEC marker expression compared to cells seeded on uncoated dishes. Collectively, our results suggest that an adequate ECM protein combination can provide a long-term culture environment for HCECs for corneal endothelium transplantation. (paper)

  8. BMP7 transfection induces in-vitro osteogenic differentiation of dental pulp mesenchymal stem cells

    Directory of Open Access Journals (Sweden)

    Ka Po John Yau

    2013-01-01

    Full Text Available Objective: To assess whether in-vitro osteogenic differentiation of human dental pulp mesenchymal stem cells can be induced by transient transfection with the gene encoding human bone morphogenic protein 7 (BMP7. Materials and Methods: A mesenchymal stem cell population was isolated from the dental pulp of two extracted permanent premolars, expanded and characterized. The human BMP7 gene, as a recombinant pcDNA3.1/V5-His-TOPO-BMP7 plasmid, was transfected into the cells. Three negative controls were used: No plasmid, empty vector, and an unrelated vector encoding green fluorescent protein. After the interval of 24 and 48 h, mRNA levels of alkaline phosphatase and osteocalcin as markers of in-vitro osteogenic differentiation were measured by real-time polymerase chain reaction and standardized against β-actin mRNA levels. Results: The level of alkaline phosphatase mRNA was significantly higher for the BMP7 group than for all three negative controls 48 h after transfection (706.9 vs. 11.24 for untransfected cells, 78.05 for empty vector, and 73.10 for green fluorescent protein vector. The level of osteocalcin mRNA was significantly higher for the BMP7 group than for all three negative controls 24 h after transfection (1.0, however, decreased after another 24 h. Conclusions: In-vitro osteoblastic differentiation of human dental pulp mesenchymal stem cells, as indicated by expression of alkaline phosphatase and osteocalcin, can be induced by transient transfection with the BMP7 gene.

  9. Knockdown of SVCT2 impairs in-vitro cell attachment, migration and wound healing in bone marrow stromal cells

    Directory of Open Access Journals (Sweden)

    Rajnikumar Sangani

    2014-03-01

    Full Text Available Bone marrow stromal cell (BMSC adhesion and migration are fundamental to a number of pathophysiologic processes, including fracture and wound healing. Vitamin C is beneficial for bone formation, fracture repair and wound healing. However, the role of the vitamin C transporter in BMSC adhesion, migration and wound healing is not known. In this study, we knocked-down the sodium-dependent vitamin C transporter, SVCT2, the only known transporter of vitamin C in BMSCs, and performed cell adhesion, migration, in-vitro scratch wound healing and F-actin re-arrangement studies. We also investigated the role of oxidative stress on the above processes. Our results demonstrate that both oxidative stress and down-regulation of SVCT2 decreased cell attachment and spreading. A trans-well cell migration assay showed that vitamin C helped in BMSC migration and that knockdown of SVCT2 decreased cell migration. In the in-vitro scratch wound healing studies, we established that oxidative stress dose-dependently impairs wound healing. Furthermore, the supplementation of vitamin C significantly rescued the BMSCs from oxidative stress and increased wound closing. The knockdown of SVCT2 in BMSCs strikingly decreased wound healing, and supplementing with vitamin C failed to rescue cells efficiently. The knockdown of SVCT2 and induction of oxidative stress in cells produced an alteration in cytoskeletal dynamics. Signaling studies showed that oxidative stress phosphorylated members of the MAP kinase family (p38 and that vitamin C inhibited their phosphorylation. Taken together, these results indicate that both the SVCT2 transporter and oxidative stress play a vital role in BMSC attachment, migration and cytoskeletal re-arrangement. BMSC-based cell therapy and modulation of SVCT2 could lead to a novel therapeutic approach that enhances bone remodeling, fracture repair and wound healing in chronic disease conditions.

  10. CXCL1 is a negative regulator of mast cell chemotaxis to airway smooth muscle cell products in vitro.

    Science.gov (United States)

    Alkhouri, H; Moir, L M; Armour, C L; Hughes, J M

    2014-03-01

    Activated mast cells (MC) numbers on airway smooth muscle (ASM) are increased in eosinophilic asthma. In vitro, asthmatic cytokine-stimulated ASM cell-conditioned medium (CM) induces more MC chemotaxis than CM from nonasthmatic ASM cells. Intriguingly the nonasthmatic ASM CM inhibits MC chemotaxis to the asthmatic ASM CM. However, the inhibitory factor(s) in the nonasthmatic ASM CM is still to be identified. To identify the factor(s) released by nonasthmatic ASM cells that inhibits MC chemotaxis. Confluent, serum-starved ASM cells from donors with and without asthma were stimulated with IL-1β and T-helper (Th)1 (TNFα and IFNγ) or Th2 (IL-4, IL-13) cytokines, or left unstimulated. CM samples were collected after 24 h, and a potential inhibitory factor identified using cytokine protein arrays. Its production was assessed using ELISA and RT-PCR and inhibitory role investigated in MC chemotaxis and Ca(2+) mobilization assays. Only CXCL1 was produced in greater amounts by nonasthmatic than asthmatic ASM cells following Th1 and Th2 cytokine stimulation. CXCL1 mRNA expression was also increased. Exogenous rh-CXCL1 significantly inhibited MC intracellular Ca(2+) mobilization and chemotaxis to either CXCL10, CXCL8 or CM collected from asthmatic ASM cells following Th1 or Th2 cytokine stimulation. Neutralizing CXCL1 in nonasthmatic ASM CM or blocking its receptor significantly promoted MC chemotaxis. CXCL1 was a major factor regulating MC chemotaxis in vitro. Its differential release by ASM cells may explain the differences observed in MC localization to the ASM of people with and without asthma. CXCL1 inhibition of MC recruitment to the ASM may lead to new targets to limit asthma pathophysiology. © 2013 John Wiley & Sons Ltd.

  11. The role of changes in the oxygen concentration in modification of reproductive death of cells in vitro

    International Nuclear Information System (INIS)

    Korystov, Yu.N.

    1983-01-01

    In this report the data are discussed and summarized concerning cell oxygenation in culture. Formulae are proposed for calculation of the oxygen concentration in suspension, monolayer and spheroid, as well as numerical parameters are submitted determining the oxygenation of cells in vitro. This permits to estimate quantitatively the oxygen concentration at the cell surface upon irradiation in different experimental conditions

  12. Activation of ribosomal RNA genes in porcine embryos produced in vitro or by somatic cell nuclear transfer

    DEFF Research Database (Denmark)

    Bjerregaard, Bolette; Pedersen, Hanne Gervi; Jakobsen, Anne Sørig

    2007-01-01

    The onset of ribosomal RNA (rRNA) synthesis occurs during the second half of the third cell cycle, that is, at the four-cell stage, in porcine embryos developed in vivo. In the present study the onset of rRNA synthesis was investigated in porcine embryos produced in vitro (IVP) or by somatic cell...

  13. The immunomodulator PSK induces in vitro cytotoxic activity in tumour cell lines via arrest of cell cycle and induction of apoptosis

    International Nuclear Information System (INIS)

    Jiménez-Medina, Eva; Berruguilla, Enrique; Romero, Irene; Algarra, Ignacio; Collado, Antonia; Garrido, Federico; Garcia-Lora, Angel

    2008-01-01

    Protein-bound polysaccharide (PSK) is derived from the CM-101 strain of the fungus Coriolus versicolor and has shown anticancer activity in vitro and in in vivo experimental models and human cancers. Several randomized clinical trials have demonstrated that PSK has great potential in adjuvant cancer therapy, with positive results in the adjuvant treatment of gastric, esophageal, colorectal, breast and lung cancers. These studies have suggested the efficacy of PSK as an immunomodulator of biological responses. The precise molecular mechanisms responsible for its biological activity have yet to be fully elucidated. The in vitro cytotoxic anti-tumour activity of PSK has been evaluated in various tumour cell lines derived from leukaemias, melanomas, fibrosarcomas and cervix, lung, pancreas and gastric cancers. Tumour cell proliferation in vitro was measured by BrdU incorporation and viable cell count. Effect of PSK on human peripheral blood lymphocyte (PBL) proliferation in vitro was also analyzed. Studies of cell cycle and apoptosis were performed in PSK-treated cells. PSK showed in vitro inhibition of tumour cell proliferation as measured by BrdU incorporation and viable cell count. The inhibition ranged from 22 to 84%. Inhibition mechanisms were identified as cell cycle arrest, with cell accumulation in G 0 /G 1 phase and increase in apoptosis and caspase-3 expression. These results indicate that PSK has a direct cytotoxic activity in vitro, inhibiting tumour cell proliferation. In contrast, PSK shows a synergistic effect with IL-2 that increases PBL proliferation. These results indicate that PSK has cytotoxic activity in vitro on tumour cell lines. This new cytotoxic activity of PSK on tumour cells is independent of its previously described immunomodulatory activity on NK cells

  14. The immunomodulator PSK induces in vitro cytotoxic activity in tumour cell lines via arrest of cell cycle and induction of apoptosis

    Directory of Open Access Journals (Sweden)

    Garrido Federico

    2008-03-01

    Full Text Available Abstract Background Protein-bound polysaccharide (PSK is derived from the CM-101 strain of the fungus Coriolus versicolor and has shown anticancer activity in vitro and in in vivo experimental models and human cancers. Several randomized clinical trials have demonstrated that PSK has great potential in adjuvant cancer therapy, with positive results in the adjuvant treatment of gastric, esophageal, colorectal, breast and lung cancers. These studies have suggested the efficacy of PSK as an immunomodulator of biological responses. The precise molecular mechanisms responsible for its biological activity have yet to be fully elucidated. Methods The in vitro cytotoxic anti-tumour activity of PSK has been evaluated in various tumour cell lines derived from leukaemias, melanomas, fibrosarcomas and cervix, lung, pancreas and gastric cancers. Tumour cell proliferation in vitro was measured by BrdU incorporation and viable cell count. Effect of PSK on human peripheral blood lymphocyte (PBL proliferation in vitro was also analyzed. Studies of cell cycle and apoptosis were performed in PSK-treated cells. Results PSK showed in vitro inhibition of tumour cell proliferation as measured by BrdU incorporation and viable cell count. The inhibition ranged from 22 to 84%. Inhibition mechanisms were identified as cell cycle arrest, with cell accumulation in G0/G1 phase and increase in apoptosis and caspase-3 expression. These results indicate that PSK has a direct cytotoxic activity in vitro, inhibiting tumour cell proliferation. In contrast, PSK shows a synergistic effect with IL-2 that increases PBL proliferation. Conclusion These results indicate that PSK has cytotoxic activity in vitro on tumour cell lines. This new cytotoxic activity of PSK on tumour cells is independent of its previously described immunomodulatory activity on NK cells.

  15. Apoptosis related genes expressed in cultured Fallopian tube epithelial cells infected in vitro with Neisseria gonorrhoeae

    Directory of Open Access Journals (Sweden)

    PAZ A REYES

    2007-01-01

    Full Text Available Background: Infection of the Fallopian tubes (FT by Neisseria gonorrhoeae (Ngo can lead to acute salpingitis, an inflammatory condition resulting in damage primarily to the ciliated cells, with loss of ciliary activity and sloughing of the cells from the epithelium. Recently, we have shown that Ngo infection induced apoptosis in FT epithelium cells by a TNF-alpha dependent mechanism that could contribute to the cell and tissue damage observed in gonococcal salpingitis. Aim: To investigate the apoptosis-related genes expressed during apoptosis induction in cultured FT epithelial cells infected in vitro by Ngo. Materials and Methods: In the current study, we used cDNA macroarrays and real time PCR to identify and determine the expression levels of apoptosis related genes during the in vitro gonococci infection of FT epithelial cells. Results: Significant apoptosis was induced following infection with Ngo. Macroarray analysis identified the expression of multiple genes of the TNF receptor family (TNFRSF1B, -4, -6, -10A, -10B and -10D and the Bcl-2 family (BAK1, BAX, BLK, HRK and MCL-1 without differences between controls and infected cells. This lack of difference was confirmed by RT-PCR of BAX, Bcl-2, TNFRS1A (TNFR-I and TNFRSF1B (TNFR-II. Conclusion: Several genes related to apoptosis are expressed in primary cultures of epithelial cells of the human Fallopian tube. Infection with Ngo induces apoptosis without changes in the pattern of gene expression of several apoptosis-related genes. Results strongly suggest that Ngo regulates apoptosis in the FT by post-transcriptional mechanisms that need to be further addressed

  16. Dual effect of insulin resistance and cadmium on human granulosa cells - In vitro study

    Energy Technology Data Exchange (ETDEWEB)

    Belani, Muskaan, E-mail: muskaanbelani@gmail.com [Department of Biochemistry, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara 390 002, Gujarat, India. (India); Shah, Preeti, E-mail: preeti.shah@novaivifertility.com [Nova IVI Fertility, Behind Xavier' s Ladies Hostel, 108, Swastik Society Rd., Navrangpura, Ahmedabad 390009, Gujarat, India. (India); Banker, Manish, E-mail: manish.banker@novaivifertility.com [Nova IVI Fertility, Behind Xavier' s Ladies Hostel, 108, Swastik Society Rd., Navrangpura, Ahmedabad 390009, Gujarat, India. (India); Gupta, Sarita, E-mail: saritagupta9@gmail.com [Department of Biochemistry, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara 390 002, Gujarat, India. (India)

    2016-12-15

    Combined exposure of cadmium (Cd) and insulin resistance (IR) might be responsible for subfertility. In the present study, we investigated the effects of Cd in vitro in IR human granulosa cells. Isolated human granulosa cells from control and polycystic ovary syndrome (PCOS) follicular fluid samples were confirmed for IR by decrease in protein expression of insulin receptor-β. Control and IR human granulosa cells were then incubated with or without 32 μM Cd. The combined effect of IR with 32 μM Cd in granulosa cells demonstrated significant decrease in expression of StAR, CYP11A1, CYP19A1, 17β-HSD, 3β-HSD, FSH-R and LH-R. Decrease was also observed in progesterone and estradiol concentrations as compared to control. Additionally, increase in protein expression of cleaved PARP-F2, active caspase-3 and a positive staining for Annexin V and PI indicated apoptosis as the mode of increased cell death ultimately leading to decreased steroidogenesis, as observed through the combined exposure. Taken together the results suggest decrease in steroidogenesis ultimately leading to abnormal development of the follicle thus compromising fertility at the level of preconception. - Highlights: • Protein expression of INSR-β in granulosa cells to differentiate PCOS-IR and NIR • Cd and IR together decrease steroidogenesis in human granulosa cells in vitro. • Cd and IR increase human granulosa cell death by increase in apoptosis. • Environment and life style are set to hamper pregnancies at preconception level.

  17. Dual effect of insulin resistance and cadmium on human granulosa cells - In vitro study

    International Nuclear Information System (INIS)

    Belani, Muskaan; Shah, Preeti; Banker, Manish; Gupta, Sarita

    2016-01-01

    Combined exposure of cadmium (Cd) and insulin resistance (IR) might be responsible for subfertility. In the present study, we investigated the effects of Cd in vitro in IR human granulosa cells. Isolated human granulosa cells from control and polycystic ovary syndrome (PCOS) follicular fluid samples were confirmed for IR by decrease in protein expression of insulin receptor-β. Control and IR human granulosa cells were then incubated with or without 32 μM Cd. The combined effect of IR with 32 μM Cd in granulosa cells demonstrated significant decrease in expression of StAR, CYP11A1, CYP19A1, 17β-HSD, 3β-HSD, FSH-R and LH-R. Decrease was also observed in progesterone and estradiol concentrations as compared to control. Additionally, increase in protein expression of cleaved PARP-F2, active caspase-3 and a positive staining for Annexin V and PI indicated apoptosis as the mode of increased cell death ultimately leading to decreased steroidogenesis, as observed through the combined exposure. Taken together the results suggest decrease in steroidogenesis ultimately leading to abnormal development of the follicle thus compromising fertility at the level of preconception. - Highlights: • Protein expression of INSR-β in granulosa cells to differentiate PCOS-IR and NIR • Cd and IR together decrease steroidogenesis in human granulosa cells in vitro. • Cd and IR increase human granulosa cell death by increase in apoptosis. • Environment and life style are set to hamper pregnancies at preconception level.

  18. Optimization of human corneal endothelial cell culture: density dependency of successful cultures in vitro.

    Science.gov (United States)

    Peh, Gary S L; Toh, Kah-Peng; Ang, Heng-Pei; Seah, Xin-Yi; George, Benjamin L; Mehta, Jodhbir S

    2013-05-03

    Global shortage of donor corneas greatly restricts the numbers of corneal transplantations performed yearly. Limited ex vivo expansion of primary human corneal endothelial cells is possible, and a considerable clinical interest exists for development of tissue-engineered constructs using cultivated corneal endothelial cells. The objective of this study was to investigate the density-dependent growth of human corneal endothelial cells isolated from paired donor corneas and to elucidate an optimal seeding density for their extended expansion in vitro whilst maintaining their unique cellular morphology. Established primary human corneal endothelial cells were propagated to the second passage (P2) before they were utilized for this study. Confluent P2 cells were dissociated and seeded at four seeding densities: 2,500 cells per cm2 ('LOW'); 5,000 cells per cm2 ('MID'); 10,000 cells per cm2 ('HIGH'); and 20,000 cells per cm2 ('HIGH(×2)'), and subsequently analyzed for their propensity to proliferate. They were also subjected to morphometric analyses comparing cell sizes, coefficient of variance, as well as cell circularity when each culture became confluent. At the two lower densities, proliferation rates were higher than cells seeded at higher densities, though not statistically significant. However, corneal endothelial cells seeded at lower densities were significantly larger in size, heterogeneous in shape and less circular (fibroblastic-like), and remained hypertrophic after one month in culture. Comparatively, cells seeded at higher densities were significantly homogeneous, compact and circular at confluence. Potentially, at an optimal seeding density of 10,000 cells per cm2, it is possible to obtain between 10 million to 25 million cells at the third passage. More importantly, these expanded human corneal endothelial cells retained their unique cellular morphology. Our results demonstrated a density dependency in the culture of primary human corneal endothelial

  19. Mesenchymal stem cells derived from normal gingival tissue inhibit the proliferation of oral cancer cells in vitro and in vivo.

    Science.gov (United States)

    Ji, Xiaoli; Zhang, Zhihui; Han, Ying; Song, Jiangyuan; Xu, Xiangliang; Jin, Jianqiu; Su, Sha; Mu, Dongdong; Liu, Xiaodan; Xu, Si; Cui, Hongwei; Zhao, Zhongfang; Wang, Yixiang; Liu, Hongwei

    2016-11-01

    The interplay between tumor cells and mesenchymal stem cells (MSCs) within tumor microenvironment plays a significant role in tumor development, and thus might be exploited for therapeutic intervention. In this study, we isolated MSCs from normal gingival tissue (GMSCs), and detected the effect of GMSCs on oral cancer cells via direct co-culture and indirect co-culture systems. The cell proliferation assay of direct co-culture showed that GMSCs could inhibit the growth of oral cancer cells. Conditioned medium derived from GMSCs (GMSCs-CM) also exerted an anticancer effect, which indicates that soluble factors in GMSCs-CM played a dominant role in GMSCs-induced cancer cell growth inhibition. To investigate the mechanism, we performed apoptosis assay by flow cytometry, and confirmed that cancer cell apoptosis induced by GMSCs could be a reason for the effect of GMSCs on the growth of oral cancer cells. Western blotting also confirmed that GMSCs could upregulate expression of pro-apoptotic genes including p-JNK, cleaved PARP, cleaved caspase-3, Bax expression and downregulate proliferation- and anti-apoptosis-related gene expression such as p-ERK1/2, Bcl-2, CDK4, cyclin D1, PCNA and survivin. Importantly, the inhibitory effect of GMSCs on cancer cells can partially be restored by blockade of JNK pathway. Moreover, animal studies showed that GMSCs exerted an anticancer effect after oral cancer cells and GMSCs were co-injected with oral cancer cells. Taken together, our data suggest that GMSCs can suppress oral cancer cell growth in vitro and in vivo via altering the surrounding microenvironment of oral cancer cells, which indicates that GMSCs have a potential use in the management of oral dysplasia and oral cancer in future.

  20. Heterogeneity in cytokine profiles of Babesia bovis-specific bovine CD4+ T cells clones activated in vitro.

    OpenAIRE

    Brown, W C; Woods, V M; Dobbelaere, D A; Logan, K S

    1993-01-01

    The central role of T cells in the immune response against hemoprotozoan parasites, both as helper cells for T cell-dependent antibody production and as effector cells acting on intracellular parasites through the elaboration of cytokines, has prompted an investigation of the bovine cellular immune response against Babesia bovis antigens. CD4+ T helper (Th) cell clones generated from four B. bovis-immune cattle by in vitro stimulation with a soluble or membrane-associated merozoite antigen we...

  1. Accumulation and biological effects of gallium in malignant cell lines in vitro

    International Nuclear Information System (INIS)

    Awano, Takayuki; Matsuzawa, Taiju

    1977-01-01

    Accumulation and biological effects of gallium (Ga) in malignant cells in vitro were studied. Biological effects were investigated cytokinetically and morphologically. The malignant cultured FM3A cells (originated from mammary carcinoma of C3H mice) accumulated 67 Ga actively. This accumulation was more intensive in proliferating cells than in non-proliferating cells. 6.5 percent of 67 Ga accumulated in the cultured FM3A cells was bound loosely at the cell surface. The colony forming capacity of C2W cells (originated from amelanotic melanoma of C57 Black mice ) was studied. The capacity decreased markedly when stable Ga was added to the medium in low concentration, but it decreased very little more in the range of rather high concentration. The growth response of FM3A cells to various concentrations of stable Ga was studied. The saturation density decreased and the doubling time became prolonged with increased Ga concentration. When 0.5 mM of stable Ga was added to the medium, the speed of proliferation changed markedly. The doubling time increased 1.7 times as compared to that before addition of Ga. The shape of the FM3A cells was usually spheroid in the medium. Swelling of the cells was observed when stable Ga was added to the culture medium. In particular, several per cent of these cells showed remarkable changes; that is, the cells were flattened and adhered to the dish and showed remarkable locomotion. It may be that these results are related to cell differentiation rather than to the cytotoxicity of stable Ga. (auth.)

  2. Impact of alemtuzumab treatment on the survival and function of human regulatory T cells in vitro

    Science.gov (United States)

    Havari, Evis; Turner, Michael J; Campos-Rivera, Juanita; Shankara, Srinivas; Nguyen, Tri-Hung; Roberts, Bruce; Siders, William; Kaplan, Johanne M

    2014-01-01

    Alemtuzumab is a humanized monoclonal antibody specific for the CD52 protein present at high levels on the surface of B and T lymphocytes. In clinical trials, alemtuzumab has shown a clinical benefit superior to that of interferon-β in relapsing–remitting multiple sclerosis patients. Treatment with alemtuzumab leads to the depletion of circulating lymphocytes followed by a repopulation process characterized by alterations in the number, proportions and properties of lymphocyte subsets. Of particular interest, an increase in the percentage of T cells with a regulatory phenotype (Treg cells) has been observed in multiple sclerosis patients after alemtuzumab. Since Treg cells play an important role in the control of autoimmune responses, the effect of alemtuzumab on Treg cells was further studied in vitro. Alemtuzumab effectively mediated complement-dependent cytolysis of human T lymphocytes and the remaining population was enriched in T cells with a regulatory phenotype. The alemtuzumab-exposed T cells displayed functional regulatory characteristics including anergy to stimulation with allogeneic dendritic cells and ability to suppress the allogeneic response of autologous T cells. Consistent with the observed increase in Treg cell frequency, the CD25hi T-cell population was necessary for the suppressive activity of alemtuzumab-exposed T cells. The mechanism of this suppression was found to be dependent on both cell–cell contact and interleukin-2 consumption. These findings suggest that an alemtuzumab-mediated increase in the proportion of Treg cells may play a role in promoting the long-term efficacy of alemtuzumab in patients with multiple sclerosis. PMID:24116901

  3. Accumulation and biological effects of gallium in malignant cell lines in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Awano, T; Matsuzawa, T [Tohoku Univ., Sendai (Japan). Research Inst. for Tuberculosis, Leprosy and Cancer

    1977-02-01

    Accumulation and biological effects of gallium (Ga) in malignant cells in vitro were studied. Biological effects were investigated cytokinetically and morphologically. The malignant cultured FM3A cells (originated from mammary carcinoma of C3H mice) accumulated /sup 67/Ga actively. This accumulation was more intensive in proliferating cells than in non-proliferating cells. 6.5 percent of /sup 67/Ga accumulated in the cultured FM3A cells was bound loosely at the cell surface. The colony forming capacity of C2W cells (originated from amelanotic melanoma of C57 Black mice ) was studied. The capacity decreased markedly when stable Ga was added to the medium in low concentration, but it decreased very little more in the range of rather high concentration. The growth response of FM3A cells to various concentrations of stable Ga was studied. The saturation density decreased and the doubling time became prolonged with increased Ga concentration. When 0.5 mM of stable Ga was added to the medium, the speed of proliferation changed markedly. The doubling time increased 1.7 times as compared to that before addition of Ga. The shape of the FM3A cells was usually spheroid in the medium. Swelling of the cells was observed when stable Ga was added to the culture medium. In particular, several per cent of these cells showed remarkable changes; that is, the cells were flattened and adhered to the dish and showed remarkable locomotion. It may be that these results are related to cell differentiation rather than to the cytotoxicity of stable Ga.

  4. In vitro toxicity assay of cisplatin on mouse acute lymphoblastic leukaemia and spermatogonial stem cells.

    Science.gov (United States)

    Shabani, R; Ashtari, K; Behnam, B; Izadyar, F; Asgari, H; Asghari Jafarabadi, M; Ashjari, M; Asadi, E; Koruji, M

    2016-06-01

    Testicular cancer is the most common cancer affecting men in reproductive age, and cisplatin is one of the major helpful chemotherapeutic agents for treatment of this cancer. In addition, exposure of testes cancer cells to cisplatin could potentially eliminate tumour cells from germ cells in patients. The aim of this study was to evaluate the effect of cisplatin on viability of mouse acute lymphoblastic leukaemia cell line (EL-4) and neonatal mouse spermatogonial cells in vitro. In this study, the isolated spermatogonial stem cells (SSC) and EL-4 were divided into six groups including control (received medium), sham (received DMSO in medium) and experimental groups which received different doses of cisplatin (0.5, 5, 10 and 15 μg ml(-1) ). Cells viability was evaluated with MTT assay. The identity of the cultured cells was confirmed by the expression of specific markers. Our finding showed that viability of both SSC and EL-4 cells was reduced with the dose of 15 μg/ml when compared to the control group (P ≤ 0.05). Also, the differences between the IC50 in doses 10 and 15 μg/ml at different time were significant (P ≤ 0.05). The number of TUNEL-positive cells was increased, and the BAX and caspase-3 expressions were upregulated in EL4 cells for group that received an effective dose of cisplatin). In conclusion, despite the dramatic effects of cisplatin on both cells, spermatogonial stem cells could form colony in culture. © 2015 Blackwell Verlag GmbH.

  5. Insulin-producing cells generated from dedifferentiated human pancreatic beta cells expanded in vitro.

    Directory of Open Access Journals (Sweden)

    Holger A Russ

    Full Text Available Expansion of beta cells from the limited number of adult human islet donors is an attractive prospect for increasing cell availability for cell therapy of diabetes. However, attempts at expanding human islet cells in tissue culture result in loss of beta-cell phenotype. Using a lineage-tracing approach we provided evidence for massive proliferation of beta-cell-derived (BCD cells within these cultures. Expansion involves dedifferentiation resembling epithelial-mesenchymal transition (EMT. Epigenetic analyses indicate that key beta-cell genes maintain open chromatin structure in expanded BCD cells, although they are not transcribed. Here we investigated whether BCD cells can be redifferentiated into beta-like cells.Redifferentiation conditions were screened by following activation of an insulin-DsRed2 reporter gene. Redifferentiated cells were characterized for gene expression, insulin content and secretion assays, and presence of secretory vesicles by electron microscopy. BCD cells were induced to redifferentiate by a combination of soluble factors. The redifferentiated cells expressed beta-cell genes, stored insulin in typical secretory vesicles, and released it in response to glucose. The redifferentiation process involved mesenchymal-epithelial transition, as judged by changes in gene expression. Moreover, inhibition of the EMT effector SLUG (SNAI2 using shRNA resulted in stimulation of redifferentiation. Lineage-traced cells also gave rise at a low rate to cells expressing other islet hormones, suggesting transition of BCD cells through an islet progenitor-like stage during redifferentiation.These findings demonstrate for the first time that expanded dedifferentiated beta cells can be induced to redifferentiate in culture. The findings suggest that ex-vivo expansion of adult human islet cells is a promising approach for generation of insulin-producing cells for transplantation, as well as basic research, toxicology studies, and drug

  6. Hematopoiesis on cellulose ester membranes (CEM). X. Effects of in vitro irradiation of stromal cells prior to application on CEM

    International Nuclear Information System (INIS)

    Knospe, W.H.; Husseini, S.G.

    1986-01-01

    Cellulose ester membranes (CEM) were coated with stromal cells from murine bone or bone marrow irradiated in vitro with 1000, 2000, or 4000 rad and then implanted i.p. in CAF1 mice for periods of six and 12 months. CEM coated with stromal cells from bone showed excellent regeneration of bone and hematopoiesis after 1000 rad in vitro irradiation. After 2000 rad, hematopoietic and bone regeneration was reduced by about 50%, and after 4000 rad it was completely absent in CEM coated with stromal cells from bone. CEM coated with stromal cells from bone marrow showed no regeneration of hematopoiesis or bone after 1000, 2000, and 4000 rad in vitro irradiation and residence i.p. for six and 12 months. These results indicate that regeneration of the hematopoietic microenvironment is dependent upon living stromal cells. A difference in radiation sensitivity is demonstrated between stromal cells from bone and from bone marrow

  7. Inflammation-Related Effects of Diesel Engine Exhaust Particles: Studies on Lung Cells In Vitro

    Science.gov (United States)

    Schwarze, P. E.; Totlandsdal, A. I.; Låg, M.; Refsnes, M.; Holme, J. A.; Øvrevik, J.

    2013-01-01

    Diesel exhaust and its particles (DEP) have been under scrutiny for health effects in humans. In the development of these effects inflammation is regarded as a key process. Overall, in vitro studies report similar DEP-induced changes in markers of inflammation, including cytokines and chemokines, as studies in vivo. In vitro studies suggest that soluble extracts of DEP have the greatest impact on the expression and release of proinflammatory markers. Main DEP mediators of effects have still not been identified and are difficult to find, as fuel and engine technology developments lead to continuously altered characteristics of emissions. Involved mechanisms remain somewhat unclear. DEP extracts appear to comprise components that are able to activate various membrane and cytosolic receptors. Through interactions with receptors, ion channels, and phosphorylation enzymes, molecules in the particle extract will trigger various cell signaling pathways that may lead to the release of inflammatory markers directly or indirectly by causing cell death. In vitro studies represent a fast and convenient system which may have implications for technology development. Furthermore, knowledge regarding how particles elicit their effects may contribute to understanding of DEP-induced health effects in vivo, with possible implications for identifying susceptible groups of people and effect biomarkers. PMID:23509760

  8. Cartilage Derived from Bone Marrow Mesenchymal Stem Cells Expresses Lubricin In Vitro and In Vivo

    Science.gov (United States)

    Nakagawa, Yusuke; Muneta, Takeshi; Otabe, Koji; Ozeki, Nobutake; Mizuno, Mitsuru; Udo, Mio; Saito, Ryusuke; Yanagisawa, Katsuaki; Ichinose, Shizuko; Koga, Hideyuki; Tsuji, Kunikazu; Sekiya, Ichiro

    2016-01-01

    Objective Lubricin expression in the superficial cartilage will be a crucial factor in the success of cartilage regeneration. Mesenchymal stem cells (MSCs) are an attractive cell source and the use of aggregates of MSCs has some advantages in terms of chondrogenic potential and efficiency of cell adhesion. Lubricin expression in transplanted MSCs has not been fully elucidated so far. Our goals were to determine (1) whether cartilage pellets of human MSCs expressed lubricin in vitro chondrogenesis, (2) whether aggregates of human MSCs promoted lubricin expression, and (3) whether aggregates of MSCs expressed lubricin in the superficial cartilage after transplantation into osteochondral defects in rats. Methods For in vitro analysis, human bone marrow (BM) MSCs were differentiated into cartilage by pellet culture, and also aggregated using the hanging drop technique. For an animal study, aggregates of BM MSCs derived from GFP transgenic rats were transplanted to the osteochondral defect in the trochlear groove of wild type rat knee joints. Lubricin expression was mainly evaluated in differentiated and regenerated cartilages. Results In in vitro analysis, lubricin was detected in the superficial zone of the pellets and conditioned medium. mRNA expression of Proteoglycan4 (Prg4), which encodes lubricin, in pellets was significantly higher than that of undifferentiated MSCs. Aggregates showed different morphological features between the superficial and deep zone, and the Prg4 mRNA expression increased after aggregate formation. Lubricin was also found in the aggregate. In a rat study, articular cartilage regeneration was significantly better in the MSC group than in the control group as shown by macroscopical and histological a