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Sample records for adherent cell cultures

  1. Automated and online characterization of adherent cell culture growth in a microfabricated bioreactor.

    PubMed

    Jaccard, Nicolas; Macown, Rhys J; Super, Alexandre; Griffin, Lewis D; Veraitch, Farlan S; Szita, Nicolas

    2014-10-01

    Adherent cell lines are widely used across all fields of biology, including drug discovery, toxicity studies, and regenerative medicine. However, adherent cell processes are often limited by a lack of advances in cell culture systems. While suspension culture processes benefit from decades of development of instrumented bioreactors, adherent cultures are typically performed in static, noninstrumented flasks and well-plates. We previously described a microfabricated bioreactor that enables a high degree of control on the microenvironment of the cells while remaining compatible with standard cell culture protocols. In this report, we describe its integration with automated image-processing capabilities, allowing the continuous monitoring of key cell culture characteristics. A machine learning-based algorithm enabled the specific detection of one cell type within a co-culture setting, such as human embryonic stem cells against the background of fibroblast cells. In addition, the algorithm did not confuse image artifacts resulting from microfabrication, such as scratches on surfaces, or dust particles, with cellular features. We demonstrate how the automation of flow control, environmental control, and image acquisition can be employed to image the whole culture area and obtain time-course data of mouse embryonic stem cell cultures, for example, for confluency.

  2. Laminin-adherent versus suspension-non-adherent cell culture conditions for the isolation of cancer stem cells in the DAOY medulloblastoma cell line.

    PubMed

    de la Rosa, Javier; Sáenz Antoñanzas, Ander; Shahi, Mehdi H; Meléndez, Bárbara; Rey, Juan A; Castresana, Javier S

    2016-09-01

    Medulloblastoma (MB) is a highly malignant tumor of childhood. MB seems to be initiated and maintained by a small group of cells, known as cancer stem cells (CSCs). The CSC hypothesis suggests that a subset of tumor cells is able to proliferate, sustain the tumor, and develop chemoresistance, all of which make of CSC an interesting target for new anticancer therapies. The MB cell line DAOY was cultured in suspension by a medullosphere traditional culturing method and in adherent conditions by laminin-pre-coated flasks and serum-free medium enriched with specific growth factors. An increase in the stem features was shown when cells were successively cultured in hypoxia conditions. By contrast, a reduction in these properties was appreciated when cells were exposed to differentiation conditions. In addition, the CD133+ and CD133- subpopulations were isolated from cells grown in laminin-pre-coated flasks, and in vitro experiments showed that the CD133+ fraction represented the stem population and it could have CSC with a higher probability than the CD133- fraction. We can conclude that the laminin culture method in adherent conditions and the medullosphere traditional culturing method in suspension are similarly good for obtaining stem-like cells in the DAOY cell line.

  3. Neurosphere and adherent culture conditions are equivalent for malignant glioma stem cell lines

    PubMed Central

    Reyner, Karina; Deleyrolle, Loic; Millette, Sebastien; Azari, Hassan; Day, Bryan W.; Stringer, Brett W.; Boyd, Andrew W.; Johns, Terrance G.; Blot, Vincent; Duggal, Rohit; Reynolds, Brent A.

    2015-01-01

    Certain limitations of the neurosphere assay (NSA) have resulted in a search for alternative culture techniques for brain tumor-initiating cells (TICs). Recently, reports have described growing glioblastoma (GBM) TICs as a monolayer using laminin. We performed a side-by-side analysis of the NSA and laminin (adherent) culture conditions to compare the growth and expansion of GBM TICs. GBM cells were grown using the NSA and adherent culture conditions. Comparisons were made using growth in culture, apoptosis assays, protein expression, limiting dilution clonal frequency assay, genetic affymetrix analysis, and tumorigenicity in vivo. In vitro expansion curves for the NSA and adherent culture conditions were virtually identical (P=0.24) and the clonogenic frequencies (5.2% for NSA vs. 5.0% for laminin, P=0.9) were similar as well. Likewise, markers of differentiation (glial fibrillary acidic protein and beta tubulin III) and proliferation (Ki67 and MCM2) revealed no statistical difference between the sphere and attachment methods. Several different methods were used to determine the numbers of dead or dying cells (trypan blue, DiIC, caspase-3, and annexin V) with none of the assays noting a meaningful variance between the two methods. In addition, genetic expression analysis with microarrays revealed no significant differences between the two groups. Finally, glioma cells derived from both methods of expansion formed large invasive tumors exhibiting GBM features when implanted in immune-compromised animals. A detailed functional, protein and genetic characterization of human GBM cells cultured in serum-free defined conditions demonstrated no statistically meaningful differences when grown using sphere (NSA) or adherent conditions. Hence, both methods are functionally equivalent and remain suitable options for expanding primary high-grade gliomas in tissue culture. PMID:25806119

  4. Specificity in calcium oxalate adherence to papillary epithelial cells in culture

    SciTech Connect

    Riese, R.J.; Riese, J.W.; Kleinman, J.G.; Wiessner, J.H.; Mandel, G.S.; Mandel, N.S. )

    1988-11-01

    Attachment of microcystallites to cellular membranes may be an important component of the pathophysiology of many diseases including urolithiasis. This study attempts to characterize the interaction of calcium oxalate (CaOx) crystals and apatite (AP) crystals with renal papillary collecting tubule (RPCT) cells in primary culture. Primary cultures of RPCT cells showed the characteristic monolayer growth with sporadically interspersed clumped cells. Cultures were incubated with ({sup 14}C)CaOx crystals, and the crystals that bound were quantified by microscopy and adherent radioactivity. Per unit of cross-sectional area, 32 times more CaOx crystals were bound to the clumps than to the monolayer. CaOx adherence demonstrated concentration-dependent saturation with a {beta} value (fraction of cell culture area binding CaOx crystals) of 0.179 and a 1/{alpha}{sub ox} value of 287 {mu}g/cm{sup 2}. On incubation with AP crystals, CaOx binding demonstrated concentration-dependent inhibition with a 1/{alpha}{sub AP} value of 93 {mu}g/cm{sup 2}. Microcystallite adherence to RPCT cells demonstrates selectivity for cellular clumps, saturation, and inhibition. These features suggest specific binding.

  5. Adherent cells in granulocyte-macrophage colony-stimulating factor-induced bone marrow-derived dendritic cell culture system are qualified dendritic cells.

    PubMed

    Li, Gong-Bo; Lu, Guang-Xiu

    2010-01-01

    A widely-used method for generating dendritic cell (DC) is to culture bone marrow cells in granulocyte-macrophage colony-stimulating factor (GM-CSF)-containing medium for 6-10 days. Usually, non-adherent cells are used as qualified dendritic cells while the adherent ones are discarded as "non-dendritic cells" or macrophages. In this study, we show that the adherent cells are nearly identical to the non-adherent cells in both dendritic cell surface markers expression and main dendritic cell-related functions, hence to prove that these "junk cells" are actually qualified dendritic cells.

  6. [Adherent and single-cell suspension culture of Madin-Darby canine kidney cells in serum-free medium].

    PubMed

    Huang, Ding; Zhao, Liang; Tan, Wensong

    2011-04-01

    In recent years, there are tremendous economic and social losses across the world because of virus-related diseases. It is well known that Madin-Darby canine kidney (MDCK) cells are easily handled, quickly amplified and efficiently infected with influenza virus. Therefore, they are considered as one of the most important cell lines for the production of influenza vaccine. In this work, we first developed a serum-free adherent culture process for MDCK cells with an in-house prepared serum-free medium MDCK-SFM. Next, we derived a cell line named ssf-MDCK, which was amenable for single-cell suspension culture in the serum-free medium. We found that during serum-free batch culture of MDCK cells, the peak viable cell density and maximum specific growth rate were 3.81 x 10(6) cells/mL and 0.056 h(-1), respectively; 3.6- and 1.6-fold increase compared with those in serum-containing adherent batch culture. In addition, we compared growth and metabolic characteristics of MDCK cells in serum-containing adherent culture, serum-free adherent culture and serum-free single-cell suspension culture. We found that less metabolic by-products were produced in both serum-free cultures. In serum-free single-cell suspension batch culture, the viable cell density was highest. These results are critical for establishing large-scale suspension culture of MDCK cells as subsequent well as large-scale influenza vaccine production.

  7. Roles of adherent myogenic cells and dynamic culture in engineered muscle function and maintenance of satellite cells.

    PubMed

    Juhas, Mark; Bursac, Nenad

    2014-11-01

    Highly functional engineered skeletal muscle constructs could serve as physiological models of muscle function and regeneration and have utility in therapeutic replacement of damaged or diseased muscle tissue. In this study, we examined the roles of different myogenic cell fractions and culturing conditions in the generation of highly functional engineered muscle. Fibrin-based muscle bundles were fabricated using either freshly-isolated myogenic cells or their adherent fraction pre-cultured for 36 h. Muscle bundles made of these cells were cultured in both static and dynamic conditions and systematically characterized with respect to early myogenic events and contractile function. Following 2 weeks of culture, we observed both individual and synergistic benefits of using the adherent cell fraction and dynamic culture on muscle formation and function. In particular, optimal culture conditions resulted in significant increase in the total cross-sectional muscle area (- 3-fold), myofiber size (- 1.6-fold), myonuclei density (- 1.2-fold), and force generation (- 9-fold) compared to traditional use of freshly-isolated cells and static culture. Curiously, we observed that only a simultaneous use of the adherent cell fraction and dynamic culture resulted in accelerated formation of differentiated myofibers which were critical for providing a niche-like environment for maintenance of a satellite cell pool early during culture. Our study identifies key parameters for engineering large-size, highly functional skeletal muscle tissues with improved ability for retention of functional satellite cells.

  8. [Modified method for whole bone marrow adherent culture of human bone marrow mesenchymal stem cells].

    PubMed

    Wang, Xiao-Qing; Zhong, Zhao-Dong; Chen, Zhi-Chao; Zou, Ping

    2014-04-01

    This study was aimed to investigate a more convenient and efficient method to cultivate the human bone marrow mesenchymal stem cells by means of natural erythrocyte sedimentation principle, based on the whole bone marrow adherent method. The bone marrow was cultured with a six-well plate instead of the flasks.Firstly, the bone marrow specimen was cultivated with the MSC complete medium for 48 h, then the upper RBC-free supernatant layer was drawn and placed into the new wells to isolate MSC. Inverted microscope was used to observe the cell morphology and to record the adherent time of first cell passage, first passaging time. The traditional whole bone marrow adherent method was used as the control. The cell cycle and cell surface markers were detected by flow cytometry,and the differentiative capacity of MSC into osteocyte and adipocyte was identified by alkaline phosphatase kit and oil red O, respectively. Besides, the proliferative curve of P1,P3,P5 of BMSC was depicted by counting method. The results showed that MSC cultured by the modified method highly expressed CD90, CD105, CD13, CD44 and lowly expressed CD14, CD45, CD34. Concerning the cell cycle feature, it was found that most of the cells were in G0/G1 phase (88.76%) , followed by G2/M phase (3.04%) and S phase (8.2%), which was in accordance with stem cell cycle characteristics. The proliferative curve showed a typical "S" type, and both the oil red O and alkaline phosphatase staining of MSC were positive. Compared with the traditional method, the modified method had the advantage of high adherence rate (P = 0.0001) and shorter passaging time for the first passage (P = 0.001), with the statistically significant difference. It is concluded that there is a large number of adherent, active and suspended MSC in the RBC-free supernatant layer after the culture of bone marrow for 48 h. Isolating MSC by the modified method is more convenient and efficient than the traditional whole bone marrow adherent method.

  9. Lipophilic rather than hydrophilic photosensitizers show strong adherence to standard cell culture microplates under cell-free conditions.

    PubMed

    Engelhardt, Victoria; Kiesslich, Tobias; Berlanda, Juergen; Hofbauer, Stefanie; Krammer, Barbara; Plaetzer, Kristjan

    2011-06-02

    Analysis of photosensitizer (PS) uptake kinetics into tumor cells is a standard cell culture experiment in photodynamic therapy (PDT) - usually performed in plastic microplates or petri dishes. Organic substances such as PS can potentially interact with the plastic surfaces. In this study, we provide a qualitative comparison of three lipophilic PS (hypericin, Foscan® and Photofrin®) and two rather hydrophilic PS formulations (PVP-hypericin and aluminum (III) phthalocyanine tetrasulfonate chloride) regarding their adherence to the surfaces of 96-well microplates obtained from four different manufacturers. For estimation of the relevance of PS adherence for cellular uptake studies we compared the fluorescence signal of the respective PS in microplates containing A431 human epithelial carcinoma cells with microplates incubated with the respective PS under cell-free conditions. We demonstrate that lipophilic PS substances show a strong adherence to microplates - in case of direct lysis and fluorescence measurement resulting in 50% up to 90% of the overall signal to be caused by adherence of the substances to the plastic materials in a cellular uptake experiment. For the hydrophilic compounds, adherence is negligible. Interestingly, adherence of PS agents to microplates takes place in a time-dependent and thus kinetic-like manner, requiring up to several hours to reach a plateau of the fluorescence signal. Furthermore, PS adherence is a function of the PS concentration applied and no saturation effect was observed for the concentrations used in this study. Taken together, this study provides a systematic analysis under which conditions PS adherence to cell culture plates may contribute to the overall fluorescence signal in - for example - PS uptake experiments.

  10. A Minimally Invasive Method for Retrieving Single Adherent Cells of Different Types from Cultures

    PubMed Central

    Zeng, Jia; Mohammadreza, Aida; Gao, Weimin; Merza, Saeed; Smith, Dean; Kelbauskas, Laimonas; Meldrum, Deirdre R.

    2014-01-01

    The field of single-cell analysis has gained a significant momentum over the last decade. Separation and isolation of individual cells is an indispensable step in almost all currently available single-cell analysis technologies. However, stress levels introduced by such manipulations remain largely unstudied. We present a method for minimally invasive retrieval of selected individual adherent cells of different types from cell cultures. The method is based on a combination of mechanical (shear flow) force and biochemical (trypsin digestion) treatment. We quantified alterations in the transcription levels of stress response genes in individual cells exposed to varying levels of shear flow and trypsinization. We report optimal temperature, RNA preservation reagents, shear force and trypsinization conditions necessary to minimize changes in the stress-related gene expression levels. The method and experimental findings are broadly applicable and can be used by a broad research community working in the field of single cell analysis. PMID:24957932

  11. Bioluminescent high-throughput assay for the bacteria adherence to the tissue culture cells.

    PubMed

    Brovko, L; Minikh, O; Piekna, A; Griffiths, M W

    2011-07-01

    The goal of this study was develop a rapid high-throughput method for the assessment of the bacterial adhesion to tissue culture cells and test this method by investigation of the adhesion and growth of pathogenic and non-pathogenic Escherichia coli strains in the presence of HeLa human epithelial cells. Fifteen strains of E. coli were transformed with a plasmid carrying the entire lux operon of Photorhabdus luminescens to make them bioluminescent. By using the Time-to-Detection approach and bioluminescence imaging in microplate format, the adherence and growth of bacteria in tissue culture medium in the presence of HeLa cells was monitored. It was observed that Eagle's minimal essential medium (EMEM) supplemented with 10% fetal bovine serum (FBS) significantly inhibited growth of E. coli. However, in the presence of HeLa cells the detected growth of E. coli was similar to the growth observed in LB medium. It was established that the initial number of E. coli cells present in the microplate directly correlated with the time necessary for the bioluminescence signal to reach the threshold level, hence allowing the accurate assessment of the adhered cells within 8-10 h. Neither bacterial adherence nor growth kinetics correlated with the pathogenicity of the strain though they were strain-specific. The developed approach provided new information on the interaction of E. coli with epithelial cells and could be used for both pathogenicity research and for the screening of potential therapeutic agents for the ability to minimize pathogen colonization of human tissues.

  12. Non-invasive and non-destructive measurements of confluence in cultured adherent cell lines

    PubMed Central

    Busschots, Steven; O’Toole, Sharon; O’Leary, John J.; Stordal, Britta

    2014-01-01

    Many protocols used for measuring the growth of adherent monolayer cells in vitro are invasive, destructive and do not allow for the continued, undisturbed growth of cells within flasks. Protocols often use indirect methods for measuring proliferation. Microscopy techniques can analyse cell proliferation in a non-invasive or non-destructive manner but often use expensive equipment and software algorithms. In this method images of cells within flasks are captured by photographing under a standard inverted phase contract light microscope using a digital camera with a camera lens adaptor. Images are analysed for confluence using ImageJ freeware resulting in a measure of confluence known as an Area Fraction (AF) output. An example of the AF method in use on OVCAR8 and UPN251 cell lines is included. • Measurements of confluence from growing adherent cell lines in cell culture flasks is obtained in a non-invasive, non-destructive, label-free manner. • The technique is quick, affordable and eliminates sample manipulation. • The technique provides an objective, consistent measure of when cells reach confluence and is highly correlated to manual counting with a haemocytometer. The average correlation co-efficient from a Spearman correlation (n = 3) was 0.99 ± 0.008 for OVCAR8 (p = 0.01) and 0.99 ± 0.01 for UPN251 (p = 0.01) cell lines. PMID:26150966

  13. Non-invasive and non-destructive measurements of confluence in cultured adherent cell lines.

    PubMed

    Busschots, Steven; O'Toole, Sharon; O'Leary, John J; Stordal, Britta

    2015-01-01

    Many protocols used for measuring the growth of adherent monolayer cells in vitro are invasive, destructive and do not allow for the continued, undisturbed growth of cells within flasks. Protocols often use indirect methods for measuring proliferation. Microscopy techniques can analyse cell proliferation in a non-invasive or non-destructive manner but often use expensive equipment and software algorithms. In this method images of cells within flasks are captured by photographing under a standard inverted phase contract light microscope using a digital camera with a camera lens adaptor. Images are analysed for confluence using ImageJ freeware resulting in a measure of confluence known as an Area Fraction (AF) output. An example of the AF method in use on OVCAR8 and UPN251 cell lines is included. •Measurements of confluence from growing adherent cell lines in cell culture flasks is obtained in a non-invasive, non-destructive, label-free manner.•The technique is quick, affordable and eliminates sample manipulation.•The technique provides an objective, consistent measure of when cells reach confluence and is highly correlated to manual counting with a haemocytometer. The average correlation co-efficient from a Spearman correlation (n = 3) was 0.99 ± 0.008 for OVCAR8 (p = 0.01) and 0.99 ± 0.01 for UPN251 (p = 0.01) cell lines.

  14. Long-term culture of mouse embryonic stem cell-derived adherent neurospheres and functional neurons.

    PubMed

    Hayashi, Mirian A F; Guerreiro, Juliano R; Cassola, Antonio C; Lizier, Nelson F; Kerkis, Alexandre; Camargo, Antonio C M; Kerkis, Irina

    2010-12-01

    Innumerous protocols, using the mouse embryonic stem (ES) cells as model for in vitro study of neurons functional properties and features, have been developed. Most of these protocols are short lasting, which, therefore, does not allow a careful analysis of the neurons maturation, aging, and death processes. We describe here a novel and efficient long-lasting protocol for in vitro ES cells differentiation into neuronal cells. It consists of obtaining embryoid bodies, followed by induction of neuronal differentiation with retinoic acid of nonadherent embryoid bodies (three-dimensional model), which further allows their adherence and formation of adherent neurospheres (AN, bi-dimensional model). The AN can be maintained for at least 12 weeks in culture under repetitive mechanical splitting, providing a constant microenvironment (in vitro niche) for the neuronal progenitor cells avoiding mechanical dissociation of AN. The expression of neuron-specific proteins, such as nestin, sox1, beta III-tubulin, microtubule-associated protein 2, neurofilament medium protein, Tau, neuronal nuclei marker, gamma-aminobutyric acid, and 5-hydroxytryptamine, were confirmed in these cells maintained during 3 months under several splitting. Additionally, expression pattern of microtubule-associated proteins, such as lissencephaly (Lis1) and nuclear distribution element-like (Ndel1), which were shown to be essential for differentiation and migration of neurons during embryogenesis, was also studied. As expected, both proteins were expressed in undifferentiated ES cells, AN, and nonrosette neurons, although presenting different spatial distribution in AN. In contrast to previous studies, using cultured neuronal cells derived from embryonic and adult tissues, only Ndel1 expression was observed in the centrosome region of early neuroblasts from AN. Mature neurons, obtained from ES cells in this work, display ionic channels and oscillations of membrane electrical potential typical of

  15. Clonogenic Assay: Adherent Cells

    PubMed Central

    Rafehi, Haloom; Orlowski, Christian; Georgiadis, George T.; Ververis, Katherine; El-Osta, Assam; Karagiannis, Tom C.

    2011-01-01

    The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant

  16. The majority of enteroaggregative Escherichia coli strains produce the E. coli common pilus when adhering to cultured epithelial cells.

    PubMed

    Avelino, Fabiola; Saldaña, Zeus; Islam, Sohidul; Monteiro-Neto, Valerio; Dall'Agnol, Monique; Eslava, Carlos A; Girón, Jorge A

    2010-11-01

    Enteroaggregative Escherichia coli (EAEC) have emerged as a significant worldwide cause of chronic diarrhea in the pediatric population and in HIV patients. The vast majority of EAEC strains do not produce the aggregative adherence fimbriae I-III (AAFs) so far reported and thus, what adherence factors are present in these strains remains unknown. Here, we investigated the prevalence of the chromosomal E. coli common pilus (ECP) genes and ECP production amongst 130 EAEC strains of diverse origin as well as the role of ECP in EAEC adherence. Through multiplex PCR analysis we found that 96% of EAEC strains contained the ecpA structural pilin gene whereas only 3.1% and 5.4% were positive for AAF fimbrial genes aggA or aafA, respectively. Among the ecpA(+) strains, 63% produced ECP when adhering to cultured epithelial cells. An ecpA mutant derived from prototypic strain 042 (AAF/II(+)) was not altered in adherence suggesting that the AAF/II, and not ECP, plays a major role in this strain. In contrast, strain 278-1 (AAF(-)) deleted of the ecpA gene was significantly reduced in adherence to cultured epithelial cells. In all, these data indicate a potential role of ECP in adherence for EAEC strains lacking the known AAFs and that in association with other adhesive determinants, ECP may contribute to their survival and persistence within the host and in the environment.

  17. Retinoid modulation of collagenase production by adherent human mononuclear cells in culture.

    PubMed Central

    Ohta, A; Louie, J S; Uitto, J

    1987-01-01

    Previous observations have suggested that retinoids might be useful for the treatment of rheumatoid arthritis. In this study we examined the effects of various retinoids on collagenase production by adherent human peripheral blood mononuclear cells in culture. We have previously shown that these cells, consisting predominantly of monocyte-macrophages, actively synthesize and secrete collagenase upon stimulation with concanavalin A. The cells were incubated in serum free medium with all-trans-retinoic acid, 13-cis-retinoic acid, all-trans-retinal, or Ro 10-9359 (trimethylmethoxyphenyl retinoic acid ethyl ester) for up to 72 hours, and the collagenase activity was determined with [3H]proline labelled type I collagen as substrate. The incubation of mononuclear cells with all-trans-retinoic acid in the concentration range 10(-7)-10(-5) mol/l resulted in a dose dependent inhibition of the collagenase production. All-trans-retinal was also a potent inhibitor, whereas 13-cis-retinoic acid and Ro 10-9359 in a concentration of 10(-5) mol/l had a lesser effect. Control experiments indicated that the inhibition of collagenase production by all-trans-retinoic acid did not result from inhibition of total protein synthesis nor could it be explained by induction of an inhibitory molecule. These results indicate that retinoids with distinct structural features can inhibit collagenase production by monocyte-macrophages, and suggest a role for retinoids in the treatment of rheumatoid arthritis. PMID:3036026

  18. Effects of Fibronectin Coating on Bacterial and Osteoblast Progenitor Cells Adherence in a Co-culture Assay.

    PubMed

    Hindié, Mathilde; Wu, Dongni; Anselme, Karine; Gallet, Olivier; Di Martino, Patrick

    2016-07-06

    Bacterial adherence to the surface of implants functionalized with cell-adhesive biomolecules is a critical first step of infection development. This study was designed to determine how the immobilization of human plasmatic fibronectin (pFN) could impact bacterial and osteoblast cells interaction with the surface during concomitant exposition to the two cell-types. Calibrated suspensions of P. aeruginosa PAOI or S. aureus CIP4.83 bacteria and STRO-1(+)A osteoblast progenitor cells were mixed, co-seeded on glass coverslips coated or not with pFN and incubated at 37 °C. After 3 h of co-culture, the presence of bacteria did not modify the STRO-1(+)A cells adherence to glass. pFN coating significantly enhanced STRO-1(+)A cells, CIP4.83 and PAOI adherence to glass and bacterial interaction with STRO-1(+)A cells. Confocal laser scanning microscopy observations revealed that cells on the pFN-coated substrate exhibited a greater spreading, better organized network of cytoskeletal filaments, and an increased cellular FN expression than cells on the uncoated substrate. The use of fluorescently labeled pFN showed that adherent STRO-1(+)A cells were able to remodel and to concentrate coated pFN at the cells surface. Thus, the use of FN coating could increase the risk of bacterial adherence to the material surface, acting either directly onto the coating layer or indirectly on adherent osteoblastic cells. This may increase the infection risk in the presence of bacterial contamination.

  19. Long-term cultures of stem/progenitor cells from lobular and ductal breast carcinomas under non-adherent conditions

    PubMed Central

    Nardone, Agostina; Corvigno, Sara; Brescia, Annalisa; D’Andrea, Daniel; Limite, Gennaro

    2010-01-01

    A small subpopulation of stem/progenitor cells can give rise to the diversity of differentiated cells that comprise the bulk of the tumor. Are proliferating cells, within the bulk of tumor, few cells with uncommon features? The cell biological approach provides a limitless model for studying the hierarchical organization of progenitor subpopulation and identifying potential therapeutic targets. Aim of the study was to expand patients’ breast cancer cells for evaluating functional cell properties, and to characterize the protein expression profile of selected cells to be compared with that of primary tumors. Breast cancer cells from estrogen receptor (ERα) positive, HER2 negative lobular (LoBS cells) and ductal (DuBS cells) histotype were cultured under non-adherent conditions to form mammospheres. Sorting of the cells by their surface expression of CD24 and CD44 gave rise to subpopulations which were propagated, enriched and characterized for the expression of epithelial and stromal markers. We found that non-adherent culture conditions generate mammospheres of slowly proliferating cells; single cells, dissociated from mammospheres, grow in soft agar; long-term cultured LoBS and DuBS cells, CD44+/CD24low, express cytokeratin 5 (CK5), α-smooth muscle actin (α-sma) and vimentin, known as markers of basal/myoepithelial cells; and ERα (only DuBS cells), HER1 (EGF-Receptor), activated HER2, and cyclinD1 as markers of luminal epithelial cell. Isolates of cells from breast cancer patients may be a tool for a marker-driven testing of targeted therapies. PMID:21188518

  20. Extending metabolome coverage for untargeted metabolite profiling of adherent cultured hepatic cells.

    PubMed

    García-Cañaveras, Juan Carlos; López, Silvia; Castell, José Vicente; Donato, M Teresa; Lahoz, Agustín

    2016-02-01

    MS-based metabolite profiling of adherent mammalian cells comprises several challenging steps such as metabolism quenching, cell detachment, cell disruption, metabolome extraction, and metabolite measurement. In LC-MS, the final metabolome coverage is strongly determined by the separation technique and the MS conditions used. Human liver-derived cell line HepG2 was chosen as adherent mammalian cell model to evaluate the performance of several commonly used procedures in both sample processing and LC-MS analysis. In a first phase, metabolite extraction and sample analysis were optimized in a combined manner. To this end, the extraction abilities of five different solvents (or combinations) were assessed by comparing the number and the levels of the metabolites comprised in each extract. Three different chromatographic methods were selected for metabolites separation. A HILIC-based method which was set to specifically separate polar metabolites and two RP-based methods focused on lipidome and wide-ranging metabolite detection, respectively. With regard to metabolite measurement, a Q-ToF instrument operating in both ESI (+) and ESI (-) was used for unbiased extract analysis. Once metabolite extraction and analysis conditions were set up, the influence of cell harvesting on metabolome coverage was also evaluated. Therefore, different protocols for cell detachment (trypsinization or scraping) and metabolism quenching were compared. This study confirmed the inconvenience of trypsinization as a harvesting technique, and the importance of using complementary extraction solvents to extend metabolome coverage, minimizing interferences and maximizing detection, thanks to the use of dedicated analytical conditions through the combination of HILIC and RP separations. The proposed workflow allowed the detection of over 300 identified metabolites from highly polar compounds to a wide range of lipids.

  1. Single cell dual adherent-suspension co-culture micro-environment for studying tumor-stromal interactions with functionally selected cancer stem-like cells.

    PubMed

    Chen, Yu-Chih; Zhang, Zhixiong; Fouladdel, Shamileh; Deol, Yadwinder; Ingram, Patrick N; McDermott, Sean P; Azizi, Ebrahim; Wicha, Max S; Yoon, Euisik

    2016-08-07

    Considerable evidence suggests that cancer stem-like cells (CSCs) are critical in tumor pathogenesis, but their rarity and transience has led to much controversy about their exact nature. Although CSCs can be functionally identified using dish-based tumorsphere assays, it is difficult to handle and monitor single cells in dish-based approaches; single cell-based microfluidic approaches offer better control and reliable single cell derived sphere formation. However, like normal stem cells, CSCs are heavily regulated by their microenvironment, requiring tumor-stromal interactions for tumorigenic and proliferative behaviors. To enable single cell derived tumorsphere formation within a stromal microenvironment, we present a dual adherent/suspension co-culture device, which combines a suspension environment for single-cell tumorsphere assays and an adherent environment for co-culturing stromal cells in close proximity by selectively patterning polyHEMA in indented microwells. By minimizing dead volume and improving cell capture efficiency, the presented platform allows for the use of small numbers of cells (<100 cells). As a proof of concept, we co-cultured single T47D (breast cancer) cells and primary cancer associated fibroblasts (CAF) on-chip for 14 days to monitor sphere formation and growth. Compared to mono-culture, co-cultured T47D have higher tumorigenic potential (sphere formation rate) and proliferation rates (larger sphere size). Furthermore, 96-multiplexed single-cell transcriptome analyses were performed to compare the gene expression of co-cultured and mono-cultured T47D cells. Phenotypic changes observed in co-culture correlated with expression changes in genes associated with proliferation, apoptotic suppression, tumorigenicity and even epithelial-to-mesechymal transition. Combining the presented platform with single cell transcriptome analysis, we successfully identified functional CSCs and investigated the phenotypic and transcriptome effects induced

  2. Extracellular mass transport considerations for space flight research concerning suspended and adherent in vitro cell cultures.

    PubMed

    Klaus, David M; Benoit, Michael R; Nelson, Emily S; Hammond, Timmothy G

    2004-03-01

    Conducting biological research in space requires consideration be given to isolating appropriate control parameters. For in vitro cell cultures, numerous environmental factors can adversely affect data interpretation. A biological response attributed to microgravity can, in theory, be explicitly correlated to a specific lack of weight or gravity-driven motion occurring to, within or around a cell. Weight can be broken down to include the formation of hydrostatic gradients, structural load (stress) or physical deformation (strain). Gravitationally induced motion within or near individual cells in a fluid includes sedimentation (or buoyancy) of the cell and associated shear forces, displacement of cytoskeleton or organelles, and factors associated with intra- or extracellular mass transport. Finally, and of particular importance for cell culture experiments, the collective effects of gravity must be considered for the overall system consisting of the cells, their environment and the device in which they are contained. This does not, however, rule out other confounding variables such as launch acceleration, on orbit vibration, transient acceleration impulses or radiation, which can be isolated using onboard centrifuges or vibration isolation techniques. A framework is offered for characterizing specific cause-and-effect relationships for gravity-dependent responses as a function of the above parameters.

  3. Extracellular mass transport considerations for space flight research concerning suspended and adherent in vitro cell cultures

    NASA Technical Reports Server (NTRS)

    Klaus, David M.; Benoit, Michael R.; Nelson, Emily S.; Hammond, Timmothy G.

    2004-01-01

    Conducting biological research in space requires consideration be given to isolating appropriate control parameters. For in vitro cell cultures, numerous environmental factors can adversely affect data interpretation. A biological response attributed to microgravity can, in theory, be explicitly correlated to a specific lack of weight or gravity-driven motion occurring to, within or around a cell. Weight can be broken down to include the formation of hydrostatic gradients, structural load (stress) or physical deformation (strain). Gravitationally induced motion within or near individual cells in a fluid includes sedimentation (or buoyancy) of the cell and associated shear forces, displacement of cytoskeleton or organelles, and factors associated with intra- or extracellular mass transport. Finally, and of particular importance for cell culture experiments, the collective effects of gravity must be considered for the overall system consisting of the cells, their environment and the device in which they are contained. This does not, however, rule out other confounding variables such as launch acceleration, on orbit vibration, transient acceleration impulses or radiation, which can be isolated using onboard centrifuges or vibration isolation techniques. A framework is offered for characterizing specific cause-and-effect relationships for gravity-dependent responses as a function of the above parameters.

  4. AFBI assay – Aptamer Fluorescence Binding and Internalization assay for cultured adherent cells

    PubMed Central

    Thiel, William H.; Giangrande, Paloma H.

    2016-01-01

    The SELEX (Systematic Evolution of Ligands by Exponential Enrichment) process allows for the enrichment of DNA or RNA aptamers from a complex nucleic acid library that are specific for a target molecule. The SELEX process has been adapted from identifying aptamers in vitro using recombinant target protein to cell-based methodologies (Cell-SELEX), where the targets are expressed on the surface of cells. One major advantage of Cell-SELEX is that the target molecules are maintained in a native confirmation. Additionally, Cell-SELEX may be used to discover novel therapeutic biomarkers by performing selections on diseased versus healthy cells. However, a caveat to Cell-SELEX is that testing of single aptamers identified in the selection is laborious, time-consuming, and expensive. The most frequently used methods to screen for aptamer binding and internalization on cells are flow cytometry and quantitative PCR (qPCR). While flow cytometry can directly assess binding of a fluorescently-labeled aptamer to a target, it requires significant starting material and is not easily scalable. qPCR-based approaches are highly sensitive but have non-negligible experiment-to-experiment variability due to the number of sample processing steps. Herein we describe a cell-based aptamer fluorescence binding and internalization (AFBI) assay. This assay requires minimal reagents and has few experimental steps/manipulations, thereby allowing for rapid screening of many aptamers and conditions simultaneously and direct quantitation of aptamer binding and internalization. PMID:26972784

  5. Non-adherent culture induces paclitaxel resistance in H460 lung cancer cells via ERK-mediated up-regulation of βIVa-tubulin.

    PubMed

    Atjanasuppat, Korakot; Lirdprapamongkol, Kriengsak; Jantaree, Phatcharida; Svasti, Jisnuson

    2015-10-23

    Circulating tumor cells (CTCs) are metastasizing epithelial cancer cells that adapt to survive when floating in bloodstream during metastasis. This condition can be mimicked in vitro by using non-adherent cell culture. The chemosensitivity of CTCs appears to correlate with the response of metastatic cancer patients to therapy, but chemoresistance is also frequently observed in advanced stage cancer patients, who have never previously received chemotherapy. We hypothesize that adaptation of epithelial cancer cells to become floating CTCs could lead to development of chemoresistance. Here, we explore whether chemoresistance is induced in epithelial cancer cells when cultured under non-adherent conditions. Increased paclitaxel-specific resistance was observed in floating cells compared to attached cells in H460, MCF-7, and HepG2 human cancer cell lines, by 15.6-, 3.9-, and 2.6-fold increases in IC50 values, respectively. qRT-PCR analysis showed that a paclitaxel-resistant β-tubulin isotype, βIVa-tubulin, was the most up-regulated gene compared with other β-tubulin isotypes in H460 floating cells, concomitant with elevated ERK activation. ERK inhibitor treatment could attenuate the up-regulation of βIVa-tubulin, and decreased the paclitaxel resistance of H460 floating cells, even though other β-tubulin isotypes were up-regulated when the ERK activation was blocked. In conclusion, we show induction of paclitaxel resistance in epithelial cancer cells, when floating in non-adherent culture, and this might occur with CTCs of cancer patients.

  6. Characterization of Three-Dimensional Retinal Tissue Derived from Human Embryonic Stem Cells in Adherent Monolayer Cultures

    PubMed Central

    Singh, Ratnesh K.; Mallela, Ramya K.; Cornuet, Pamela K.; Reifler, Aaron N.; Chervenak, Andrew P.; West, Michael D.; Wong, Kwoon Y.; Nasonkin, Igor O.

    2015-01-01

    Stem cell-based therapy of retinal degenerative conditions is a promising modality to treat blindness, but requires new strategies to improve the number of functionally integrating cells. Grafting semidifferentiated retinal tissue rather than progenitors allows preservation of tissue structure and connectivity in retinal grafts, mandatory for vision restoration. Using human embryonic stem cells (hESCs), we derived retinal tissue growing in adherent conditions consisting of conjoined neural retina and retinal pigment epithelial (RPE) cells and evaluated cell fate determination and maturation in this tissue. We found that deriving such tissue in adherent conditions robustly induces all eye field genes (RX, PAX6, LHX2, SIX3, SIX6) and produces four layers of pure populations of retinal cells: RPE (expressing NHERF1, EZRIN, RPE65, DCT, TYR, TYRP, MITF, PMEL), early photoreceptors (PRs) (coexpressing CRX and RCVRN), inner nuclear layer neurons (expressing CALB2), and retinal ganglion cells [RGCs, expressing BRN3B and Neurofilament (NF) 200]. Furthermore, we found that retinal progenitors divide at the apical side of the hESC-derived retinal tissue (next to the RPE layer) and then migrate toward the basal side, similar to that found during embryonic retinogenesis. We detected synaptogenesis in hESC-derived retinal tissue, and found neurons containing many synaptophysin-positive boutons within the RGC and PR layers. We also observed long NF200-positive axons projected by RGCs toward the apical side. Whole-cell recordings demonstrated that putative amacrine and/or ganglion cells exhibited electrophysiological responses reminiscent of those in normal retinal neurons. These responses included voltage-gated Na+ and K+ currents, depolarization-induced spiking, and responses to neurotransmitter receptor agonists. Differentiation in adherent conditions allows generation of long and flexible pieces of 3D retinal tissue suitable for isolating transplantable slices of tissue for

  7. Differentiation of cultured keratinocytes promotes the adherence of Streptococcus pyogenes.

    PubMed Central

    Darmstadt, G L; Fleckman, P; Jonas, M; Chi, E; Rubens, C E

    1998-01-01

    Based on a consideration of the histopathology of nonbullous impetigo that shows localization of Streptococcus pyogenes to highly differentiated, subcorneal keratinocytes, we hypothesized that adherence of an impetigo strain of S. pyogenes would be promoted by terminal differentiation of keratinocytes. An assay was developed in which S. pyogenes adhered via pilus-like projections from the cell wall to the surface of cultured human keratinocytes in a time- and inoculum-dependent manner suggestive of a receptor-mediated process. Terminal differentiation of keratinocytes was induced by increasing the calcium concentration in the growth medium, and was confirmed by morphologic analysis using electron microscopy. Adherence of S. pyogenes was three and fourfold greater to keratinocytes differentiated in 1.0 and 1.5 mM calcium, respectively, compared with undifferentiated keratinocytes in 0.15 mM calcium. The presence of calcium during the adherence assay further enhanced adherence nearly twofold. Adherence occurred preferentially to sites of contact between adjacent keratinocytes, suggesting that the keratinocyte receptor may be a molecule involved in cell-to-cell adhesion. In contrast, nonpathogenic Streptococcus gordonii adhered poorly to keratinocytes regardless of their state of terminal differentiation, and adherence of a pharyngeal strain of S. pyogenes was twofold greater to undifferentiated than differentiated keratinocytes. This is the first report of in vitro adherence of S. pyogenes to keratinocytes in a manner that emulates human impetigo. Adherence of only the impetigo strain, and not the pharyngeal strain of S. pyogenes or the nonpathogenic S. gorgonii isolate, was promoted by keratinocyte differentiation. This result provides a model system for investigating the molecular pathogenesis of streptococcal skin infections. PMID:9421474

  8. Mitigation of Lethal Radiation Syndrome in Mice by Intramuscular Injection of 3D Cultured Adherent Human Placental Stromal Cells.

    PubMed

    Gaberman, Elena; Pinzur, Lena; Levdansky, Lilia; Tsirlin, Maria; Netzer, Nir; Aberman, Zami; Gorodetsky, Raphael

    2013-01-01

    Exposure to high lethal dose of ionizing radiation results in acute radiation syndrome with deleterious systemic effects to different organs. A primary target is the highly sensitive bone marrow and the hematopoietic system. In the current study C3H/HeN mice were total body irradiated by 7.7 Gy. Twenty four hrs and 5 days after irradiation 2×10(6) cells from different preparations of human derived 3D expanded adherent placental stromal cells (PLX) were injected intramuscularly. Treatment with batches consisting of pure maternal cell preparations (PLX-Mat) increased the survival of the irradiated mice from ∼27% to 68% (P<0.001), while cell preparations with a mixture of maternal and fetal derived cells (PLX-RAD) increased the survival to ∼98% (P<0.0001). The dose modifying factor of this treatment for both 50% and 37% survival (DMF50 and DMF37) was∼1.23. Initiation of the more effective treatment with PLX-RAD injection could be delayed for up to 48 hrs after irradiation with similar effect. A delayed treatment by 72 hrs had lower, but still significantly effect (p<0.05). A faster recovery of the BM and improved reconstitution of all blood cell lineages in the PLX-RAD treated mice during the follow-up explains the increased survival of the cells treated irradiated mice. The number of CD45+/SCA1+ hematopoietic progenitor cells within the fast recovering population of nucleated BM cells in the irradiated mice was also elevated in the PLX-RAD treated mice. Our study suggests that IM treatment with PLX-RAD cells may serve as a highly effective "off the shelf" therapy to treat BM failure following total body exposure to high doses of radiation. The results suggest that similar treatments may be beneficial also for clinical conditions associated with severe BM aplasia and pancytopenia.

  9. Mitigation of Lethal Radiation Syndrome in Mice by Intramuscular Injection of 3D Cultured Adherent Human Placental Stromal Cells

    PubMed Central

    Gaberman, Elena; Pinzur, Lena; Levdansky, Lilia; Tsirlin, Maria; Netzer, Nir; Aberman, Zami; Gorodetsky, Raphael

    2013-01-01

    Exposure to high lethal dose of ionizing radiation results in acute radiation syndrome with deleterious systemic effects to different organs. A primary target is the highly sensitive bone marrow and the hematopoietic system. In the current study C3H/HeN mice were total body irradiated by 7.7 Gy. Twenty four hrs and 5 days after irradiation 2×106 cells from different preparations of human derived 3D expanded adherent placental stromal cells (PLX) were injected intramuscularly. Treatment with batches consisting of pure maternal cell preparations (PLX-Mat) increased the survival of the irradiated mice from ∼27% to 68% (P<0.001), while cell preparations with a mixture of maternal and fetal derived cells (PLX-RAD) increased the survival to ∼98% (P<0.0001). The dose modifying factor of this treatment for both 50% and 37% survival (DMF50 and DMF37) was∼1.23. Initiation of the more effective treatment with PLX-RAD injection could be delayed for up to 48 hrs after irradiation with similar effect. A delayed treatment by 72 hrs had lower, but still significantly effect (p<0.05). A faster recovery of the BM and improved reconstitution of all blood cell lineages in the PLX-RAD treated mice during the follow-up explains the increased survival of the cells treated irradiated mice. The number of CD45+/SCA1+ hematopoietic progenitor cells within the fast recovering population of nucleated BM cells in the irradiated mice was also elevated in the PLX-RAD treated mice. Our study suggests that IM treatment with PLX-RAD cells may serve as a highly effective “off the shelf” therapy to treat BM failure following total body exposure to high doses of radiation. The results suggest that similar treatments may be beneficial also for clinical conditions associated with severe BM aplasia and pancytopenia. PMID:23823334

  10. Topography Influences Adherent Cell Regulation of Osteoclastogenesis.

    PubMed

    Nagasawa, M; Cooper, L F; Ogino, Y; Mendonca, D; Liang, R; Yang, S; Mendonca, G; Uoshima, K

    2016-03-01

    The importance of osteoclast-mediated bone resorption in the process of osseointegration has not been widely considered. In this study, cell culture was used to investigate the hypothesis that the function of implant-adherent bone marrow stromal cells (BMSCs) in osteoclastogenesis is influenced by surface topography. BMSCs isolated from femur and tibia of Sprague-Dawley rats were seeded onto 3 types of titanium surfaces (smooth, micro, and nano) and a control surface (tissue culture plastic) with or without osteogenic supplements. After 3 to 14 d, conditioned medium (CM) was collected. Subsequently, rat bone marrow-derived macrophages (BMMs) were cultured in media supplemented with soluble receptor activator of NF-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) as well as BMSC CM from each of the 4 surfaces. Gene expression levels of soluble RANKL, osteoprotegerin, tumor necrosis factor α, and M-CSF in cultured BMSCs at different time points were measured by real-time polymerase chain reaction. The number of differentiated osteoclastic cells was determined after tartrate-resistant acid phosphatase staining. Analysis of variance and t test were used for statistical analysis. The expression of prominent osteoclast-promoting factors tumor necrosis factor α and M-CSF was increased by BMSCs cultured on both micro- and nanoscale titanium topographies (P < 0.01). BMSC CM contained a heat-labile factor that increased BMMs osteoclastogenesis. CM from both micro- and nanoscale surface-adherent BMSCs increased the osteoclast number (P < 0.01). Difference in surface topography altered BMSC phenotype and influenced BMM osteoclastogenesis. Local signaling by implant-adherent cells at the implant-bone interface may indirectly control osteoclastogenesis and bone accrual around endosseous implants.

  11. Hydrocortisone differentially affects the ability of murine stromal cells and human marrow-derived adherent cells to promote the differentiation of CD34++/CD38- long-term culture-initiating cells.

    PubMed

    Croisille, L; Auffray, I; Katz, A; Izac, B; Vainchenker, W; Coulombel, L

    1994-12-15

    Very primitive human hematopoietic progenitor cells are identified indirectly by their ability to give rise to clonogenic progenitors in the presence of either human or murine stromal cells. These long-term culture-initiating cell (LTC-IC) assays are usually performed in the presence of hydrocortisone based on the initial observation that hydrocortisone was required for prolonged hematopoiesis in standard long-term bone marrow cultures. In this report, we investigated the role of hydrocortisone in LTC-IC assays initiated with CD34++/CD38- cells seeded onto either human bone marrow LTC-derived adherent cells or a murine marrow-derived stromal cell line, MS-5. It was found that weekly addition of hydrocortisone to the cultures reduced the frequency of LTC-IC (from 1/5 to 1/20) calculated from limiting dilution experiments and also reduced fivefold to 10-fold the number of their progeny clonogenic cells detected after 4 to 5 weeks. In contrast, the frequency and differentiative potential of CD34++/CD38- grown in the presence of human marrow feeders was unaltered by the addition of glucocorticoids. Data are consistent with the hypothesis that hydrocortisone inhibited LTC-IC differentiation by downregulating the expression of a synergistic factor produced by MS-5 cells. (1) In the absence of hydrocortisone, the number of clonogenic progenitors generated by LTC-IC was much higher in cultures seeded on MS-5 than in cultures seeded on human marrow adherent cells, which was also true when cytokines were added to the cocultures. However, based on the phenotype of the colonies, progenitors produced in MS-5 cocultures were more mature than those generated on human marrow adherent cells. (2) Hydrocortisone counteracted the stimulatory effect of recombinant human cytokines (interleukin-3, interleukin-6, and steel factor) in assays performed on MS-5 but not on human marrow feeders. (3) Hydrocortisone led to a 50% decrease in the numbers of colony-forming units

  12. Non-viral engineering of skin precursor-derived Schwann cells for enhanced NT-3 production in adherent and microcarrier culture.

    PubMed

    Shakhbazau, A; Shcharbin, D; Bryszewska, M; Kumar, R; Wobma, H M; Kallos, M S; Goncharova, N; Seviaryn, I; Kosmacheva, S; Potapnev, M; Midha, R

    2012-01-01

    Genetic engineering of stem cells and their derivatives has the potential to enhance their regenerative capabilities. Here, dendrimer- and lipofection-based approaches were used for non-viral neurotrophin-3 (NT-3) over-expression in Schwann cells differentiated from skin precursors (SKP-SCs). A variety of dendrimers were first tested for transfection efficiency on HEK 293T cells, with PAMAMNH2 G4 found most effective and used subsequently for SKP-SCs transfection. Plasmid-based expression resulted in increased NT-3 release from SKP-SCs in both adherent and microcarrier-based culture. In a proof-of-concept study, the microcarrier/SKP-SCs were implanted into the injured nerve, and transfected cells were shown to detach, integrate into the nerve tissue and associate with regenerating axons. Virus-free systems for transient neurotrophin expression are a feasible and biologically safe option to increase the therapeutic value of stem cells and stem cell-derived cells in nerve repair strategies. Further work to develop bioprocesses for expansion of SKP-SCs on microcarriers in bioreactors is still needed.

  13. Graphene oxide sheets-based platform for induced pluripotent stem cells culture: toxicity, adherence, growth and application

    NASA Astrophysics Data System (ADS)

    Durán, Marcela; Andrade, Patricia F.; Durán, Nelson; Luzo, Angela C. M.; Fávaro, Wagner J.

    2015-05-01

    It was prepared the graphene oxide (GO) sheets by suspension of GO in ultrapure deionized water or in Pluronic F-68 using a ultrasonicator bath. Total characterization of GO sheets was carried out. The results on suspension of GO in water showed excellent growth and cell adhesion. GO/Pluronic F-68 platform for the growth and adhesion of adipose-derived stem cells (ASCs) that exhibits excellent properties for these processes. GO in water suspension exhibited an inhibition of the cell growth over 5 μg/mL In vivo study with GO suspended in water (100 μg/mL) on Fisher 344 rats via i.p. administration showed low toxicity. Despite GO particle accumulates in the intraperitoneal cavity, this fact did not interfere with the final absorption of GO. The AST (aspartate aminotransferase) and ALT (alanine aminotransferase) levels (liver function) did not differ statistically in all experimental groups. Also, creatinine and urea levels (renal function) did not differ statistically in all experimental groups. Taking together, the data suggest the great potential of graphene oxide sheets as platform to ACSs, as well as, new material for treatment several urological diseases.

  14. Adherence of Tritrichomonas foetus to bovine vaginal epithelial cells.

    PubMed Central

    Corbeil, L B; Hodgson, J L; Jones, D W; Corbeil, R R; Widders, P R; Stephens, L R

    1989-01-01

    Adherence of Tritrichomonas foetus to bovine vaginal epithelial cells (VECs) in vitro was investigated with fresh washed bovine VECs and log-phase cultures of T. foetus. Observation under phase-contrast microscopy showed that T. foetus usually adhered first by the posterior flagellum and later by the body. Significantly more keratinized squamous epithelial cells were detected with attached parasites than nonkeratinized round epithelial cells. The optimal pH range for attachment was 6.0 to 7.5, with peak attachment at pH 6.5 for squamous VECs. Surface-reactive bovine antiserum to T. foetus prevented adherence to bovine squamous VECs. Inhibition of adherence occurred at nonagglutinating, nonimmobilizing serum dilutions. Antiserum fractions enriched for immunoglobulin G1 inhibited adherence, but fractions enriched for immunoglobulin G2 did not. The inhibitory antiserum was specific for several medium- to high-molecular-weight membrane antigens as detected in Western blots (immunoblots). The ability of surface-reactive antibodies to prevent adherence and to agglutinate and immobilize T. foetus indicates that they may be protective. Images PMID:2471692

  15. Cell Phone Intervention to Improve Adherence

    PubMed Central

    Marciel, Kristen K.; Saiman, Lisa; Quittell, Lynne M.; Dawkins, Kevin; Quittner, Alexandra L.

    2010-01-01

    Summary Background Treatment regimens for patients with cystic fibrosis (CF) are time-consuming and complex, resulting in consistently low adherence rates. To date, few studies have evaluated innovative technologies to improve adherence in this population. Current infection control guidelines for patients with CF seek to minimize patient-to-patient transmission of potential pathogens. Thus, interventions must avoid face-to-face contact and be delivered individually, limiting opportunities for peer support. This study aimed to develop and assess a web-enabled cell phone, CFFONE™, designed to provide CF information and social support to improve adherence in adolescents with CF. Methods The acceptability, feasibility, and utility of CFFONE™ were evaluated with health care professionals (n = 17) adolescents with CF aged 11–18 years old (n = 12), adults with CF aged 21–36 years old (n = 6), parents of adolescents with CF (n = 12), and technology experts (n = 8). Adolescents also tested a prototype of CFFONE™ (n = 9). Qualitative and quantitative data were collected. Results Focus group data with health care = professionals indicated a need for this intervention, and indicated that CFFONE™ would be likely to improve knowledge and social support, and somewhat likely to improve adherence. Adolescent, adults, and parents all rated CFFONE™ as likely to improve adherence. Technology experts rated the prototype design and format as appropriate. Conclusions The current study provided some support from key stakeholders for this intervention to improve adherence in adolescents with CF. Next steps include a multi-center trial of the efficacy and safety of CFFONE™. PMID:20054860

  16. Evaluation of a Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) assay (Keystone Sym)

    EPA Science Inventory

    Our goal is to establish an in vitro model system to evaluate chemical effects using a single stem cell culture technique that would improve throughput and provide quantitative markers of differentiation and cell number. To this end, we have used an adherent cell differentiation ...

  17. Factors involved in adherence of lactobacilli to human Caco-2 cells.

    PubMed Central

    Greene, J D; Klaenhammer, T R

    1994-01-01

    A quantitative assay performed with bacterial cells labelled with [3H]thymidine was used to investigate factors involved in the adherence of human isolates Lactobacillus acidophilus BG2FO4 and NCFM/N2 and Lactobacillus gasseri ADH to human Caco-2 intestinal cells. For all three strains, adherence was concentration dependent, greater at acidic pH values, and significantly greater than adherence of a control dairy isolate, Lactobacillus delbrueckii subsp. bulgaricus 1489. Adherence of L. acidophilus BG2FO4 and NCFM/N2 was decreased by protease treatment of the bacterial cells, whereas adherence of L. gasseri ADH either was not affected or was enhanced by protease treatment. Putative surface layer proteins were identified on L. acidophilus BG2FO4 and NCFM/N2 cells but were not involved in adherence. Periodate oxidation of bacterial cell surface carbohydrates significantly reduced adherence of L. gasseri ADH, moderately reduced adherence of L. acidophilus BG2FO4, and had no effect on adherence of L. acidophilus NCFM/N2. These results indicate that Lactobacillus species adhere to human intestinal cells via mechanisms which involve different combinations of carbohydrate and protein factors on the bacterial cell surface. The involvement of a secreted bridging protein, which has been proposed as the primary mediator of adherence of L. acidophilus BG2FO4 in spent culture supernatant (M.-H. Coconnier, T. R. Klaenhammer, S. Kernéis, M.-F. Bernet, and A. L. Servin, Appl. Environ. Microbiol. 58:2034-2039, 1992), was not confirmed in this study. Rather, a pH effect on Caco-2 cells contributed significantly to the adherence of this strain in spent culture supernatant.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:7811085

  18. Acidic fibroblast growth factor modulates Staphylococcus aureus adherence to human endothelial cells.

    PubMed Central

    Blumberg, E A; Hatcher, V B; Lowy, F D

    1988-01-01

    Alteration of human endothelial cells may increase their susceptibility to staphylococcal invasion and thus may contribute to the development of intravascular staphylococcal disease. Acidic fibroblast growth factor, a potent regulator of endothelial cell function, had a significant effect on Staphylococcus aureus infection of cultured human endothelial cells. Three of four S. aureus strains had diminished adherence to endothelial cells when the latter were grown in the presence of acidic fibroblast growth factor (P less than 0.05). The diminished adherence was time dependent, maximal at 72 h, and independent of the initial bacterial inoculum. A twofold enhancement of S. aureus adherence was observed when endothelial cells were pretreated with heparitinase. Adherence was unaffected by endothelial cell activation by interleukin-1 or endotoxin. Thus, acidic fibroblast growth factor exerted a protective effect, deterring S. aureus adherence to cultured endothelial cells. Endothelial cell heparan sulfate was also directly involved in the adherence process. Subtle modulations of endothelial cells can significantly affect the ability of S. aureus to adhere to and then infect these cells. Similar alterations may contribute to the ability of S. aureus to infect endovascular tissue in vivo. PMID:3259546

  19. Multiple roles of Activin/Nodal, bone morphogenetic protein, fibroblast growth factor and Wnt/β-catenin signalling in the anterior neural patterning of adherent human embryonic stem cell cultures

    PubMed Central

    Lupo, Giuseppe; Novorol, Claire; Smith, Joseph R.; Vallier, Ludovic; Miranda, Elena; Alexander, Morgan; Biagioni, Stefano; Pedersen, Roger A.; Harris, William A.

    2013-01-01

    Several studies have successfully produced a variety of neural cell types from human embryonic stem cells (hESCs), but there has been limited systematic analysis of how different regional identities are established using well-defined differentiation conditions. We have used adherent, chemically defined cultures to analyse the roles of Activin/Nodal, bone morphogenetic protein (BMP), fibroblast growth factor (FGF) and Wnt/β-catenin signalling in neural induction, anteroposterior patterning and eye field specification in hESCs. We show that either BMP inhibition or activation of FGF signalling is required for effective neural induction, but these two pathways have distinct outcomes on rostrocaudal patterning. While BMP inhibition leads to specification of forebrain/midbrain positional identities, FGF-dependent neural induction is associated with strong posteriorization towards hindbrain/spinal cord fates. We also demonstrate that Wnt/β-catenin signalling is activated during neural induction and promotes acquisition of neural fates posterior to forebrain. Therefore, inhibition of this pathway is needed for efficient forebrain specification. Finally, we provide evidence that the levels of Activin/Nodal and BMP signalling have a marked influence on further forebrain patterning and that constitutive inhibition of these pathways represses expression of eye field genes. These results show that the key mechanisms controlling neural patterning in model vertebrate species are preserved in adherent, chemically defined hESC cultures and reveal new insights into the signals regulating eye field specification. PMID:23576785

  20. Adherent neural stem (NS) cells from fetal and adult forebrain.

    PubMed

    Pollard, Steven M; Conti, Luciano; Sun, Yirui; Goffredo, Donato; Smith, Austin

    2006-07-01

    Stable in vitro propagation of central nervous system (CNS) stem cells would offer expanded opportunities to dissect basic molecular, cellular, and developmental processes and to model neurodegenerative disease. CNS stem cells could also provide a source of material for drug discovery assays and cell replacement therapies. We have recently reported the generation of adherent, symmetrically expandable, neural stem (NS) cell lines derived both from mouse and human embryonic stem cells and from fetal forebrain (Conti L, Pollard SM, Gorba T, Reitano E, Toselli M, Biella G, Sun Y, Sanzone S, Ying QL, Cattaneo E, Smith A. 2005. Niche-independent symmetrical self-renewal of a mammalian tissue stem cell. PLoS Biol 3(9):e283). These NS cells retain neuronal and glial differentiation potential after prolonged passaging and are transplantable. NS cells are likely to comprise the resident stem cell population within heterogeneous neurosphere cultures. Here we demonstrate that similar NS cell cultures can be established from the adult mouse brain. We also characterize the growth factor requirements for NS cell derivation and self-renewal. We discuss our current understanding of the relationship of NS cell lines to physiological progenitor cells of fetal and adult CNS.

  1. Adherence of skin bacteria to human epithelial cells.

    PubMed Central

    Romero-Steiner, S; Witek, T; Balish, E

    1990-01-01

    Aerobic and anaerobic bacteria isolated from human axillae were tested for their capacity to adhere to buccal epithelial cells, immortalized human epithelial (HEp-2) cells, and undifferentiated and differentiated human epithelial cells. In general, both aerobic and anaerobic diphtheroids adhered better to differentiated human epithelial cells than to HEp-2 and undifferentiated human epithelial cells (P less than 0.05). Mannose, galactose, fucose, N-acetyl-D-glucosamine, and fibronectin were also assayed for their capacity to inhibit the adherence of diphtheroids to human epithelial cells. A great deal of variability was observed in the capacity of the latter compounds to inhibit the attachment of aerobic diphtheroids to undifferentiated and differentiated epithelial cells. Overall, mannose appeared to be best at inhibiting the adherence of the aerobic diphtheroids to undifferentiated human epithelial cells. Galactose, fucose, N-acetyl-D-glucosamine, and fibronectin showed a greater capacity to inhibit attachment of aerobic diphtheroids to differentiated than to undifferentiated human epithelial cells. The inhibition of adherence to differentiated human epithelial cells varied with the microorganism and the compound tested; however, the highest and most consistent inhibition of adherence (76.1 to 88.6%) was observed with a 5% solution of N-acetyl-D-glucosamine. The in vitro adherence and adherence inhibition assays presented here demonstrate that a number of adhesins and receptors are involved in the adherence of skin bacteria to human epithelial cells and receptors on human epithelial cells are apparently altered during differentiation. PMID:2298877

  2. SPI-9 of Salmonella enterica serovar Typhi is constituted by an operon positively regulated by RpoS and contributes to adherence to epithelial cells in culture.

    PubMed

    Velásquez, Juan C; Hidalgo, Alejandro A; Villagra, Nicolás; Santiviago, Carlos A; Mora, Guido C; Fuentes, Juan A

    2016-08-01

    The genomic island 9 (SPI-9) from Salmonella enterica serovar Typhi (S. Typhi) carries three ORFs (STY2876, STY2877, STY2878) presenting 98 % identity with a type 1 secretory apparatus (T1SS), and a single ORF (STY2875) similar to a large RTX-like protein exhibiting repeated Ig domains. BapA, the Salmonella enterica serovar Enteritidis orthologous to S. Typhi STY2875, has been associated with biofilm formation, and is described as a virulence factor in mice. Preliminary in silico analyses revealed that S. Typhi STY2875 ORF has a 600 bp deletion compared with S. Enteritidis bapA, suggesting that S. Typhi STY2875 might be non-functional. At present, SPI-9 has not been studied in S. Typhi. We found that the genes constituting SPI-9 are arranged in an operon whose promoter was up-regulated in high osmolarity and low pH in a RpoS-dependent manner. All the proteins encoded by S. Typhi SPI-9 were located at the membrane fraction, consistent with their putative role as T1SS. Furthermore, SPI-9 contributed to adherence of S. Typhi to epithelial cells when bacteria were grown under high osmolarity or low pH. Under the test conditions, S. Typhi SPI-9 did not participate in biofilm formation. SPI-9 is functional in S. Typhi and encodes an adhesin induced under conditions normally found in the intestine, such as high osmolarity. Hence, this is an example of a locus that might be designated a pseudogene by computational approaches but not by direct biological assays.

  3. Hepatitis B virus efficiently infects non-adherent hepatoma cells via human sodium taurocholate cotransporting polypeptide

    PubMed Central

    Okuyama-Dobashi, Kaori; Kasai, Hirotake; Tanaka, Tomohisa; Yamashita, Atsuya; Yasumoto, Jun; Chen, Wenjia; Okamoto, Toru; Maekawa, Shinya; Watashi, Koichi; Wakita, Takaji; Ryo, Akihide; Suzuki, Tetsuro; Matsuura, Yoshiharu; Enomoto, Nobuyuki; Moriishi, Kohji

    2015-01-01

    Sodium taurocholate cotransporting polypeptide (NTCP) has been reported as a functional receptor for hepatitis B virus (HBV) infection. However, HBV could not efficiently infect HepG2 cells expressing NTCP (NTCP-HepG2 cells) under adherent monolayer-cell conditions. In this study, NTCP was mainly detected in the basolateral membrane region, but not the apical site, of monolayer NTCP-HepG2 cells. We hypothesized that non-adherent cell conditions of infection would enhance HBV infectivity. Non-adherent NTCP-HepG2 cells were prepared by treatment with trypsin and EDTA, which did not degrade NTCP in the membrane fraction. HBV successfully infected NTCP-HepG2 cells at a viral dose 10 times lower in non-adherent phase than in adherent phase. Efficient infection of non-adherent NTCP-HepG2 cells with blood-borne or cell-culture-derived HBV was observed and was remarkably impaired in the presence of the myristoylated preS1 peptide. HBV could also efficiently infect HepaRG cells under non-adherent cell conditions. We screened several compounds using our culture system and identified proscillaridin A as a potent anti-HBV agent with an IC50 value of 7.2 nM. In conclusion, non-adherent host cell conditions of infection augmented HBV infectivity in an NTCP-dependent manner, thus providing a novel strategy to identify anti-HBV drugs and investigate the mechanism of HBV infection. PMID:26592202

  4. Neuronal-like cell differentiation of non-adherent bone marrow cell-derived mesenchymal stem cells.

    PubMed

    Wu, Yuxin; Zhang, Jinghan; Ben, Xiaoming

    2013-08-05

    Non-adherent bone marrow cell-derived mesenchymal stem cells from C57BL/6J mice were separated and cultured using the "pour-off" method. Non-adherent bone marrow cell-derived mesenchymal stem cells developed colony-forming unit-fibroblasts, and could be expanded by supplementation with epidermal growth factor. Immunocytochemistry showed that the non-adherent bone marrow cell-derived mesenchymal stem cells exposed to basic fibroblast growth factor/epidermal growth factor/nerve growth factor expressed the neuron specific markers, neurofilament-200 and NeuN, in vitro. Non-adherent bone marrow cell-derived mesenchymal stem cells from β-galactosidase transgenic mice were also transplanted into focal ischemic brain (right corpus striatum) of C57BL/6J mice. At 8 weeks, cells positive for LacZ and β-galactosidase staining were observed in the ischemic tissues, and cells co-labeled with both β-galactosidase and NeuN were seen by double immunohistochemical staining. These findings suggest that the non-adherent bone marrow cell-derived mesenchymal stem cells could differentiate into neuronal-like cells in vitro and in vivo.

  5. Titanium surface topography affects collagen biosynthesis of adherent cells.

    PubMed

    Mendonça, Daniela B S; Miguez, Patrícia A; Mendonça, Gustavo; Yamauchi, Mitsuo; Aragão, Francisco J L; Cooper, Lyndon F

    2011-09-01

    Collagen-dependent microstructure and physicochemical properties of newly formed bone around implant surfaces represent key determinants of implant biomechanics. This study investigated the effects of implant surface topography on collagen biosynthesis of adherent human mesenchymal stem cells (hMSCs). hMSCs were grown for 0 to 42 days on titanium disks (20.0 × 1.0 mm) with smooth or rough surfaces. Cell attachment and spreading were evaluated by incubating cells with Texas-Red-conjugated phalloidin antibody. Quantitative real-time PCR was used to measure the mRNA levels of Col1α1 and collagen modifying genes including prolyl hydroxylases (PHs), lysyl oxidases (LOXs) and lysyl hydroxylases (LHs). Osteogenesis was assessed at the level of osteoblast specific gene expression and alizarin red staining for mineralization. Cell layer-associated matrix and collagen content were determined by amino acid analysis. At 4h, 100% cells were flattened on both surfaces, however the cells on smooth surface had a fibroblast-like shape, while cells on rough surface lacked any defined long axis. PH, LH, and most LOX mRNA levels were greater in hMSCs grown on rough surfaces for 3 days. The mineralized area was greater for rough surface at 28 and 42 days. The collagen content (percent total protein) was also greater at rough surface compared to smooth surface at 28 (36% versus 26%) and 42 days (46% versus 29%), respectively (p<.05). In a cell culture model, rough surface topography positively modulates collagen biosynthesis and accumulation and the expression of genes associated with collagen cross-linking in adherent hMSC. The altered biosynthesis of the collagen-rich ECM adjacent to endosseous implants may influence the biomechanical properties of osseointegrated endosseous implants.

  6. 3-D measurement of osmotic dehydration of isolated and adhered PC-3 cells.

    PubMed

    Yoshimori, Takashi; Takamatsu, Hiroshi

    2009-02-01

    Cell dehydration during freezing results from an elevated concentration of electrolytes in the extracellular medium that is deeply involved in cellular injury. We undertook real-time threedimensional (3-D) observation of osmotic dehydration of cells, motivated by a comparison of cellular responses between isolated cells in suspension and cultured cells adhering to a surface since several studies have suggested a difference in freeze tolerance between cell suspensions and monolayers. A laser confocal scanner was used with a perfusion microscope to capture sectional images of chloromethylbenzamido (DiI)-stained PC-3 cells that were exposed to an increase in NaCl concentration from 0.15 to 0.5M at 23 degrees C. Change in cell volume was determined from reconstructed 3-D images taken every 2.5s. When cells were exposed to an elevated NaCl concentration, isolated cells contracted and markedly distorted from their original spherical shape. In contrast, adhered cells showed only a reduction in height and kept their basal area constant. Apparent membrane hydraulic conductivity did not vary considerably between isolated and adhered cells, suggesting a negligible effect of the cytoskeletal structure on the rate of water transport. The surface area that contributed to water transport in adhered PC-3 cells was nearly equal to or slightly smaller than that present in isolated cells. Therefore, the similarity in properties and dimensions between isolated and adhered cells indicate that there will be similar extents of dehydration, resulting in a similar degree of supercooling during freezing.

  7. Isolation of dendritic cells from umbilical cord blood using magnetic activated cell sorting or adherence.

    PubMed

    Bie, Yachun; Xu, Qiuxiang; Zhang, Zhenyu

    2015-07-01

    Dendritic cells (DCs) are a highly specialized type of antigen-presenting cell. The present study describes and compares two methods for preparing DCs from umbilical cord blood. The first method involves the isolation of DCs by magnetic activated cell sorting (MACS). This technique isolates CD34(+) cells from cord blood and induces the formation of DCs by the addition of cytokines, granulocyte macrophage colony-stimulating factor and interleukin-4. The second method involves the generation of large numbers of DCs from cord blood using an adherent method, which isolates umbilical cord blood mononuclear cells and induces DCs in the same conditions as those used in MACS. The DCs were harvested following 7 days of incubation and observed with an inverted microscope. The phenotype of the cells was then analyzed by flow cytometry. The results revealed that, subsequent to 7 days of incubation, the differentiated DCs obtained using the adherent method were more mature than those isolated using MACS. However, these cells were unable to be maintained in culture for more than 9-10 days. By contrast, the DCs derived from CD34(+) cells by MACS were phenotypically stable and could be maintained for up to 3 weeks in culture. Either method produced DCs from cord blood. However, the DCs isolated using the MACS method demonstrated higher homogeneity, yield and viability than those obtained using the adherent method. Due to the various compositions of the monocyte subsets isolated, isolation methods affect the phenotypes and functions of the resultant DCs.

  8. Piracy of decay-accelerating factor (CD55) signal transduction by the diffusely adhering strain Escherichia coli C1845 promotes cytoskeletal F-actin rearrangements in cultured human intestinal INT407 cells.

    PubMed

    Peiffer, I; Servin, A L; Bernet-Camard, M F

    1998-09-01

    Diffusely adhering Escherichia coli (DAEC) C1845 (clinical isolate) harboring the fimbrial adhesin F1845 can infect cultured human differentiated intestinal epithelial cells; this process is followed by the disassembly of the actin network in the apical domain. The aim of this study was to examine the mechanism by which DAEC C1845 promotes F-actin rearrangements. For this purpose, we used a human embryonic intestinal cell line (INT407) expressing the membrane-associated glycosylphosphatidylinositol (GPI) protein-anchored decay-accelerating factor (DAF), the receptor of the F1845 adhesin. We show here that infection of INT407 cells by DAEC C1845 can provoke dramatic F-actin rearrangements without cell entry. Clustering of phosphotyrosines was observed, revealing that the DAEC C1845-DAF interaction involves the recruitment of signal transduction molecules. A pharmacological approach with a subset of inhibitors of signal transduction molecules was used to identify the cascade of signal transduction molecules that are coupled to the DAF, that are activated upon infection, and that promote the F-actin rearrangements. DAEC C1845-induced F-actin rearrangements can be blocked dose dependently by protein tyrosine kinase, phospholipase Cgamma, phosphatidylinositol 3-kinase, protein kinase C, and Ca2+ inhibitors. F-actin rearrangements and blocking by inhibitors were observed after infection of the cells with two E. coli recombinants carrying the plasmids containing the fimbrial adhesin F1845 or the fimbrial hemagglutinin Dr, belonging to the same family of adhesins. These findings show that the DAEC Dr family of pathogens promotes alterations in the intestinal cell cytoskeleton by piracy of the DAF-GPI signal cascade without bacterial cell entry.

  9. Neutral red uptake inhibition in adhered and adhering rat hepatoma-derived Fa32 cells to predict human toxicity.

    PubMed

    Dierickx, Paul J; Scheers, Ellen M

    2002-01-01

    The cytotoxicity of the MEIC (Multicentre Evaluation of In vitro Cytotoxicity) reference chemicals was investigated by measuring the neutral red uptake inhibition in adhered and adhering rat hepatoma-derived Fa32 cells. The adhered cells were seeded and then treated and the adhering cells were treated simultaneously upon seeding. Five of the 44 test chemicals were twofold more toxic in adhering cells; ethylene glycol was 28-fold more toxic and mercuric chloride was 5.2-fold more toxic than in adhered cells. The cytotoxicity of dithiothreitol was altered in the same way as that of ethylene glycol, probably by interacting with calcium. When the neutral red uptake inhibition was compared with human toxicity, the correlation coefficient for adhering cells was almost identical to that obtained previously in human hepatoma-derived Hep G2 cells and slightly higher for adhered cells. The Hep G2 assay was the best acute in vitro assay for the prediction of human toxicity within the MEIC study. An obviously better correlation was obtained when the strong intoxicant mercuric chloride was withdrawn from the comparison, both for the adhered and the adhering cells. Altogether, the results can be integrated very well with the basal cytotoxicity concept.

  10. Advances in cell culture

    SciTech Connect

    Maramorosch, K. )

    1987-01-01

    This book presents papers on advances in cell culture. Topics covered include: Genetic changes in the influenza viruses during growth in cultured cells; The biochemistry and genetics of mosquito cells in culture; and Tree tissue culture applications.

  11. Image and flow cytometry: companion techniques for adherent and non-adherent cell analysis and sorting.

    PubMed

    Métézeau, P

    1993-01-01

    Flow cytometry (FMC) is an analytical and preparative technique whereas image analysis is only applied to cell analysis. Recently, image analysis has been adapted as a preparative method using a new technique: image cytometry for analysis and sorting (ICAS). FCM and ICAS are complementary. Flow cytometry allows rapid, quantitative and precise study of fluorescence and light scattering in a large number of cells in suspension, while ICAS analyses fewer cells (adherent cells or tissue) on the basis of fluorescence, morphology and size. ICAS can use these criteria to destroy unwanted cells and hence sort selected cells. ICAS can also be used for confocal microscopy and laser surgery.

  12. Oscillating Cell Culture Bioreactor

    NASA Technical Reports Server (NTRS)

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

    2010-01-01

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

  13. Comparative study of the radiobiological effects induced on adherent vs suspended cells by BNCT, neutrons and gamma rays treatments.

    PubMed

    Cansolino, L; Clerici, A M; Zonta, C; Dionigi, P; Mazzini, G; Di Liberto, R; Altieri, S; Ballarini, F; Bortolussi, S; Carante, M P; Ferrari, M; González, S J; Postuma, I; Protti, N; Santa Cruz, G A; Ferrari, C

    2015-12-01

    The present work is part of a preclinical in vitro study to assess the efficacy of BNCT applied to liver or lung coloncarcinoma metastases and to limb osteosarcoma. Adherent growing cell lines can be irradiated as adherent to the culture flasks or as cell suspensions, differences in radio-sensitivity of the two modalities of radiation exposure have been investigated. Dose related cell survival and cell cycle perturbation results evidenced that the radiosensitivity of adherent cells is higher than that of the suspended ones.

  14. Glycogen synthase kinase 3 (GSK3)-inhibitor SB216763 promotes the conversion of human umbilical cord mesenchymal stem cells into neural precursors in adherent culture.

    PubMed

    Gao, Liyang; Zhao, Mingyan; Li, Peng; Kong, Junchao; Liu, Zhijun; Chen, Yonghua; Huang, Rui; Chu, Jiaqi; Quan, Juanhua; Zeng, Rong

    2017-01-01

    The ability to generate neural progenitor cells from human umbilical cord mesenchymal stem cells (hUC-MSCs) has provided an option to treat neurodegenerative diseases. To establish a method for this purpose, we characterized the early neural markers of hUC-MSCs-derived cells under different conditions. We found that neither the elimination of signals for alternative fate nor N2 supplement was sufficient to differentiate hUC-MSCs into neural precursor cells, but the GSK3 inhibitor SB216763 could promote an efficient neural commitment of hUC-MSCs. The results indicated that Wnt/β-catenin might play an important role during the early neural differentiation of hUC-MSCs. Here, we report a method for hUC-MSCs to commit efficiently into a neural fate within a short period of time. This protocol provides an efficient method for hUC-MSCs-based neural regeneration.

  15. Modulating temporal and spatial oxygenation over adherent cellular cultures.

    PubMed

    Oppegard, Shawn C; Nam, Ki-Hwan; Carr, Janai R; Skaalure, Stacey C; Eddington, David T

    2009-09-03

    Oxygen is a key modulator of many cellular pathways, but current devices permitting in vitro oxygen modulation fail to meet the needs of biomedical research. A microfabricated insert for multiwell plates has been developed to more effectively control the temporal and spatial oxygen concentration to better model physiological phenomena found in vivo. The platform consists of a polydimethylsiloxane insert that nests into a standard multiwell plate and serves as a passive microfluidic gas network with a gas-permeable membrane aimed to modulate oxygen delivery to adherent cells. Equilibration time is on the order of minutes and a wide variety of oxygen profiles can be attained based on the device design, such as the cyclic profile achieved in this study, and even oxygen gradients to mimic those found in vivo. The proper biological consequences of the device's oxygen delivery were confirmed in cellular models via a proliferation assay and western analysis of the upregulation of hypoxia inducible transcription factor-1alpha. These experiments serve as a demonstration for the platform as a viable tool to increase experimental throughput and permit novel experimental possibilities in any biomedical research lab.

  16. Sonoporation of adherent cells under regulated ultrasound cavitation conditions.

    PubMed

    Muleki Seya, Pauline; Fouqueray, Manuela; Ngo, Jacqueline; Poizat, Adrien; Inserra, Claude; Béra, Jean-Christophe

    2015-04-01

    A sonoporation device dedicated to the adherent cell monolayer has been implemented with a regulation process allowing the real-time monitoring and control of inertial cavitation activity. Use of the cavitation-regulated device revealed first that adherent cell sonoporation efficiency is related to inertial cavitation activity, without inducing additional cell mortality. Reproducibility is enhanced for the highest sonoporation rates (up to 17%); sonoporation efficiency can reach 26% when advantage is taken of the standing wave acoustic configuration by applying a frequency sweep with ultrasound frequency tuned to the modal acoustic modes of the cavity. This device allows sonoporation of adherent and suspended cells, and the use of regulation allows some environmental parameters such as the temperature of the medium to be overcome, resulting in the possibility of cell sonoporation even at ambient temperature.

  17. Permeabilization of adhered cells using an inert gas jet.

    PubMed

    Cooper, Scott; Jonak, Paul; Chouinard-Pelletier, Guillaume; Coulombe, Sylvain; Jones, Elizabeth; Leask, Richard L

    2013-09-04

    Various cell transfection techniques exist and these can be broken down to three broad categories: viral, chemical and mechanical. This protocol describes a mechanical method to temporally permeabilize adherent cells using an inert gas jet that can facilitate the transfer of normally non-permeable macromolecules into cells. We believe this technique works by imparting shear forces on the plasma membrane of adherent cells, resulting in the temporary formation of micropores. Once these pores are created, the cells are then permeable to genetic material and other biomolecules. The mechanical forces involved do run the risk of permanently damaging or detaching cells from their substrate. There is, therefore, a narrow range of inert gas dynamics where the technique is effective. An inert gas jet has proven efficient at permeabilizing various adherent cell lines including HeLa, HEK293 and human abdominal aortic endothelial cells. This protocol is appropriate for the permeabilization of adherent cells both in vitro and, as we have demonstrated, in vivo, showing it may be used for research and potentially in future clinical applications. It also has the advantage of permeabilizing cells in a spatially restrictive manner, which could prove to be a valuable research tool.

  18. Proliferative activity of vervet monkey bone marrow-derived adherent cells

    SciTech Connect

    Kramvis, A.; Garnett, H.M.

    1987-11-01

    Vervet monkey bone marrow-derived adherent cell population cultured in Fischer's medium supplemented with 12.5% fetal calf serum and 12.5% horse serum consists of two cell shapes: fusiform (type I) and polygonal (type II). Limiting-dilution cloning of the cells suggested that the two morphologically distinct cell types belong to the same cellular system even though they differ in their proliferative capabilities. The labeling index of type II cells, as measured by autoradiography, was found to be consistently lower than that of type I cells. It is probable that these two phenotypes represent different stages of differentiation, where progenitor type I gives rise to type II cells. The bone marrow-derived adherent cells were found to be cytokinetically at rest in vivo, using the thymidine suicide test, and relatively radioresistant with a D0 = 2.1 Gy and n = 2.36 at the time of explantation from the bone. Furthermore, in culture these cells are characterized by a relatively long cell cycle of 60 h, where the length of the S phase is 30 h, G2 is 12 h, M is 6 h, and G1 is 12 h. Thus, the vervet monkey bone marrow-derived adherent cells represent a cell population with a low turnover rate both in vivo and in vitro.

  19. A Selective and Purification-Free Strategy for Labeling Adherent Cells with Inorganic Nanoparticles.

    PubMed

    Gao, Yu; Lim, Jing; Yeo, David Chen Loong; Liao, Shanshan; Lans, Malin; Wang, Yaqi; Teoh, Swee-Hin; Goh, Bee Tin; Xu, Chenjie

    2016-03-01

    Cellular labeling with inorganic nanoparticles such as magnetic iron oxide nanoparticles, quantum dots, and fluorescent silica nanoparticles is an important method for the noninvasive visualization of cells using various imaging modalities. Currently, this is mainly achieved through the incubation of cultured cells with the nanoparticles that eventually reach the intracellular compartment through specific or nonspecific internalization. This classic method is advantageous in terms of simplicity and convenience, but it suffers from issues such as difficulties in fully removing free nanoparticles (suspended in solution) and the lack of selectivity on cell types. This article reports an innovative strategy for the specific labeling of adherent cells without the concern of freely suspended nanoparticles. This method relies on a nanocomposite film that is prepared by homogeneously dispersing nanoparticles within a biodegradable polymeric film. When adherent cells are seeded on the film, they adhere, spread, and filtrate into the film through the micropores formed during the film fabrication. The pre-embedded nanoparticles are thus internalized by the cells during this infiltration process. As an example, fluorescent silica nanoparticles were homogeneously distributed within a polycaprolactone film by utilizing cryomilling and heat pressing. Upon incubation within physiological buffer, no silica nanoparticles were released from the nanocomposite film even after 20 d of incubation. However, when adherent cells (e.g., human mesenchymal stem cells) were grown on the film, they became fluorescent after 3 d, which suggests internalization of silica nanoparticles by cells. In comparison, the suspension cells (e.g., monocytes) in the medium remained nonfluorescent no matter whether there was the presence of adherent cells or not. This strategy eventually allowed the selective and concomitant labeling of mesenchymal stem cells during their harvest from bone marrow aspiration.

  20. Adherence, accumulation, and cell division of a natural adherent bacterial population.

    PubMed Central

    Bloomquist, C G; Reilly, B E; Liljemark, W F

    1996-01-01

    Developing dental bacterial plaques formed in vivo on enamel surfaces were examined in specimens from 18 adult volunteers during the first day of plaque formation. An intraoral model placing enamel pieces onto teeth was used to study bacterial plaque populations developing naturally to various cell densities per square millimeter of surface area of the enamel (W. F. Liljemark, C. G. Bloomquist, C. L. Bandt, B. L. Philstrom, J. E. Hinrichs, and L. F. Wolff, Oral Microbiol. Immunol. 8:5-15, 1993). Radiolabeled nucleoside incorporation was used to measure DNA synthesis concurrent with the taking of standard viable cell counts of the plaque samples. Results showed that in vivo plaque formation began with the rapid adherence of bacteria until ca. 12 to 32% of the enamel's salivary pellicle was saturated (ca. 2.5 x 10(5) to 6.3 x 10(5) cells per mm2). The pioneer adherent species were predominantly those of the "sanguis streptococci." At the above-noted density, the bacteria present on the salivary pellicle incorporated low levels of radiolabeled nucleoside per viable cell. As bacterial numbers reached densities between 8.0 x 10(5) and 2.0 x 10(6) cells per mm2, there was a small increase in the incorporation of radiolabeled nucleosides per cell. At 2.5 x 10(6) to 4.0 x 10(6) cells per mm2 of enamel surface, there was a marked increase in the incorporation of radiolabeled nucleosides per cell which appeared to be cell-density dependent. The predominant species group in developing dental plaque films during density-dependent growth was the sanguis streptococci; however, most other species present showed similar patterns of increased DNA synthesis as the density noted above approached 2.5 x 10(6) to 4.0 x 10(6) cells per mm2. PMID:8576054

  1. Cultural Rationales Guiding Medication Adherence Among African American with HIV/AIDS

    PubMed Central

    Neufeld, Stewart; Berry, Rico; Luborsky, Mark

    2011-01-01

    Abstract To date, only modest gains have been achieved in explaining adherence to medical regimens, limiting effective interventions. This is a particularly important issue for African Americans who are disproportionately affected by the HIV epidemic. Few studies have focused on intragroup variation among African Americans in adherence to ART. The aim of this study was to identify and describe the cultural rationales guiding African American patients' formulation and evaluation of adherence. Rationales are key features of purposeful human action. In-depth interviews with 80 seropositive African Americans were tape recorded, transcribed, and analyzed. Participant CD4, viral load and medical histories were collected at each data point. Analysis of four waves of panel data identified three types of adherence rationales: Authoritative Knowledge Rationale (AKR; n=29, 36.3%), Following Doctors' Orders Rationale (DOR; n=24, 30.0%) and Individualized Adherence Rationale (IAR; n=27, 33.8%). Differences in mean reported adherence between the rationale groups did not achieve statistical significance. However, the fraction reporting low adherence (<70%), although not different by rationale group at the first interview (T1), was significantly higher for the IAR group by the fourth interview (T4). Objective clinical markers (CD4 and viral load) improved over time (from T1 to T4) for AKR and DOR groups, but remained unchanged for the IAR group, yet self-reported adherence declined for all groups over the course of the four interviews. PMID:21777141

  2. The relation between growth phases, cell volume changes and metabolism of adherent cells during cultivation.

    PubMed

    Rehberg, M; Ritter, J B; Genzel, Y; Flockerzi, D; Reichl, U

    2013-04-15

    In biotechnology, mathematical models often consider changes in cell numbers as well as in metabolite conversion to describe different cell growth phases. It has been frequently observed that the cell number is only a delayed indicator of cell growth compared to the biomass, which challenges the principle structure of corresponding models. Here, we evaluate adherent cell growth phases in terms of cell number and biomass increase on the basis of detailed experimental data of three independent cultivations for Madin Darby canine kidney cells. We develop a model linking cell numbers and mean cell diameters to estimate cell volume changes during growth without the need for diameter distribution measurements. It simultaneously describes the delay between cell number and cell volume increase, cell-specific volume changes and the transition from growth to maintenance metabolism while taking different pre-culture conditions, which affect the cell diameter, into account. In addition, inspection of metabolite uptake and release rates reveals that glucose is mainly used for generation of cellular energy and glutamine is not required for cellular maintenance. Finally, we conclude that changes in cell number, cell diameter and metabolite uptake during cultivation contribute to the understanding of the time course of intracellular metabolites during the cultivation process.

  3. Magnetic labeling of non-phagocytic adherent cells with iron oxide nanoparticles: a comprehensive study.

    PubMed

    Boutry, Sébastien; Brunin, Stéphanie; Mahieu, Isabelle; Laurent, Sophie; Vander Elst, Luce; Muller, Robert N

    2008-01-01

    Small particles of iron oxide (SPIO) and ultrasmall particles of iron oxide (USPIO), inducing a strong negative contrast on T(2) and T(2)*-weighted MR images, are the most commonly used systems for the magnetic labeling of cultured cells and their subsequent detection by magnetic resonance imaging (MRI). The purpose of this work is to study the influence of iron incubation concentration, nanoparticle size and nanoparticle coating on the magnetic labeling and the viability of non-phagocytic adherent cells in culture. The magnetic labeling of 3T6 fibroblasts was studied by T(2)-weighted MRI at 4.7 T and by dosing-or cytochemical revealing-of iron through methods based on Perl's Prussian blue staining. Cells were incubated for 48 h with increasing iron concentrations of SPIO (25-1000 microg Fe/ml Endorem. Sinerem, a USPIO (20-40 nm) coated with neutral dextran, and Resovist (65 nm), a SPIO bearing an anionic carboxydextran coating, were compared with Endorem (dextran-coated, 80-150 nm) as magnetic tags. The iron loading of marrow stromal cell primary cultures (MSCs) isolated from rat femurs was compared with that of 3T6 fibroblasts. The SPIO-labeling of cells with Endorem was found to be dependent on the iron incubation concentration. MSCs, more sparsely distributed in the culture, exhibited higher iron contents than more densely populated 3T6 fibroblast cultures. A larger iron loading was achieved with Resovist than with Endorem, which in turn was more efficient than Sinerem as a magnetic tag. The magnetic labeling of cultured non-phagocytic adherent cells with iron oxide nanoparticles was thus found to be dependent on the relative concentration of the magnetic tag and of the cells in culture, on the nanoparticle size, and on the coating type. The viability of cells, estimated by methods assessing cell membrane permeability, was not affected by magnetic labeling in the conditions used in this work.

  4. A Localized Adherence-Like Pattern as a Second Pattern of Adherence of Classic Enteropathogenic Escherichia coli to HEp-2 Cells That Is Associated with Infantile Diarrhea

    PubMed Central

    Scaletsky, Isabel C. A.; Pedroso, Margareth Z.; Oliva, Carlos A. G.; Carvalho, Rozane L. B.; Morais, Mauro B.; Fagundes-Neto, Ulysses

    1999-01-01

    Escherichia coli strains that cause nonbloody diarrhea in infants are known to present three distinct patterns of adherence to epithelial cells, namely, localized (LA), diffuse (DA), and aggregative (AA) adherence. Strains with LA (typical Enteropathogenic Escherichia coli [EPEC]) are well recognized as a cause of secretory diarrhea, but the role of strains with DA (DAEC) is controversial, and strains with AA (EAEC) have been more frequently related to persistent diarrhea whereas its relationship with acute diarrhea is not well defined. To determine the relationship of the different types of E. coli adherence patterns with acute diarrhea (lasting less than 14 days) and persistent diarrhea (lasting more than 14 days) in São Paulo, Brazil, we studied stool specimens from 40 infants under 1 year of age with diarrhea and 40 age-matched control infants without any gastrointestinal symptoms. Twenty-eight (35.0%) of eighty cases yielded adherent E. coli (HEp-2 cells). Strains with localized and aggregative adherence were associated with acute and persistent diarrhea. A total of 11.2% of the adherent strains were typical EPEC serotypes and hybridized with the enteroadherence factor probe; 5.0% were EAEC and hybridized with the EAEC probe. DAEC strains were isolated from 10.0% of patients and 7.5% of controls and did not hybridize with the two probes used (daaC and AIDA-I). Strains with a localized adherence-like pattern (atypical EPEC) were found significantly more frequently (P = 0.028) in cultures from children with diarrhea (17.5%) than in controls (2.5%). PMID:10377120

  5. Patterned Thermoresponsive Microgel Coatings for Noninvasive Processing of Adherent Cells.

    PubMed

    Uhlig, Katja; Wegener, Thomas; He, Jian; Zeiser, Michael; Bookhold, Johannes; Dewald, Inna; Godino, Neus; Jaeger, Magnus; Hellweg, Thomas; Fery, Andreas; Duschl, Claus

    2016-03-14

    Cultivation of adherently growing cells in artificial environments is of utmost importance in medicine and biotechnology to accomplish in vitro drug screening or to investigate disease mechanisms. Precise cell manipulation, like localized control over adhesion, is required to expand cells, to establish cell models for novel therapies and to perform noninvasive cell experiments. To this end, we developed a method of gentle, local lift-off of mammalian cells using polymer surfaces, which are reversibly and repeatedly switchable between a cell-attractive and a cell-repellent state. This property was introduced through micropatterned thermoresponsive polymer coatings formed from colloidal microgels. Patterning was obtained through automated nanodispensing or microcontact printing, making use of unspecific electrostatic interactions between microgels and substrates. This process is much more robust against ambient conditions than covalent coupling, thus lending itself to up-scaling. As an example, wound healing assays were accomplished at 37 °C with highly increased precision in microfluidic environments.

  6. Adherence of Pseudomonas aeruginosa to tracheal cells injured by influenza infection or by endotracheal intubation.

    PubMed

    Ramphal, R; Small, P M; Shands, J W; Fischlschweiger, W; Small, P A

    1980-02-01

    Adherence of Pseudomonas aeruginosa to normal, injured, and regenerating tracheal mucosa was examined by scanning electron microscopy. Uninfected and influenza-infected murine tracheas were exposed to six strains of P. aeruginosa isolated from human sources and one strain of platn origin. All of the strains tested adhered to desquamating cells of the infected tracheas, but not to normal mucosa, the basal cell layer, or the regenerating epithelium. Adherence increased when the incubation time of the bacteria with the trachea was prolonged. Strains isolated from human tracheas appeared to adhere better than strains derived from the urinary tract. After endotracheal intubation of ferrets, P. aeruginosa adhered only to the injured cells and to areas of exposed basement membrane. We call this phenomenon "opportunistic adherence" and propose that alteration of the cell surfaces or cell injury facilitates the adherence of this bacterium and that adherence to injured cells may be a key to the pathogenesis of opportunistic Pseudomonas infections.

  7. Isolation and Characterization of Multipotent Mesenchymal Stem Cells Adhering to Adipocytes in Canine Bone Marrow.

    PubMed

    Lin, Hsing-Yi; Fujita, Naoki; Endo, Kentaro; Morita, Maresuke; Takeda, Tae; Nakagawa, Takayuki; Nishimura, Ryohei

    2017-03-15

    The ceiling culture method has been used to isolate mature adipocytes from adipose tissue that can be dedifferentiated into fibroblastic cells, also known as dedifferentiated fat (DFAT) cells that self-renew and are multipotent, with much higher homogeneity and colony-forming efficiency than those of adipose tissue-derived mesenchymal stem cells. We cultured adipocytes from canine bone marrow using this technique, with the expectation of obtaining DFAT cells. However, contrary to our expectations, continuous monitoring of ceiling cultures by time-lapse microscopy revealed many small cells adhering to adipocytes that proliferated rapidly into cells with a fibroblastic morphology and without any dedifferentiation from adipocytes. We named these cells bone marrow peri-adipocyte cells (BM-PACs) and demonstrated the multipotent properties of BM-PACs compared to that of conventionally cultured canine bone marrow mesenchymal stem cells (BMMSCs). BM-PACs showed significantly greater clonogenicity and proliferation ability than BMMSCs. An in vitro trilineage differentiation assay revealed that BM-PACs possess adipogenic, osteogenic, and chondrogenic capacities superior to those of BMMSCs. Flow cytometric analysis revealed that the expression of CD73, which plays an important role in cell growth and differentiation, was significantly higher in BM-PACs than in BMMSCs. These results indicate that canine BM-PACs have stem cell characteristics that are superior to those of BMMSCs, and that these mesenchymal stem cells (MSCs) appear to be a feasible source for cell-based therapies in dogs.

  8. Dynamic mechanical measurement of the viscoelasticity of single adherent cells

    NASA Astrophysics Data System (ADS)

    Corbin, Elise A.; Adeniba, Olaoluwa O.; Ewoldt, Randy H.; Bashir, Rashid

    2016-02-01

    Many recent studies on the viscoelasticity of individual cells link mechanics with cellular function and health. Here, we introduce a measurement of the viscoelastic properties of individual human colon cancer cells (HT-29) using silicon pedestal microelectromechanical systems (MEMS) resonant sensors. We demonstrate that the viscoelastic properties of single adherent cells can be extracted by measuring a difference in vibrational amplitude of our resonant sensor platform. The magnitude of vibration of the pedestal sensor is measured using a laser Doppler vibrometer (LDV). A change in amplitude of the sensor, compared with the driving amplitude (amplitude ratio), is influenced by the mechanical properties of the adhered cells. The amplitude ratio of the fixed cells was greater than the live cells, with a p-value <0.0001. By combining the amplitude shift with the resonant frequency shift measure, we determined the elastic modulus and viscosity values of 100 Pa and 0.0031 Pa s, respectively. Our method using the change in amplitude of resonant MEMS devices can enable the determination of a refined solution space and could improve measuring the stiffness of cells.

  9. Nonadherent culture method downregulates stem cell antigen-1 expression in mouse bone marrow mesenchymal stem cells

    PubMed Central

    DENG, BAOPING; DENG, WEIPING; XIAO, PINGNAN; ZENG, KUAN; ZHANG, SHINING; ZHANG, HONGWU; DENG, DAVID YB; YANG, YANQI

    2015-01-01

    Mesenchymal stem cells (MSCs) are primarily isolated by their adherence to plastic and their in vitro growth characteristics. Expansion of these cells from an adherent culture is the only method to obtain a sufficient number of cells for use in clinical practice and research. However, little is known with regard to the effect of adherence to plastic on the phenotype of the cells. In the present study, bone marrow CD45−CD31−CD44− stem cell antigen (Sca)-1+ MSCs were sorted by flow cytometry and expanded in adherent cultures. The expression levels of the adhesion molecule, Sca-1, in the adherent cultures were compared with those from nonadherent cultures at different time points. The flow cytometry results indicated that the expression levels of Sca-1 decreased in the MSCs in the nonadherent cultures grown in ultra-low-adherent plates. Furthermore, the result was confirmed by quantitative polymerase chain reaction at the same time points. Therefore, the results demonstrated that the loss of plastic adherence downregulated the expression of Sca-1. The observations may provide novel insights into the molecular mechanisms underlying plastic adherent culture. PMID:26170908

  10. Evidence that extracellular components function in adherence of Actinobacillus actinomycetemcomitans to epithelial cells.

    PubMed Central

    Meyer, D H; Fives-Taylor, P M

    1993-01-01

    Extracellular microvesicles and a highly proteinaceous polymer associated with a leukotoxin-producing strain, Actinobacillus actinomycetemcomitans SUNY 75, were shown to increase adherence of other weakly adherent A. actinomycetemcomitans strains to KB epithelial cells. Images PMID:8406899

  11. Advances in cell culture: anchorage dependence

    PubMed Central

    Merten, Otto-Wilhelm

    2015-01-01

    Anchorage-dependent cells are of great interest for various biotechnological applications. (i) They represent a formidable production means of viruses for vaccination purposes at very large scales (in 1000–6000 l reactors) using microcarriers, and in the last decade many more novel viral vaccines have been developed using this production technology. (ii) With the advent of stem cells and their use/potential use in clinics for cell therapy and regenerative medicine purposes, the development of novel culture devices and technologies for adherent cells has accelerated greatly with a view to the large-scale expansion of these cells. Presently, the really scalable systems—microcarrier/microcarrier-clump cultures using stirred-tank reactors—for the expansion of stem cells are still in their infancy. Only laboratory scale reactors of maximally 2.5 l working volume have been evaluated because thorough knowledge and basic understanding of critical issues with respect to cell expansion while retaining pluripotency and differentiation potential, and the impact of the culture environment on stem cell fate, etc., are still lacking and require further studies. This article gives an overview on critical issues common to all cell culture systems for adherent cells as well as specifics for different types of stem cells in view of small- and large-scale cell expansion and production processes. PMID:25533097

  12. Fabrication and operation of an oxygen insert for adherent cellular cultures.

    PubMed

    Oppegard, Shawn; Sinkala, Elly; Eddington, David

    2010-01-06

    Oxygen is a key modulator of many cellular pathways, but current devices permitting in vitro oxygen modulation fail to meet the needs of biomedical research. The hypoxic chamber offers a simple system to control oxygenation in standard culture vessels, but lacks precise temporal and spatial control over the oxygen concentration at the cell surface, preventing its application in studying a variety of physiological phenomena. Other systems have improved upon the hypoxic chamber, but require specialized knowledge and equipment for their operation, making them intimidating for the average researcher. A microfabricated insert for multiwell plates has been developed to more effectively control the temporal and spatial oxygen concentration to better model physiological phenomena found in vivo. The platform consists of a polydimethylsiloxane insert that nests into a standard multiwell plate and serves as a passive microfluidic gas network with a gas-permeable membrane aimed to modulate oxygen delivery to adherent cells. The device is simple to use and is connected to gas cylinders that provide the pressure to introduce the desired oxygen concentration into the platform. Fabrication involves a combination of standard SU-8 photolithography, replica molding, and defined PDMS spinning on a silicon wafer. The components of the device are bonded after surface treatment using a hand-held plasma system. Validation is accomplished with a planar fluorescent oxygen sensor. Equilibration time is on the order of minutes and a wide variety of oxygen profiles can be attained based on the device design, such as the cyclic profile achieved in this study, and even oxygen gradients to mimic those found in vivo. The device can be sterilized for cell culture using common methods without loss of function. The device's applicability to studying the in vitro wound healing response will be demonstrated.

  13. Cell Culture Made Easy.

    ERIC Educational Resources Information Center

    Dye, Frank J.

    1985-01-01

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

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

    PubMed Central

    Shukla, Shakti D; Fairbairn, Rory L; Gell, David A; Latham, Roger D; Sohal, Sukhwinder S; Walters, Eugene H; O’Toole, Ronan F

    2016-01-01

    Background COPD is emerging as the third largest cause of human mortality worldwide after heart disease and stroke. Tobacco smoking, the primary risk factor for the development of COPD, induces increased expression of platelet-activating factor receptor (PAFr) in the lung epithelium. Nontypeable Haemophilus influenzae (NTHi) and Streptococcus pneumoniae adhere to PAFr on the luminal surface of human respiratory tract epithelial cells. Objective To investigate PAFr as a potential drug target for the prevention of infections caused by the main bacterial drivers of acute exacerbations in COPD patients, NTHi and S. pneumoniae. Methods Human bronchial epithelial BEAS-2B cells were exposed to cigarette smoke extract (CSE). PAFr expression levels were determined using immunocytochemistry and quantitative polymerase chain reaction. The epithelial cells were challenged with either NTHi or S. pneumoniae labeled with fluorescein isothiocyanate, and bacterial adhesion was measured using immunofluorescence. The effect of a well-evaluated antagonist of PAFr, WEB-2086, on binding of the bacterial pathogens to BEAS-2B cells was then assessed. In silico studies of the tertiary structure of PAFr and the binding pocket for PAF and its antagonist WEB-2086 were undertaken. Results PAFr expression by bronchial epithelial cells was upregulated by CSE, and significantly associated with increased bacterial adhesion. WEB-2086 reduced the epithelial adhesion by both NTHi and S. pneumoniae to levels observed for non-CSE-exposed cells. Furthermore, it was nontoxic toward the bronchial epithelial cells. In silico analyses identified a binding pocket for PAF/WEB-2086 in the predicted PAFr structure. Conclusion WEB-2086 represents an innovative class of candidate drugs for inhibiting PAFr-dependent lung infections caused by the main bacterial drivers of smoking-related COPD. PMID:27524890

  15. Infection with human coronavirus NL63 enhances streptococcal adherence to epithelial cells.

    PubMed

    Golda, Anna; Malek, Natalia; Dudek, Bartosz; Zeglen, Slawomir; Wojarski, Jacek; Ochman, Marek; Kucewicz, Ewa; Zembala, Marian; Potempa, Jan; Pyrc, Krzysztof

    2011-06-01

    Understanding the mechanisms of augmented bacterial pathogenicity in post-viral infections is the first step in the development of an effective therapy. This study assessed the effect of human coronavirus NL63 (HCoV-NL63) on the adherence of bacterial pathogens associated with respiratory tract illnesses. It was shown that HCoV-NL63 infection resulted in an increased adherence of Streptococcus pneumoniae to virus-infected cell lines and fully differentiated primary human airway epithelium cultures. The enhanced binding of bacteria correlated with an increased expression level of the platelet-activating factor receptor (PAF-R), but detailed evaluation of the bacterium-PAF-R interaction revealed a limited relevance of this process.

  16. A visual targeting system for the microinjection of unstained adherent cells.

    PubMed

    Becattini, Gabriele; Mattos, Leonardo S; Caldwell, Darwin G

    2013-02-01

    Automatic localization and targeting are critical steps in automating the process of microinjecting adherent cells. This process is currently performed manually by highly trained operators and is characterized as a laborious task with low success rate. Therefore, automation is desired to increase the efficiency and consistency of the operations. This research offers a contribution to this procedure through the development of a vision system for a robotic microinjection setup. Its goals are to automatically locate adherent cells in a culture dish and target them for a microinjection. Here the major concern was the achievement of an error-free targeting system to guarantee high consistency in microinjection experiments. To accomplish this, a novel visual targeting algorithm integrating different image processing techniques was proposed. This framework employed defocusing microscopy to highlight cell features and improve cell segmentation and targeting reliability. Three main image processing techniques, operating at three different focus levels in a bright field (BF) microscope, were used: an anisotropic contour completion (ACC) method, a local intensity variation background-foreground classifier, and a grayscale threshold-based segmentation. The proposed framework combined information gathered by each of these methods using a validation map and this was shown to provide reliable cell targeting results. Experiments conducted with sets of real images from two different cell lines (CHO-K1 and HEK), which contained a total of more than 650 cells, yielded flawless targeting results along with a cell detection ratio greater than 50%.

  17. Computational Tension Mapping of Adherent Cells Based on Actin Imaging.

    PubMed

    Manifacier, Ian; Milan, Jean-Louis; Jeanneau, Charlotte; Chmilewsky, Fanny; Chabrand, Patrick; About, Imad

    2016-01-01

    Forces transiting through the cytoskeleton are known to play a role in adherent cell activity. Up to now few approaches haves been able to determine theses intracellular forces. We thus developed a computational mechanical model based on a reconstruction of the cytoskeleton of an adherent cell from fluorescence staining of the actin network and focal adhesions (FA). Our custom made algorithm converted the 2D image of an actin network into a map of contractile interactions inside a 2D node grid, each node representing a group of pixels. We assumed that actin filaments observed under fluorescence microscopy, appear brighter when thicker, we thus presumed that nodes corresponding to pixels with higher actin density were linked by stiffer interactions. This enabled us to create a system of heterogeneous interactions which represent the spatial organization of the contractile actin network. The contractility of this interaction system was then adapted to match the level of force the cell truly exerted on focal adhesions; forces on focal adhesions were estimated from their vinculin expressed size. This enabled the model to compute consistent mechanical forces transiting throughout the cell. After computation, we applied a graphical approach on the original actin image, which enabled us to calculate tension forces throughout the cell, or in a particular region or even in single stress fibers. It also enabled us to study different scenarios which may indicate the mechanical role of other cytoskeletal components such as microtubules. For instance, our results stated that the ratio between intra and extra cellular compression is inversely proportional to intracellular tension.

  18. Computational Tension Mapping of Adherent Cells Based on Actin Imaging

    PubMed Central

    Manifacier, Ian; Milan, Jean-Louis; Jeanneau, Charlotte; Chmilewsky, Fanny; Chabrand, Patrick; About, Imad

    2016-01-01

    Forces transiting through the cytoskeleton are known to play a role in adherent cell activity. Up to now few approaches haves been able to determine theses intracellular forces. We thus developed a computational mechanical model based on a reconstruction of the cytoskeleton of an adherent cell from fluorescence staining of the actin network and focal adhesions (FA). Our custom made algorithm converted the 2D image of an actin network into a map of contractile interactions inside a 2D node grid, each node representing a group of pixels. We assumed that actin filaments observed under fluorescence microscopy, appear brighter when thicker, we thus presumed that nodes corresponding to pixels with higher actin density were linked by stiffer interactions. This enabled us to create a system of heterogeneous interactions which represent the spatial organization of the contractile actin network. The contractility of this interaction system was then adapted to match the level of force the cell truly exerted on focal adhesions; forces on focal adhesions were estimated from their vinculin expressed size. This enabled the model to compute consistent mechanical forces transiting throughout the cell. After computation, we applied a graphical approach on the original actin image, which enabled us to calculate tension forces throughout the cell, or in a particular region or even in single stress fibers. It also enabled us to study different scenarios which may indicate the mechanical role of other cytoskeletal components such as microtubules. For instance, our results stated that the ratio between intra and extra cellular compression is inversely proportional to intracellular tension. PMID:26812601

  19. Microfabricated elastomeric stencils for micropatterning cell cultures.

    PubMed

    Folch, A; Jo, B H; Hurtado, O; Beebe, D J; Toner, M

    2000-11-01

    Here we present an inexpensive method to fabricate microscopic cellular cultures, which does not require any surface modification of the substrate prior to cell seeding. The method utilizes a reusable elastomeric stencil (i.e., a membrane containing thru holes) which seals spontaneously against the surface. The stencil is applied to the cell-culture substrate before seeding. During seeding, the stencil prevents the substrate from being exposed to the cell suspension except on the hole areas. After cells are allowed to attach and the stencil is peeled off, cellular islands with a shape similar to the holes remain on the cell-culture substrate. This solvent-free method can be combined with a wide range of substrates (including biocompatible polymers, homogeneous or nonplanar surfaces, microelectronic chips, and gels), biomolecules, and virtually any adherent cell type.

  20. Spheroid Culture of Mesenchymal Stem Cells

    PubMed Central

    Cesarz, Zoe; Tamama, Kenichi

    2016-01-01

    Compared with traditional 2D adherent cell culture, 3D spheroidal cell aggregates, or spheroids, are regarded as more physiological, and this technique has been exploited in the field of oncology, stem cell biology, and tissue engineering. Mesenchymal stem cells (MSCs) cultured in spheroids have enhanced anti-inflammatory, angiogenic, and tissue reparative/regenerative effects with improved cell survival after transplantation. Cytoskeletal reorganization and drastic changes in cell morphology in MSC spheroids indicate a major difference in mechanophysical properties compared with 2D culture. Enhanced multidifferentiation potential, upregulated expression of pluripotency marker genes, and delayed replicative senescence indicate enhanced stemness in MSC spheroids. Furthermore, spheroid formation causes drastic changes in the gene expression profile of MSC in microarray analyses. In spite of these significant changes, underlying molecular mechanisms and signaling pathways triggering and sustaining these changes are largely unknown. PMID:26649054

  1. Development of micropatterning technology for cultured cells.

    PubMed

    Matsuda, T; Inoue, K; Sugawara, T

    1990-01-01

    The manipulation of regional cell adhesiveness by surface design could provide micropatterned cell culturing. Based on the photoreactive chemistry of a phenylazide group, a novel surface micropatterning technology for cultured cells was successfully developed. The principle is as follows: 1) a photoreactive hydrophilic co-polymer with phenylazide was cast on a hydrophobic matrix surface, 2) a photoreactive hydrophobic co-polymer was cast on a hydrophilic matrix; 3) a photomask with a given pattern was tightly placed on the cast film; and 4) after UV irradiation and subsequent washing, bovine endothelial cells (ECs) were seeded and cultured. ECs adhered and grew only on nonhydrophilic regions, eventually resulting in micropatterning of ECs. The micropatterns of cultured ECs prepared by 1) and 2) were negative- and positive-type patterns to that of the photomask used, respectively.

  2. Fucoidans Disrupt Adherence of Helicobacter pylori to AGS Cells In Vitro

    PubMed Central

    Chua, Eng-Guan; Verbrugghe, Phebe; Perkins, Timothy T.; Tay, Chin-Yen

    2015-01-01

    Fucoidans are complex sulphated polysaccharides derived from abundant and edible marine algae. Helicobacter pylori is a stomach pathogen that persists in the hostile milieu of the human stomach unless treated with antibiotics. This study aims to provide preliminary data to determine, in vitro, if fucoidans can inhibit the growth of H. pylori and its ability to adhere to gastric epithelial cells (AGS). We analysed the activity of three different fucoidan preparations (Fucus A, Fucus B, and Undaria extracts). Bacterial growth was not arrested or inhibited by the fucoidan preparations supplemented into culture media. All fucoidans, when supplemented into tissue culture media at 1000 µg mL−1, were toxic to AGS cells and reduced the viable cell count significantly. Fucoidan preparations at 100 µg mL−1 were shown to significantly reduce the number of adherent H. pylori. These in vitro findings provide the basis for further studies on the clinical use of sulphated polysaccharides as complementary therapeutic agents. PMID:26604968

  3. Dynamic culture improves cell reprogramming efficiency.

    PubMed

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

    2016-06-01

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

  4. Mammalian Cell Culture Simplified.

    ERIC Educational Resources Information Center

    Moss, Robert; Solomon, Sondra

    1991-01-01

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

  5. Surface modification of closed plastic bags for adherent cell cultivation

    NASA Astrophysics Data System (ADS)

    Lachmann, K.; Dohse, A.; Thomas, M.; Pohl, S.; Meyring, W.; Dittmar, K. E. J.; Lindenmeier, W.; Klages, C.-P.

    2011-07-01

    In modern medicine human mesenchymal stem cells are becoming increasingly important. However, a successful cultivation of this type of cells is only possible under very specific conditions. Of great importance, for instance, are the absence of contaminants such as foreign microbiological organisms, i.e., sterility, and the chemical functionalization of the ground on which the cells are grown. As cultivation of these cells makes high demands, a new procedure for cell cultivation has been developed in which closed plastic bags are used. For adherent cell growth chemical functional groups have to be introduced on the inner surface of the plastic bag. This can be achieved by a new, atmospheric-pressure plasma-based method presented in this paper. The method which was developed jointly by the Fraunhofer IST and the Helmholtz HZI can be implemented in automated equipment as is also shown in this contribution. Plasma process gases used include helium or helium-based gas mixtures (He + N2 + H2) and vapors of suitable film-forming agents or precursors such as APTMS, DACH, and TMOS in helium. The effect of plasma treatment is investigated by FTIR-ATR spectroscopy as well as surface tension determination based on contact angle measurements and XPS. Plasma treatment in nominally pure helium increases the surface tension of the polymer foil due to the presence of oxygen traces in the gas and oxygen diffusing through the gas-permeable foil, respectively, reacting with surface radical centers formed during contact with the discharge. Primary amino groups are obtained on the inner surface by treatment in mixtures with nitrogen and hydrogen albeit their amount is comparably small due to diffusion of oxygen through the gas-permeable bag, interfering with the plasma-amination process. Surface modifications introducing amino groups on the inner surface turned out to be most efficient in the promotion of cell growth.

  6. Adherence of Candida albicans and Candida parapsilosis to epithelial cells correlates with fungal cell surface carbohydrates.

    PubMed

    Lima-Neto, Reginaldo G; Beltrão, Eduardo I C; Oliveira, Patrícia C; Neves, Rejane P

    2011-01-01

    Many studies have described the adherence of Candida albicans to epithelial cells but little is known about Candida parapsilosis adhesion and its role in host cell surface recognition. This study was designed to evaluate the correlation between the adherence of 20 C. albicans and 12 C. parapsilosis strains to human buccal epithelial cells and the expression of fungal cell surface carbohydrates using lectin histochemistry. Adherence assays were carried out by incubating epithelial cells in yeast suspensions (10(7) cells ml(-1) ) and peroxidase conjugated lectins (Con A, WGA, UEA I and PNA at 25 μg ml(-1) ) were used for lectin histochemistry. The results showed that adherence was overall greater for C. albicans than for C. parapsilosis (P < 0.01) and that the individual strain differences correlated with a high content of cell surface α-l-fucose residues as indicated by the UEA I staining pattern. Based on the saccharide specificity of the lectins used, these results suggest that l-fucose residues on cell surface glycoconjugates may represent recognition molecules for interactions between the yeast strain studied and the host (r = 0.6985, P = 0.0045). In addition, our results indicated the presence of α-d-glucose/α-d-mannose, N-acetyl-D-glucosamine/N-acetylneuraminic acid and D-galactose/N-acetyl-D-galactosamine in fungal cell wall.

  7. Role of pili in the adherence of Pseudomonas aeruginosa to mouse epidermal cells.

    PubMed Central

    Sato, H; Okinaga, K

    1987-01-01

    Pili have been demonstrated to be the adhesins of Pseudomonas aeruginosa for mouse epidermal cells. The mechanisms of adhesion of P. aeruginosa to mouse epidermal cells was studied by using four mutants derived from a single strain: flagellated and piliated (F+P+), flagellated and nonpiliated (F+P-), nonflagellated and piliated (F-P+), and nonflagellated and nonpiliated (F-P-) mutants. F+P+ and F-P+ bacteria efficiently adhered to mouse epidermal cells, while F+P- and F-P- bacteria hardly adhered to mouse epidermal cells. The number of F+P+ bacteria that adhered to mouse epidermal cells was almost the same as that of F-P+ bacteria. The number of F+P- bacteria that adhered to mouse epidermal cells was almost the same as that of F-P- bacteria. The adhesion of P+ (F+P+ and F-P+) bacteria was inhibited by antipilus serum, while that of P- (F+P- and F-P-) bacteria was not inhibited by antipilus serum. There were no significant differences between the number of bacteria adhering to mouse epidermal cells isolated from normal skin and those adhering to cells isolated from burned skin. Heating of the mouse epidermal cell suspension had no effect on the adhesion of P. aeruginosa. These results suggest that pili mediate the adhesion of P. aeruginosa to mouse epidermal cells and that P. aeruginosa adheres efficiently to mouse epidermal cells despite the loss of cell viability caused by burning. PMID:2886430

  8. Interleukin-3 greatly expands non-adherent endothelial forming cells with pro-angiogenic properties.

    PubMed

    Moldenhauer, Lachlan M; Cockshell, Michaelia P; Frost, Lachlan; Parham, Kate A; Tvorogov, Denis; Tan, Lih Y; Ebert, Lisa M; Tooley, Katie; Worthley, Stephen; Lopez, Angel F; Bonder, Claudine S

    2015-05-01

    Circulating endothelial progenitor cells (EPCs) provide revascularisation for cardiovascular disease and the expansion of these cells opens up the possibility of their use as a cell therapy. Herein we show that interleukin-3 (IL3) strongly expands a population of human non-adherent endothelial forming cells (EXnaEFCs) with low immunogenicity as well as pro-angiogenic capabilities in vivo, making their therapeutic utilisation a realistic option. Non-adherent CD133(+) EFCs isolated from human umbilical cord blood and cultured under different conditions were maximally expanded by day 12 in the presence of IL3 at which time a 350-fold increase in cell number was obtained. Cell surface marker phenotyping confirmed expression of the hematopoietic progenitor cell markers CD133, CD117 and CD34, vascular cell markers VEGFR2 and CD31, dim expression of CD45 and absence of myeloid markers CD14 and CD11b. Functional experiments revealed that EXnaEFCs exhibited classical properties of endothelial cells (ECs), namely binding of Ulex europaeus lectin, up-take of acetylated-low density lipoprotein and contribution to EC tube formation in vitro. These EXnaEFCs demonstrated a pro-angiogenic phenotype within two independent in vivo rodent models. Firstly, a Matrigel plug assay showed increased vascularisation in mice. Secondly, a rat model of acute myocardial infarction demonstrated reduced heart damage as determined by lower levels of serum creatinine and a modest increase in heart functionality. Taken together, these studies show IL3 as a potent growth factor for human CD133(+) cell expansion with clear pro-angiogenic properties (in vitro and in vivo) and thus may provide clinical utility for humans in the future.

  9. A validated measure of adherence to antibiotic prophylaxis in children with sickle cell disease

    PubMed Central

    Duncan, Natalie A; Kronenberger, William G; Hampton, Kisha C; Bloom, Ellen M; Rampersad, Angeli G; Roberson, Christopher P; Shapiro, Amy D

    2016-01-01

    Background Antibiotic prophylaxis is a mainstay in sickle cell disease management. However, adherence is estimated at only 66%. This study aimed to develop and validate a Sickle Cell Antibiotic Adherence Level Evaluation (SCAALE) to promote systematic and detailed adherence evaluation. Methods A 28-item questionnaire was created, covering seven adherence areas. General Adherence Ratings from the parent and one health care provider and medication possession ratios were obtained as validation measures. Results Internal consistency was very good to excellent for the total SCAALE (α=0.89) and four of the seven subscales. Correlations between SCAALE scores and validation measures were strong for the total SCAALE and five of the seven subscales. Conclusion The SCAALE provides a detailed, quantitative, multidimensional, and global measurement of adherence and can promote clinical care and research. PMID:27354768

  10. Adherence to Asian Cultural Values and Cultural Fit in Korean American Undergraduates' Help-Seeking Attitudes

    ERIC Educational Resources Information Center

    Gloria, Alberta M.; Castellanos, Jeanett; Park, Yong Sue; Kim, Daniel

    2008-01-01

    Differences in and relationships of Asian cultural values, cultural congruity, perception of the university environment, and help-seeking attitudes for 1st- and 2nd-generation Korean American undergraduates (N = 228) were examined. Women reported significantly higher cultural congruity and more positive help-seeking attitudes than did men. Asian…

  11. Adherence of Pseudomonas aeruginosa to tracheal cells injured by influenza infection or by endotracheal intubation.

    PubMed Central

    Ramphal, R; Small, P M; Shands, J W; Fischlschweiger, W; Small, P A

    1980-01-01

    Adherence of Pseudomonas aeruginosa to normal, injured, and regenerating tracheal mucosa was examined by scanning electron microscopy. Uninfected and influenza-infected murine tracheas were exposed to six strains of P. aeruginosa isolated from human sources and one strain of platn origin. All of the strains tested adhered to desquamating cells of the infected tracheas, but not to normal mucosa, the basal cell layer, or the regenerating epithelium. Adherence increased when the incubation time of the bacteria with the trachea was prolonged. Strains isolated from human tracheas appeared to adhere better than strains derived from the urinary tract. After endotracheal intubation of ferrets, P. aeruginosa adhered only to the injured cells and to areas of exposed basement membrane. We call this phenomenon "opportunistic adherence" and propose that alteration of the cell surfaces or cell injury facilitates the adherence of this bacterium and that adherence to injured cells may be a key to the pathogenesis of opportunistic Pseudomonas infections. Images Fig. 1 Fig. 5 PMID:6769805

  12. Basic cell culture.

    PubMed

    Pollard, J W

    1990-01-01

    This article will describe the basic techniques required for successful cell culture. It will also act to introduce some of the other chapters in this volume. It is not intended, as this volume is not, to describe the establishment of a tissue culture laboratory, nor to provide a historical or theoretical survey of cell culture. There are several books that adequately cover these areas, including the now somewhat dated but still valuable volume by Paul (1), the multi-authored Methods in Enzymology volume edited by Jakoby and Pastan (2), and the new edition of Freshney (3). Instead, this chapter's focus will be on the techniques for establishing primary rodent cell cultures from embryos and adult skin, maintaining and subculturing these fibro-blasts and their transformed derivatives, and the isolation of genetically pure strains. The cells described are all derived from Chinese hamsters since, to date, these cells, have proved to be the most useful for somatic cell genetics (4,5). The techniques, however, are generally applicable to most fibroblastic cell types.

  13. An acid phosphatase assay for quantifying the growth of adherent and nonadherent cells.

    PubMed

    Yang, T T; Sinai, P; Kain, S R

    1996-10-01

    We describe an acid phosphatase assay for determination of cell growth based on quantification of cytosolic acid phosphatase activity. The assay is based on the hydrolysis of the p-nitrophenyl phosphate by intracellular acid phosphatases in viable cells to produce p-nitrophenol. For all cell types examined, absorbance of p-nitrophenol at 405 nm is directly proportional to the cell number in the range of 10(3)-10(5) cells. The assay can quantify as few as 1000 cells per well in 96-well microtiter plates. The acid phosphatase assay was used to count various adherent and nonadherent cells, including human tumors, L6, and HT-2 cells. We also demonstrate the utility of this assay for analysis of growth factor and cytokine bioactivity on mammalian cells in culture. In comparison to [3H]thymidine incorporation, the acid phosphatase assay has similar sensitivity but a wider linear response range. The method also shows higher sensitivity and reproducibility in comparison to cell proliferation assays based on the reduction of tetrazolium salts. Because of the ease of use, sensitivity, and low cost, the acid phosphatase method is especially suited to applications where a large number of samples are assayed.

  14. Method of making a membrane having hydrophilic and hydrophobic surfaces for adhering cells or antibodies by using atomic oxygen or hydroxyl radicals

    NASA Technical Reports Server (NTRS)

    Koontz, Steven L. (Inventor); Spaulding, Glenn F. (Inventor)

    1994-01-01

    A portion of an organic polymer article such as a membrane is made hydrophilic by exposing a hydrophobic surface of the article to a depth of about 50 to about 5000 angstroms to atomic oxygen or hydroxyl radicals at a temperature below 100C., preferably below 40 C, to form a hydrophilic uniform surface layer of hydrophilic hydroxyl groups. The atomic oxygen and hydroxyl radicals are generated by a flowing afterglow microwave discharge, and the surface is outside of a plasma produced by the discharge. A membrane having both hydrophilic and hydrophobic surfaces can be used in an immunoassay by adhering antibodies to the hydrophobic surface. In another embodiment, the membrane is used in cell culturing where cells adhere to the hydrophilic surface. Prior to adhering cells, the hydrophilic surface may be grafted with a compatibilizing compound. A plurality of hydrophilic regions bounded by adjacent hydrophobic regions can be produced such that a maximum of one cell per each hydrophilic region adheres.

  15. A novel method for high-pressure freezing of adherent cells for frozen hydrated sectioning and CEMOVIS.

    PubMed

    Mesman, R J

    2013-09-01

    With the development of Cryo Electron Microscopy Of Vitreous Sections (CEMOVIS), imaging cells in a close to native state has become a reality. However with the commonly used carriers for high-pressure freezing and cryo-sectioning, adherent grown cells either need to be detached from their substrate. Here a new method is presented for high-pressure freezing adherent growing cells for frozen-hydrated sectioning and CEMOVIS. Cells are cultured on golden grids, containing a carbon coated Formvar film, and frozen on a membrane carrier which provides the grids with the structural support needed to withstand the strain of trimming and cryo-sectioning. This method was successfully tested for the two different types of high-pressure freezers, those using a pressure chamber (HPM010, EMHPF, Wohlwend Compact 01/02, HPM100) and those directly pressurizing the sample (EMPact series).

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

    PubMed Central

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

    2013-01-01

    In vitro cell culture systems from molluscs have significantly contributed to our basic understanding of complex physiological processes occurring within or between tissue-specific cells, yielding information unattainable using intact animal models. In vitro cultures of neuronal cells from gastropods show how simplified cell models can inform our understanding of complex networks in intact organisms. Primary cell cultures from marine and freshwater bivalve and gastropod species are used as biomonitors for environmental contaminants, as models for gene transfer technologies, and for studies of innate immunity and neoplastic disease. Despite efforts to isolate proliferative cell lines from molluscs, the snail Biomphalaria glabrata Say, 1818 embryonic (Bge) cell line is the only existing cell line originating from any molluscan species. Taking an organ systems approach, this review summarizes efforts to establish molluscan cell cultures and describes the varied applications of primary cell cultures in research. Because of the unique status of the Bge cell line, an account is presented of the establishment of this cell line, and of how these cells have contributed to our understanding of snail host-parasite interactions. Finally, we detail the difficulties commonly encountered in efforts to establish cell lines from molluscs and discuss how these difficulties might be overcome. PMID:24198436

  17. Quantum dot labeling of butyrylcholinesterase maintains substrate and inhibitor interactions and cell adherence features.

    PubMed

    Waiskopf, Nir; Shweky, Itzhak; Lieberman, Itai; Banin, Uri; Soreq, Hermona

    2011-03-16

    Butyrylcholinesterase (BChE) is the major acetylcholine hydrolyzing enzyme in peripheral mammalian systems. It can either reside in the circulation or adhere to cells and tissues and protect them from anticholinesterases, including insecticides and poisonous nerve gases. In humans, impaired cholinesterase functioning is causally involved in many pathologies, including Alzheimer's and Parkinson's diseases, trait anxiety, and post stroke conditions. Recombinant cholinesterases have been developed for therapeutic use; therefore, it is important to follow their in vivo path, location, and interactions. Traditional labeling methods, such as fluorescent dyes and proteins, generally suffer from sensitivity to environmental conditions, from proximity to different molecules or special enzymes which can alter them, and from relatively fast photobleaching. In contrast, emerging development in synthesis and surface engineering of semiconductor nanocrystals enable their use to detect and follow molecules in biological milieus at high sensitivity and in real time. Therefore, we developed a platform for conjugating highly purified recombinant human BChE dimers (rhBChE) to CdSe/CdZnS quantum dots (QDs). We report the development and characterization of highly fluorescent aqueous soluble QD-rhBChE conjugates, present maintenance of hydrolytic activity, inhibitor sensitivity, and adherence to the membrane of cultured live cells of these conjugates, and outline their advantageous features for diverse biological applications.

  18. Quantum Dot Labeling of Butyrylcholinesterase Maintains Substrate and Inhibitor Interactions and Cell Adherence Features

    PubMed Central

    2010-01-01

    Butyrylcholinesterase (BChE) is the major acetylcholine hydrolyzing enzyme in peripheral mammalian systems. It can either reside in the circulation or adhere to cells and tissues and protect them from anticholinesterases, including insecticides and poisonous nerve gases. In humans, impaired cholinesterase functioning is causally involved in many pathologies, including Alzheimer’s and Parkinson’s diseases, trait anxiety, and post stroke conditions. Recombinant cholinesterases have been developed for therapeutic use; therefore, it is important to follow their in vivo path, location, and interactions. Traditional labeling methods, such as fluorescent dyes and proteins, generally suffer from sensitivity to environmental conditions, from proximity to different molecules or special enzymes which can alter them, and from relatively fast photobleaching. In contrast, emerging development in synthesis and surface engineering of semiconductor nanocrystals enable their use to detect and follow molecules in biological milieus at high sensitivity and in real time. Therefore, we developed a platform for conjugating highly purified recombinant human BChE dimers (rhBChE) to CdSe/CdZnS quantum dots (QDs). We report the development and characterization of highly fluorescent aqueous soluble QD-rhBChE conjugates, present maintenance of hydrolytic activity, inhibitor sensitivity, and adherence to the membrane of cultured live cells of these conjugates, and outline their advantageous features for diverse biological applications. PMID:22778863

  19. Cell Culturing of Cytoskeleton

    NASA Technical Reports Server (NTRS)

    2004-01-01

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

  20. Cell Culturing of Cytoskeleton

    NASA Technical Reports Server (NTRS)

    2004-01-01

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

  1. Inhibition of Pneumococcal Adherence to Human Nasopharyngeal Epithelial Cells by Anti-PsaA Antibodies

    PubMed Central

    Romero-Steiner, Sandra; Pilishvili, Tamar; Sampson, Jacquelyn S.; Johnson, Scott E.; Stinson, Annie; Carlone, George M.; Ades, Edwin W.

    2003-01-01

    The role of pneumococcal (Pnc) surface adhesin A (PsaA) in the adherence of Streptococcus pneumoniae (pneumococcus) to host cells is not well defined. We examined the effect of anti-PsaA antibodies in an inhibition of adherence assay using Detroit 562 nasopharyngeal human epithelial cells. Rabbit polyclonal (Pab) anti-recombinant PsaA (rPsaA) sera, a purified mouse monoclonal antibody (MAb) (MAb 6F62G8E12), and 22 healthy adult sera with known anti-PsaA IgG levels (obtained by enzyme-linked immunosorbent assay) were evaluated for their abilities to inhibit Pnc adherence to confluent monolayers (measured as percent reduction in CFU counts compared to those of uninhibited controls). Pnc adherence was dependent on capsular phenotype (no or low adherence for opaque strains). With an inoculum of 104 to 105 bacteria/well, the mean ± standard deviation count in controls was 163 ± 32 CFU/well for transparent strains. Low adherence was observed for a PsaA-minus mutant even at higher inoculum doses. Mean percent inhibitions of adherence with Pab and MAb were 54 and 50%, respectively. Adult sera showed inhibition in a dose-response fashion with a range of 98 to 8%, depending on the serum anti-PsaA antibody concentration. Absorption of Pab with rPsaA restored Pnc adherence to control levels. Absorption of sera with a PsaA-minus mutant did not result in a significant decrease (P >0.05) of inhibition of adherence activity. Additionally, nearly 100% of Pnc adherence was inhibited by lipidated rPsaA at 2.5 μg/ml. Our data support the argument that PsaA is an adhesin that mediates Pnc adherence to human nasopharyngeal cells. This functional assay may be useful in evaluating antibodies elicited in response to PsaA vaccination. PMID:12626450

  2. Neonatal rat heart cells cultured in simulated microgravity

    NASA Technical Reports Server (NTRS)

    Akins, Robert E.; Schroedl, Nancy A.; Gonda, Steve R.; Hartzell, Charles R.

    1994-01-01

    In vitro characteristics of cardiac cells cultured in simulated microgravity are reported. Tissue culture methods performed at unit gravity constrain cells to propagate, differentiate, and interact in a two dimensional (2D) plane. Neonatal rat cardiac cells in 2D culture organize predominantly as bundles of cardiomyocytes with the intervening areas filled by non-myocyte cell types. Such cardiac cell cultures respond predictably to the addition of exogenous compounds, and in many ways they represent an excellent in vitro model system. The gravity-induced 2D organization of the cells, however, does not accurately reflect the distribution of cells in the intact tissue. We have begun characterizations of a three-dimensional (3D) culturing system designed to mimic microgravity. The NASA designed High-Aspect-Ratio-Vessel (HARV) bioreactors provide a low shear environment which allows cells to be cultured in static suspension. HARV-3D cultures were prepared on microcarrier beads and compared to control-2D cultures using a combination of microscopic and biochemical techniques. Both systems were uniformly inoculated and medium exchanged at standard intervals. Cells in control cultures adhered to the polystyrene surface of the tissue culture dishes and exhibited typical 2D organization. Cells in cultured in HARV's adhered to microcarrier beads, the beads aggregated into defined clusters containing 8 to 15 beads per cluster, and the clusters exhibited distinct 3D layers: myocytes and fibroblasts appeared attached to the surfaces of beads and were overlaid by an outer cell type. In addition, cultures prepared in HARV's using alternative support matrices also displayed morphological formations not seen in control cultures. Generally, the cells prepared in HARV and control cultures were similar, however, the dramatic alterations in 3D organization recommend the HARV as an ideal vessel for the generation of tissue-like organizations of cardiac cells in simulated microgravity.

  3. Neonatal rat heart cells cultured in simulated microgravity

    NASA Technical Reports Server (NTRS)

    Akins, R. E.; Schroedl, N. A.; Gonda, S. R.; Hartzell, C. R.

    1997-01-01

    In vitro characteristics of cardiac cells cultured in simulated microgravity are reported. Tissue culture methods performed at unit gravity constrain cells to propagate, differentiate, and interact in a two-dimensional (2D) plane. Neonatal rat cardiac cells in 2D culture organize predominantly as bundles of cardiomyocytes with the intervening areas filled by nonmyocyte cell types. Such cardiac cell cultures respond predictably to the addition of exogenous compounds, and in many ways they represent an excellent in vitro model system. The gravity-induced 2D organization of the cells, however, does not accurately reflect the distribution of cells in the intact tissue. We have begun characterizations of a three-dimensional (3D) culturing system designed to mimic microgravity. The NASA-designed High-Aspect Ratio Vessel (HARV) bioreactors provide a low shear environment that allows cells to be cultured in static suspension. HARV-3D cultures were prepared on microcarrier beads and compared to control-2D cultures using a combination of microscopic and biochemical techniques. Both systems were uniformly inoculated and medium exchanged at standard intervals. Cells in control cultures adhered to the polystyrene surface of the tissue culture dishes and exhibited typical 2D organization. Cells cultured in HARVs adhered to microcarrier beads, the beads aggregated into defined clusters containing 8 to 15 beads per cluster, and the clusters exhibited distinct 3D layers: myocytes and fibroblasts appeared attached to the surfaces of beads and were overlaid by an outer cell type. In addition, cultures prepared in HARVs using alternative support matrices also displayed morphological formations not seen in control cultures. Generally, the cells prepared in HARV and control cultures were similar; however, the dramatic alterations in 3D organization recommend the HARV as an ideal vessel for the generation of tissuelike organization of cardiac cells in vitro.

  4. Localized electroporation effect on adherent cells in modified electric cell-substrate impedance sensing circuits

    NASA Astrophysics Data System (ADS)

    Kim, Yu Jin; Ram Song, Ka; Kim, Hee-Dae; Park, Bum Chul; Kim, Young Keun; Kang, Chi Jung

    2016-10-01

    Electroporation is a physical transfection method for introducing foreign genes or drugs into cells. It does not require toxic reagents or transfection vectors. However, its applications have been limited because of cell damage and nonspecific transport. Here, we present an effective method for selective and localized electroporation using atomic force microscopy. This electroporation method is applied to adherent cells on substrates, instead of conventionally used suspended cells, and offers relatively effective cell transfection. Moreover, this method enables localized transfection into targeted areas at the single-cell level.

  5. Pathogen and host differences in bacterial adherence to human buccal epithelial cells in a northeast Brazilian community.

    PubMed Central

    Walser, B L; Newman, R D; Lima, A A; Guerrant, R L

    1992-01-01

    The adherence of several strains of Escherichia coli to human buccal epithelial cells was studied, using cells obtained from five groups: healthy adults, healthy children, children with acute diarrhea, children with persistent diarrhea associated with cryptosporidial parasites, and children with noncryptosporidial persistent diarrhea. All groups lived or worked in an urban slum in northeastern Brazil. Samples of buccal epithelial cells from subjects in each of these groups were incubated with wild-type E. coli K-12 (strain C600), the enteroaggregative E. coli strains 17-2 and PDAS 30-5, CFA/II-positive E. coli 1392+ and its plasmid-cured derivative 1392-, and hydrophobic E. coli 132-3. Samples were evaluated microscopically to determine background contamination and the percentage of cells with more than 15% of their surface area obscured by adherent bacteria after incubation and washing. The assay was tested under field conditions and was shown to produce reliable and consistent results. Both enteroaggregative strains of E. coli were shown to adhere to a significantly higher percentage of all groups of human buccal epithelial cells than any of the other tested strains. In addition, buccal epithelial cells from children with nonparasitic persistent diarrhea showed substantially more bacterial adherence in both the native state and with all tested strains of E. coli than did cells from children with persistent cryptosporidial diarrhea or acute diarrhea or from healthy controls. This study provides evidence that enteroaggregative strains of E. coli demonstrate increased adherence to human buccal epithelial cells (as well as to cultured HEp-2 cells) and that buccal epithelial cells from children with noncryptosporidial persistent diarrhea appear to be more susceptible to bacterial adherence and colonization than buccal epithelial cells from control groups. These findings suggest that host differences as well as pathogen differences are important in the pathogenesis of

  6. Laser-generated Micro-bubbles for Molecular Delivery to Adherent Cells

    NASA Astrophysics Data System (ADS)

    Genc, Suzanne Lee

    We examine the use of optical breakdown in aqueous media as a means to deliver molecules into live adherent cell cultures. This process, called optoinjection (OI), is affected both by the media composition and the cellular exposure to hydrodynamic stresses associated with the cavitation bubble formed by the optical breakdown process. Here we explore the possibility of performing OI using laser microbeams focused at low numerical aperture to provide conditions where OI can be performed at high-throughput. We first investigate the effect of media composition on plasma and cavitation bubble formation. We make the discovery that irradiation of minimal essential media, supports the formation of low-density plasmas (LDP) resulting in the generation of small (2--20 mum radius) cavitation bubbles. This provides gentle specific hydrodynamic perturbations to single or small groups of cells. The addition of supplemental fetal bovine serum to the medium prevents the formation LDPs and the resulting avalanche ionization generates larger (> 100 mum radius) bubbles and more violent hydrodynamic effects. Second, using high-speed photography we provide the first visualization of LDP-generated cavitation bubbles at precise offset locations relative to a boundary on which a cell monolayer can be cultured. These images depict the cellular exposure to different hydrodynamic conditions depending on the normalized offset distance (gamma = s/Rmax) and show how it affects the cellular exposure to shear stresses upon bubble expansion and different distributions of bubble energy upon collapse. Lastly, we examine the effects of pulse energy, parameters, and single vs. multiple laser exposures on the ability to deliver 3-5 kDa dextrans into adherent cells using both small (< 20 mum) and large (100mu m) radius bubbles. For single exposures, we identify several conditions under which OI can be optimized: (a) conditions where cell viability is maximized (˜90%) but optoinjection of viable cells

  7. Acute myelogenous leukemia cells with the MLL-ELL translocation convert morphologically and functionally into adherent myofibroblasts

    SciTech Connect

    Tashiro, Haruko; Mizutani-Noguchi, Mitsuho; Shirasaki, Ryosuke

    2010-01-01

    Bone marrow-myofibroblasts, a major component of bone marrow-stroma, are reported to originate from hematopoietic stem cells. We show in this paper that non-adherent leukemia blasts can change into myofibroblasts. When myeloblasts from two cases of acute myelogenous leukemia with a fusion product comprising mixed lineage leukemia and RNA polymerase II elongation factor, were cultured long term, their morphology changed to that of myofibroblasts with similar molecular characteristics to the parental myeloblasts. The original leukemia blasts, when cultured on the leukemia blast-derived myofibroblasts, grew extensively. Leukemia blasts can create their own microenvironment for proliferation.

  8. Immunoregulatory adherent cells in human tuberculosis: radiation-sensitive antigen-specific suppression by monocytes

    SciTech Connect

    Kleinhenz, M.E.; Ellner, J.J.

    1985-07-01

    In human tuberculosis, adherent mononuclear cells (AMC) selectively depress in vitro responses to the mycobacterial antigen tuberculin purified protein derivative (PPD). The phenotype of this antigen-specific adherent suppressor cell was characterized by examining the functional activity of adherent cells after selective depletion of sheep erythrocyte-rosetting T cells or OKM1-reactive monocytes. Adherent cell suppression was studied in the (/sup 3/H)thymidine-incorporation microculture assay by using T cells rigorously depleted of T cells with surface receptors for the Fc portion of IgG (T gamma cells) as antigen-responsive cells. PPD-induced (/sup 3/H)thymidine incorporation by these non gamma T cells was uniformly reduced (mean, 42% +/- 10% (SD)) when autologous AMC were added to non gamma T cells at a ratio of 1:2. Antigen-specific suppression by AMC was not altered by depletion of sheep erythrocyte-rosetting T cells or treatment with indomethacin. However, AMC treated with OKM1 and complement or gamma irradiation (1,500 rads) no longer suppressed tuberculin responses in vitro. These studies identify the antigen-specific adherent suppressor cell in tuberculosis as an OKM1-reactive, non-erythrocyte-rosetting monocyte. The radiosensitivity of this monocyte immunoregulatory function may facilitate its further definition.

  9. On-chip integrated lensless microscopy module for optical monitoring of adherent growing mammalian cells.

    PubMed

    Li, Wei; Knoll, Thorsten; Thielecke, Hagen

    2010-01-01

    Lab-on-a-chip systems are increasingly applied in cell-based assays for toxicology and drug testing. In this paper, an on-chip integrated lensless microscopy module using a direct projection method for optical monitoring of the shadow images of adherent growing mammalian cells is presented. The biological cells are conserved and interfaced by a microfabricated cavity chip with a 1 microm thick silicon nitride (Si(3)N(4)) substrate onto the surface of a 5 megapixel CMOS image sensor with 2.2 microm pixel size. The optical resolution of the assembly is estimated by the contact/proximate printing theory from optical lithography. Further characterization is made by imaging microbeads in chips with the Si(3)N(4)-membrane as well as in cavity chips with membranes made from dry film resist (DFR, thickness 20, 40 and 60 microm). The module represents a 3 × optical microscope for cell morphology imaging. The function is demonstrated by the growth monitoring of L929 cells cultured in cavity chips with Si(3)N(4) substrate for 2 days and by checking the colorimetric staining of cells with a compromised membrane.

  10. Cell fusion through a microslit between adhered cells and observation of their nuclear behavior.

    PubMed

    Wada, Ken-Ichi; Hosokawa, Kazuo; Kondo, Eitaro; Ito, Yoshihiro; Maeda, Mizuo

    2014-07-01

    This paper describes a novel cell fusion method which induces cell fusion between adhered cells through a microslit for preventing nuclear mixing. For this purpose, a microfluidic device which had ∼ 100 cell pairing structures (CPSs) making cell pairs through microslits with 2.1 ± 0.3 µm width was fabricated. After trapping NIH3T3 cells with hydrodynamic forces at the CPSs, the cells were fused through the microslit by the Sendai virus envelope method. With following timelapse observation, we discovered that the spread cells were much less susceptible to nuclear migration passing through the microslit compared with round cells, and that cytoplasmic fraction containing mitochondria was transferred through the microslit without nuclear mixing. These findings will provide an effective method for cell fusion without nuclear mixing, and will lead to an efficient method for reprograming and transdifferentiation of target cells toward regenerative medicine.

  11. An approach to collaborative care and consultation: interviewing, cultural competence, and enhancing rapport and adherence.

    PubMed

    Beck, B J; Gordon, Christopher

    2010-11-01

    Although changes in the US health care system promote a population-based approach, increases in population diversity emphasize the need for culturally competent, patient-centered, participatory care. Despite this perceived conflict, the global view has improved the recognition of mental health issues as a driver of overall health as well as health care spending. This recognition, along with the many forces that keep mental health care in the primary care sector, actually encourages the development of collaborative models that capitalize on the primary care provider's opportunity to leverage their rapport with the patient to improve access to, and comfort with, specialty mental health services. Engaging patients in their own path to recovery or well-being improves engagement in, and adherence to, the treatment plan and ultimately improves outcomes.

  12. Fibronectin-binding protein of Streptococcus pyogenes: sequence of the binding domain involved in adherence of streptococci to epithelial cells.

    PubMed Central

    Talay, S R; Valentin-Weigand, P; Jerlström, P G; Timmis, K N; Chhatwal, G S

    1992-01-01

    The sequence of the fibronectin-binding domain of the fibronectin-binding protein of Streptococcus pyogenes (Sfb protein) was determined, and its role in streptococcal adherence was investigated by use of an Sfb fusion protein in adherence studies. A 1-kb DNA fragment coding for the binding domain of Sfb protein was cloned into the expression vector pEX31 to produce an Sfb fusion protein consisting of the N-terminal part of MS2 polymerase and a C-terminal fragment of the streptococcal protein. Induction of the vector promoter resulted in hyperexpression of fibronectin-binding fusion protein in the cytoplasm of the recombinant Escherichia coli cells. Sequence determination of the cloned 1-kb fragment revealed an in-frame reading frame for a 268-amino-acid peptide composed of a 37-amino-acid sequence which is completely repeated three times and incompletely repeated a fourth time. Cloning of one repeat into pEX31 resulted in expression of small fusion peptides that show fibronectin-binding activity, indicating that one repeat contains at least one binding domain. Each repeat exhibits two charged domains and shows high homology with the 38-amino-acid D3 repeat of the fibronectin-binding protein of Staphylococcus aureus. Sequence comparison with other streptococcal ligand-binding surface proteins, including M protein, failed to reveal significant homology, which suggests that Sfb protein represents a novel type of functional protein in S. pyogenes. The Sfb fusion protein isolated from the cytoplasm of recombinant cells was purified by fast protein liquid chromatography. It showed a strong competitive inhibition of fibronectin binding to S. pyogenes and of the adherence of bacteria to cultured epithelial cells. In contrast, purified streptococcal lipoteichoic acid showed only a weak inhibition of fibronectin binding and streptococcal adherence. These results demonstrate that Sfb protein is directly involved in the fibronectin-mediated adherence of S. pyogenes to

  13. High efficiency, site-specific transfection of adherent cells with siRNA using microelectrode arrays (MEA).

    PubMed

    Patel, Chetan; Muthuswamy, Jit

    2012-09-13

    The discovery of RNAi pathway in eukaryotes and the subsequent development of RNAi agents, such as siRNA and shRNA, have achieved a potent method for silencing specific genes for functional genomics and therapeutics. A major challenge involved in RNAi based studies is the delivery of RNAi agents to targeted cells. Traditional non-viral delivery techniques, such as bulk electroporation and chemical transfection methods often lack the necessary spatial control over delivery and afford poor transfection efficiencies. Recent advances in chemical transfection methods such as cationic lipids, cationic polymers and nanoparticles have resulted in highly enhanced transfection efficiencies. However, these techniques still fail to offer precise spatial control over delivery that can immensely benefit miniaturized high-throughput technologies, single cell studies and investigation of cell-cell interactions. Recent technological advances in gene delivery have enabled high-throughput transfection of adherent cells, a majority of which use microscale electroporation. Microscale electroporation offers precise spatio-temporal control over delivery (up to single cells) and has been shown to achieve high efficiencies. Additionally, electroporation based approaches do not require a prolonged period of incubation (typically 4 hours) with siRNA and DNA complexes as necessary in chemical based transfection methods and lead to direct entry of naked siRNA and DNA molecules into the cell cytoplasm. As a consequence gene expression can be achieved as early as six hours after transfection. Our lab has previously demonstrated the use of microelectrode arrays (MEA) for site-specific transfection in adherent mammalian cell cultures. In the MEA based approach, delivery of genetic payload is achieved via localized micro-scale electroporation of cells. An application of electric pulse to selected electrodes generates local electric field that leads to electroporation of cells present in the region

  14. Microfluidic Cell Culture Device

    NASA Technical Reports Server (NTRS)

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

    2014-01-01

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

  15. Inhibition of mitogenesis induced by phytohemagglutinin and Lens culinaris lectin in adherent-cell supernatants treated with protein extract of Mycobacterium tuberculosis.

    PubMed Central

    Parra, C; Montaño, L F; Huesca, M; Rayón, I; Willms, K; Goodsaid, F

    1986-01-01

    Specific stimulation of T cells by phytohemagglutinin and Lens culinaris lectin was inhibited by a soluble factor(s) secreted by normal adherent cells stimulated with culture filtrate protein extract (CFPE) derived from bacterial cultures of Mycobacterium tuberculosis H37Ra (avirulent) and H37Rv (virulent). The induction of the inhibitory factor was blocked by the presence of hyperimmune antisera to H37Rv or H37Ra CFPE. The inhibitory factor did not seem to be a CFPE reprocessed by the adherent cells. Inhibitory activity was maximal in supernatants of adherent-cell cultures incubated for 48 h; the inhibitory factor was heat labile, and its production was dependent on the concentration of M. tuberculosis CFPE. A mouse monocyte-macrophage cell line, ATCC J774A.1, produced an identical inhibitory factor, thus excluding a non-macrophage-contaminating adherent cell as the source of the factor. This inhibitory factor also interfered with the recognition of phytohemagglutinin and Lens culinaris lectin by T cells. PMID:3082760

  16. Surface glycosaminoglycans mediate adherence between HeLa cells and Lactobacillus salivarius Lv72

    PubMed Central

    2013-01-01

    Background The adhesion of lactobacilli to the vaginal surface is of paramount importance to develop their probiotic functions. For this reason, the role of HeLa cell surface proteoglycans in the attachment of Lactobacillus salivarius Lv72, a mutualistic strain of vaginal origin, was investigated. Results Incubation of cultures with a variety of glycosaminoglycans (chondroitin sulfate A and C, heparin and heparan sulfate) resulted in marked binding interference. However, no single glycosaminoglycan was able to completely abolish cell binding, the sum of all having an additive effect that suggests cooperation between them and recognition of specific adhesins on the bacterial surface. In contrast, chondroitin sulfate B enhanced cell to cell attachment, showing the relevance of the stereochemistry of the uronic acid and the sulfation pattern on binding. Elimination of the HeLa surface glycosaminoglycans with lyases also resulted in severe adherence impairment. Advantage was taken of the Lactobacillus-glycosaminoglycans interaction to identify an adhesin from the bacterial surface. This protein, identify as a soluble binding protein of an ABC transporter system (OppA) by MALDI-TOF/(MS), was overproduced in Escherichia coli, purified and shown to interfere with L. salivarius Lv72 adhesion to HeLa cells. Conclusions These data suggest that glycosaminoglycans play a fundamental role in attachment of mutualistic bacteria to the epithelium that lines the cavities where the normal microbiota thrives, OppA being a bacterial adhesin involved in the process. PMID:24044741

  17. A novel multi-coaxial hollow fiber bioreactor for adherent cell types. Part 1: hydrodynamic studies.

    PubMed

    Wolfe, Stephen P; Hsu, Edward; Reid, Lola M; Macdonald, Jeffrey M

    2002-01-05

    A novel multi-coaxial bioreactor for three-dimensional cultures of adherent cell types, such as liver, is described. It is composed of four tubes of increasing diameter placed one inside the other, creating four spatially isolated compartments. Liver acinar structure and physiological parameters are mimicked by sandwiching cells in the space between the two innermost semi-permeable tubes, or hollows fibers, and creating a radial flow of media from an outer compartment (ECC), through the cell mass compartment, and to an inner compartment (ICC). The outermost compartment is created by gas-permeable tubing, and the housing is used to oxygenate the perfusion media to periportal levels in the ECC. Experiments were performed using distilled water to correlate the radial flow rate (Q(r)) with (1) the pressure drop (DeltaP) between the media compartments that sandwich the cell compartment and (2) the pressure in the cell compartment (P(c)). These results were compared with the theoretical profile calculated based on the hydraulic permeability of the two innermost fibers. Phase-contrast velocity-encoded magnetic resonance imaging was used to visualize directly the axial velocities inside the bioreactor and confirm the assumptions of laminar flow and zero axial velocity at the boundaries of each compartment in the bioreactor. Axial flow rates were calculated from the magnetic resonance imaging results and were similar to the measured axial flow rates for the previously described experiments.

  18. Proteolytic processing of reovirus is required for adherence to intestinal M cells.

    PubMed Central

    Amerongen, H M; Wilson, G A; Fields, B N; Neutra, M R

    1994-01-01

    Reovirus adheres specifically to apical membranes of mouse intestinal M cells and exploits M-cell transepithelial transport activity to enter Peyer's patch mucosa, where replication occurs. Proteolytic conversion of native reovirus to intermediate subviral particles (ISVPs) occurs in the intestine, but it is not known whether conversion is essential for interaction of virus with M cells. We tested the capacity of native virions, ISVPs, and cores (that lack outer capsid proteins) to bind to intestinal epithelial cells in vivo and found that only ISVPs adhered to M cells. Thus, intraluminal conversion of native reovirus to ISVPs is a prerequisite for M-cell adherence, and outer capsid proteins unique to ISVPs (either sigma 1 or products of mu 1) mediate interaction of virus with M-cell apical membranes. Images PMID:7525989

  19. Non-invasive, label-free cell counting and quantitative analysis of adherent cells using digital holography.

    PubMed

    Mölder, A; Sebesta, M; Gustafsson, M; Gisselson, L; Wingren, A Gjörloff; Alm, K

    2008-11-01

    Manual cell counting is time consuming and requires a high degree of skill on behalf of the person performing the count. Here we use a technique that utilizes digital holography, allowing label-free and completely non-invasive cell counting directly in cell culture vessels with adherent viable cells. The images produced can provide both quantitative and qualitative phase information from a single hologram. The recently constructed microscope Holomonitor (Phase Holographic Imaging AB, Lund, Sweden) combines the commonly used phase contrast microscope with digital holography, the latter giving us the possibility of achieving quantitative information on cellular shape, area, confluence and optical thickness. This project aimed at determining the accuracy and repeatability of cell counting measurements using digital holography compared to the conventional manual cell counting method using a haemocytometer. The collected data were also used to determine cell size and cellular optical thickness. The results show that digital holography can be used for non-invasive automatic cell counting as precisely as conventional manual cell counting.

  20. Monitoring of adherent live cells morphology using the undecimated wavelet transform multivariate image analysis (UWT-MIA).

    PubMed

    Juneau, Pierre-Marc; Garnier, Alain; Duchesne, Carl

    2017-01-01

    Cell morphology is an important macroscopic indicator of cellular physiology and is increasingly used as a mean of probing culture state in vitro. Phase contrast microscopy (PCM) is a valuable tool for observing live cells morphology over long periods of time with minimal culture artifact. Two general approaches are commonly used to analyze images: individual object segmentation and characterization by pattern recognition. Single-cell segmentation is difficult to achieve in PCM images of adherent cells since their contour is often irregular and blurry, and the cells bundle together when the culture reaches confluence. Alternatively, pattern recognition approaches such as the undecimated wavelet transform multivariate image analysis (UWT-MIA), allow extracting textural features from PCM images that are correlated with cellular morphology. A partial least squares (PLS) regression model built using textural features from a set of 200 ground truth images was shown to predict the distribution of cellular morphological features (major and minor axes length, orientation, and roundness) with good accuracy for most images. The PLS models were then applied on a large dataset of 631,136 images collected from live myoblast cell cultures acquired under different conditions and grown in two different culture media. The method was found sensitive to morphological changes due to cell growth (culture time) and those introduced by the use of different culture media, and was able to distinguish both sources of variations. The proposed approach is promising for application on large datasets of PCM live-cell images to assess cellular morphology and growth kinetics in real-time which could be beneficial for high-throughput screening as well as automated cell culture kinetics assessment and control applications. Biotechnol. Bioeng. 2017;114: 141-153. © 2016 Wiley Periodicals, Inc.

  1. Highly Efficient Neural Conversion of Human Pluripotent Stem Cells in Adherent and Animal-Free Conditions.

    PubMed

    Lukovic, Dunja; Diez Lloret, Andrea; Stojkovic, Petra; Rodríguez-Martínez, Daniel; Perez Arago, Maria Amparo; Rodriguez-Jimenez, Francisco Javier; González-Rodríguez, Patricia; López-Barneo, José; Sykova, Eva; Jendelova, Pavla; Kostic, Jelena; Moreno-Manzano, Victoria; Stojkovic, Miodrag; Bhattacharya, Shomi S; Erceg, Slaven

    2017-04-01

    Neural differentiation of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) can produce a valuable and robust source of human neural cell subtypes, holding great promise for the study of neurogenesis and development, and for treating neurological diseases. However, current hESCs and hiPSCs neural differentiation protocols require either animal factors or embryoid body formation, which decreases efficiency and yield, and strongly limits medical applications. Here we develop a simple, animal-free protocol for neural conversion of both hESCs and hiPSCs in adherent culture conditions. A simple medium formula including insulin induces the direct conversion of >98% of hESCs and hiPSCs into expandable, transplantable, and functional neural progenitors with neural rosette characteristics. Further differentiation of neural progenitors into dopaminergic and spinal motoneurons as well as astrocytes and oligodendrocytes indicates that these neural progenitors retain responsiveness to instructive cues revealing the robust applicability of the protocol in the treatment of different neurodegenerative diseases. The fact that this protocol includes animal-free medium and human extracellular matrix components avoiding embryoid bodies makes this protocol suitable for the use in clinic. Stem Cells Translational Medicine 2017;6:1217-1226.

  2. Adherence to human lung microvascular endothelial cells (HMVEC-L) of Plasmodium vivax isolates from Colombia

    PubMed Central

    2013-01-01

    Background For years Plasmodium vivax has been considered the cause of benign malaria. Nevertheless, it has been observed that this parasite can produce a severe disease comparable to Plasmodium falciparum. It has been suggested that some physiopathogenic processes might be shared by these two species, such as cytoadherence. Recently, it has been demonstrated that P. vivax-infected erythrocytes (Pv-iEs) have the capacity to adhere to endothelial cells, in which intercellular adhesion molecule-1 (ICAM-1) seems to be involved in this process. Methods Adherence capacity of 21 Colombian isolates, from patients with P. vivax mono-infection to a microvascular line of human lung endothelium (HMVEC-L) was assessed in static conditions and binding was evaluated at basal levels or in tumor necrosis factor (TNF) stimulated cells. The adherence specificity for the ICAM-1 receptor was determined through inhibition with an anti-CD54 monoclonal antibody. Results The majority of P. vivax isolates, 13 out of 21 (61.9%), adhered to the HMVEC-L cells, but P. vivax adherence was at least seven times lower when compared to the four P. falciparum isolates. Moreover, HMVEC-L stimulation with TNF led to an increase of 1.6-fold in P. vivax cytoadhesion, similar to P. falciparum isolates (1.8-fold) at comparable conditions. Also, blockage of ICAM-1 receptor with specific antibodies showed a significant 50% adherence reduction. Conclusions Plasmodium vivax isolates found in Colombia are also capable of adhering specifically in vitro to lung endothelial cells, via ICAM-1 cell receptor, both at basal state and after cell stimulation with TNF. Collectively, these findings reinforce the concept of cytoadherence for P. vivax, but here, to a different endothelial cell line and using geographical distinct isolates, thus contributing to understanding P. vivax biology. PMID:24080027

  3. Supervised classification of etoposide-treated in vitro adherent cells based on noninvasive imaging morphology.

    PubMed

    Mölder, Anna Leida; Persson, Johan; El-Schich, Zahra; Czanner, Silvester; Gjörloff-Wingren, Anette

    2017-04-01

    Single-cell studies using noninvasive imaging is a challenging, yet appealing way to study cellular characteristics over extended periods of time, for instance to follow cell interactions and the behavior of different cell types within the same sample. In some cases, e.g., transplantation culturing, real-time cellular monitoring, stem cell studies, in vivo studies, and embryo growth studies, it is also crucial to keep the sample intact and invasive imaging using fluorophores or dyes is not an option. Computerized methods are needed to improve throughput of image-based analysis and for use with noninvasive microscopy such methods are poorly developed. By combining a set of well-documented image analysis and classification tools with noninvasive microscopy, we demonstrate the ability for long-term image-based analysis of morphological changes in single cells as induced by a toxin, and show how these changes can be used to indicate changes in biological function. In this study, adherent cell cultures of DU-145 treated with low-concentration (LC) etoposide were imaged during 3 days. Single cells were identified by image segmentation and subsequently classified on image features, extracted for each cell. In parallel with image analysis, an MTS assay was performed to allow comparison between metabolic activity and morphological changes after long-term low-level drug response. Results show a decrease in proliferation rate for LC etoposide, accompanied by changes in cell morphology, primarily leading to an increase in cell area and textural changes. It is shown that changes detected by image analysis are already visible on day 1 for [Formula: see text] etoposide, whereas effects on MTS and viability are detected only on day 3 for [Formula: see text] etoposide concentration, leading to the conclusion that the morphological changes observed occur before and at lower concentrations than a reduction in cell metabolic activity or viability. Three classifiers are compared and we

  4. Preparing Adherent Cells for X-ray Fluorescence Imaging by Chemical Fixation

    PubMed Central

    Finney, Lydia A.; Jin, Qiaoling

    2015-01-01

    X-ray fluorescence imaging allows us to non-destructively measure the spatial distribution and concentration of multiple elements simultaneously over large or small sample areas. It has been applied in many areas of science, including materials science, geoscience, studying works of cultural heritage, and in chemical biology. In the case of chemical biology, for example, visualizing the metal distributions within cells allows us to study both naturally-occurring metal ions in the cells, as well as exogenously-introduced metals such as drugs and nanoparticles. Due to the fully hydrated nature of nearly all biological samples, cryo-fixation followed by imaging under cryogenic temperature represents the ideal imaging modality currently available. However, under the circumstances that such a combination is not easily accessible or practical, aldehyde based chemical fixation remains useful and sometimes inevitable. This article describes in as much detail as possible in the preparation of adherent mammalian cells by chemical fixation for X-ray fluorescent imaging. PMID:25867691

  5. Adherence of group B streptococci to adult and neonatal epithelial cells mediated by lipoteichoic acid.

    PubMed Central

    Teti, G; Tomasello, F; Chiofalo, M S; Orefici, G; Mastroeni, P

    1987-01-01

    We have investigated the role of lipoteichoic acid in mediating the adherence of different serotypes of group B streptococci to human adult and neonatal epithelial cells. Pretreatment of neonatal buccal and vaginal epithelial cells with lipoteichoic acid, but not with deacylated lipoteichoic acid, induced a marked inhibition in the adherence of all strains tested. Pretreatment of bacteria with substances known to bind lipoteichoic acid, such as monoclonal and polyclonal antipolyglycerophosphate antibodies and albumin, also resulted in adherence inhibition. Group B streptococci adhered in 6- to 10-fold-higher numbers to buccal epithelial cells from neonates older than 3 days than to those from neonates less than 1 day old. This increase in receptiveness for group B streptococci was paralleled by an increased ability of epithelial cells from older neonates to bind group B streptococcal lipoteichoic acid. These data suggest a role for the lipid portion of lipoteichoic acid in the adherence of different serotypes of group B streptococci to vaginal and neonatal epithelial cells. PMID:3316030

  6. Cell culture's spider silk road.

    PubMed

    Perkel, Jeffrey

    2014-06-01

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

  7. Attitudes to cosmetic surgery among ethnic minority groups in Britain: cultural mistrust, adherence to traditional cultural values, and ethnic identity salience as protective factors.

    PubMed

    Swami, Viren; Hendrikse, Sinead

    2013-01-01

    Previous work has suggested that ethnic minority women have more negative attitudes to cosmetic surgery than British Whites, but reasons for this are not fully understood. To overcome this dearth in the literature, the present study asked 250 British Asian and 250 African Caribbean university students to complete measures of attitudes to cosmetic surgery, cultural mistrust, adherence to traditional cultural values, ethnic identity salience, self-esteem, and demographics. Preliminary analyses showed that there were significant between-group differences only on cultural mistrust and self-esteem, although effect sizes were small (d values = .21-.37). Further analyses showed that more negative attitudes to cosmetic surgery were associated with greater cultural mistrust, stronger adherence to traditional values, and stronger ethnic identity salience, although these relationships were weaker for African Caribbean women than for British Asians. These results are discussed in relation to perceptions of cosmetic surgery among ethnic minority women.

  8. Kingella kingae expresses type IV pili that mediate adherence to respiratory epithelial and synovial cells.

    PubMed

    Kehl-Fie, Thomas E; Miller, Sara E; St Geme, Joseph W

    2008-11-01

    Kingella kingae is a gram-negative bacterium that colonizes the respiratory tract and is a common cause of septic arthritis and osteomyelitis. Despite the increasing frequency of K. kingae disease, little is known about the mechanism by which this organism adheres to respiratory epithelium and seeds joints and bones. Previous work showed that K. kingae expresses long surface fibers that vary in surface density. In the current study, we found that these fibers are type IV pili and are necessary for efficient adherence to respiratory epithelial and synovial cells and that the number of pili expressed by the bacterium correlates with the level of adherence to synovial cells but not with the level of adherence to respiratory cells. In addition, we established that the major pilin subunit is encoded by a pilA homolog in a conserved region of the chromosome that also contains a second pilin gene and a type IV pilus accessory gene, both of which are dispensable for pilus assembly and pilus-mediated adherence. Upon examination of the K. kingae genome, we identified two genes in physically separate locations on the chromosome that encode homologs of the Neisseria PilC proteins and that have only a low level homology to each other. Examination of mutant strains revealed that both of the K. kingae PilC homologs are essential for a wild-type level of adherence to both respiratory epithelial and synovial cells. Taken together, these results demonstrate that type IV pili and the two PilC homologs play important roles in mediating K. kingae adherence.

  9. Three-dimensional (3D) hydrodynamic focusing for continuous sampling and analysis of adherent cells.

    PubMed

    Xu, Chunxiu; Wang, Min; Yin, Xuefeng

    2011-10-07

    A simple three-dimensional (3D) hydrodynamic focusing microfluidic device integrated with continuous sampling, rapid dynamic lysis, capillary electrophoretic (CE) separation and detection of intracellular content is presented. One of the major difficulties in microfluidic cell analysis for adherent cells is that the cells are prone to attaching to the channel surface. To solve this problem, a cross microfluidic chip with three sheath-flow channels located on both sides of and below the sampling channel was developed. With the three sheath flows around the sample solution-containing cells, the formed soft fluid wall prevents the cells from adhering to the channel surface. Labeled cells were 3D hydrodynamically focused by the sheath-flow streams and smoothly introduced into the cross-section one by one. The introduction of sheath-flow streams not only ensured single-cell sampling but avoided blockage of the sampling channel by adherent cells as well. The maximum rate for introduction of individual cells into the separation channel was about 151 cells min(-1). With electric field applied on the separation channel, the aligned cells were driven into the separation channel and rapidly lysed within 400 ms at the entry of the channel by sodium dodecylsulfate (SDS) added in the sheath-flow solution. The microfluidic system was evaluated by analysis of reduced glutathione (GSH) and reactive oxygen species (ROS) in single HepG2 cells. The average analysis throughput of ROS and GSH in single cells was 16-18 cells min(-1).

  10. Identification of Corynebacterium diphtheriae gene involved in adherence to epithelial cells.

    PubMed

    Kolodkina, Valentina; Denisevich, Tatyana; Titov, Leonid

    2011-03-01

    Corynebacterium diphtheriae the causative pathogen of human diphtheria infects the nasopharynx or skin. Although diphtheria has been extensively studied, little is known about the two key aspects of C. diphtheriae invasiveness: colonization and invasion. The role of adhesive properties in establishing the infection of C. diphtheriae strains, independent of toxin production, still needs to be clarified. In this study, we describe a novel gene involved in adherence to epithelial cells. Transformation of C. diphtheriae 225, biotype gravis, ribotype St-Petersburg by EZ:TN(KAN-2)Tnp Transposome was undertaken. A C. diphtheriae 225 Tn5 insertion library of 2800 mutants was created. Five hundred and eighty five transformants were qualitatively screened for reduced adherence to HEp-2 cells by an adherence assay. One mutant strain consistently exhibiting 15.2% of the wild-type adherence was isolated. The DNA flanking the transposon was identified by inverse PCR and subsequent sequencing. The disrupted gene was 94% identical to the C. diphtheriae DIP1621 gene that belongs to unclassified genes. In conclusion, the disruption of the C. diphtheriae DIP1621 gene led to decreased adherence to epithelial cells; its exact function remains to be established.

  11. Apa is a trimeric autotransporter adhesin of Actinobacillus pleuropneumoniae responsible for autoagglutination and host cell adherence.

    PubMed

    Xiao, Longwen; Zhou, Liang; Sun, Changjiang; Feng, Xin; Du, ChongTao; Gao, Yu; Ji, Qun; Yang, Shuxin; Wang, Yu; Han, Wenyu; Langford, P R; Lei, Liancheng

    2012-10-01

    Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia, and adherence to host cells is a key step in the pathogenic process. Although trimeric autotransporter adhesins (TAAs) were identified in many pathogenic bacteria in recent years, none in A. pleuropneumoniae have been characterized. In this study, we identified a TAA from A. pleuropneumoniae, Apa, and characterized the contribution of its amino acid residues to the adhesion process. Sequence analysis of the C-terminal amino acid residues of Apa revealed the presence of a putative translocator domain and six conserved HsfBD1-like or HsfBD2-like binding domains. Western blot analysis revealed that the 126 C-terminal amino acids of Apa could form trimeric molecules. By confocal laser scanning microscopy, one of these six domains (ApaBD3) was determined to mediate adherence to epithelial cells. Adherence assays and adherence inhibition assays using a recombinant E. coli- ApaBD3 strain which expressed ApaBD3 on the surface of E. coli confirmed that this domain was responsible for the adhesion activity. Moreover, cellular enzyme-linked immunosorbent assays demonstrated that ApaBD3 mediated high-level adherence to epithelial cell lines. Intriguingly, autoagglutination was observed with the E. coli- ApaBD3 strain, and this phenomenon was dependent upon the association of the expressed ApaBD3 with the C-terminal translocator domain.

  12. Role of different classes of mammalian cell surface molecules in adherence of coagulase positive and coagulase negative staphylococci.

    PubMed

    Hafez, Mohamed M; Aboulwafa, Mohammad M; Yassien, Mahmoud A; Hassouna, Nadia A

    2008-10-01

    In the present study the role of different mammalian cell receptors in adherence of the coagulase positive pathogen, Staphylococcus aureus and some coagulase negative staphylococci, namely Staphylococcus epidermidis and Staphylococcus saprophyticus was investigated. Upon testing the adherence to Vero and Hep-2 cells, S. aureus isolates showed an adherence to both cell lines while S. epidermidis and S. saprophyticus isolates adhered to Vero cells only. According to the obtained results, both O-linked and N-linked mammalian cell surface glycoproteins are involved in the adherence of S. aureus isolates to Vero and Hep-2 cells, whereas only the O-linked ones serve as receptors for adherence of S. epidermidis and S. saprophyticus isolates to Vero cells. Of the O-linked glycoproteins, GAG-like receptors are involved in adherence of all tested isolates to Vero cells. The coagulase positive staphylococci preferred to adhere to the highly sulphated GAGs (Heparin and chondroitin sulphate B) while the coagulase negative isolates showed higher affinity to the less sulphated ones (Chondroitin sulphate A and C). Mucin like receptors appeared to be important for the adherence of all tested staphylococci. The role exhibited by fibronectin- and fibrinogen-like receptors was detected with S. aureus and S. epidermidis but not with S. saprophyticus isolates. While, collagen and gelatin were found to contribute to the adherence of S. aureus isolates only. Neither carbohydrate moieties of the glycoconjugates nor lipid molecules on the mammalian cell surface played a role in the adherence of the tested staphylococcal isolates. Taken together, the results revealed that both coagulase negative and coagulase positive staphylococcal tested isolates adhere to the same classes of mammalian cell surface receptors such as mucin-like, fibrinogen-like, fibronectin-like and GAG-like receptors. However, the tested isolates exhibited different degrees of affinities to such receptors.

  13. S-carboxymethylcysteine inhibits adherence of Streptococcus pneumoniae to human alveolar epithelial cells.

    PubMed

    Sumitomo, Tomoko; Nakata, Masanobu; Yamaguchi, Masaya; Terao, Yutaka; Kawabata, Shigetada

    2012-01-01

    Streptococcus pneumoniae is a major pathogen of respiratory infections that utilizes platelet-activating factor receptor (PAFR) for firm adherence to host cells. The mucolytic agent S-carboxymethylcysteine (S-CMC) has been shown to exert inhibitory effects against infection by several respiratory pathogens including S. pneumoniae in vitro and in vivo. Moreover, clinical studies have implicated the benefits of S-CMC in preventing exacerbation of chronic obstructive pulmonary disease, which is considered to be related to respiratory infections. In this study, to assess whether the potency of S-CMC is attributable to inhibition of pneumococcal adherence to host cells, an alveolar epithelial cell line stimulated with interleukin-1α was used as a model of inflamed epithelial cells. Despite upregulation of PAFR by inflammatory activation, treatment with S-CMC efficiently inhibited pneumococcal adherence to host epithelial cells. In order to gain insight into the inhibitory mechanism, the effects of S-CMC on PAFR expression were also investigated. Following treatment with S-CMC, PAFR expression was reduced at both mRNA and post-transcriptional levels. Interestingly, S-CMC was also effective in inhibiting pneumococcal adherence to cells transfected with PAFR small interfering RNAs. These results indicate S-CMC as a probable inhibitor targeting numerous epithelial receptors that interact with S. pneumoniae.

  14. A simple method for establishing adherent ex vivo explant cultures from human eye pathologies for use in subsequent calcium imaging and inflammatory studies.

    PubMed

    Andjelic, Sofija; Lumi, Xhevat; Veréb, Zoltán; Josifovska, Natasha; Facskó, Andrea; Hawlina, Marko; Petrovski, Goran

    2014-01-01

    A novel, simple, and reproducible method for cultivating pathological tissues obtained from human eyes during surgery was developed using viscoelastic material as a tissue adherent to facilitate cell attachment and expansion and calcium imaging of cultured cells challenged by mechanical and acetylcholine (ACh) stimulation as well as inflammatory studies. Anterior lens capsule-lens epithelial cells (aLC-LECs) from cataract surgery and proliferative diabetic retinopathy (PDR) fibrovascular epiretinal membranes (fvERMs) from human eyes were used in the study. We hereby show calcium signaling in aLC-LECs by mechanical and acetylcholine (ACh) stimulation and indicate presence of ACh receptors in these cells. Furthermore, an ex vivo study model was established for measuring the inflammatory response in fvERMs and aLC-LECs upon TNFα treatment.

  15. A Simple Method for Establishing Adherent Ex Vivo Explant Cultures from Human Eye Pathologies for Use in Subsequent Calcium Imaging and Inflammatory Studies

    PubMed Central

    Veréb, Zoltán; Facskó, Andrea; Hawlina, Marko

    2014-01-01

    A novel, simple, and reproducible method for cultivating pathological tissues obtained from human eyes during surgery was developed using viscoelastic material as a tissue adherent to facilitate cell attachment and expansion and calcium imaging of cultured cells challenged by mechanical and acetylcholine (ACh) stimulation as well as inflammatory studies. Anterior lens capsule-lens epithelial cells (aLC-LECs) from cataract surgery and proliferative diabetic retinopathy (PDR) fibrovascular epiretinal membranes (fvERMs) from human eyes were used in the study. We hereby show calcium signaling in aLC-LECs by mechanical and acetylcholine (ACh) stimulation and indicate presence of ACh receptors in these cells. Furthermore, an ex vivo study model was established for measuring the inflammatory response in fvERMs and aLC-LECs upon TNFα treatment. PMID:25276840

  16. Catechin-based procyanidins from Peumus boldus Mol. aqueous extract inhibit Helicobacter pylori urease and adherence to adenocarcinoma gastric cells.

    PubMed

    Pastene, Edgar; Parada, Víctor; Avello, Marcia; Ruiz, Antonieta; García, Apolinaria

    2014-11-01

    In this work, the anti-Helicobacter pylori effect of an aqueous extract from dried leaves of Peumus boldus Mol. (Monimiaceae) was evaluated. This extract displayed high inhibitory activity against H. pylori urease. Therefore, in order to clarify the type of substances responsible for such effect, a bioassay-guided fractionation strategy was carried out. The active compounds in the fractions were characterized through different chromatographic methods (RP-HPLC; HILIC-HPLC). The fraction named F5 (mDP = 7.8) from aqueous extract was the most active against H. pylori urease with an IC50  = 15.9 µg gallic acid equivalents (GAE)/mL. HPLC analysis evidenced that F5 was composed mainly by catechin-derived proanthocyanidins (LC-MS and phloroglucinolysis). The anti-adherent effect of boldo was assessed by co-culture of H. pylori and AGS cells. Both the aqueous extract and F5 showed an anti-adherent effect in a concentration-dependent manner. An 89.3% of inhibition was reached at 2.0 mg GAE/mL of boldo extract. In conjunction, our results suggest that boldo extract has a potent anti-urease activity and anti-adherent effect against H. pylori, properties directly linked with the presence of catechin-derived proanthocyanidins.

  17. A Serine-Threonine Kinase (StkP) Regulates Expression of the Pneumococcal Pilus and Modulates Bacterial Adherence to Human Epithelial and Endothelial Cells In Vitro.

    PubMed

    Herbert, Jenny A; Mitchell, Andrea M; Mitchell, Timothy J

    2015-01-01

    The pneumococcal serine threonine protein kinase (StkP) acts as a global regulator in the pneumococcus. Bacterial mutants deficient in StkP are less virulent in animal models of infection. The gene for this regulator is located adjacent to the gene for its cognate phosphatase in the pneumococcal genome. The phosphatase dephosphorylates proteins phosphorylated by StkP and has been shown to regulate a number of key pneumococcal virulence factors and to modulate adherence to eukaryotic cells. The role of StkP in adherence of pneumococci to human cells has not previously been reported. In this study we show StkP represses the pneumococcal pilus, a virulence factor known to be important for bacterial adhesion. In a serotype 4 strain regulation of the pilus by StkP modulates adherence to human brain microvascular endothelial cells (HBMEC) and human lung epithelial cells. This suggests that the pneumococcal pilus may play a role in adherence during infections such as meningitis and pneumonia. We show that regulation of the pilus occurs at the population level as StkP alters the number of pili-positive cells within a single culture. As far as we are aware this is the first gene identified outside of the pilus islet that regulates the biphasic expression of the pilus. These findings suggest StkPs role in cell division may be linked to regulation of expression of a cell surface adhesin.

  18. Silver colloidal nanoparticles: antifungal effect against adhered cells and biofilms of Candida albicans and Candida glabrata.

    PubMed

    Monteiro, D R; Gorup, L F; Silva, S; Negri, M; de Camargo, E R; Oliveira, R; Barbosa, D B; Henriques, M

    2011-08-01

    The aim of this study was to evaluate the effect of silver nanoparticles (SN) against Candida albicans and Candida glabrata adhered cells and biofilms. SN (average diameter 5 nm) were synthesized by silver nitrate reduction with sodium citrate and stabilized with ammonia. Minimal inhibitory concentration (MIC) tests were performed for C. albicans (n = 2) and C. glabrata (n = 2) grown in suspension following the Clinical Laboratory Standards Institute microbroth dilution method. SN were applied to adhered cells (2 h) or biofilms (48 h) and after 24 h of contact their effect was assessed by enumeration of colony forming units (CFUs) and quantification of total biomass (by crystal violet staining). The MIC results showed that SN were fungicidal against all strains tested at very low concentrations (0.4-3.3 μg ml(-1)). Furthermore, SN were more effective in reducing biofilm biomass when applied to adhered cells (2 h) than to pre-formed biofilms (48 h), with the exception of C. glabrata ATCC, which in both cases showed a reduction ∼90%. Regarding cell viability, SN were highly effective on adhered C. glabrata and respective biofilms. On C. albicans the effect was not so evident but there was also a reduction in the number of viable biofilm cells. In summary, SN may have the potential to be an effective alternative to conventional antifungal agents for future therapies in Candida-associated denture stomatitis.

  19. In vitro inhibition of Helicobacter pylori growth and adherence to gastric mucosal cells by Pycnogenol.

    PubMed

    Rohdewald, Peter; Beil, Winfried

    2008-05-01

    The emergence of antibiotic resistant H. pylori strains has necessitated the identification of alternative additive therapies for the treatment of this infection. The study tested whether a specific pine bark extract (Pycnogenol is effective in inhibiting the growth and adherence of H. pylori in vitro. Inhibition of H. pylori growth by Pycnogenol was tested in liquid medium as well as in an in vitro model by using sessile bacteria attached to AGS cells. Adherence was determined by co-incubation of gastric cells with Pycnogenol and H. pylori in vitro. Pycnogenol inhibited H. pylori growth in suspension with an MIC(50) of 12.5 microg/mL. Growth of H. pylori in infected cells was reduced to 10% of the control value by 125 microg/mL Pycnogenol. Adherence of H. pylori to gastric cells was reduced by 70% after 3 h incubation with 125 microg/mL Pycnogenol. The results show a significant, yet limited inhibition of growth and adherence of H. pylori to gastric cells by Pycnogenol. In vivo studies have to demonstrate the clinical relevance of these findings.

  20. Slow-Adhering Stem Cells Derived from Injured Skeletal Muscle Have Improved Regenerative Capacity

    DTIC Science & Technology

    2011-08-01

    levels represent a major determi- nant in the regenerative capacity of muscle stem cells. Mol Biol Cell 2009, 20:509–520 43. Quintero AJ, Wright VJ, Fu...injury on their characteristics and engraftment potential has yet to be described. In the present study, slow-adhering stem cells (SASCs) from both...laceration-injured and control noninjured skeletal muscles in mice were iso- lated and studied. Migration and proliferation rates, multidifferentiation

  1. Cell prestress. I. Stiffness and prestress are closely associated in adherent contractile cells

    NASA Technical Reports Server (NTRS)

    Wang, Ning; Tolic-Norrelykke, Iva Marija; Chen, Jianxin; Mijailovich, Srboljub M.; Butler, James P.; Fredberg, Jeffrey J.; Stamenovic, Dimitrije; Ingber, D. E. (Principal Investigator)

    2002-01-01

    The tensegrity hypothesis holds that the cytoskeleton is a structure whose shape is stabilized predominantly by the tensile stresses borne by filamentous structures. Accordingly, cell stiffness must increase in proportion with the level of the tensile stress, which is called the prestress. Here we have tested that prediction in adherent human airway smooth muscle (HASM) cells. Traction microscopy was used to measure the distribution of contractile stresses arising at the interface between each cell and its substrate; this distribution is called the traction field. Because the traction field must be balanced by tensile stresses within the cell body, the prestress could be computed. Cell stiffness (G) was measured by oscillatory magnetic twisting cytometry. As the contractile state of the cell was modulated with graded concentrations of relaxing or contracting agonists (isoproterenol or histamine, respectively), the mean prestress ((t)) ranged from 350 to 1,900 Pa. Over that range, cell stiffness increased linearly with the prestress: G (Pa) = 0.18(t) + 92. While this association does not necessarily preclude other interpretations, it is the hallmark of systems that secure shape stability mainly through the prestress. Regardless of mechanism, these data establish a strong association between stiffness of HASM cells and the level of tensile stress within the cytoskeleton.

  2. Screening ToxCast™ Phase I Chemicals in a Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) Assay

    EPA Science Inventory

    An Adherent Cell Differentiation and Cytotoxicity (ACDC) in vitro assay with mouse embryonic stem cells was used to screen the ToxCast Phase I chemical library for effects on cellular differentiation and cell number. The U.S. Environmental Protection Agency (EPA) established the ...

  3. Epidermal cells adhere preferentially to type IV (basement membrane) collagen

    PubMed Central

    1979-01-01

    Epidermal cells from adult guinea pig skin attach and differentiate preferentially on substrates of type IV (basement membrane) collagen, compared to those of types I--III collagen. In contrast, guinea pig dermal fibroblasts attach equally well to all four collagen substrates. Fibronectin mediates the attachment of fibroblasts but not of epidermal cells to collagen. PMID:422650

  4. Bovine recto-anal junction squamous epithelial (RSE) cell adhesion assay for studying Escherichia coli O157 adherence

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An adherence assay, using recto-anal junction squamous epithelial cells (RSEC), was developed for Escherichia coli O157 and related organisms. The assay was standardized in comparison with the routinely used HEp-2 cell adherence assay, in this “proof of concept” study. The novel RSEC adhesion assay ...

  5. High density cell culture system

    NASA Technical Reports Server (NTRS)

    Spaulding, Glenn F. (Inventor)

    1994-01-01

    An annular culture vessel for growing mammalian cells is constructed in a one piece integral and annular configuration with an open end which is closed by an endcap. The culture vessel is rotatable about a horizontal axis by use of conventional roller systems commonly used in culture laboratories. The end wall of the endcap has tapered access ports to frictionally and sealingly receive the ends of hypodermic syringes. The syringes permit the introduction of fresh nutrient and withdrawal of spent nutrients. The walls are made of conventional polymeric cell culture material and are subjected to neutron bombardment to form minute gas permeable perforations in the walls.

  6. Human IgA inhibits adherence of Acanthamoeba polyphaga to epithelial cells and contact lenses.

    PubMed

    Campos-Rodríguez, Rafael; Oliver-Aguillón, Gabriela; Vega-Pérez, Luz M; Jarillo-Luna, Adriana; Hernández-Martínez, Dolores; Rojas-Hernández, Saúl; Rodríguez-Monroy, Marco A; Rivera-Aguilar, Víctor; González-Robles, Arturo

    2004-09-01

    Specific anti-Acanthamoeba IgA antibodies have been detected in the serum and tears of patients and healthy individuals. However, the role of human secretory IgA antibodies in inhibiting the adherence of Acanthamoeba had not been previously investigated. Therefore, the purpose of this study was to purify secretory IgA from human colostrum and analyze its effect on the adherence of Acanthamoeba trophozoites to contact lenses and Madin-Darby canine kidney (MDCK) cells. IgA antibodies to Acanthamoeba polyphaga in colostrum of healthy women as well as in saliva and serum of healthy subjects were analyzed by ELISA and Western blot analysis. In serum, saliva, and colostrum, we detected IgA antibodies that recognized several antigens of A. polyphaga. In addition, colostrum and IgA antibodies purified from it inhibited adherence of A. polyphaga trophozoites to contact lenses and MDCK cells. These results suggest that IgA antibodies may participate in the resistance to the amoebic infection, probably by inhibiting the adherence of the trophozoites to contact lenses and corneal epithelial cells.

  7. Brief behavioral self-regulation counseling for HIV treatment adherence delivered by cell phone: an initial test of concept trial.

    PubMed

    Kalichman, Seth C; Kalichman, Moira O; Cherry, Chauncey; Swetzes, Connie; Amaral, Christina M; White, Denise; Jones, Mich'l; Grebler, Tamar; Eaton, Lisa

    2011-05-01

    Affordable and effective antiretroviral therapy (ART) adherence interventions are needed for many patients to promote positive treatment outcomes and prevent viral resistance. We conducted a two-arm randomized trial (n = 40 men and women receiving and less than 95% adherent to ART) to test a single office session followed by four biweekly cell phone counseling sessions that were grounded in behavioral self-management model of medication adherence using data from phone-based unannounced pill counts to provide feedback-guided adherence strategies. The control condition received usual care and matched office and cell phone/pill count contacts. Participants were baseline assessed and followed with biweekly unannounced pill counts and 4-month from baseline computerized interviews (39/40 retained). Results showed that the self-regulation counseling delivered by cell phone demonstrated significant improvements in adherence compared to the control condition; adherence improved from 87% of pills taken at baseline to 94% adherence 4 months after baseline, p < 0.01. The observed effect sizes ranged from moderate (d = 0.45) to large (d = 0.80). Gains in adherence were paralleled with increased self-efficacy (p < 0.05) and use of behavioral strategies for ART adherence (p < 0.05). We conclude that the outcomes from this test of concept trial warrant further research on cell phone-delivered self-regulation counseling in a larger and more rigorous trial.

  8. Optical painting and fluorescence activated sorting of single adherent cells labelled with photoswitchable Pdots

    PubMed Central

    Kuo, Chun-Ting; Thompson, Alison M.; Gallina, Maria Elena; Ye, Fangmao; Johnson, Eleanor S.; Sun, Wei; Zhao, Mengxia; Yu, Jiangbo; Wu, I-Che; Fujimoto, Bryant; DuFort, Christopher C.; Carlson, Markus A.; Hingorani, Sunil R.; Paguirigan, Amy L.; Radich, Jerald P.; Chiu, Daniel T.

    2016-01-01

    The efficient selection and isolation of individual cells of interest from a mixed population is desired in many biomedical and clinical applications. Here we show the concept of using photoswitchable semiconducting polymer dots (Pdots) as an optical ‘painting' tool, which enables the selection of certain adherent cells based on their fluorescence, and their spatial and morphological features, under a microscope. We first develop a Pdot that can switch between the bright (ON) and dark (OFF) states reversibly with a 150-fold contrast ratio on irradiation with ultraviolet or red light. With a focused 633-nm laser beam that acts as a ‘paintbrush' and the photoswitchable Pdots as the ‘paint', we select and ‘paint' individual Pdot-labelled adherent cells by turning on their fluorescence, then proceed to sort and recover the optically marked cells (with 90% recovery and near 100% purity), followed by genetic analysis. PMID:27118210

  9. Principles of cancer cell culture.

    PubMed

    Cree, Ian A

    2011-01-01

    The basics of cell culture are now relatively common, though it was not always so. The pioneers of cell culture would envy our simple access to manufactured plastics, media and equipment for such studies. The prerequisites for cell culture are a well lit and suitably ventilated laboratory with a laminar flow hood (Class II), CO(2) incubator, benchtop centrifuge, microscope, plasticware (flasks and plates) and a supply of media with or without serum supplements. Not only can all of this be ordered easily over the internet, but large numbers of well-characterised cell lines are available from libraries maintained to a very high standard allowing the researcher to commence experiments rapidly and economically. Attention to safety and disposal is important, and maintenance of equipment remains essential. This chapter should enable researchers with little prior knowledge to set up a suitable laboratory to do basic cell culture, but there is still no substitute for experience within an existing well-run laboratory.

  10. Development of cystic embryoid bodies with visceral yolk-sac-like structures from mouse embryonic stem cells using low-adherence 96-well plate.

    PubMed

    Yasuda, Emiko; Seki, Yuji; Higuchi, Takatoshi; Nakashima, Fumio; Noda, Tomozumi; Kurosawa, Hiroshi

    2009-04-01

    Cystic embryoid bodies with visceral yolk-sac-like structure (cystic EB-Vs) are used as a model for the study of early extraembryonic tissue formation containing visceral endoderm-like derivatives. In this study, we optimized the cell density of embryonic stem (ES) cells for developing cystic EB-Vs in a low-adherence 96-well plate. When ES cells were seeded at a density of 4000 cells/well, the cystic EB-Vs were most efficiently developed from ES cells via forming multicellular spherical aggregates called embryoid bodies (EBs). The suspension culture in the low-adherence plate was preferable for developing EBs into cystic EB-Vs rather than the attachment culture in the plate coated with 0.1% gelatin. The seeding cell density of 4000 cells/well was always superior to 1000 cells/well in the efficiency of cystic EB-V development. Because the high-cell density culture generally raises the limitation of oxygen and nutrient supplies, we investigated the effects of low-oxygen and low-nutrient conditions on the development of cystic EB-Vs. It was found that low oxygen tension was not a factor for promoting the development of cystic EB-Vs. It was suggested that a low-nutrient medium is preferred for developing cystic EB-Vs rather than a sufficient-nutrient medium. In conclusion, the suspension culture in the low-adherence 96-well plate seeded with 4000 ES cells/well was optimum for developing cystic EB-Vs. The low-nutrient condition may be one of the factors for promoting the development of cystic EB-Vs.

  11. Interleukin-8 secretion by epithelial cells infected with diffusely adherent Escherichia coli possessing Afa adhesin-coding genes.

    PubMed

    Arikawa, Kentaro; Meraz, Ismail Mustafa; Nishikawa, Yoshikazu; Ogasawara, Jun; Hase, Atsushi

    2005-01-01

    Escherichia coli that adhere sparsely to human epithelial (HEp-2) cells are known as diffusely adherent E. coli(DAEC) and considered potentially diarrheagenic. The role of the afimbrial adhesive sheath (Afa)-identified originally as a uropathogenic factor-in diffuse adhesion is now understood. However, the role of DAEC in diarrheal disease remains controversial. Recently, ability to induce interleukin-8 (IL-8) secretion from intestinal epithelial cells has been suggested as one of the properties of enterovirulent bacteria. In this study, we examined whether DAEC strains possessing Afa genes induced IL-8 in cultures of human carcinoma epithelial cells (e.g., HEp-2, Caco-2, and T84). Nineteen afa-positive DAEC strains were examined for their ability to induce IL-8 secretion, and only 7 strains (37%; 7/19) induced IL-8 as much as enteroaggregative E. coli did. No marked differences in adhesion were observed between high and low inducers. Diffusive adhesiveness itself is unlikely to be sufficient to induce IL-8. All high inducers were motile and others were nonmotile. Additional stimulation by flagella may be required to cause high levels of chemokine induction. Motility or presence of flagella can be an important criterion to predict DAEC diarrheagenicity at clinical laboratories.

  12. Capsule reduces adherence of enterotoxigenic Escherichia coli to isolated intestinal epithelial cells of pigs.

    PubMed Central

    Runnels, P L; Moon, H W

    1984-01-01

    Previous reports have demonstrated that heat-stable (A-type) capsule on piliated enterotoxigenic Escherichia coli enhances colonization of enterotoxigenic E. coli in the small intestine and enhances virulence of enterotoxigenic E. coli. In this report, four encapsulated enterotoxigenic E. coli strains and one encapsulated nonenterotoxigenic strain of E. coli and their nonencapsulated mutants were tested for adhesion to isolated intestinal epithelial cells or brush borders from neonatal pigs. The enterotoxigenic E. coli also expressed the K99 pilus antigen. The nonencapsulated mutants of the four enterotoxigenic E. coli adhered in higher numbers than did the encapsulated parental strains. Both the encapsulated and nonencapsulated forms of enterotoxigenic E. coli 431 grown at 18 degrees C (K99 production suppressed) adhered poorly to the isolated cells. The nonenterotoxigenic E. coli 1793 which does not express K99 antigen also adhered poorly in both encapsulated and nonencapsulated forms. Fab fragments of anticapsular immunoglobulin G failed to block the effect of capsule on adherence of strain 431. The results indicated that K99 was the principal mediator of in vitro adhesion of the enterotoxigenic E. coli strains and that capsule impedes the in vitro adhesion. They also suggested that the capsular enhancement of colonization by such strains in vivo probably is by some mechanism other than enhanced adhesion to epithelium. PMID:6147310

  13. Capsule reduces adherence of enterotoxigenic Escherichia coli to isolated intestinal epithelial cells of pigs.

    PubMed

    Runnels, P L; Moon, H W

    1984-09-01

    Previous reports have demonstrated that heat-stable (A-type) capsule on piliated enterotoxigenic Escherichia coli enhances colonization of enterotoxigenic E. coli in the small intestine and enhances virulence of enterotoxigenic E. coli. In this report, four encapsulated enterotoxigenic E. coli strains and one encapsulated nonenterotoxigenic strain of E. coli and their nonencapsulated mutants were tested for adhesion to isolated intestinal epithelial cells or brush borders from neonatal pigs. The enterotoxigenic E. coli also expressed the K99 pilus antigen. The nonencapsulated mutants of the four enterotoxigenic E. coli adhered in higher numbers than did the encapsulated parental strains. Both the encapsulated and nonencapsulated forms of enterotoxigenic E. coli 431 grown at 18 degrees C (K99 production suppressed) adhered poorly to the isolated cells. The nonenterotoxigenic E. coli 1793 which does not express K99 antigen also adhered poorly in both encapsulated and nonencapsulated forms. Fab fragments of anticapsular immunoglobulin G failed to block the effect of capsule on adherence of strain 431. The results indicated that K99 was the principal mediator of in vitro adhesion of the enterotoxigenic E. coli strains and that capsule impedes the in vitro adhesion. They also suggested that the capsular enhancement of colonization by such strains in vivo probably is by some mechanism other than enhanced adhesion to epithelium.

  14. Sphere Culture of Murine Lung Cancer Cell Lines Are Enriched with Cancer Initiating Cells

    PubMed Central

    Morrison, Brian J.

    2012-01-01

    Cancer initiating cells (CICs) represent a unique cell population essential for the maintenance and growth of tumors. Most in vivo studies of CICs utilize human tumor xenografts in immunodeficient mice. These models provide limited information on the interaction of CICs with the host immune system and are of limited value in assessing therapies targeting CICs, especially immune-based therapies. To assess this, a syngeneic cancer model is needed. We examined the sphere-forming capacity of thirteen murine lung cancer cell lines and identified TC-1 and a metastatic subclone of Lewis lung carcinoma (HM-LLC) as cell lines that readily formed and maintained spheres over multiple passages. TC-1 tumorspheres were not enriched for expression of CD133 or CD44, putative CIC markers, nor did they demonstrate Hoechst 33342 side population staining or Aldefluor activity compared to adherent TC-1 cells. However, in tumorsphere culture, these cells exhibited self-renewal and long-term symmetric division capacity and expressed more Oct-4 compared to adherent cells. HM-LLC sphere-derived cells exhibited increased Oct-4, CD133, and CD44 expression, demonstrated a Hoechst 33342 side population and Aldefluor activity compared to adherent cells or a low metastatic subclone of LLC (LM-LLC). In syngeneic mice, HM-LLC sphere-derived cells required fewer cells to initiate tumorigenesis compared to adherent or LM-LLC cells. Similarly TC-1 sphere-derived cells were more tumorigenic than adherent cells in syngeneic mice. In contrast, in immunocompromised mice, less than 500 sphere or adherent TC-1 cells and less than 1,000 sphere or adherent LLC cells were required to initiate a tumor. We suggest that no single phenotypic marker can identify CICs in murine lung cancer cell lines. Tumorsphere culture may provide an alternative approach to identify and enrich for murine lung CICs. Furthermore, we propose that assessing tumorigenicity of murine lung CICs in syngeneic mice better models the

  15. Effects of the Campylobacter jejuni CJIE1 prophage homologs on adherence and invasion in culture, patient symptoms, and source of infection

    PubMed Central

    2012-01-01

    Background Prophages of enteric bacteria are frequently of key importance for the biology, virulence, or host adaptation of their host. Some C. jejuni isolates carry homologs of the CJIE1 (CMLP 1) prophage that carry cargo genes potentially involved in virulence. Possible role(s) of CJIE1 homologs in the biology and virulence of C. jejuni were therefore investigated by using in vitro cell culture assays and by assessing the association of C. jejuni isolates with and without these prophages with patients’ symptoms, with source, and with clonal lineages within the C. jejuni population. Results Four C. jejuni isolates, three carrying the CJIE1-like prophage and one without, were tested in cell culture assays for adherence and invasion. Both adherence and invasion of C. jejuni to cells in culture were increased by the presence of the CJIE1-family prophage. Differences in motility and growth rate did not appear to be responsible. The CJIE1 prophage was present in 23% of isolates from human and non-human sources combined that were obtained through sentinel-site surveillance, and the distribution of CJIE1 in this population showed modest clonal associations. There was no correlation between the presence of the CJIE1 prophage in C. jejuni and patient symptoms, although there was some statistical support for lower rates of abdominal pain and fever when the prophage was present. Little evidence was found for a role of the prophage in host adaptation or host specificity. Conclusion These biological effects suggest that the presence of the prophage may be a marker for differential virulence of some C. jejuni isolates. Ongoing research into the effects of the prophage on protein expression may provide additional insights into the roles the prophage may play in the biology of its host bacterium. PMID:23167543

  16. Specific Mechanical and Structural Responses of Cortical and Cytosolic Cytoskeleton in Living Adherent Cells

    NASA Astrophysics Data System (ADS)

    Laurent, Valérie M.; Fodil, Redouane; Cañadas, Patrick; Planus, Emmanuelle; Isabey, Daniel

    We studied the relation between actin structural changes and cytoskeleton mechanical properties in living adherent epithelial alveolar cells, before and during actin depolymerization. The mechanical response of adherent alveolar epithelial cells was measured using magnetic twisting cytometry and a two-component model representing the cortical and the cytosolic elastic components at equilibrium. Chemiluminescent staining of the actin cytoskeleton was performed in the same living cells to estimate the intracellular actin density distribution for each cytoskeleton component. We found that (i) cytoskeleton alterations induced by actin depolymerization differed between the cortical and cytosolic cytoskeleton components (e.g., -30% and -49%, respectively, at a stress of 31 Pa) and that (ii) the concomitant change in actin distribution was also different (e.g., actin volume decrease was -7% and -19% for the cortical and cytosolic components, respectively).

  17. Chitosan nanoparticles affect the acid tolerance response in adhered cells of Streptococcus mutans.

    PubMed

    Neilands, J; Sutherland, D; Resin, A; Wejse, P L; Chávez de Paz, L E

    2011-01-01

    In this study we evaluated the effect of chitosan nanoparticles on the acid tolerance response (ATR) of adhered Streptococcus mutans. An ATR was induced by exposing S. mutans to pH 5.5 for 2 h and confirmed by exposing the acid-adapted cells to pH 3.5 for 30 min, with the majority of cells appearing viable according to the LIVE/DEAD® technique. However, when chitosan nanoparticles were present during the exposure to pH 5.5, no ATR occurred as most cells appeared dead after the pH 3.5 shock. We conclude that the chitosan nanoparticles tested had the ability to hinder ATR induction in adhered S. mutans.

  18. Cell culture purity issues and DFAT cells

    SciTech Connect

    Wei, Shengjuan; Bergen, Werner G.; Zan, Linsen; Dodson, Michael V.

    2013-04-12

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

  19. Expression of intercellular adhesion molecule 1 (ICAM-1) on the human oviductal epithelium and mediation of lymphoid cell adherence.

    PubMed

    Utreras, E; Ossandon, P; Acuña-Castillo, C; Varela-Nallar, L; Müller, C; Arraztoa, J A; Cardenas, H; Imarai, M

    2000-09-01

    The epithelium of the human oviduct expresses the major histocompatibility complex (MHC) class II and shows endocytic properties towards luminal antigens. Therefore, the epithelial cells might behave as antigen-presenting cells, inducing a local immune response. The activation of antigen-specific T cells not only requires presentation of the peptide antigen by MHC class II, but also the presence of co-stimulatory molecules in the antigen-presenting cells. Therefore, the expression of the intercellular adhesion molecule 1 (ICAM-1) was examined in the epithelium of the human oviduct. Most oviducts showed epithelial ICAM-1 expression, as assessed by immunocytochemistry, western blot analysis and RT-PCR assay, and the expression was restricted to the luminal border of ciliated and secretory cells. Interferon gamma, interleukin 1 and lipopolysaccharide treatments increased the percentage of ICAM-1-positive cells in primary cultures, indicating that the expression of ICAM-1 in the oviduct might be upregulated in vivo by inflammatory cytokines or bacterial infections. Binding assays between allogenic phytohaemagglutinin-activated lymphocytes and epithelial monolayers expressing ICAM-1 demonstrated that this molecule stimulated lymphocyte adherence. The presence of ICAM-1, in addition to MHC class II, supports the putative role of the oviductal epithelium in antigen presentation. The exclusive apical distribution of ICAM-1 indicates that T-cell activation would occur in a polarized manner. Binding of lymphoid cells to the surface of the oviductal epithelium may help to retain these immune cells that are required for the clearance of pathogens.

  20. Impact Mediated Loading Cytoplasmic Loading of Macromolecules into Adherent Cells

    NASA Technical Reports Server (NTRS)

    Clarke, Mark S. F.; Feeback, Daniel L.; Vanderburg, Charles R.

    2003-01-01

    The advent of modern molecular biology, including the development of gene array technologies, has resulted in an explosion of information concerning the specific genes activated during normal cellular development, as well as those associated with a variety of pathological conditions. These techniques have served as a highly efficient, broacI.-based screening approach for those specific genes involved. in regulating normal cellular physiology and identifying candidate genes directly associated with the etiology of specific disease states. However, this approach provides information at the transcriptional' level only and does not necessarily indicate . that the gene in question is in fact translated ito a protein, or whether or not post-translational modification of the protein occurs. The critical importance of post-translational modification (i.e. phosphorylation, glycosylation, sialyation, etc.) to protein function has been recognized with regard to a number of proteins involved in a variety of important disease states. For example, altered glycosylation of beta-amyloid precursor protein results in an increase in the amount of beta-amyloid peptide generated and hence secreted as insoluble extracellular amyloid deposits (Georgopoulou, McLaughlin et al. 2001; Walter, Fluhrer et al. 2001), a pathological hal1nark of Alzheimer's disease. Abnormal phosphorylaion of synapsin I has been linked to alterations in synaptic vesicle trafficking leading to defective neurotransmission in Huntington's disease (Lievens, Woodman et al. 2002). Altered phosphorylation of the TAU protein involved in microtubule function has been linked to a number of neurodegenative diseases such as Alzheimer's disease (Billingsley and Kincaid 1997; Sanchez, Alvarez-Tllada et a1. 2001). Aberrant siaIyation of cell/I surface antigens has been detected in a number of different tumor cell types and has been linked to the acquisition of a neoplastic phenotype (Sell 1990), while improper' sia1yation of

  1. Relationship between cell surface composition of Candida albicans and adherence to acrylic after growth on different carbon sources.

    PubMed Central

    McCourtie, J; Douglas, L J

    1981-01-01

    The adherence of Candida albicans to acrylic was measured in vitro after growth of the yeast to stationary phase in defined medium containing glucose, sucrose, galactose, fructose, or maltose as the carbon source. In each case, yeast adherence was proportional to the concentration of sugar in the growth medium, but equimolar concentrations of different sugars promoted adherence to different extents. In vitro adherence was further increased by the addition of divalent cations to assay mixtures but was inhibited when saliva-treated acrylic strips were used or when yeasts were suspended in mixed saliva during the assay. The rate of spheroplast formation of yeasts grown in media containing a 500 mM concentration of the different sugars correlated well with the relative adherence of the cells to acrylic. Galactose-grown yeasts were most resistant to spheroplast formation with Zymolyase-5000 and most adherent to acrylic, whereas fructose-grown organisms were least resistant to spheroplast formation and least adherent to acrylic. These results indicate that when grown to stationary phase in media containing high concentrations of certain sugars, C. albicans undergoes a change in cell surface composition which facilitates its adherence to acrylic surfaces. Electron microscopy of yeasts harvested from such media revealed the presence of an additional surface layer which may be responsible for this enhanced adherence. Images PMID:7019091

  2. Development and Cultural Adaptation Of the Spanish Version of the End Stage Renal Disease Adherence Questionnaire (SESRD-AQ)

    PubMed Central

    Kim, Youngmee; Evangelista, Lorraine S.

    2015-01-01

    We previously developed and validated the End-Stage Renal Disease Adherence Questionnaire (ESRD-AQ) to measure adherence behaviors (e.g., hemodialysis attendance, medication use, fluid restrictions, and diet) of patients on maintenance hemodialysis. To determine whether the ESRD-AQ can be used to measure adherence behaviors in non- English-speaking patients, we translated and adapted the ESRD-AQ into Spanish (SESRD-AQ) using forward and backward translation and cultural adaptation of the content. Validity and reliability were measured using item-level content validity indexes, intraclass correlation coefficients, and known-group analysis. All validity indices were within an acceptable range; strong test-retest stability existed across all items, with intraclass correlation coefficients ranging from 0.82 to 1.00. The developed SESRD-AQ is a valid assessment tool for use among Spanish-speaking patients on maintenance hemodialysis. This instrument refinement and validation process can be replicated with other maintenance hemodialysis population groups. PMID:24579396

  3. Hydrodynamic Determinants of Cell Necrosis and Molecular Delivery Produced by Pulsed Laser Microbeam Irradiation of Adherent Cells

    PubMed Central

    Compton, Jonathan L.; Hellman, Amy N.; Venugopalan, Vasan

    2013-01-01

    Time-resolved imaging, fluorescence microscopy, and hydrodynamic modeling were used to examine cell lysis and molecular delivery produced by picosecond and nanosecond pulsed laser microbeam irradiation in adherent cell cultures. Pulsed laser microbeam radiation at λ = 532 nm was delivered to confluent monolayers of PtK2 cells via a 40×, 0.8 NA microscope objective. Using laser microbeam pulse durations of 180–1100 ps and pulse energies of 0.5–10.5 μJ, we examined the resulting plasma formation and cavitation bubble dynamics that lead to laser-induced cell lysis, necrosis, and molecular delivery. The cavitation bubble dynamics are imaged at times of 0.5 ns to 50 μs after the pulsed laser microbeam irradiation, and fluorescence assays assess the resulting cell viability and molecular delivery of 3 kDa dextran molecules. Reductions in both the threshold laser microbeam pulse energy for plasma formation and the cavitation bubble energy are observed with decreasing pulse duration. These energy reductions provide for increased precision of laser-based cellular manipulation including cell lysis, cell necrosis, and molecular delivery. Hydrodynamic analysis reveals critical values for the shear-stress impulse generated by the cavitation bubble dynamics governs the location and spatial extent of cell necrosis and molecular delivery independent of pulse duration and pulse energy. Specifically, cellular exposure to a shear-stress impulse J≳0.1 Pa s ensures cell lysis or necrosis, whereas exposures in the range of 0.035≲J≲0.1 Pa s preserve cell viability while also enabling molecular delivery of 3 kDa dextran. Exposure to shear-stress impulses of J≲0.035 Pa s leaves the cells unaffected. Hydrodynamic analysis of these data, combined with data from studies of 6 ns microbeam irradiation, demonstrates the primacy of shear-stress impulse in determining cellular outcome resulting from pulsed laser microbeam irradiation spanning a nearly two

  4. Temperature-induced labelling of Fluo-3 AM selectively yields brighter nucleus in adherent cells

    SciTech Connect

    Meng, Guixian; Pan, Leiting; Li, Cunbo; Hu, Fen; Shi, Xuechen; Lee, Imshik; Drevenšek-Olenik, Irena; Zhang, Xinzheng; Xu, Jingjun

    2014-01-17

    Highlights: •We detailedly examine temperature effects of Fluo-3 AM labelling in adherent cells. •4 °C Loading and 20 °C de-esterification of Fluo-3 AM yields brighter nuclei. •Brighter nuclei labelling by Fluo-3 AM also depends on cell adhesion quality. •A qualitative model of the brighter nucleus is proposed. -- Abstract: Fluo-3 is widely used to study cell calcium. Two traditional approaches: (1) direct injection and (2) Fluo-3 acetoxymethyl ester (AM) loading, often bring conflicting results in cytoplasmic calcium ([Ca{sup 2+}]{sub c}) and nuclear calcium ([Ca{sup 2+}]{sub n}) imaging. AM loading usually yields a darker nucleus than in cytoplasm, while direct injection always induces a brighter nucleus which is more responsive to [Ca{sup 2+}]{sub n} detection. In this work, we detailedly investigated the effects of loading and de-esterification temperatures on the fluorescence intensity of Fluo-3 in response to [Ca{sup 2+}]{sub n} and [Ca{sup 2+}]{sub c} in adherent cells, including osteoblast, HeLa and BV2 cells. Interestingly, it showed that fluorescence intensity of nucleus in osteoblast cells was about two times larger than that of cytoplasm when cells were loaded with Fluo-3 AM at 4 °C and allowed a subsequent step for de-esterification at 20 °C. Brighter nuclei were also acquired in HeLa and BV2 cells using the same experimental condition. Furthermore, loading time and adhesion quality of cells had effect on fluorescence intensity. Taken together, cold loading and room temperature de-esterification treatment of Fluo-3 AM selectively yielded brighter nucleus in adherent cells.

  5. Co-elimination of mec and spa genes in Staphylococcus aureus and the effect of agr and protein A production on bacterial adherence to cell monolayers.

    PubMed

    Poston, S M; Glancey, G R; Wyatt, J E; Hogan, T; Foster, T J

    1993-12-01

    Phenotypic loss of protein A production was tested in six methicillin-resistant (McR) Staphylococcus aureus (MRSA) isolates and their isogenic methicillin-sensitive (McS) variants by a radiolabelled IgG-binding assay with washed cells and by Western blotting of supernates prepared from lysed washed cells. Genomic DNA was probed for homology with the protein A gene (spa) in EcoRI digests and for homology to the methicillin resistance gene (mec) in HindIII digests. The McS variants had lost homology with mec. An isogenic pair of McR and McS strains, and derivatives of S. aureus 8325-4 with site-specific mutations of the accessory gene regulator locus (agr) and spa, were tested for adherence to human peritoneal mesothelial cells in monolayer culture. The isogenic pair were also tested for adherence to HEp-2 and Vero cell monolayers in assays with 3H thymidine-labelled bacteria. McR isolates produced protein A which was absent from three strains that had become McS. This correlated with deletion of the spa locus. Spa homology, but reduced production of protein A, was retained in one McS strain which also showed reduced adherence to HEp-2, Vero and mesothelial cells (p < 0.05) compared with the parent McR strain. A spa mutation in strain 8325-4 did not significantly affect adherence to mesothelial cells but mutation in agr increased adherence significantly in both Spa+ and Spa- strains.

  6. Cell culture purity issues and DFAT cells.

    PubMed

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

    2013-04-12

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

  7. Study protocol for a randomized controlled trial to assess the feasibility of an open label intervention to improve hydroxyurea adherence in youth with sickle cell disease

    PubMed Central

    Smaldone, Arlene; Findley, Sally; Bakken, Suzanne; Matiz, L. Adriana; Rosenthal, Susan L.; Jia, Haomiao; Matos, Sergio; Manwani, Deepa; Green, Nancy S.

    2016-01-01

    Background Community health workers (CHW) are increasingly recognized as a strategy to improve health outcomes for the underserved with chronic diseases but has not been formally explored in adolescents with sickle cell disease (SCD). SCD primarily affects African American, Hispanic and other traditionally underserved populations. Hydroxyurea (HU), an oral, once-daily medication, is the only approved therapeutic drug for sickle cell disease and markedly reduces symptoms, morbidity and mortality and improves quality of life largely by increasing hemoglobin F blood levels. This paper presents the rationale, study design and protocol for an open label randomized controlled trial to improve parent-youth partnerships in self-management and medication adherence to HU in adolescents with SCD. Methods/Design A CHW intervention augmented by text messaging was designed for adolescents with SCD ages 10–18 years and their parents to improve daily HU adherence. Thirty adolescent parent dyads will be randomized with 2:1 intervention group allocation. Intervention dyads will establish a relationship with a culturally aligned CHW to identify barriers to HU use, identify cues to build a habit, and develop a dyad partnership to improve daily HU adherence and achieve their individualized “personal best” hemoglobin F target. Intervention feasibility, acceptability and efficacy will be assessed via a 2-site trial. Outcomes of interest are HU adherence, dyad self-management communication, quality of life, and resource use. Discussion Despite known benefits, poor HU adherence is common. If feasible and acceptable, the proposed intervention may improve health of underserved adolescents with SCD by enhancing long-term HU adherence. PMID:27327779

  8. Enhancement of adherence of Helicobacter pylori to host cells by virus: possible mechanism of development of symptoms of gastric disease.

    PubMed

    Wu, Hong; Nakano, Takashi; Suzuki, Youichi; Ooi, Yukimasa; Sano, Kouichi

    2017-03-10

    It remains unclear why gastric disease does not develop in all cases of Helicobacter pylori infection. In this study, we analyzed whether simian virus 5 (SV5) enhanced adherence of H. pylori to adenocarcinoma epithelial cells (AGS). H. pylori in AGS (harboring SV5) and SV5-infected Vero cells, and an agglutination of H. pylori mixed with SV5 were observed by light microscopy, scanning and transmission electron microscopies. The adherent rate of H. pylori to SV5-infected Vero cells and treated with an anti-SV5 antibody was determined. H. pylori adhered to the surface of AGS cells near SV5 particles, as shown by scanning and transmission electron microscopies. The adherence of H. pylori to SV5-infected Vero cells was significantly enhanced compared with that to Vero cells. In contrast, the adherence of H. pylori to Vero cells was decreased by treatment with the anti-SV5 antibody. Agglutination of H. pylori mixed with SV5 was observed by scanning and transmission electron microscopies. Agglutination did not occur when SV5 was treated with the anti-SV5 antibody before mixing. These findings demonstrated that SV5 enhanced the adherence of H. pylori to host cells, suggesting that a persistently infected virus may be a factor enhancing the pathogenicity of H. pylori in humans.

  9. Evidence for a bladder cell glycolipid receptor for Escherichia coli and the effect of neuraminic acid and colominic acid on adherence.

    PubMed Central

    Davis, C P; Avots-Avotins, A E; Fader, R C

    1981-01-01

    The rat bladder epithelial cell receptors involved in mannose-sensitive adherence of Escherichia coli strains were studied. Sodium metaperiodate and lipase pretreatment of epithelial cells significantly reduced bacterial adherence to cells whereas trypsin and phospholipase C had a marginal or insignificant effect on adherence. Neuraminidase and colominic acid significantly increased adherence, whereas N-acetylneuraminic acid significantly reduced adherence. These data suggest that the rat bladder epithelial cell receptors involved in mannose-sensitive adherence are glycolipids. In addition, the data suggested that sialic acid on bladder epithelial cells acts as a nonspecific inhibitor of adherence, whereas colominic acid, a component of some E. coli K1 capsules, may act as a promoter of adherence. PMID:6277793

  10. Towards high-throughput automated targeted femtosecond laser-based transfection of adherent cells

    NASA Astrophysics Data System (ADS)

    Antkowiak, Maciej; Torres-Mapa, Maria Leilani; Gunn-Moore, Frank; Dholakia, Kishan

    2011-03-01

    Femtosecond laser induced cell membrane poration has proven to be an attractive alternative to the classical methods of drug and gene delivery. It is a selective, sterile, non-contact technique that offers a highly localized operation, low toxicity and consistent performance. However, its broader application still requires the development of robust, high-throughput and user-friendly systems. We present a system capable of unassisted enhanced targeted optoinjection and phototransfection of adherent mammalian cells with a femtosecond laser. We demonstrate the advantages of a dynamic diffractive optical element, namely a spatial light modulator (SLM) for precise three dimensional positioning of the beam. It enables the implementation of a "point-and-shoot" system in which using the software interface a user simply points at the cell and a predefined sequence of precisely positioned doses can be applied. We show that irradiation in three axial positions alleviates the problem of exact beam positioning on the cell membrane and doubles the number of viably optoinjected cells when compared with a single dose. The presented system enables untargeted raster scan irradiation which provides transfection of adherent cells at the throughput of 1 cell per second.

  11. Engineering cell-compatible paper chips for cell culturing, drug screening, and mass spectrometric sensing.

    PubMed

    Chen, Qiushui; He, Ziyi; Liu, Wu; Lin, Xuexia; Wu, Jing; Li, Haifang; Lin, Jin-Ming

    2015-10-28

    Paper-supported cell culture is an unprecedented development for advanced bioassays. This study reports a strategy for in vitro engineering of cell-compatible paper chips that allow for adherent cell culture, quantitative assessment of drug efficiency, and label-free sensing of intracellular molecules via paper spray mass spectrometry. The polycarbonate paper is employed as an excellent alternative bioscaffold for cell distribution, adhesion, and growth, as well as allowing for fluorescence imaging without light scattering. The cell-cultured paper chips are thus amenable to fabricate 3D tissue construction and cocultures by flexible deformation, stacks and assembly by layers of cells. As a result, the successful development of cell-compatible paper chips subsequently offers a uniquely flexible approach for in situ sensing of live cell components by paper spray mass spectrometry, allowing profiling the cellular lipids and quantitative measurement of drug metabolism with minimum sample pretreatment. Consequently, the developed paper chips for adherent cell culture are inexpensive for one-time use, compatible with high throughputs, and amenable to label-free and rapid analysis.

  12. Pathogenic hantaviruses direct the adherence of quiescent platelets to infected endothelial cells.

    PubMed

    Gavrilovskaya, Irina N; Gorbunova, Elena E; Mackow, Erich R

    2010-05-01

    Hantavirus infections are noted for their ability to infect endothelial cells, cause acute thrombocytopenia, and trigger 2 vascular-permeability-based diseases. However, hantavirus infections are not lytic, and the mechanisms by which hantaviruses cause capillary permeability and thrombocytopenia are only partially understood. The role of beta(3) integrins in hemostasis and the inactivation of beta(3) integrin receptors by pathogenic hantaviruses suggest the involvement of hantaviruses in altered platelet and endothelial cell functions that regulate permeability. Here, we determined that pathogenic hantaviruses bind to quiescent platelets via a beta(3) integrin-dependent mechanism. This suggests that platelets may contribute to hantavirus dissemination within infected patients and provides a means by which hantavirus binding to beta(3) integrin receptors prevents platelet activation. The ability of hantaviruses to bind platelets further suggested that cell-associated hantaviruses might recruit platelets to the endothelial cell surface. Our findings indicate that Andes virus (ANDV)- or Hantaan virus (HTNV)-infected endothelial cells specifically direct the adherence of calcein-labeled platelets. In contrast, cells comparably infected with nonpathogenic Tula virus (TULV) failed to recruit platelets to the endothelial cell surface. Platelet adherence was dependent on endothelial cell beta(3) integrins and neutralized by the addition of the anti-beta(3) Fab fragment, c7E3, or specific ANDV- or HTNV-neutralizing antibodies. These findings indicate that pathogenic hantaviruses displayed on the surface of infected endothelial cells bind platelets and that a platelet layer covers the surface of infected endothelial cells. This fundamentally changes the appearance of endothelial cells and has the potential to alter cellular immune responses, platelet activation, and endothelial cell functions that affect vascular permeability. Hantavirus-directed platelet quiescence and

  13. Electroporation-induced formation of individual calcium entry sites in the cell body and processes of adherent cells.

    PubMed Central

    Teruel, M N; Meyer, T

    1997-01-01

    Electroporation is a widely used method for introducing macromolecules into cells. We developed an electroporation device that requires only 1 microl of sample to load adherent cells in a 10-mm2 surface area while retaining greater than 90% cell survivability. To better understand this device, field-induced permeabilization of adherent rat basophilic leukemia and neocortical neuroblastoma cells was investigated by using fluorescent calcium and voltage indicators. Rectangular field pulses led to the formation of only a few calcium entry sites, preferentially in the hyperpolarized parts of the cell body and processes. Individual entry sites were formed at the same locations when field pulses were repeated. Before calcium entry, a partial breakdown of the membrane potential was observed in both polar regions. Based on our results, a model is proposed for the formation and closure of macromolecule entry sites in adherent cells. First, the rapid formation of a large number of small pores leads to a partial membrane potential breakdown in both polar regions of the cell. Second, over tens of milliseconds, a few entry sites for macromolecules are formed, preferentially in the hyperpolarized part of cell body and processes, at locations defined by the local membrane structure. These entry sites reseal on a time scale of 50 ms to several seconds, with residual small pores remaining open for several minutes. Images FIGURE 1 FIGURE 2 FIGURE 3 FIGURE 4 FIGURE 5 FIGURE 6 FIGURE 8 FIGURE 9 PMID:9336174

  14. Aseptic technique for cell culture.

    PubMed

    Coté, R J

    2001-05-01

    This unit describes some of the ways that a laboratory can deal with the constant threat of microbial contamination in cell cultures. A protocol on aseptic technique is described first. This catch-all term universally appears in any set of instructions pertaining to procedures in which noncontaminating conditions must be maintained. In reality, aseptic technique encompasses all aspects of environmental control, personal hygiene, equipment and media sterilization, and associated quality control procedures needed to ensure that a procedure is, indeed, performed with aseptic, noncontaminating technique. Although cell culture can theoretically be carried out on an open bench in a low-traffic area, most cell culture work is carried out using a horizontal laminar-flow clean bench or a vertical laminar-flow biosafety cabinet. Both are described here.

  15. Subinhibitory concentrations of triclosan promote Streptococcus mutans biofilm formation and adherence to oral epithelial cells.

    PubMed

    Bedran, Telma Blanca Lombardo; Grignon, Louis; Spolidorio, Denise Palomari; Grenier, Daniel

    2014-01-01

    Triclosan is a general membrane-active agent with a broad-spectrum antimicrobial activity that is commonly used in oral care products. In this study, we investigated the effect of sub-minimum inhibitory concentrations (MICs) of triclosan on the capacity of the cariogenic bacterium Streptococcus mutans to form biofilm and adhere to oral epithelial cells. As quantified by crystal violet staining, biofilm formation by two reference strains of S. mutans was dose-dependently promoted, in the range of 2.2- to 6.2-fold, by 1/2 and 1/4 MIC of triclosan. Observations by scanning electron microscopy revealed the presence of a dense biofilm attached to the polystyrene surface. Growth of S. mutans in the presence of triclosan at sub-MICs also increased its capacity to adhere to a monolayer of gingival epithelial cells. The expression of several genes involved in adherence and biofilm formation in S. mutans was investigated by quantitative RT-PCR. It was found that sub-MICs of triclosan significantly increased the expression of comD, gtfC, and luxS, and to a lesser extent of gtfB and atlA genes. These findings stress the importance of maintaining effective bactericidal concentrations of therapeutic triclosan since sub-MICs may promote colonization of the oral cavity by S. mutans.

  16. Anti-adherence potential of Enterococcus durans cells and its cell-free supernatant on plastic and stainless steel against foodborne pathogens.

    PubMed

    Amel, Ait Meddour; Farida, Bendali; Djamila, Sadoun

    2015-07-01

    It is demonstrated that numerous bacteria are able to attach to surfaces of equipment used for food handling or processing. In this study, a strain of Enterococcus durans, originally isolated from a milking machine surface, was firstly studied for its biofilm formation potential on plastic and stainless steel supports. The strain was found to be a biofilm producer either at 25, 30 or 37 °C on polystyrene microtitre plates, with a best adherence level observed at 25 °C. En. durans showed a strong adhesion to stainless steel AISI-304. Antibacterial and anti-adherence activities of En. durans were tested against four foodborne pathogens (Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853 and Listeria innocua CLIP 74915) which were shown as biofilm producers on both plastic and stainless steel. En. durans cells and cell-free culture supernatant showed a significant (P < 0.05) inhibition potential of the pathogens either on solid media or in broth co-cultures. Characterization of the antibacterial substances indicated their proteinaceous nature which assigned them most probably to bacteriocins group.

  17. Cultured Human Renal Cortical Cells

    NASA Technical Reports Server (NTRS)

    1998-01-01

    During the STS-90 shuttle flight in April 1998, cultured renal cortical cells revealed new information about genes. Timothy Hammond, an investigator in NASA's microgravity biotechnology program was interested in culturing kidney tissue to study the expression of proteins useful in the treatment of kidney diseases. Protein expression is linked to the level of differentiation of the kidney cells, and Hammond had difficulty maintaining differentiated cells in vitro. Intrigued by the improvement in cell differentiation that he observed in rat renal cells cultured in NASA's rotating wall vessel (a bioreactor that simulates some aspects of microgravity) and during an experiment performed on the Russian Space Station Mir, Hammond decided to sleuth out which genes were responsible for controlling differentiation of kidney cells. To do this, he compared the gene activity of human renal cells in a variety of gravitational environments, including the microgravity of the space shuttle and the high-gravity environment of a centrifuge. Hammond found that 1,632 genes out of 10,000 analyzed changed their activity level in microgravity, more than in any of the other environments. These results have important implications for kidney research as well as for understanding the basic mechanism for controlling cell differentiation.

  18. In Vitro Binding of Agrobacterium tumefaciens to Plant Cells from Suspension Culture 1

    PubMed Central

    Ohyama, Kanji; Pelcher, Lawrence E.; Schaefer, Angelika; Fowke, Larry C.

    1979-01-01

    In vitro binding experiments were carried out using 32P-labeled cells of the virulent Agrobacterium tumefaciens strain B6 and Datura innoxia cells from suspension culture. Binding kinetics showed that adherence of bacteria to Datura cells increased gradually during the first 60 minutes and attained a maximum level within 120 minutes of incubation. Maximum binding occurred at pH 6.0. The presence of Ca2+ and Mg2+ reduced binding slightly and EDTA had little effect at concentrations of 0.1 to 10 millimolar. The binding of bacteria to Datura cells was temperature-dependent. Escherichia coli, Salmonella typhimurium, Rhizobium japonicum, and Micrococcus lysodeikticus did not compete with virulent A. tumefaciens strain B6 for binding to Datura cells. The admixture of avirulent A. tumefaciens strain IIBNV6 enhanced adherence of virulent A. tumefaciens strain B6 to Datura cells. Octopine had no effect on the binding of virulent A. tumefaciens strain B6 to Datura cells, but 10 millimolar canavanine was inhibitory. Arginine enhanced the adherence of the bacteria at concentrations higher than 0.1 millimolar. Incubation with DNase, RNase, and lipase did not affect the binding, but protease stimulated the adherence of bacteria to Datura cells. Concanavaline A and soybean lectin had little effect whereas lecithin and lysolecithin enhanced binding slightly. Poly-l-lysine markedly stimulated the bacteria-plant cell adherence. Cells from suspension cultures of pea, vetch, and soybean had a 2- to 3-fold higher binding capacity than Datura cells, whereas cells from wheat, corn, rice, and sorghum had a considerably lower affinity for binding with virulent A. tumefaciens strain B6. Bacterial adherence to plant cells was confirmed by autoradiography and electron microscopy. Autoradiographic analysis showed that bacteria were associated with the cell wall, and that often binding of bacteria was localized. Electron micrographs clearly illustrated a tight association of virulent A

  19. Toxicity Minimized Cryoprotectant Addition and Removal Procedures for Adherent Endothelial Cells

    PubMed Central

    Davidson, Allyson Fry; Glasscock, Cameron; McClanahan, Danielle R.; Benson, James D.; Higgins, Adam Z.

    2015-01-01

    Ice-free cryopreservation, known as vitrification, is an appealing approach for banking of adherent cells and tissues because it prevents dissociation and morphological damage that may result from ice crystal formation. However, current vitrification methods are often limited by the cytotoxicity of the concentrated cryoprotective agent (CPA) solutions that are required to suppress ice formation. Recently, we described a mathematical strategy for identifying minimally toxic CPA equilibration procedures based on the minimization of a toxicity cost function. Here we provide direct experimental support for the feasibility of these methods when applied to adherent endothelial cells. We first developed a concentration- and temperature-dependent toxicity cost function by exposing the cells to a range of glycerol concentrations at 21°C and 37°C, and fitting the resulting viability data to a first order cell death model. This cost function was then numerically minimized in our state constrained optimization routine to determine addition and removal procedures for 17 molal (mol/kg water) glycerol solutions. Using these predicted optimal procedures, we obtained 81% recovery after exposure to vitrification solutions, as well as successful vitrification with the relatively slow cooling and warming rates of 50°C/min and 130°C/min. In comparison, conventional multistep CPA equilibration procedures resulted in much lower cell yields of about 10%. Our results demonstrate the potential for rational design of minimally toxic vitrification procedures and pave the way for extension of our optimization approach to other adherent cell types as well as more complex systems such as tissues and organs. PMID:26605546

  20. Cell culture compositions

    DOEpatents

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

    2014-03-18

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

  1. Ormocomp-modified glass increases collagen binding and promotes the adherence and maturation of human embryonic stem cell-derived retinal pigment epithelial cells.

    PubMed

    Käpylä, Elli; Sorkio, Anni; Teymouri, Shokoufeh; Lahtonen, Kimmo; Vuori, Leena; Valden, Mika; Skottman, Heli; Kellomäki, Minna; Juuti-Uusitalo, Kati

    2014-12-09

    In in vitro live-cell imaging, it would be beneficial to grow and assess human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells on thin, transparent, rigid surfaces such as cover glasses. In this study, we assessed how the silanization of glass with 3-aminopropyltriethoxysilane (APTES), 3-(trimethoxysilyl)propyl methacrylate (MAPTMS), or polymer-ceramic material Ormocomp affects the surface properties, protein binding, and maturation of hESC-RPE cells. The surface properties were studied by contact angle measurements, X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), and a protein binding assay. The cell adherence and proliferation were evaluated by culturing hESCRPE cells on collagen IV-coated untreated or silanized surfaces for 42 days. The Ormocomp treatment significantly increased the hydrophobicity and roughness of glass surfaces compared to the APTES and MAPTMS treatments. The XPS results indicated that the Ormocomp treatment changes the chemical composition of the glass surface by increasing the carbon content and the number of C-O/═O bonds. The protein-binding test confirmed that the Ormocomp-treated surfaces bound more collagen IV than did APTES- or MAPTMS-treated surfaces. All of the silane treatments increased the number of cells: after 42 days of culture, Ormocomp had 0.38, APTES had 0.16, MAPTMS had 0.19, and untreated glass had only 0.062, all presented as million cells cm(-2). There were no differences in cell numbers compared to smoother to rougher Ormocomp surfaces, suggesting that the surface chemistry and, more specifically, the collagen binding in combination with Ormocomp are beneficial to hESC-RPE cell culture. This study clearly demonstrates that Ormocomp treatment combined with collagen coating significantly increases hESC-RPE cell attachment compared to commonly used silanizing agents APTES and MAPTMS. Ormocomp silanization could thus enable the use of microscopic live cell imaging methods for h

  2. Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) assay-Book Chapter*

    EPA Science Inventory

    There are thousands of environmental chemicals for which there is limited toxicological information, motivating the development and application of in vitro systems to profile the biological effects of xenobiotic exposure and predict their potential developmental hazard. An adhere...

  3. Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) Assay: Book Chapter

    EPA Science Inventory

    There are thousands of environmental chemicals for which there is limited toxicological information, motivating the development and application of in vitro systems to profile the biological effects of xenobiotic exposure and predict their potential developmental hazard. An adher...

  4. Integrin VLA-3: ultrastructural localization at cell-cell contact sites of human cell cultures

    PubMed Central

    1989-01-01

    The integrin VLA-3 is a cell surface receptor, which binds to fibronectin, laminin, collagen type I and VI (Takada, Y., E. A. Wayner, W. G. Carter, and M. E. Hemler. 1988. J. Cell. Biochem. 37:385-393) and is highly expressed in substrate adherent cultures of almost all human cell types. The ligand specificity of VLA-3 and the inhibition of cell adhesion by anti-VLA-3 monoclonal antibodies suggest its involvement in cell-substrate interaction. In normal tissues, VLA-3 is restricted to few cell types, notably the kidney glomeruli and basal cells of the epidermis. In the epidermis, VLA-3 is generally strongly expressed on the entire plasma membrane of basal cells and is not polarized towards the basement membrane (Klein, C. E., C. Cardon-Cardo, R. Soehnchen, R. J. Cote, H. F. Oettgen, M. Eisinger, and L. J. Old. 1987. J. Invest. Dermatol. 89:500-507). Based on this finding we speculated that, in addition to a role of VLA-3 for adhesion of cells to substrate, it could also be relevant for cell-cell interaction. To investigate this, we ultrastructurally localized VLA-3 on the surface of cultured cells by immunoelectron microscopy. In accordance with our concept, we found VLA-3 strongly associated with intercellular contact sites. Interestingly, very little immunoreactivity was detected at the under- surface of cells which had been cultured for 18-32 h. This observation was unexpected but is consistent with previous findings (Kantor, R. R. S., M. J. Mattes, K. D. Lloyd, L. J. Old, and A. P. Albino. 1987. J. Biol. Chem. 262:15158-15165) which suggest that the association of VLA- 3 with the basal surface of substrate adherent tumor cells is a late event occurring after days of culture under confluent conditions. However, we cannot formally rule out VLA-3 expression at the undersurface of cells under our experimental conditions, since VLA-3 molecules at this location could be inaccessible for in situ labeling of unfixed cells because of spatial interferences. In conclusion

  5. Cell Phone-Based and Adherence Device Technologies for HIV Care and Treatment in Resource-Limited Settings: Recent Advances.

    PubMed

    Campbell, Jeffrey I; Haberer, Jessica E

    2015-12-01

    Numerous cell phone-based and adherence monitoring technologies have been developed to address barriers to effective HIV prevention, testing, and treatment. Because most people living with HIV and AIDS reside in resource-limited settings (RLS), it is important to understand the development and use of these technologies in RLS. Recent research on cell phone-based technologies has focused on HIV education, linkage to and retention in care, disease tracking, and antiretroviral therapy adherence reminders. Advances in adherence devices have focused on real-time adherence monitors, which have been used for both antiretroviral therapy and pre-exposure prophylaxis. Real-time monitoring has recently been combined with cell phone-based technologies to create real-time adherence interventions using short message service (SMS). New developments in adherence technologies are exploring ingestion monitoring and metabolite detection to confirm adherence. This article provides an overview of recent advances in these two families of technologies and includes research on their acceptability and cost-effectiveness when available. It additionally outlines key challenges and needed research as use of these technologies continues to expand and evolve.

  6. Comparisons of rabbit bone marrow mesenchymal stem cell isolation and culture methods in vitro.

    PubMed

    Zhang, Weidong; Zhang, Fangbiao; Shi, Hongcan; Tan, Rongbang; Han, Shi; Ye, Gang; Pan, Shu; Sun, Fei; Liu, Xingchen

    2014-01-01

    Bone marrow mesenchymal stem cells (BMSCs) have great potential in tissue engineering and clinical therapy, and various methods for isolation and cultivation of BMSCs have been reported. However, the best techniques are still uncertain. Therefore, we sought the most suitable among the four most common methods for BMSC separation from rabbits. BMSCs were obtained from untreated whole bone marrow (BM) adherent cultures, 3 volumes of red blood cells (RBC) lysed with ammonium chloride, 6 volumes of RBC lysed with ammonium chloride, and Ficoll density gradient centrifugation. Then, isolated BMSCs were evaluated with respect to primary cell yield, number of CFU-F colonies, proliferative capacity, cell phenotype, and chondrogenic differentiation potential. Our data show that BMSCs were successfully isolated by all four methods, and each method was similar with regard to cell morphology, phenotype, and differentiation potential. However, BMSCs from untreated whole BM adherent cultures had greater primary cell yields, larger colonies, and the shortest primary culture time (P<0.05). Moreover, the 4(th) generation of cultured cells had the strongest proliferative activity, the fastest growth rate and the most numerous cells compared with other cell passage generations (P<0.05). In conclusion, untreated whole BM adherent cultures are best for rabbit BMSC isolation and the 4(th) generation of cells has the strongest proliferation capacity.

  7. A mechanical model of actin stress fiber formation and substrate elasticity sensing in adherent cells.

    PubMed

    Walcott, Sam; Sun, Sean X

    2010-04-27

    Tissue cells sense and respond to the stiffness of the surface on which they adhere. Precisely how cells sense surface stiffness remains an open question, though various biochemical pathways are critical for a proper stiffness response. Here, based on a simple mechanochemical model of biological friction, we propose a model for cell mechanosensation as opposed to previous more biochemically based models. Our model of adhesion complexes predicts that these cell-surface interactions provide a viscous drag that increases with the elastic modulus of the surface. The force-velocity relation of myosin II implies that myosin generates greater force when the adhesion complexes slide slowly. Then, using a simple cytoskeleton model, we show that an external force applied to the cytoskeleton causes actin filaments to aggregate and orient parallel to the direction of force application. The greater the external force, the faster this aggregation occurs. As the steady-state probability of forming these bundles reflects a balance between the time scale of bundle formation and destruction (because of actin turnover), more bundles are formed when the cytoskeleton time-scale is small (i.e., on stiff surfaces), in agreement with experiment. As these large bundles of actin, called stress fibers, appear preferentially on stiff surfaces, our mechanical model provides a mechanism for stress fiber formation and stiffness sensing in cells adhered to a compliant surface.

  8. Soluble suppressor factors in patients with acquired immune deficiency syndrome and its prodrome. Elaboration in vitro by T lymphocyte-adherent cell interactions.

    PubMed Central

    Laurence, J; Gottlieb, A B; Kunkel, H G

    1983-01-01

    Supernatants from peripheral blood mononuclear cells obtained from certain patients with the acquired immune deficiency syndrome (AIDS) or its prodrome were capable of depressing spontaneous and pokeweed mitogen-driven B lymphocyte differentiation into plasmacytes, and the proliferative responses of T cells to specific antigen. These soluble suppressor factors (SSF) were present in uniquely high concentrations, with significant differences from healthy controls and from patients with various other conditions previously associated with factor-mediated immunosuppression. T cell-independent functions were not modified by SSF. Suppression was not genetically constrained, and did not appear to be mediated by cytotoxicity, prostaglandin, or alpha or gamma interferons. SSF was a product of the interaction of T lymphocytes with adherent cells. T cells or T cell factors from AIDS patients, but not from normal controls, could collaborate with control adherent cells in the formation of SSF. Restoration of DNA synthesis-independent differentiation of B lymphocytes into plasmacytes in SSF-treated cultures was realized by addition of reducing agents, such as 2-mercaptoethanol, on culture initiation. These data suggest inhibitory mechanisms possibly related to that of concanavalin A-induced soluble immune response suppression, and perhaps offer clues to clinically applicable substances which are potentially capable of mitigating such responses. PMID:6605980

  9. Experimental evidence for the role of lipids in adherence of Candida spp. to human buccal epithelial cells.

    PubMed Central

    Ghannoum, M A; Burns, G R; Elteen, K A; Radwan, S S

    1986-01-01

    Lipids extracted from Candida albicans and C. tropicalis, but not from the weakly adherent C. pseudotropicalis, significantly blocked in vitro adherence of the respective yeast cells to buccal epithelial cells. The percentage of reduction from control values ranged between 16.4 and 42.1%, depending on the species, the strain, and the solvent used for lipid extraction. The constituent lipid classes of both the acetone and chloroform-methanol extracts of C. albicans ATCC 10231 were qualitatively and quantitatively analyzed. The individual classes were isolated by preparative thin-layer chromatography and then tested for their effects on the adherence of this strain to buccal epithelial cells. Individual phospholipids, sterols, and steryl esters blocked adherence significantly (between 15.5 and 55.7% reduction). Triacylglycerols and free fatty acids showed no effect whatsoever. The same results were obtained when standard lipid samples were investigated. Images PMID:3759234

  10. Impact of a mutator phenotype on motility and cell adherence in Salmonella Heidelberg.

    PubMed

    Le Bars, Hervé; Le Gall-David, Sandrine; Renoux, Virginie Madeleine; Bonnaure-Mallet, Martine; Jolivet-Gougeon, Anne; Bousarghin, Latifa

    2012-09-14

    In this study, we investigated adherence and motility of the hypermutator Salmonella enterica Heidelberg B182 bovine strain related to a 12bp deletion in mutS. This mutator phenotype was associated with increased adherence to epithelial cells and with high expression of fimA as shown by real-time RT-PCR. Motility studies showed that fliC were up-regulated in the B182 strain, while fljA and fljB were down-regulated. In order to determine if mutated mutS is implicated in this genes expression, isogenic strains, derived from a WT strain, containing the 12bp deletion in mutS (Δ12bpmutS) or an inactivated mutS (ΔmutS) were generated. Δ12bpmutS and ΔmutS strains showed a spontaneous mutation rate similar to the environmental strain B182, but exhibited lower adherence capacity and fimA expression. In contrast to the fimbriae genes, in Δ12bpmutS, fliC expression was up-regulated, but fljA and fljB expression were decreased, as in the B182 strain. Only fljB expression was increased in ΔmutS mutants. Taken together, our data suggest that mutS alteration does not influence fimbriae expression but can impact flagella genes.

  11. Synergistic role of curli and cellulose in cell adherence and biofilm formation of attaching and effacing Escherichia coli and identification of Fis as a negative regulator of curli

    PubMed Central

    Saldaña, Zeus; Xicohtencatl-Cortes, Juan; Avelino, Fabiola; Phillips, Alan D.; Kaper, James B.; Puente, José L.; Girón, Jorge A.

    2009-01-01

    Summary Curli are adhesive fimbriae of Escherichia coli and Salmonella enterica. Expression of curli (csgA) and cellulose (bcsA) is co-activated by the transcriptional activator CsgD. In this study, we investigated the contribution of curli and cellulose to the adhesive properties of enterohemorragic (EHEC) O157:H7 and enteropathogenic E. coli (EPEC) O127:H6. While single mutations in csgA, csgD, or bcsA in EPEC and EHEC had no dramatic effect on cell adherence, double csgAbcsA mutants were significantly less adherent than the single mutants or wild-type strains to human colonic HT-29 epithelial cells or to cow colon tissue in vitro. Over-expression of csgD (carried on plasmid pCP994) in a csgD mutant, but not in the single csgA or bscA mutants, led to significant increase in adherence and biofilm formation in EPEC and EHEC, suggesting that synchronized over-production of curli and cellulose enhances bacterial adherence. In line with this finding, csgD transcription was activated significantly in the presence of cultured epithelial cells as compared to growth in tissue culture medium. Analysis of the influence of virulence and global regulators in the production of curli in EPEC identified Fis (factor for inversion stimulation) as a, heretofore unrecognized, negative transcriptional regulator of csgA expression. An EPEC E2348/69Δfis produced abundant amounts of curli whereas a double fiscsgD mutant yielded no detectable curli production. Our data suggest that curli and cellulose act in concert to favor host colonization, biofilm formation, and survival in different environments. PMID:19187284

  12. Modulation of Kingella kingae Adherence to Human Epithelial Cells by Type IV Pili, Capsule, and a Novel Trimeric Autotransporter

    PubMed Central

    Porsch, Eric A.; Kehl-Fie, Thomas E.; Geme, Joseph W. St.

    2012-01-01

    ABSTRACT Kingella kingae is an emerging bacterial pathogen that is being recognized increasingly as an important etiology of septic arthritis, osteomyelitis, and bacteremia, especially in young children. Colonization of the posterior pharynx is a key step in the pathogenesis of K. kingae disease. Previous work established that type IV pili are necessary for K. kingae adherence to the respiratory epithelium. In this study, we set out to identify additional factors that influence K. kingae interactions with human epithelial cells. We found that genetic disruption of the gene encoding a predicted trimeric autotransporter protein called Knh (Kingella NhhA homolog) resulted in reduced adherence to human epithelial cells. In addition, we established that K. kingae elaborates a surface-associated polysaccharide capsule that requires a predicted ABC-type transporter export operon called ctrABCD for surface presentation. Furthermore, we discovered that the presence of a surface capsule interferes with Knh-mediated adherence to human epithelial cells by nonpiliated organisms and that maximal adherence in the presence of a capsule requires the predicted type IV pilus retraction machinery, PilT/PilU. On the basis of the data presented here, we propose a novel adherence mechanism that allows K. kingae to adhere efficiently to human epithelial cells while remaining encapsulated and more resistant to immune clearance. PMID:23093386

  13. Inhibition by yeast-derived mannoproteins of adherence to and invasion of Caco-2 cells by Campylobacter jejuni.

    PubMed

    Ganan, M; Carrascosa, A V; de Pascual-Teresa, S; Martinez-Rodriguez, A J

    2009-01-01

    The main objective of the present work was to study the influence of yeast-derived mannoproteins on the adherence to and invasion of Caco-2 cells by Campylobacter jejuni. Mannoprotein fractions were prepared by enzymatic and thermal extraction methods. The method used to prepare the mannoprotein extracts influenced their composition and determined the efficacy of the extract against C. jejuni adherence and/or invasion. The availability of mannose in the mannoprotein fraction seemed to be important for inhibiting effective adherence and invasion of Caco-2-cells by C. jejuni, although protein moieties also played a role in the process. The study of the mechanisms involved in the inhibition of C. jejuni adherence and invasion by mannoproteins may have further implications in the control of this foodborne pathogen.

  14. Phosphorylcholine and SpaA, a choline-binding protein, are involved in the adherence of Erysipelothrix rhusiopathiae to porcine endothelial cells, but this adherence is not mediated by the PAF receptor.

    PubMed

    Harada, Tomoyuki; Ogawa, Yohsuke; Eguchi, Masahiro; Shi, Fang; Sato, Masumi; Uchida, Kazuyuki; Nakayama, Hiroyuki; Shimoji, Yoshihiro

    2014-08-06

    A crucial event in the initiation of many bacterial infections is the adherence of the bacteria to host cells, and bacterial surface structures and their interactions with host cell receptors play an important role in this process. Erysipelothrix rhusiopathiae is the causative agent of swine erysipelas, which may cause acute septicemia or chronic endocarditis and polyarthritis. To study the pathogenic mechanism of the widespread vascular disease observed in the acute form of swine erysipelas, we investigated the role of phosphorylcholine (PCho), a component of the E. rhusiopathiae capsule, in bacterial adherence to porcine endothelial cells (PECs) in vitro. We found that adherence of E. rhusiopathiae strain Fujisawa to PECs was twice that of adherence to control COS-7 cells and that the adherence rates of PCho-defective mutants were approximately 30-50% lower than those of the Fujisawa strain. The adherence of the Fujisawa strain to COS-7 cells transfected with the porcine platelet-activating factor receptor (PAFR) gene, which encodes a G protein-coupled receptor that has been shown to directly bind to Streptococcus pneumoniae via PCho in the bacterial cell wall, was not enhanced. Treatment with a PAFR antagonist (WEB-2086) did not inhibit bacterial adherence to PECs. Incubation of the bacterial cells with an antibody against PCho or SpaA, a choline-binding protein anchored to PCho of the Fujisawa strain, reduced the adherence of the strain to PECs. This effect was not observed when PCho-defective mutants were used. These results suggest that E. rhusiopathiae adheres to PECs via PCho and SpaA and that the PCho-mediated adherence is independent of PAFR.

  15. Adherence to and Invasion of Host Cells by Spotted Fever Group Rickettsia Species

    PubMed Central

    Chan, Yvonne Gar-Yun; Riley, Sean Phillip; Martinez, Juan Jose

    2010-01-01

    The pathogenic lifecycle of obligate intracellular bacteria presents a superb opportunity to develop understanding of the interaction between the bacteria and host under the pretext that disruption of these processes will likely lead to death of the pathogen and prevention of associated disease. Species of the genus Rickettsia contain some of the most hazardous of the obligate intracellular bacteria, including Rickettsia rickettsii and R. conorii the causative agents of Rocky Mountain and Mediterranean spotted fevers, respectively. Spotted fever group Rickettsia species commonly invade and thrive within cells of the host circulatory system whereby the endothelial cells are severely perturbed. The subsequent disruption of circulatory continuity results in much of the severe morbidity and mortality associated with these diseases, including macropapular dermal rash, interstitial pneumonia, acute renal failure, pulmonary edema, and other multisystem manifestations. This review describes current knowledge of the essential pathogenic processes of adherence to and invasion of host cells, efforts to disrupt these processes, and potential for disease prevention through vaccination with recently identified bacterial adherence and invasion proteins. A more complete understanding of these bacterial proteins will provide an opportunity for prevention and treatment of spotted fever group Rickettsia infections. PMID:21687751

  16. Deleterious effects of endotoxin on cultured endothelial cells: an in vitro model of vascular injury

    SciTech Connect

    Yamada, O.; Moldow, C.F.; Sacks, T.; Craddock, P.R.; Boogaerts, M.A.; Jacob, H.S.

    1981-06-01

    The effects of endotoxin-triggered granulocytes on the viability of endothelial cells in vitro was investigated. Endotoxin or its lipid A component caused granulocytes to adhere to and significantly damage cultured endothelial cells. Fresh serum is not necessary but does amplify both adherence and endothelial injury. Much of the endothelial injury was inhibited by free-radical scavengers or by blocking granulocyte adhesion to endothelial cells and appears to result from free radical production by the stimulated granulocyte. Studies in this model suggest a pathogenic role for the endotoxin-triggered granulocyte in the Shwartzman reaction and perhaps related clinical disorders.

  17. African-Americans' perceptions of health care provider cultural competence that promote HIV medical self-care and antiretroviral medication adherence.

    PubMed

    Gaston, Gina B

    2013-01-01

    Most studies of cultural competence in healthcare examine healthcare providers' definitions of cultural competence practices. This study is unique in that it examines the relationship between African-American patients' perceptions of the cultural competence of their HIV healthcare providers and the adherence of these patients to medical self-care and antiretroviral therapy (ART). This cross-sectional, exploratory, descriptive study was conducted at the Ruth Rothstein CORE Center in Chicago, Illinois. The sample consisted of 202 HIV-positive African-Americans who completed surveys during clinic visits. Multiple measures were used, including the Patient Assessments of Cultural Competency survey instrument developed by the Department of Health and Human Services Agency for Healthcare Research and Quality. Medical self-care was measured using the advice and instructions scale and the self-care symptom management for people living with HIV/AIDS categorical scale. ART adherence was measured using the Adherence Behaviors Self-Report and Adherence Self-Report scales. The data revealed many significant correlations between variables. The more patients believed that providers should integrate culture in HIV treatment; the better their reported health (F1,138=0.151, P=0.05) and the more they followed their provider's advice and instructions (medical self-care; F1,138=0.029, P=0.05). Participants who trusted their providers engaged in more medical self-care (F1,138=0.280, P=0.01). More shared treatment decisions were reported among participants who had higher levels of education (F1,127=0.337, P=0.05). Findings of this study indicate the need for increased attention to the role of cultural competence in HIV/AIDS care. Understanding patient perceptions of provider cultural competence has the potential to improve HIV treatment adherence and health outcomes.

  18. Acinetobacter baumannii and A. pittii clinical isolates lack adherence and cytotoxicity to lung epithelial cells in vitro.

    PubMed

    Lázaro-Díez, María; Navascués-Lejarza, Teresa; Remuzgo-Martínez, Sara; Navas, Jesús; Icardo, José Manuel; Acosta, Felix; Martínez-Martínez, Luis; Ramos-Vivas, José

    2016-09-01

    The molecular and genetic basis of Acinetobacter baumannii and Acinetobacter pittii virulence remains poorly understood, and there is still lack of knowledge in host cell response to these bacteria. In this study, we have used eleven clinical Acinetobacter strains (A. baumannii n = 5; A. pittii n = 6) to unravel bacterial adherence, invasion and cytotoxicity to human lung epithelial cells. Our results showed that adherence to epithelial cells by Acinetobacter strains is scarce and cellular invasion was not truly detected. In addition, all Acinetobacter strains failed to induce any cytotoxic effect on A549 cells.

  19. In vitro adherence patterns of Shigella serogroups to bovine recto-anal junction squamous epithelial (RSE) cells are similar to those of Escherichia coli O157

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The aim of this study was to determine whether Shigella species, which are human gastrointestinal pathogens, can adhere to cattle recto-anal junction squamous epithelial (RSE) cells using a recently standardized adherence assay, and to compare their adherence patterns to that of Escherichia coli O15...

  20. SRY gene transferred by extracellular vesicles accelerates atherosclerosis by promotion of leucocyte adherence to endothelial cells.

    PubMed

    Cai, Jin; Guan, Weiwei; Tan, Xiaorong; Chen, Caiyu; Li, Liangpeng; Wang, Na; Zou, Xue; Zhou, Faying; Wang, Jialiang; Pei, Fang; Chen, Xinjian; Luo, Hao; Wang, Xinquan; He, Duofen; Zhou, Lin; Jose, Pedro A; Zeng, Chunyu

    2015-08-01

    We set out to investigate whether and how SRY (sex-determining region, Y) DNAs in plasma EVs (extracellular vesicles) is involved in the pathogenesis of atherosclerosis. PCR and gene sequencing found the SRY gene fragment in plasma EVs from male, but not female, patients; EVs from male patients with CAD (coronary artery disease) had a higher SRY GCN (gene copy number) than healthy subjects. Additional studies found that leucocytes, the major source of plasma EVs, had higher SRY GCN and mRNA and protein expression in male CAD patients than controls. After incubation with EVs from SRY-transfected HEK (human embryonic kidney)-293 cells, monocytes (THP-1) and HUVECs (human umbilical vein endothelial cells), which do not endogenously express SRY protein, were found to express newly synthesized SRY protein. This resulted in an increase in the adherence factors CD11-a in THP-1 cells and ICAM-1 (intercellular adhesion molecule 1) in HUVECs. EMSA showed that SRY protein increased the promoter activity of CD11-a in THP-1 cells and ICAM-1 in HUVECs. There was an increase in THP-1 cells adherent to HUVECs after incubation with SRY-EVs. SRY DNAs transferred from EVs have pathophysiological significance in vivo; injection of SRY EVs into ApoE-/- (apolipoprotein-knockout) mice accelerated atherosclerosis. The SRY gene in plasma EVs transferred to vascular endothelial cells may play an important role in the pathogenesis of atherosclerosis; this mechanism provides a new approach to the understanding of inheritable CAD in men.

  1. Magnetic levitating polymeric nano/microparticular substrates for three-dimensional tumor cell culture.

    PubMed

    Lee, Woong Ryeol; Oh, Kyung Taek; Park, So Young; Yoo, Na Young; Ahn, Yong Sik; Lee, Don Haeng; Youn, Yu Seok; Lee, Deok-Keun; Cha, Kyung-Hoi; Lee, Eun Seong

    2011-07-01

    Herein, we describe magnetic cell levitation models using conventional polymeric microparticles or nanoparticles as a substrate for the three-dimensional tumor cell culture. When the magnetic force originating from the ring-shaped magnets overcame the gravitational force, the magnetic field-levitated KB tumor cells adhered to the surface area of magnetic iron oxide (Fe(3)O(4))-encapsulated nano/microparticles and concentrated clusters of levitated cells, ultimately developing tumor cells to tumor spheroids. These simple cell culture models may prove useful for the screening of anticancer drugs and their formulations.

  2. Lactobacilli Interfere with Streptococcus pyogenes Hemolytic Activity and Adherence to Host Epithelial Cells

    PubMed Central

    Saroj, Sunil D.; Maudsdotter, Lisa; Tavares, Raquel; Jonsson, Ann-Beth

    2016-01-01

    Streptococcus pyogenes [Group A streptococcus (GAS)], a frequent colonizer of the respiratory tract mucosal surface, causes a variety of human diseases, ranging from pharyngitis to the life-threatening streptococcal toxic shock-like syndrome. Lactobacilli have been demonstrated to colonize the respiratory tract. In this study, we investigated the interference of lactobacilli with the virulence phenotypes of GAS. The Lactobacillus strains L. rhamnosus Kx151A1 and L. reuteri PTA-5289, but not L. salivarius LMG9477, inhibited the hemolytic activity of S. pyogenes S165. The inhibition of hemolytic activity was attributed to a decrease in the production of streptolysin S (SLS). Conditioned medium (CM) from the growth of L. rhamnosus Kx151A1 and L. reuteri PTA-5289 was sufficient to down-regulate the expression of the sag operon, encoding SLS. The Lactobacillus strains L. rhamnosus Kx151A1, L. reuteri PTA-5289, and L. salivarius LMG9477 inhibited the initial adherence of GAS to host epithelial cells. Intriguingly, competition with a combination of Lactobacillus species reduced GAS adherence to host cells most efficiently. The data suggest that an effector molecule released from certain Lactobacillus strains attenuates the production of SLS at the transcriptional level and that combinations of Lactobacillus strains may protect the pharyngeal mucosa more efficiently from the initial colonization of GAS. The effector molecules released from Lactobacillus strains affecting the virulence phenotypes of pathogens hold potential in the development of a new generation of therapeutics. PMID:27524981

  3. AHCC Activation and Selection of Human Lymphocytes via Genotypic and Phenotypic Changes to an Adherent Cell Type: A Possible Novel Mechanism of T Cell Activation

    PubMed Central

    Olamigoke, Loretta; Mansoor, Elvedina; Mann, Vivek; Ellis, Ivory; Okoro, Elvis; Wakame, Koji; Fuji, Hajime; Kulkarni, Anil; Francoise Doursout, Marie; Sundaresan, Alamelu

    2015-01-01

    Active Hexose Correlated Compound (AHCC) is a fermented mushroom extract and immune supplement that has been used to treat a wide range of health conditions. It helps in augmentation of the natural immune response and affects immune cell activation and outcomes. The goal of this project was to study and understand the role and mechanisms of AHCC supplementation in the prevention of immunosuppression through T cell activation. The method described here involves “in vitro” culturing of lymphocytes, exposing them to different concentrations of AHCC (0 μg/mL, 50 μg/mL, 100 μg/mL, 250 μg/mL, and 500 μg/mL) at 0 hours. Interestingly, clumping and aggregation of the cells were seen between 24 and 72 hours of incubation. The cells lay down extracellular matrix, which become adherent, and phenotypical changes from small rounded lymphocytes to large macrophage-like, spindle shaped, elongated, fibroblast-like cells even beyond 360 hours were observed. These are probably translated from genotypic changes in the cells since the cells propagate for at least 3 to 6 generations (present observations). RNA isolated was subjected to gene array analysis. We hypothesize that cell adhesion is an activation and survival pathway in lymphocytes and this could be the mechanism of AHCC activation in human lymphocytes. PMID:26788109

  4. Role of specific determinants in mannan of Candida albicans serotype A in adherence to human buccal epithelial cells.

    PubMed Central

    Miyakawa, Y; Kuribayashi, T; Kagaya, K; Suzuki, M; Nakase, T; Fukazawa, Y

    1992-01-01

    Candida albicans serotype A (C. albicans A) possesses a specific antigen, designated antigen 6, which resides in mannans on the cell surface. To determine the role of the mannan moiety of the C. albicans cell wall in adherence to buccal epithelial cells, we used antigen 6-deficient mutants which had been isolated by screening with an agglutinating monoclonal antibody against antigen 6 (MAb-6). 1H nuclear magnetic resonance spectral analysis of the purified mannans from the mutants showed a loss of the signals related to that beta-linkage of the side chains. Moreover, acetolyzed fragments of the mutant mannans showed a decreased amount of mannohexaose and mannopentaose. The mutant yeast cells exhibited significantly reduced ability to adhere both to exfoliated buccal epithelial cells and to a human buccal cell line. A number of strains of C. albicans A, C. tropicalis, and C. glabrata, all of which bear antigen 6, showed significantly higher adherence to the cell line than did those of C. albicans serotype B, which lack antigen 6. The whole mannan from the C. albicans A parent inhibited the adherence of C. albicans A to epithelial cells dose dependently, whereas mannan from a mutant strains did not. Moreover, C. albicans A treated with MAb-6 or polyclonal factor 6 serum showed reduced adherence. A close correlation was found between adhesive ability and agglutinability with MAb-6 in the C. albicans A parent, the antigenic mutants, and their spontaneous revertants. These results suggest that so far as mannan adhesion is concerned, serotype A-specific determinants are largely involved in the mechanisms of adherence of C. albicans A to human buccal epithelial cells. PMID:1375200

  5. Effects of retinol on proliferation, cell adherence and extracellular matrix synthesis in a liver myofibroblast or lipocyte cell line (GRX).

    PubMed Central

    Margis, R.; Pinheiro-Margis, M.; da Silva, L. C.; Borojevic, R.

    1992-01-01

    We have studied the effect of retinol on an established murine cell line (GRX), representative of liver connective tissue cells. This cell line has myofibroblast characteristics; under retinol treatment it is induced into the lipocyte (Ito-cell) phenotype. Retinol decreased the proliferation rate in the entire cell population. It increased cell adherence to the substrate, which was correlated with the increased secretion of fibronectin. Collagen secretion was specifically decreased, whilst the total protein secretion remained stable. Heparan sulphate was decreased in the pericellular compartment, but other glycosaminoglycans were not affected by retinol treatment. Modulations of pericellular components induced by retinol may alter the relations among liver mesenchymal cells, and may be related to vitamin-A-induced modifications of the homoeostasis of hepatic connective tissue and hepatic fibrosis. Images Fig. 6 PMID:1571273

  6. Muscle-derived stem cells isolated as non-adherent population give rise to cardiac, skeletal muscle and neural lineages

    SciTech Connect

    Arsic, Nikola; Mamaeva, Daria; Lamb, Ned J.; Fernandez, Anne

    2008-04-01

    Stem cells with the ability to differentiate in specialized cell types can be extracted from a wide array of adult tissues including skeletal muscle. Here we have analyzed a population of cells isolated from skeletal muscle on the basis of their poor adherence on uncoated or collagen-coated dishes that show multi-lineage differentiation in vitro. When analysed under proliferative conditions, these cells express stem cell surface markers Sca-1 (65%) and Bcrp-1 (80%) but also MyoD (15%), Neuronal {beta} III-tubulin (25%), GFAP (30%) or Nkx2.5 (1%). Although capable of growing as non-attached spheres for months, when given an appropriate matrix, these cells adhere giving rise to skeletal muscle, neuronal and cardiac muscle cell lineages. A similar cell population could not be isolated from either bone marrow or cardiac tissue suggesting their specificity to skeletal muscle. When injected into damaged muscle, these non-adherent muscle-derived cells are retrieved expressing Pax7, in a sublaminar position characterizing satellite cells and participate in forming new myofibers. These data show that a non-adherent stem cell population can be specifically isolated and expanded from skeletal muscle and upon attachment to a matrix spontaneously differentiate into muscle, cardiac and neuronal lineages in vitro. Although competing with resident satellite cells, these cells are shown to significantly contribute to repair of injured muscle in vivo supporting that a similar muscle-derived non-adherent cell population from human muscle may be useful in treatment of neuromuscular disorders.

  7. The effects of copper additives on the quantity and cell viability of adherent Staphylococcus epidermidis in silicone implants.

    PubMed

    Gosau, Martin; Prantl, Lukas; Feldmann, Martina; Kokott, Andreas; Hahnel, Sebastian; Burgers, Ralf

    2010-04-01

    This in vitro study evaluated the antibacterial effect of copper additives in silicone implants. Specimens of a standard silicone material used in breast augmentation and modified copper-loaded silicone specimens were prepared and incubated in a Staphylococcus epidermidis suspension (2 h, 37 degrees C). After the quantification of adhering staphylococci using a biofluorescence assay (Resazurin), the viability of the adhering bacterial cells was quantified by live or dead cell labeling in combination with fluorescence microscopy. In the Resazurin fluorometric quantification, a higher amount of adhering S. epidermidis cells was detected on pure silicone (4612 [2319/7540] relative fluorescence units [rfu]) than on silicone with copper additives (2701 [2158/4153] rfu). Additionally, a significantly higher amount of adhering bacterial cells (5.07% [2.03%/8.93%]) was found for pure silicone than for silicone with copper additives (1.72% [1.26%/2.32%]); (p < 0.001). Calculations from live or dead staining showed that the percentage of dead S. epidermidis cells adhered on pure silicone (52.1%) was significantly lower than on silicone with copper additives (79.7%); (p < 0.001). In vitro, silicone material with copper additives showed antibacterial effects against S. epidermidis. Copper-loaded silicone may prevent bacterial colonization, resulting in lower infection rates of silicone implants.

  8. Characterization of natural killer cells cultured from human bone marrow cells

    SciTech Connect

    Yoda, Y.; Kawakami, Z.; Shibuya, A.; Abe, T.

    1988-09-01

    Human bone marrow (BM) cells, depleted of nylon wool-adherent cells, T cells, and natural killer (NK) cells, were cultured in medium containing recombinant interleukin 2 (rIL2). After 21 or 24 days in culture, numerous lymphoid cells with multiple azurophilic granules and a morphology similar to large granular lymphocytes (LGL) were found. Two-color analysis of surface phenotype showed many of these cells to be NKH1-positive and a limited number of cells had other NK markers such as CD16, CD2, or CD8. The CD3 antigen was not coexpressed with NKH1. The cultured BM cells were cytotoxic for K562, Daudi, and Raji cell lines. The NKH1+, CD2-, CD3-, CD16- cells were sorted and, in addition to having the LGL morphology, were found to be cytotoxic for K562 cells (NK (K562)). The generation of NK(K562) activity was significantly suppressed by 5-bromodeoxyuridine plus ultraviolet light treatment, indicating that DNA synthesis is required. These experiments suggest that the described culture conditions allow differentiation of progenitor cells, into immature, but functionally active, NK cells.

  9. 9 CFR 101.6 - Cell cultures.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Cell cultures. 101.6 Section 101.6..., SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS DEFINITIONS § 101.6 Cell cultures. When used in conjunction with or in reference to cell cultures, which may be referred to as tissue...

  10. 9 CFR 101.6 - Cell cultures.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Cell cultures. 101.6 Section 101.6..., SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS DEFINITIONS § 101.6 Cell cultures. When used in conjunction with or in reference to cell cultures, which may be referred to as tissue...

  11. 9 CFR 101.6 - Cell cultures.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Cell cultures. 101.6 Section 101.6..., SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS DEFINITIONS § 101.6 Cell cultures. When used in conjunction with or in reference to cell cultures, which may be referred to as tissue...

  12. 9 CFR 101.6 - Cell cultures.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Cell cultures. 101.6 Section 101.6..., SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS DEFINITIONS § 101.6 Cell cultures. When used in conjunction with or in reference to cell cultures, which may be referred to as tissue...

  13. 9 CFR 101.6 - Cell cultures.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

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

  14. Metabolic glycoengineering of Staphylococcus aureus reduces its adherence to human T24 bladder carcinoma cells.

    PubMed

    Memmel, Elisabeth; Homann, Arne; Oelschlaeger, Tobias A; Seibel, Jürgen

    2013-08-25

    The Gram-positive bacterium Staphylococcus aureus is a human pathogen increasingly causing severe infections, especially in hospital environments. Moreover, strains which are resistant against various types of antibiotics are developing and spreading widely as in the case of the community-acquired MRSA (methicillin resistant S. aureus). In this study metabolic glycoengineering with N-azidoacetyl-glucosamine (GlcNAz) has been successfully applied to S. aureus for the first time. The following bioorthogonal Mendal-Sharpless-Huisgen click reaction between the azido-functionalized S. aureus cells and alkyne dyes enabled staining of these bacteria and reduced their adherence to human T24 bladder carcinoma cells by 48%. The results are of urgent interest to study S. aureus infections.

  15. NLRP3 protects alveolar barrier integrity by an inflammasome-independent increase of epithelial cell adherence

    PubMed Central

    Kostadinova, Elena; Chaput, Catherine; Gutbier, Birgitt; Lippmann, Juliane; Sander, Leif E.; Mitchell, Timothy J.; Suttorp, Norbert; Witzenrath, Martin; Opitz, Bastian

    2016-01-01

    Bacterial pneumonia is a major cause of acute lung injury and acute respiratory distress syndrome, characterized by alveolar barrier disruption. NLRP3 is best known for its ability to form inflammasomes and to regulate IL-1β and IL-18 production in myeloid cells. Here we show that NLRP3 protects the integrity of the alveolar barrier in a mouse model of Streptococcus pneumoniae-induced pneumonia, and ex vivo upon treatment of isolated perfused and ventilated lungs with the purified bacterial toxin, pneumolysin. We reveal that the preserving effect of NLRP3 on the lung barrier is independent of inflammasomes, IL-1β and IL-18. NLRP3 improves the integrity of alveolar epithelial cell monolayers by enhancing cellular adherence. Collectively, our study uncovers a novel function of NLRP3 by demonstrating that it protects epithelial barrier function independently of inflammasomes. PMID:27476670

  16. In vitro adherence patterns of Shigella serogroups to bovine recto-anal junction squamous epithelial (RSE) cells are similar to those of Escherichia coli O157.

    PubMed

    Kudva, Indira T

    2012-04-01

    The aims of this study were to determine whether Shigella species, which are human gastrointestinal pathogens, can adhere to cattle recto-anal junction squamous epithelial (RSE) cells using a recently standardized in vitro adherence assay, and to compare their adherence patterns with that of Escherichia coli O157. Shigella dysenteriae (serogroup A), S. flexneri (serogroup B), S. boydii (serogroup C), and S. sonnei (serogroup D) were tested in adherence assays using both RSE and HEp-2 cells, in the presence or absence of D+mannose. Escherichia coli O157, which adheres to RSE cells in a Type I fimbriae-independent manner, was used as a positive control. Shigella serogroups A, B, D, but not C adhered to RSE cells with distinct adherence patterns in the presence of D+mannose. No such distinction could be made between the four Shigella serogroups based on the HEp-2 cell adherence patterns. Thus, this study provides evidence that certain Shigella serogroups adhere to RSE cells in a manner that is similar to the adherence pattern of E. coli O157. These unexpected observations of in vitro binding of these foodborne human pathogens to cells of the bovine gastrointestinal tract warrant evaluation of Shigella carriage by cattle using both experimental and observational studies, especially for serogroups B and D. Such studies are currently underway.

  17. Microplate cell-retaining methodology for high-content analysis of individual non-adherent unanchored cells in a population.

    PubMed

    Deutsch, Assaf; Zurgil, Naomi; Hurevich, Ihar; Shafran, Yana; Afrimzon, Elena; Lebovich, Pnina; Deutsch, Mordechai

    2006-12-01

    A high throughput Microtiter plate Cell Retainer (MCR) has been developed to enable, for the first time, high-content, time-dependent analysis of the same single non-adherent and non-anchored cells in a large cell population, while bio-manipulating the cells. The identity of each cell in the investigated population is secured, even during bio-manipulation, by cell retention in a specially designed concave microlens, acting as a picoliter well (PW). The MCR technique combines micro-optical features and microtiter plate methodology. The array of PWs serves as the bottom of a microtiter plate, fitted with a unique flow damper element. The latter enables rapid fluid exchange without dislodging the cells from their original PWs, thus maintaining the cells' identity. Loading cell suspensions and reagents into the MCR is performed by simple pouring, followed by gravitational sedimentation and settling of cells into the PWs. Cell viability and cell division within the MCR were shown to be similar to those obtained under similar conditions in a standard microtiter plate. The efficiency of single cell occupancy in the MCR exceeded 90%. No cell dislodging was observed when comparing images before and after bio-manipulations (rinsing, staining, etc.). The MCR permits the performance of kinetic measurements on an individual cell basis. Data acquisition is governed by software, controlling microscope performance, stage position and image acquisition and analysis. The PW's unique micro-optical features enable rapid, simultaneous signal analysis of each individual cell, bypassing lengthy image analysis.

  18. The MP65 gene is required for cell wall integrity, adherence to epithelial cells and biofilm formation in Candida albicans

    PubMed Central

    2011-01-01

    Background The MP65 gene of Candida albicans (orf19.1779) encodes a putative β-glucanase mannoprotein of 65 kDa, which plays a main role in a host-fungus relationship, morphogenesis and pathogenicity. In this study, we performed an extensive analysis of a mp65Δ mutant to assess the role of this protein in cell wall integrity, adherence to epithelial cells and biofilm formation. Results The mp65Δ mutant showed a high sensitivity to a range of cell wall-perturbing and degrading agents, especially Congo red, which induced morphological changes such as swelling, clumping and formation of hyphae. The mp65Δ mutant showed an activation of two MAPKs (Mkc1p and Cek1p), a high level of expression of two stress-related genes (DDR48 and SOD5), and a modulated expression of β-glucan epitopes, but no gross changes in cell wall polysaccharide composition. Interestingly, the mp65Δ mutant displayed a marked reduction in adhesion to BEC and Caco-2 cells and severe defects in biofilm formation when compared to the wild type. All of the mentioned properties were totally or partially recovered in a revertant strain, demonstrating the specificity of gene deletion. Conclusions We demonstrate that the MP65 gene of Candida albicans plays a significant role in maintaining cell wall integrity, as well as in adherence to epithelia and biofilm formation, which are major virulence attributes of this fungus. PMID:21575184

  19. The minotaur proteome: avoiding cross-species identifications deriving from bovine serum in cell culture models.

    PubMed

    Bunkenborg, Jakob; García, Guadalupe Espadas; Paz, Marcia Ivonne Peña; Andersen, Jens S; Molina, Henrik

    2010-08-01

    Cell culture is a fundamental tool in proteomics where mammalian cells are cultured in vitro using a growth medium often supplemented with 5-15% FBS. Contamination by bovine proteins is difficult to avoid because of adherence to the plastic vessel and the cultured cells. We have generated peptides from bovine serum using four sample preparation methods and analyzed the peptides by high mass accuracy LC-MS/MS. Distinguishing between bovine and human peptides is difficult because of a considerable overlap of identical tryptic peptide sequences. Pitfalls in interpretation, different database search strategies to minimize erroneous identifications and an augmented contaminant database are presented.

  20. Curli modulates adherence of Escherichia coli O157:H7 to bovine recto-anal junction squamous epithelial cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Our recent studies have shown that Intimin and the Locus of Enterocyte Effacement-encoded proteins do not play a role in Escherichia coli O157 (O157) adherence to the bovine recto-anal junction squamous epithelial cells (RSE) cells. Hence, to define factors that play a contributory role, we investi...

  1. Microstructured multi-well plate for three-dimensional packed cell seeding and hepatocyte cell culture.

    PubMed

    Goral, Vasiliy N; Au, Sam H; Faris, Ronald A; Yuen, Po Ki

    2014-07-01

    In this article, we present a microstructured multi-well plate for enabling three-dimensional (3D) high density seeding and culture of cells through the use of a standard laboratory centrifuge to promote and maintain 3D tissue-like cellular morphology and cell-specific functionality in vitro without the addition of animal derived or synthetic matrices or coagulants. Each well has microfeatures on the bottom that are comprised of a series of ditches/open microchannels. The dimensions of the microchannels promote and maintain 3D tissue-like cellular morphology and cell-specific functionality in vitro. After cell seeding with a standard pipette, the microstructured multi-well plates were centrifuged to tightly pack cells inside the ditches in order to enhance cell-cell interactions and induce formation of 3D cellular structures during cell culture. Cell-cell interactions were optimized based on cell packing by considering dimensions of the ditches/open microchannels, orientation of the microstructured multi-well plate during centrifugation, cell seeding density, and the centrifugal force and time. With the optimized cell packing conditions, we demonstrated that after 7 days of cell culture, primary human hepatocytes adhered tightly together to form cord-like structures that resembled 3D tissue-like cellular architecture. Importantly, cell membrane polarity was restored without the addition of animal derived or synthetic matrices or coagulants.

  2. Basic techniques in mammalian cell tissue culture.

    PubMed

    Phelan, Katy; May, Kristin M

    2015-03-02

    Cultured mammalian cells are used extensively in cell biology studies. It requires a number of special skills in order to be able to preserve the structure, function, behavior, and biology of the cells in culture. This unit describes the basic skills required to maintain and preserve cell cultures: maintaining aseptic technique, preparing media with the appropriate characteristics, passaging, freezing and storage, recovering frozen stocks, and counting viable cells.

  3. Visceral leishmaniasis in congenic mice of susceptible and resistant phenotypes: immunosuppression by adherent spleen cells.

    PubMed Central

    Nickol, A D; Bonventre, P F

    1985-01-01

    . Removal of adherent cells from the suppressive spleen cell populations restored normal mitogen responses. On the basis of adherence characteristics, phagocytosis, and morphology, the suppressor was identified as a macrophage population which appears to be responsible for a nonspecific immunosuppression of Lshs mice with significant parasite burdens of L. donovani. PMID:2931376

  4. Enhanced adherence of mouse fibroblast and vascular cells to plasma modified polyethylene.

    PubMed

    Reznickova, Alena; Novotna, Zdenka; Kolska, Zdenka; Kasalkova, Nikola Slepickova; Rimpelova, Silvie; Svorcik, Vaclav

    2015-01-01

    Since the last decade, tissue engineering has shown a sensational promise in providing more viable alternatives to surgical procedures for harvested tissues, implants and prostheses. Biomedical polymers, such as low-density polyethylene (LDPE), high-density polyethylene (HDPE) and ultra-high molecular weight polyethylene (UHMWPE), were activated by Ar plasma discharge. Degradation of polymer chains was examined by determination of the thickness of ablated layer. The amount of an ablated polymer layer was measured by gravimetry. Contact angle, measured by goniometry, was studied as a function of plasma exposure and post-exposure aging times. Chemical structure of modified polymers was characterized by angle resolved X-ray photoelectron spectroscopy. Surface chemistry and polarity of the samples were investigated by electrokinetic analysis. Changes in surface morphology were followed using atomic force microscopy. Cytocompatibility of plasma activated polyethylene foils was studied using two distinct model cell lines; VSMCs (vascular smooth muscle cells) as a model for vascular graft testing and connective tissue cells L929 (mouse fibroblasts) approved for standardized material cytotoxicity testing. Specifically, the cell number, morphology, and metabolic activity of the adhered and proliferated cells on the polyethylene matrices were studied in vitro. It was found that the plasma treatment caused ablation of the polymers, resulting in dramatic changes in their surface morphology and roughness. ARXPS and electrokinetic measurements revealed oxidation of the polymer surface. It was found that plasma activation has a positive effect on the adhesion and proliferation of VSMCs and L929 cells.

  5. Adherent-phagocytic cells influence suppressed concanavalin-A induced proliferation of spleen lymphoid cells in copper deficient rats

    SciTech Connect

    Kramer, T.R.; Briske-Anderson, M.; Johnson, W.T.

    1986-03-01

    Weanling male Lewis rats (N = 10/group) were fed ad-libitum for 42 days diets based on AIN standards containing 21% casein, 5% safflower oil, and deficient (0.6 ..mu..g/g) or adequate (5.6 ..mu..g/g) levels of cu. Cu-deficient rats showed typical biochemical and hematological changes. Immunological changes exhibited by Cu-deficient rats were influenced by the presence of splenic adherent-phagocytic cells (macrophage-like), but not by cytochrome-c oxidase activity of spleen lymphoid cells (SLC). Decreased proliferation was exhibited by concanavalin-A (Con-A) stimulated SLC of Cu-deficient rats. Following removal of plastic-adherent phagocytic cells from the SLC suspensions, equivalent proliferation was exhibited by Con-A stimulated nonadherent-SLC of Cu-deficient and Cu-adequate rats. Decreased cytochrome-c oxidase activity was exhibited by both unstimulated SLC and nonadherent-SLC of Cu-deficient rats, but decreased proliferation was exhibited only in Con-A stimulated SLC of Cu-deficient rats. These findings indicate that nonadherent splenic T-lymphocytes of Cu-deficient rats are not impaired in their ability to proliferate, and that cytochrome-c oxidase activity in unstimulated lymphoid cells of Cu-deficient rats is apparently not related to levels of proliferation by the Con-A stimulated cells.

  6. Sialidases Affect the Host Cell Adherence and Epsilon Toxin-Induced Cytotoxicity of Clostridium perfringens Type D Strain CN3718

    PubMed Central

    Li, Jihong; Sayeed, Sameera; Robertson, Susan; Chen, Jianming; McClane, Bruce A.

    2011-01-01

    Clostridium perfringens type B or D isolates, which cause enterotoxemias or enteritis in livestock, produce epsilon toxin (ETX). ETX is exceptionally potent, earning it a listing as a CDC class B select toxin. Most C. perfringens strains also express up to three different sialidases, although the possible contributions of those enzymes to type B or D pathogenesis remain unclear. Type D isolate CN3718 was found to carry two genes (nanI and nanJ) encoding secreted sialidases and one gene (nanH) encoding a cytoplasmic sialidase. Construction in CN3718 of single nanI, nanJ and nanH null mutants, as well as a nanI/nanJ double null mutant and a triple sialidase null mutant, identified NanI as the major secreted sialidase of this strain. Pretreating MDCK cells with NanI sialidase, or with culture supernatants of BMC206 (an isogenic CN3718 etx null mutant that still produces sialidases) enhanced the subsequent binding and cytotoxic effects of purified ETX. Complementation of BMC207 (an etx/nanH/nanI/nanJ null mutant) showed this effect is mainly attributable to NanI production. Contact between BMC206 and certain mammalian cells (e.g., enterocyte-like Caco-2 cells) resulted in more rapid sialidase production and this effect involved increased transcription of BMC206 nanI gene. BMC206 was shown to adhere to some (e.g. Caco-2 cells), but not all mammalian cells, and this effect was dependent upon sialidase, particularly NanI, expression. Finally, the sialidase activity of NanI (but not NanJ or NanH) could be enhanced by trypsin. Collectively these in vitro findings suggest that, during type D disease originating in the intestines, trypsin may activate NanI, which (in turn) could contribute to intestinal colonization by C. perfringens type D isolates and also increase ETX action. PMID:22174687

  7. Streptococcus pneumoniae ClpL Modulates Adherence to A549 Human Lung Cells through Rap1/Rac1 Activation

    PubMed Central

    Nguyen, Cuong Thach; Le, Nhat-Tu; Tran, Thao Dang-Hien; Kim, Eun-Hye; Park, Sang-Sang; Luong, Truc Thanh; Chung, Kyung-Tae; Pyo, Suhkneung

    2014-01-01

    Caseinolytic protease L (ClpL) is a member of the HSP100/Clp chaperone family, which is found mainly in Gram-positive bacteria. ClpL is highly expressed during infection for refolding of stress-induced denatured proteins, some of which are important for adherence. However, the role of ClpL in modulating pneumococcal virulence is poorly understood. Here, we show that ClpL impairs pneumococcal adherence to A549 lung cells by inducing and activating Rap1 and Rac1, thus increasing phosphorylation of cofilin (inactive form). Moreover, infection with a clpL mutant (ΔclpL) causes a greater degree of filopodium formation than D39 wild-type (WT) infection. Inhibition of Rap1 and Rac1 impairs filopodium formation and pneumococcal adherence. Therefore, ClpL can reduce pneumococcal adherence to A549 cells, likely via modulation of Rap1- and Rac1-mediated filopodium formation. These results demonstrate a potential role for ClpL in pneumococcal resistance to host cell adherence during infection. This study provides insight into further understanding the interactions between hosts and pathogens. PMID:24980975

  8. Real-time monitoring of specific oxygen uptake rates of embryonic stem cells in a microfluidic cell culture device.

    PubMed

    Super, Alexandre; Jaccard, Nicolas; Cardoso Marques, Marco Paulo; Macown, Rhys Jarred; Griffin, Lewis Donald; Veraitch, Farlan Singh; Szita, Nicolas

    2016-09-01

    Oxygen plays a key role in stem cell biology as a signaling molecule and as an indicator of cell energy metabolism. Quantification of cellular oxygen kinetics, i.e. the determination of specific oxygen uptake rates (sOURs), is routinely used to understand metabolic shifts. However current methods to determine sOUR in adherent cell cultures rely on cell sampling, which impacts on cellular phenotype. We present real-time monitoring of cell growth from phase contrast microscopy images, and of respiration using optical sensors for dissolved oxygen. Time-course data for bulk and peri-cellular oxygen concentrations obtained for Chinese hamster ovary (CHO) and mouse embryonic stem cell (mESCs) cultures successfully demonstrated this non-invasive and label-free approach. Additionally, we confirmed non-invasive detection of cellular responses to rapidly changing culture conditions by exposing the cells to mitochondrial inhibiting and uncoupling agents. For the CHO and mESCs, sOUR values between 8 and 60 amol cell(-1) s(-1) , and 5 and 35 amol cell(-1) s(-1) were obtained, respectively. These values compare favorably with literature data. The capability to monitor oxygen tensions, cell growth, and sOUR, of adherent stem cell cultures, non-invasively and in real time, will be of significant benefit for future studies in stem cell biology and stem cell-based therapies.

  9. Identification of a population of cells with hematopoietic stem cell properties in mouse aorta-gonad-mesonephros cultures

    SciTech Connect

    Nobuhisa, Ikuo; Ohtsu, Naoki; Okada, Seiji; Nakagata, Naomi; Taga, Tetsuya . E-mail: taga@kaiju.medic.kumamoto-u.ac.jp

    2007-03-10

    The aorta-gonad-mesonephros (AGM) region is a primary source of definitive hematopoietic cells in the midgestation mouse embryo. In cultures of dispersed AGM regions, adherent cells containing endothelial cells are observed first, and then non-adherent hematopoietic cells are produced. Here we report on the characterization of hematopoietic cells that emerge in the AGM culture. Based on the expression profiles of CD45 and c-Kit, we defined three cell populations: CD45{sup low} c-Kit{sup +} cells that had the ability to form hematopoietic cell colonies in methylcellulose media and in co-cultures with stromal cells; CD45{sup low} c-Kit{sup -} cells that showed a granulocyte morphology; CD45{sup high} c-Kit{sup low/-} that exhibited a macrophage morphology. In co-cultures of OP9 stromal cells and freshly prepared AGM cultures, CD45{sup low} c-Kit{sup +} cells from the AGM culture had the abilities to reproduce CD45{sup low} c-Kit{sup +} cells and differentiate into CD45{sup low} c-Kit{sup -} and CD45{sup high} c-Kit{sup low/-} cells, whereas CD45{sup low} c-Kit{sup -} and CD45{sup high} c-Kit{sup low/-} did not produce CD45{sup low} c-Kit{sup +} cells. Furthermore, CD45{sup low} c-Kit{sup +} cells displayed a long-term repopulating activity in adult hematopoietic tissue when transplanted into the liver of irradiated newborn mice. These results indicate that CD45{sup low} c-Kit{sup +} cells from the AGM culture have the potential to reconstitute multi-lineage hematopoietic cells.

  10. Raman micro-spectroscopy study of living SH-SY5Y cells adhering on different substrates.

    PubMed

    Caponi, S; Mattana, S; Ricci, M; Sagini, K; Urbanelli, L; Sassi, P; Morresi, A; Emiliani, C; Dalla Serra, M; Iannotta, S; Musio, C; Fioretto, D

    2016-01-01

    In this paper we test the ability of Raman micro-spectroscopy and Raman mapping to investigate the status of cells grown in adhesion on different substrates. The spectra of immortalized SH-SY5Y cells, grown on silicon and on metallic substrates are compared with those obtained for the same type of cells adhering on organic polyaniline (PANI), a memristive substrate chosen to achieve a living bio-hybrid system. Raman spectra give information on the status of the single cell, its local biochemical composition, and on the modifications induced by the substrate interaction. The good agreement between Raman spectra collected from cells adhering on different substrates confirms that the PANI, besides allowing the cell growth, doesn't strongly affect the general biochemical properties of the cell. The investigation of the cellular state in a label free condition is challenging and the obtained results confirm the Raman ability to achieve this information.

  11. Adherent cell assay results affected by variable z-position mixing.

    PubMed

    Carramanzana, Nelson; Ross, Sandra; Biddlecombe, Gloria; Lin, Chi-Hwei; Johnson, Michael

    2010-04-01

    We demonstrate that modifying mixing dynamics after addition of organic solute into aqueous buffers dramatically affects cell morphology and protein expression. Variable z-position (VZP) or varying the height of aspiration and dispense positions during mixing eliminates artifactual effects. Here, we tested 4 adherent cell types and show effects of VZP on quantitative imaging, protein expression, viability, and morphology. The result: The quantitation of cytoplasmic fluorescence within the fields of interest of the phalloidin-actin stain assay improved by 47% and fluorescence variability emitted by cells expressing green fluorescence protein (GFP) fusion proteins decreased by 15%. Assays that perform measurement by averaged reading of the entire well are somewhat susceptible. For example, protein production decreased 8% on the hypoxia response element (HRE)-luciferase assay. VZP did not affect quantitative cell viability, deviate the half maximal effective dose concentration (EC(50)) values or alter expected curve patterns. VZP is a valuable systematic process for cellular assay workflows as it efficiently folds organic solute into the aqueous solution.

  12. A computational model of the response of adherent cells to stretch and changes in substrate stiffness.

    PubMed

    Parameswaran, Harikrishnan; Lutchen, Kenneth R; Suki, Béla

    2014-04-01

    Cells in the body exist in a dynamic mechanical environment where they are subject to mechanical stretch as well as changes in composition and stiffness of the underlying extracellular matrix (ECM). However, the underlying mechanisms by which cells sense and adapt to their dynamic mechanical environment, in particular to stretch, are not well understood. In this study, we hypothesized that emergent phenomena at the level of the actin network arising from active structural rearrangements driven by nonmuscle myosin II molecular motors play a major role in the cellular response to both stretch and changes in ECM stiffness. To test this hypothesis, we introduce a simple network model of actin-myosin interactions that links active self-organization of the actin network to the stiffness of the network and the traction forces generated by the network. We demonstrate that such a network replicates not only the effect of changes in substrate stiffness on cellular traction and stiffness and the dependence of rate of force development by a cell on the stiffness of its substrate, but also explains the physical response of adherent cells to transient and cyclic stretch. Our results provide strong indication that network phenomena governed by the active reorganization of the actin-myosin structure plays an important role in cellular mechanosensing and response to both changes in ECM stiffness and externally applied mechanical stretch.

  13. Soft Micro-Channels for Cell Culturing and Migration Studies

    NASA Astrophysics Data System (ADS)

    Abbasirazgaleh, Sara

    Various techniques and methods have been studied and developed to aid nerve regeneration and repairing nerve injuries. Among all, nerve grafting is the gold standard for bridging the gap between the injured nerve stumps. Despite the advantages of this technique, there are also various drawbacks that have encouraged the exploration of alternative, less invasive methods for promoting nerve regeneration. In this thesis, we have fabricated soft micro-channels for cell culturing and migration studies which could act as an interface capable of long-term, reliable, and high-resolution stimulation device for nerve regeneration. Micro-channels fabrication is performed using a combination of photolithography technique and physical vapor deposition (PVD) methods. Initially, the surfaces of the micro-channels are treated with oxygen plasma to convert the surface of PDMS from hydrophobic to hydrophilic and to further provide an optimal environment for cells to adhere and grow. Next, in vitro studies were performed on the fabricated micro-channels to demonstrate feasibility of the platform to promote adherence and growth of PC12 cells (cell line derived from a pheochromocytomas of the rat adrenal medulla).

  14. Effect of Chitosan and Sodium Alginate on the adherence of autochthonous C. Albicans to oral epithelial cells (in vitro).

    PubMed

    Barembaum, Silvina; Virga, Carolina; Bojanich, Alejandra; Cornejo, Lila; Calamari, Silvia; Pontón, José; Dorronsoro, Susana

    2003-01-01

    The aim of the present study was to evaluate the effect of Heavy Molecular Weight Chitosan (HMWCh) and Sodium Alginate (NaAl) on fungal adherence. C albicans was identified and isolated from non-stimulated saliva extracted from male and female healthy adults. The minimum inhibitory concentration (MIC) was determined for each of the biopolymers. MIC values were 0.25 % (W/V) for HMWCh and 0.10 % (W/V) for NaAl. Fungal cell hydrophobicity was evaluated against xylene in the presence of HMWCh. Statistically significant differences between the control (without HMWCh) and the different HMWCh concentrations in fungal suspension were observed (P< 0.05). The fact that HMWCh and NaAl impaired fungal adherence to buccal epithelial cells (BEC) as compared to control revealed that polymers inhibit Candida albicans adherence to BEC (HMWCh and NaAl: P= 0.00001), NaAl being more effective than HMWCh (P = 0.00001). HMWCh dettached and aggregated C. albicans, including the fungi and BEC in the mesh. NaAl inhibited adherence, aggregated and entrapped the fungi in the mesh, excluding BEC. We may conclude that both biopolymers are effective. However, NaAl is a stronger inhibitor of adherence. Thus, in combination or alone, these biopolymers could be used in the treatment of oral candidosis.

  15. Recognition of laminin by Paracoccidioides brasiliensis conidia: a possible mechanism of adherence to human type II alveolar cells.

    PubMed

    Caro, Erika; Gonzalez, Angel; Muñoz, César; Urán, Marta E; Restrepo, Angela; John Hamilton, Andrew; Elena Cano, Luz

    2008-12-01

    This study addresses the recognition of laminin by Paracoccidioides brasiliensis conidia, as well as its possible role in the adherence of conidia to A549 cells. Adherence of conidia to immobilized laminin was shown to be specific, as anti-laminin antibodies, soluble laminin or the laminin-derived peptides IKVAV and CDPGYIGSR inhibited this interaction. RGD containing peptides and various monosaccharides had no effect on adherence, with the exception of N-acetylneuraminic acid. Pre-treatment of conidia with fibrinogen and fibronectin, but not with BSA, also resulted in significant inhibition, suggesting that P. brasiliensis conidia might cross-recognize host proteins involved in colonization. In assays using transmission electron microscopy, we observed internalization of conidia 30 min after exposition to A549 cells. Laminin present on the surface of A549 cells shown to serve as mediator of this interaction, with a significant decrease in fungal adherence when the epithelial cells were pre-treated with anti-laminin antibodies or when conidia were pre-incubated with either soluble laminin or the laminin-specific peptides. Together these results suggest that the recognition of laminin by P. brasiliensis conidia is a key process in the interaction with pulmonary epithelial cells, where this extracellular matrix protein acts as bridging molecule.

  16. The Chinese Life-Steps Program: A Cultural Adaptation of a Cognitive-Behavioral Intervention to Enhance HIV Medication Adherence

    ERIC Educational Resources Information Center

    Shiu, Cheng-Shi; Chen, Wei-Ti; Simoni, Jane; Fredriksen-Goldsen, Karen; Zhang, Fujie; Zhou, Hongxin

    2013-01-01

    China is considered to be the new frontier of the global AIDS pandemic. Although effective treatment for HIV is becoming widely available in China, adherence to treatment remains a challenge. This study aimed to adapt an intervention promoting HIV-medication adherence--favorably evaluated in the West--for Chinese HIV-positive patients. The…

  17. Cultural adaptation of a cognitive-behavioural intervention to improve adherence to antiretroviral therapy among people living with HIV/AIDS in Zimbabwe: Nzira Itsva.

    PubMed

    Bere, Tarisai; Nyamayaro, Primrose; Magidson, Jessica F; Chibanda, Dixon; Chingono, Alfred; Munjoma, Ronald; Macpherson, Kirsty; Ndhlovu, Chiratidzo Ellen; O'Cleirigh, Conall; Kidia, Khameer; Safren, Steven A; Abas, Melanie

    2016-02-18

    Few evidence-based interventions to improve adherence to antiretroviral therapy have been adapted for use in Africa. We selected, culturally adapted and tested the feasibility of a cognitive-behavioural intervention for adherence and for delivery in a clinic setting in Harare, Zimbabwe. The intervention consisted of a single, 50-minute problem-solving cognitive-behavioural intervention session with four skill-based booster sessions, delivered by four lay adherence counsellors in the context of HIV care. Adaptation followed a theoretically driven approach to intervention adaptation, Assessment-Decision-Administration-Production-Topical Experts-Integration-Training-Testing (ADAPT-ITT), and included modifications to language, session length, tailoring content for delivery by lay counsellors and inclusion of culturally competent probes. The feasibility of the intervention was evaluated using a mixed-methods assessment, including ratings of provider fidelity of intervention delivery, and qualitative assessments of feasibility using individual semi-structured interviews with counsellors (n = 4) and patients (n = 15). The intervention was feasible and acceptable when administered to 42 patients and resulted in improved self-reported adherence in a subset of 15 patients who were followed up after 6 months. Next steps from this study include conducting a randomised control trial to evaluate the adapted intervention compared to standard of care in a larger sample over a long-term follow-up.

  18. The Culture of Cancer Cell Lines as Tumorspheres Does Not Systematically Result in Cancer Stem Cell Enrichment

    PubMed Central

    Calvet, Christophe Y.; André, Franck M.; Mir, Lluis M.

    2014-01-01

    Cancer stem cells (CSC) have raised great excitement during the last decade and are promising targets for an efficient treatment of tumors without relapses and metastases. Among the various methods that enable to enrich cancer cell lines in CSC, tumorspheres culture has been predominantly used. In this report, we attempted to generate tumorspheres from several murine and human cancer cell lines: B16-F10, HT-29, MCF-7 and MDA-MB-231 cells. Tumorspheres were obtained with variable efficiencies from all cell lines except from MDA-MB-231 cells. Then, we studied several CSC characteristics in both tumorspheres and adherent cultures of the B16-F10, HT-29 and MCF-7 cells. Unexpectedly, tumorspheres-forming cells were less clonogenic and, in the case of B16-F10, less proliferative than attached cells. In addition, we did not observe any enrichment in the population expressing CSC surface markers in tumorspheres from B16-F10 (CD133, CD44 and CD24 markers) or MCF-7 (CD44 and CD24 markers) cells. On the contrary, tumorspheres culture of HT-29 cells appeared to enrich in cells expressing colon CSC markers, i.e. CD133 and CD44 proteins. For the B16-F10 cell line, when 1 000 cells were injected in syngenic C57BL/6 mice, tumorspheres-forming cells displayed a significantly lower tumorigenic potential than adherent cells. Finally, tumorspheres culture of B16-F10 cells induced a down-regulation of vimentin which could explain, at least partially, the lower tumorigenicity of tumorspheres-forming cells. All these results, along with the literature, indicate that tumorspheres culture of cancer cell lines can induce an enrichment in CSC but in a cell line-dependent manner. In conclusion, extensive characterization of CSC properties in tumorspheres derived from any cancer cell line or cancer tissue must be performed in order to ensure that the generated tumorspheres are actually enriched in CSC. PMID:24586931

  19. A microfluidic device with removable packaging for the real time visualisation of intracellular effects of nanosecond electrical pulses on adherent cells.

    PubMed

    Dalmay, C; De Menorval, M A; Français, O; Mir, L M; Le Pioufle, B

    2012-11-21

    The biological mechanisms induced by the application of nanosecond pulsed electric fields (nsPEFs: high electrical field amplitude during very short duration) on cells remain partly misunderstood. In this context, there is an increasing need for tools that allow the delivering of such pulses with the possibility to monitor their effects in real-time. Thanks to miniaturization and technology capabilities, microtechnologies offer great potential to address this issue. We report here the design and fabrication of a microfluidic device optimized for the delivery of ultra short (10 ns) and intense (up to 280 kV cm(-1)) electrical pulses on adherent cells, and the real time monitoring of their intracellular effects. Ultra short electric field pulses (nsPEFs or nanopulses) affect both the cell membrane and the intracellular organelles of the cells. In particular, intracellular release of calcium from the endoplasmic reticulum was detected in real time using the device, after exposure of adherent cells to these nsPEFs. The high intensity and spatial homogeneity of the electric field could be achieved in the device thanks to the miniaturization and the use of thick (25 μm) electroplated electrodes, disposed on a quartz substrate whose transparency allowed real time monitoring of the nsPEFs effects. The proposed biochip is compatible with cell culture glass slides that can be placed on the chip after separate culture of several days prior to exposure. This device allows the easy exposure of almost any kind of attached cells and the monitoring in real time while exposed to nsPEFs, opening large possibilities for potential use of the developed biochips.

  20. Selective adherence of non-typeable Haemophilus influenzae (NTHi) to mucus or epithelial cells in the chinchilla eustachian tube and middle ear.

    PubMed

    Miyamoto, N; Bakaletz, L O

    1996-11-01

    Frozen sections of chinchilla Eustachian tube (ET) and middle ear mucosa were incubated with either FITC-labeled non-typeable Haemophilus influenzae (NTHi) or Bordetella pertussis. The number of bacteria adherent to "roof" vs "floor" regions was compared for each of three anatomic portions of the ET and for middle ear epithelium noting whether bacteria adhered to mucus or to epithelial cells. NTHi strains adhered significantly greater to mucus in the ET lumen whereas B. pertussis preferentially adhered to epithelial cells lining the ET (P < or = 0.05). A non-fimbriated isogenic mutant of NTHi adhered significantly less to mucus than the parental isolate at all sites of the ET floor (P < or = 0.05). Isolated fimbrin protein adhered to ET mucus and blocked adherence of whole organisms. Treatment with the mucolytic agent N-acetyl-L-cysteine resulted in significantly reduced adherence of NTHi to mucus (P < or = 0.001) and eliminated the ability to detect binding of isolated fimbrin protein. N-acetyl-L-cysteine treatment did not affect adherence of either B. pertussis or NTHi to epithelial cells. These data indicated that NTHi may mediate ascension of the ET from the nasopharynx primarily via adherence to and growth in mucus overlying the floor region of the tubal lumen. The OMP P5-homologous fimbriae were shown to contribute to this binding.

  1. Prevalence of Escherichia coli strains with localized, diffuse, and aggregative adherence to HeLa cells in infants with diarrhea and matched controls.

    PubMed Central

    Gomes, T A; Blake, P A; Trabulsi, L R

    1989-01-01

    To determine the possible role of Escherichia coli strains with three different patterns of adherence to HeLa cells in causing diarrhea in infants in São Paulo, Brazil, we studied stool specimens from 100 infants up to 1 year of age with acute diarrheal illnesses and 100 age-matched control infants without recent diarrhea. E. coli with localized adherence to HeLa cells was much more common in patients (23%) than in controls (2%) (P less than 0.0001) and was detected more frequently than rotavirus (19%) was in patients, even though the study was conducted during the coldest months of the year. Most (80%) of the E. coli colonies with localized adherence were of traditional enteropathogenic E. coli serotypes. Little difference was found between patients and controls in the rate of isolation of E. coli with diffuse adherence (31 and 32%, respectively) or aggregative adherence (10 and 8%, respectively). A genetic probe used to detect a plasmid-mediated adhesin which confers expression of localized adherence proved to be 100% sensitive and 99.9% specific in detecting E. coli with localized adherence to HeLa cells. Although E. coli strains with localized adherence have now been shown to be enteric pathogens in several parts of the world, the role of strains showing diffuse adherence and aggregative adherence is still uncertain. PMID:2563383

  2. Neisseria cinerea isolates can adhere to human epithelial cells by type IV pilus-independent mechanisms

    PubMed Central

    Wörmann, Mirka E.; Horien, Corey L.; Johnson, Errin; Liu, Guangyu; Aho, Ellen; Tang, Christoph M.

    2016-01-01

    In pathogenic Neisseria species the type IV pili (Tfp) are of primary importance in host–pathogen interactions. Tfp mediate initial bacterial attachment to cell surfaces and formation of microcolonies via pilus–pilus interactions. Based on genome analysis, many non-pathogenic Neisseria species are predicted to express Tfp, but aside from studies on Neisseria elongata, relatively little is known about the formation and function of pili in these organisms. Here, we have analysed pilin expression and the role of Tfp in Neisseria cinerea. This non-pathogenic species shares a close taxonomic relationship to the pathogen Neisseria meningitidis and also colonizes the human oropharyngeal cavity. Through analysis of non-pathogenic Neisseria genomes we identified two genes with homology to pilE, which encodes the major pilin of N. meningitidis. We show which of the two genes is required for Tfp expression in N. cinerea and that Tfp in this species are required for DNA competence, similar to other Neisseria. However, in contrast to the meningococcus, deletion of the pilin gene did not impact the association of N. cinerea to human epithelial cells, demonstrating that N. cinerea isolates can adhere to human epithelial cells by Tfp-independent mechanisms. PMID:26813911

  3. Neisseria cinerea isolates can adhere to human epithelial cells by type IV pilus-independent mechanisms.

    PubMed

    Wörmann, Mirka E; Horien, Corey L; Johnson, Errin; Liu, Guangyu; Aho, Ellen; Tang, Christoph M; Exley, Rachel M

    2016-03-01

    In pathogenic Neisseria species the type IV pili (Tfp) are of primary importance in host-pathogen interactions. Tfp mediate initial bacterial attachment to cell surfaces and formation of microcolonies via pilus-pilus interactions. Based on genome analysis, many non-pathogenic Neisseria species are predicted to express Tfp, but aside from studies on Neisseria elongata, relatively little is known about the formation and function of pili in these organisms. Here, we have analysed pilin expression and the role of Tfp in Neisseria cinerea. This non-pathogenic species shares a close taxonomic relationship to the pathogen Neisseria meningitidis and also colonizes the human oropharyngeal cavity. Through analysis of non-pathogenic Neisseria genomes we identified two genes with homology to pilE, which encodes the major pilin of N. meningitidis. We show which of the two genes is required for Tfp expression in N. cinerea and that Tfp in this species are required for DNA competence, similar to other Neisseria. However, in contrast to the meningococcus, deletion of the pilin gene did not impact the association of N. cinerea to human epithelial cells, demonstrating that N. cinerea isolates can adhere to human epithelial cells by Tfp-independent mechanisms.

  4. Cell Culture as an Alternative in Education.

    ERIC Educational Resources Information Center

    Nardone, Roland M.

    1990-01-01

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

  5. Cell culture techniques in honey bee research

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  6. The role of Listeria monocytogenes cell wall surface anchor protein LapB in virulence, adherence, and intracellular replication

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lmof2365_2117 is a Listeria monocytogenes putative cell wall surface anchor protein with a conserved domain found in collagen binding proteins. We constructed a deletion mutation in lmof2365_2117 in serotype 4b strain F2365, evaluated its virulence, and determined its ability to adhere and invade co...

  7. Heat-transfer-method-based cell culture quality assay through cell detection by surface imprinted polymers.

    PubMed

    Eersels, Kasper; van Grinsven, Bart; Khorshid, Mehran; Somers, Veerle; Püttmann, Christiane; Stein, Christoph; Barth, Stefan; Diliën, Hanne; Bos, Gerard M J; Germeraad, Wilfred T V; Cleij, Thomas J; Thoelen, Ronald; De Ceuninck, Ward; Wagner, Patrick

    2015-02-17

    Previous work has indicated that surface imprinted polymers (SIPs) allow for highly specific cell detection through macromolecular cell imprints. The combination of SIPs with a heat-transfer-based read-out technique has led to the development of a selective, label-free, low-cost, and user-friendly cell detection assay. In this study, the breast cancer cell line ZR-75-1 is used to assess the potential of the platform for monitoring the quality of a cell culture in time. For this purpose, we show that the proposed methodology is able to discriminate between the original cell line (adherent growth, ZR-75-1a) and a descendant cell line (suspension growth, ZR-75-1s). Moreover, ZR-75-1a cells were cultured for a prolonged period of time and analyzed using the heat-transfer method (HTM) at regular time intervals. The results of these experiments demonstrate that the thermal resistance (Rth) signal decays after a certain number of cell culture passages. This can likely be attributed to a compromised quality of the cell culture due to cross-contamination with the ZR-75-1s cell line, a finding that was confirmed by classical STR DNA profiling. The cells do not express the same functional groups on their membrane, resulting in a weaker bond between cell and imprint, enabling cell removal by mechanical friction, provided by flushing the measuring chamber with buffer solution. These findings were further confirmed by HTM and illustrate that the biomimetic sensor platform can be used as an assay for monitoring the quality of cell cultures in time.

  8. Real‐time monitoring of specific oxygen uptake rates of embryonic stem cells in a microfluidic cell culture device

    PubMed Central

    Super, Alexandre; Jaccard, Nicolas; Cardoso Marques, Marco Paulo; Macown, Rhys Jarred; Griffin, Lewis Donald; Veraitch, Farlan Singh

    2016-01-01

    Abstract Oxygen plays a key role in stem cell biology as a signaling molecule and as an indicator of cell energy metabolism. Quantification of cellular oxygen kinetics, i.e. the determination of specific oxygen uptake rates (sOURs), is routinely used to understand metabolic shifts. However current methods to determine sOUR in adherent cell cultures rely on cell sampling, which impacts on cellular phenotype. We present real‐time monitoring of cell growth from phase contrast microscopy images, and of respiration using optical sensors for dissolved oxygen. Time‐course data for bulk and peri‐cellular oxygen concentrations obtained for Chinese hamster ovary (CHO) and mouse embryonic stem cell (mESCs) cultures successfully demonstrated this non‐invasive and label‐free approach. Additionally, we confirmed non‐invasive detection of cellular responses to rapidly changing culture conditions by exposing the cells to mitochondrial inhibiting and uncoupling agents. For the CHO and mESCs, sOUR values between 8 and 60 amol cell−1 s−1, and 5 and 35 amol cell−1 s−1 were obtained, respectively. These values compare favorably with literature data. The capability to monitor oxygen tensions, cell growth, and sOUR, of adherent stem cell cultures, non‐invasively and in real time, will be of significant benefit for future studies in stem cell biology and stem cell‐based therapies. PMID:27214658

  9. Story-based scales: development and validation of questionnaires to measure subjective health status and cultural adherence in British Bangladeshis with diabetes.

    PubMed

    Greenhalgh, Trisha; Chowdhury, Mu'min; Wood, Gary W

    2006-11-01

    Questionnaires that measure subjective health status are increasingly used in clinical trials. But scales based on the quantification of subjective traits ("rate your feelings on a scale of 1 to 5") and initially developed in western population samples may not be valid for use in minority ethnic groups, even if accurately translated. The measurement of cultural adaptation and assimilation in immigrant groups is important for health research but has well documented methodological challenges. The aim of this study was to develop valid and reliable questionnaires to measure subjective health status and cultural adherence in a minority ethnic group, using the story as the unit of inquiry. The design was a multi-phase study involving (a) narrative interview, (b) vignette construction, (c) questionnaire development, and (d) questionnaire validation in relation to two scales (well-being and cultural adherence) in British Bangladeshis with diabetes. Using data from in-depth narrative interviews (i.e., a non-directive research technique in which the participant is invited to "tell me the story about your diabetes, starting with when you first noticed anything wrong", and the only prompts used are "tell me more about that" or "what happened next?"; Greenhalgh, Helman, & Chowdhury, 1998; Muller, 1999), we constructed culturally congruent vignettes to depict different subjective health states and behaviours. We refined these items in focus group interviews and validated the instruments on 98 Bangladeshi participants, randomly sampled from GP diabetes registers in inner London and interviewed by a Bangladeshi anthropologist. We used factor analysis to explore the underlying structure in the responses to questionnaire items, plus Cronbach alpha tests to measure internal consistency of scales. The questionnaires were acceptable and credible to Bangladeshi participants with diabetes. Ninety of 98 participants were able and willing to complete them with interviewer assistance

  10. Arsenite maintains germinative state in cultured human epidermal cells

    SciTech Connect

    Patterson, Timothy J.; Reznikova, Tatiana V.; Phillips, Marjorie A.; Rice, Robert H. . E-mail: rhrice@ucdavis.edu

    2005-08-22

    Arsenic is a well-known carcinogen for human skin, but its mechanism of action and proximal macromolecular targets remain to be elucidated. In the present study, low micromolar concentrations of sodium arsenite maintained the proliferative potential of epidermal keratinocytes, decreasing their exit from the germinative compartment under conditions that promote differentiation of untreated cells. This effect was observed in suspension and in post-confluent surface cultures as measured by colony-forming ability and by proportion of rapidly adhering colony-forming cells. Arsenite-treated cultures exhibited elevated levels of {beta}1-integrin and {beta}-catenin, two proteins enriched in cells with high proliferative potential. Levels of phosphorylated (inactive) glycogen synthase kinase 3{beta} were higher in the treated cultures, likely accounting for the increased levels of transcriptionally available {beta}-catenin. These findings suggest that arsenic could have co-carcinogenic and tumor co-promoting activities in the epidermis as a result of increasing the population and persistence of germinative cells targeted by tumor initiators and promoters. These findings also identify a critical signal transduction pathway meriting further exploration in pursuit of this phenomenon.

  11. Different Use of Cell Surface Glycosaminoglycans As Adherence Receptors to Corneal Cells by Gram Positive and Gram Negative Pathogens

    PubMed Central

    García, Beatriz; Merayo-Lloves, Jesús; Rodríguez, David; Alcalde, Ignacio; García-Suárez, Olivia; Alfonso, José F.; Baamonde, Begoña; Fernández-Vega, Andrés; Vazquez, Fernando; Quirós, Luis M.

    2016-01-01

    The epithelium of the cornea is continuously exposed to pathogens, and adhesion to epithelial cells is regarded as an essential first step in bacterial pathogenesis. In this article, the involvement of glycosaminoglycans in the adhesion of various pathogenic bacteria to corneal epithelial cells is analyzed. All microorganisms use glycosaminoglycans as receptors, but arranged in different patterns depending on the Gram-type of the bacterium. The heparan sulfate chains of syndecans are the main receptors, though other molecular species also seem to be involved, particularly in Gram-negative bacteria. Adherence is inhibited differentially by peptides, including heparin binding sequences, indicating the participation of various groups of Gram-positive, and -negative adhesins. The length of the saccharides produces a major effect, and low molecular weight chains inhibit the binding of Gram-negative microorganisms but increase the adherence of Gram-positives. Pathogen adhesion appears to occur preferentially through sulfated domains, and is very dependent on N- and 6-O-sulfation of the glucosamine residue and, to a lesser extent, 2-O sulfation of uronic acid. These data show the differential use of corneal receptors, which could facilitate the development of new anti-infective strategies. PMID:27965938

  12. Agrobacterium tumefaciens Interaction with Suspension-Cultured Tomato Cells 1

    PubMed Central

    Neff, Nicola T.; Binns, Andrew N.

    1985-01-01

    Adherence of Agrobacterium tumefaciens to suspension-cultured tomato cells has been characterized using a quantitative binding assay. Saturable binding of radiolabeled A. tumefaciens to plant cells resulted in 100 to 300 bacteria bound per cell. Specificity of A. tumefaciens binding was also inferred from two additional results: (a) an initial incubation of plant cells with A. tumefaciens reduced subsequent binding of radiolabeled A. tumefaciens by 60% to 75%; (b) tomato cells bound less than three E. coli per cell. Protease treatment of plant cells had no effect on subsequent bacterial binding, but prior treatment of plant cells with pectinolytic enzymes increased binding 2- to 3-fold. Pectin-enriched and neutral polymer-enriched fractions were obtained from tomato cell walls. The soluble pectin-enriched fraction inhibited binding of bacteria to plant cells by 85% to 95%, whereas the neutral polymer fraction only partially inhibited binding. Preliminary characterization of the activity showed it is heat stable, partially inactivated by protease treatment, and substantially inactivated by acid hydrolysis. Images Fig. 2 PMID:16664024

  13. Curli Temper Adherence of Escherichia coli O157:H7 to Squamous Epithelial Cells from the Bovine Recto-Anal Junction in a Strain-Dependent Manner.

    PubMed

    Kudva, Indira T; Carter, Michelle Q; Sharma, Vijay K; Stasko, Judith A; Giron, Jorge A

    2017-01-01

    Our recent studies have shown that intimin and the locus of enterocyte effacement-encoded proteins do not play a role in Escherichia coli O157:H7 (O157) adherence to the bovine recto-anal junction squamous epithelial (RSE) cells. To define factors that play a contributory role, we investigated the role of curli, fimbrial adhesins commonly implicated in adherence to various fomites and plant and human epithelial cells, in O157 adherence to RSE cells. Specifically, we examined (i) wild-type strains of O157; (ii) curli variants of O157 strains; (iii) isogenic curli deletion mutants of O157; and (iv) adherence inhibition of O157 using anti-curlin sera. Results of these experiments conducted under stringent conditions suggest that curli do not solely contribute to O157 adherence to RSE cells and in fact demonstrate a modulating effect on O157 adherence to RSE cells in contrast to HEp-2 cells (human epidermoid carcinoma of the larynx cells with HeLa contamination). The absence of curli and presence of blocking anti-curli antibodies enhanced O157-RSE cell interactions among some strains, thus alluding to a spatial, tempering effect of curli on O157 adherence to RSE cells when present. At the same time, the presence or absence of curli did not alter RSE cell adherence patterns of another O157 strain. These observations are at variance with the reported role of curli in O157 adherence to human cell lines such as HEp-2 and need to be factored in when developing anti-adherence modalities for preharvest control of O157 in cattle.

  14. Metabolomics of adherent mammalian cells by capillary electrophoresis-mass spectrometry: HT-29 cells as case study.

    PubMed

    Ibáñez, Clara; Simó, Carolina; Valdés, Alberto; Campone, Luca; Piccinelli, Anna Lisa; García-Cañas, Virginia; Cifuentes, Alejandro

    2015-06-10

    In this work, the optimization of an effective protocol for cell metabolomics is described with special emphasis in the sample preparation and subsequent analysis of intracellular metabolites from adherent mammalian cells by capillary electrophoresis-mass spectrometry. As case study, colon cancer HT-29 cells, a human cell model to investigate colon cancer, are employed. The feasibility of the whole method for cell metabolomics is demonstrated via a fast and sensitive profiling of the intracellular metabolites HT-29 cells by capillary electrophoresis-time-of-flight mass spectrometry (CE-TOF MS). The suitability of this methodology is further corroborated through the examination of the metabolic changes in the polyamines pathway produced in colon cancer HT-29 cells by difluoromethylornithine (DFMO), a known potent ornithine decarboxylase inhibitor. The selection of the optimum extraction conditions allowed a higher sample volume injection that led to an increase in CE-TOF MS sensitivity. Following a non-targeted metabolomics approach, 10 metabolites (namely, putrescine, ornithine, gamma-aminobutyric acid (GABA), oxidized and reduced glutathione, 5'-deoxy-5'-(methylthio)adenosine, N-acetylputrescine, cysteinyl-glycine, spermidine and an unknown compound) were found to be significantly altered by DFMO (p<0.05) in HT-29 cells. In addition to the effect of DFMO on polyamine metabolism, minor modifications of other metabolic pathways (e.g., related to intracellular thiol redox state) were observed.

  15. QID74 Cell wall protein of Trichoderma harzianum is involved in cell protection and adherence to hydrophobic surfaces.

    PubMed

    Rosado, Iván V; Rey, Manuel; Codón, Antonio C; Govantes, Javier; Moreno-Mateos, Miguel A; Benítez, Tahía

    2007-10-01

    Trichoderma is widely used as biocontrol agent against phytopathogenic fungi, and as biofertilizer because of its ability to establish mycorriza-like association with plants. The key factor to the ecological success of this genus is the combination of very active mycoparasitic mechanisms plus effective defense strategies induced in plants. This work, different from most of the studies carried out that address the attacking mechanisms, focuses on elucidating how Trichoderma is able to tolerate hostile conditions. A gene from Trichoderma harzianum CECT 2413, qid74, was strongly expressed during starvation of carbon or nitrogen sources; it encoded a cell wall protein of 74kDa that plays a significant role in mycelium protection. qid74 was originally isolated and characterized, in a previous work, by a differential hybridization approach under simulated mycoparasitism conditions. Heterologous expression of Qid74 in Saccharomyces cerevisiae indicated that the protein, located in the cell wall, interfered with mating and sporulation but not with cell integrity. The qid74 gene was disrupted by homologous recombination and it was overexpressed by isolating transformants selected for the amdS gene that carried several copies of qid74 gene under the control of the pki promoter. Disruptants and transformants showed similar growth rate and viability when they were cultivated in different media, temperatures and osmolarities, or were subjected to different abiotic stress conditions. However, disruptants produced about 70% mass yield under any condition and were substantially more sensitive than the wild type to cell wall degradation by different lytic preparations. Transformants had similar mass yield and were more resistant to lytic enzymes but more sensitive to copper sulfate than the wild type. When experiments of adherence to hydrophobic surfaces were carried out, the disruptants had a reduced capacity to adhere, whereas that capacity in the overproducer transformants was

  16. Single molecule microscopy in 3D cell cultures and tissues.

    PubMed

    Lauer, Florian M; Kaemmerer, Elke; Meckel, Tobias

    2014-12-15

    From the onset of the first microscopic visualization of single fluorescent molecules in living cells at the beginning of this century, to the present, almost routine application of single molecule microscopy, the method has well-proven its ability to contribute unmatched detailed insight into the heterogeneous and dynamic molecular world life is composed of. Except for investigations on bacteria and yeast, almost the entire story of success is based on studies on adherent mammalian 2D cell cultures. However, despite this continuous progress, the technique was not able to keep pace with the move of the cell biology community to adapt 3D cell culture models for basic research, regenerative medicine, or drug development and screening. In this review, we will summarize the progress, which only recently allowed for the application of single molecule microscopy to 3D cell systems and give an overview of the technical advances that led to it. While initially posing a challenge, we finally conclude that relevant 3D cell models will become an integral part of the on-going success of single molecule microscopy.

  17. Cell-Culture Reactor Having a Porous Organic Polymer Membrane

    NASA Technical Reports Server (NTRS)

    Koontz, Steven L. (Inventor)

    2000-01-01

    A method for making a biocompatible polymer article using a uniform atomic oxygen treatment is disclosed. The substrate may be subsequently optionally grated with a compatibilizing compound. Compatibilizing compounds may include proteins, phosphory1choline groups, platelet adhesion preventing polymers, albumin adhesion promoters, and the like. The compatibilized substrate may also have a living cell layer adhered thereto. The atomic oxygen is preferably produced by a flowing afterglow microwave discharge, wherein the substrate resides in a sidearm out of the plasma. Also, methods for culturing cells for various purposes using the various membranes are disclosed as well. Also disclosed are porous organic polymers having a distributed pore chemistry (DPC) comprising hydrophilic and hydrophobic regions, and a method for making the DPC by exposing the polymer to atomic oxygen wherein the rate of hydrophilization is greater than the rate of mass loss.

  18. Culture of Cells from Amphibian Embryos.

    ERIC Educational Resources Information Center

    Stanisstreet, Martin

    1983-01-01

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

  19. Exosome Adherence and Internalization by Hepatic Stellate Cells Triggers Sphingosine 1-Phosphate-dependent Migration.

    PubMed

    Wang, Ruisi; Ding, Qian; Yaqoob, Usman; de Assuncao, Thiago M; Verma, Vikas K; Hirsova, Petra; Cao, Sheng; Mukhopadhyay, Debabrata; Huebert, Robert C; Shah, Vijay H

    2015-12-25

    Exosomes are cell-derived extracellular vesicles thought to promote intercellular communication by delivering specific content to target cells. The aim of this study was to determine whether endothelial cell (EC)-derived exosomes could regulate the phenotype of hepatic stellate cells (HSCs). Initial microarray studies showed that fibroblast growth factor 2 induced a 2.4-fold increase in mRNA levels of sphingosine kinase 1 (SK1). Exosomes derived from an SK1-overexpressing EC line increased HSC migration 3.2-fold. Migration was not conferred by the dominant negative SK1 exosome. Incubation of HSCs with exosomes was also associated with an 8.3-fold increase in phosphorylation of AKT and 2.5-fold increase in migration. Exosomes were found to express the matrix protein and integrin ligand fibronectin (FN) by Western blot analysis and transmission electron microscopy. Blockade of the FN-integrin interaction with a CD29 neutralizing antibody or the RGD peptide attenuated exosome-induced HSC AKT phosphorylation and migration. Inhibition of endocytosis with transfection of dynamin siRNA, the dominant negative dynamin GTPase construct Dyn2K44A, or the pharmacological inhibitor Dynasore significantly attenuated exosome-induced AKT phosphorylation. SK1 levels were increased in serum exosomes derived from mice with experimental liver fibrosis, and SK1 mRNA levels were up-regulated 2.5-fold in human liver cirrhosis patient samples. Finally, S1PR2 inhibition protected mice from CCl4-induced liver fibrosis. Therefore, EC-derived SK1-containing exosomes regulate HSC signaling and migration through FN-integrin-dependent exosome adherence and dynamin-dependent exosome internalization. These findings advance our understanding of EC/HSC cross-talk and identify exosomes as a potential target to attenuate pathobiology signals.

  20. Exosome Adherence and Internalization by Hepatic Stellate Cells Triggers Sphingosine 1-Phosphate-dependent Migration*

    PubMed Central

    Wang, Ruisi; Ding, Qian; Yaqoob, Usman; de Assuncao, Thiago M.; Verma, Vikas K.; Hirsova, Petra; Cao, Sheng; Mukhopadhyay, Debabrata; Huebert, Robert C.; Shah, Vijay H.

    2015-01-01

    Exosomes are cell-derived extracellular vesicles thought to promote intercellular communication by delivering specific content to target cells. The aim of this study was to determine whether endothelial cell (EC)-derived exosomes could regulate the phenotype of hepatic stellate cells (HSCs). Initial microarray studies showed that fibroblast growth factor 2 induced a 2.4-fold increase in mRNA levels of sphingosine kinase 1 (SK1). Exosomes derived from an SK1-overexpressing EC line increased HSC migration 3.2-fold. Migration was not conferred by the dominant negative SK1 exosome. Incubation of HSCs with exosomes was also associated with an 8.3-fold increase in phosphorylation of AKT and 2.5-fold increase in migration. Exosomes were found to express the matrix protein and integrin ligand fibronectin (FN) by Western blot analysis and transmission electron microscopy. Blockade of the FN-integrin interaction with a CD29 neutralizing antibody or the RGD peptide attenuated exosome-induced HSC AKT phosphorylation and migration. Inhibition of endocytosis with transfection of dynamin siRNA, the dominant negative dynamin GTPase construct Dyn2K44A, or the pharmacological inhibitor Dynasore significantly attenuated exosome-induced AKT phosphorylation. SK1 levels were increased in serum exosomes derived from mice with experimental liver fibrosis, and SK1 mRNA levels were up-regulated 2.5-fold in human liver cirrhosis patient samples. Finally, S1PR2 inhibition protected mice from CCl4-induced liver fibrosis. Therefore, EC-derived SK1-containing exosomes regulate HSC signaling and migration through FN-integrin-dependent exosome adherence and dynamin-dependent exosome internalization. These findings advance our understanding of EC/HSC cross-talk and identify exosomes as a potential target to attenuate pathobiology signals. PMID:26534962

  1. Evaluation of a Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) assay (SOT)

    EPA Science Inventory

    The Embryonic Stem Cell Test (EST) has been used to evaluate the effects of xenobiotics using three endpoints, stem cell differentiation, stem cell viability and 3T3-cell viability. Our research goal is to establish amodel system that would evaluate chemical effects using a singl...

  2. Actinobacillus actinomycetemcomitans adheres to human gingival fibroblasts and modifies cytoskeletal organization.

    PubMed

    Gutiérrez-Venegas, Gloria; Kawasaki-Cárdenas, Perla; Garcés, Carla Portillo; Román-Alvárez, Patricia; Barajas-Torres, Carolina; Contreras-Marmolejo, Luis Arturo

    2007-09-01

    Adherence of Actinobacillus actinomycetemcomitans to human gingival fibroblast cells induces cytoskeletal reorganization. A. actinomycetemcomitans is considered a pathogenic bacteria involved in localized aggressive periodontitis. Studies with epithelial cells have shown an adherent capacity of bacteria that is increased under anaerobic conditions. For adherence to take place, there is a need for interaction between extracellular vesicles and bacterial fimbriae. However, molecular events associated with the adherence process are still unknown. The aim of this study was to investigate whether A. actinomycetemcomitans adherence to human gingival fibroblasts promotes cytoskeletal reorganization. Adherence was determined with light microscopy and scanning electron microscopy. For F-actin visualization, cells were treated with fluorescein-isothiocyanate-phalloidin and samples were examined with epifluorescence optics. Fluorescent was recorded on Kodak T-Max 400 film. We showed that A. actinomycetemcomitans adheres to human gingival fibroblast primary cultures, this property stimulating an increase in the intracellular calcium levels. In human gingival fibroblast primary cultures, we observed that maximal A. actinomycetemcomitans adherence took place 1.5h after culture infection occurred and remained for 6h. The adherence was associated with morphologic alterations and an increased in the intracellular calcium levels. These experiments suggest that A. actinomycetemcomitans adherence cause morphological alterations, induce actin stress fibers and recruitment of intracellular calcium levels.

  3. The Role of Fibronectin in the Adherence and Inflammatory Response Induced by Enteroaggregative Escherichia coli on Epithelial Cells

    PubMed Central

    Yáñez, Dominique; Izquierdo, Mariana; Ruiz-Perez, Fernando; Nataro, James P.; Girón, Jorge A.; Vidal, Roberto M.; Farfan, Mauricio J.

    2016-01-01

    Enteroaggregative Escherichia coli (EAEC) infections are still one of the most important etiologic pathogens of diarrhea in children worldwide. EAEC pathogenesis comprises three stages: adherence and colonization, production of toxins, and diarrhea followed by inflammation. Previous studies have demonstrated that EAEC strains have the ability to bind to fibronectin (FN); however, the role this extracellular matrix protein plays in the inflammatory response induced by EAEC remains unknown. In this study, we postulated that FN-mediated adherence of EAEC strains to epithelial cells increases the expression of pro-inflammatory genes. To verify this hypothesis, we infected HEp-2 and HT-29 cells, in both the presence and absence of FN, with EAEC reference strain 042. We quantified IL-8 secretion and the relative expression of a set of genes regulated by the NF-κB pathway. Although FN increased EAEC adherence, no changes in IL-8 protein secretion or IL8 gene expression were observed. Similar observations were found in HEp-2 cells transfected with FN-siRNA and infected with EAEC. To evaluate the involvement of AAF/II fimbriae, we infected HEp-2 and HT-29 cells, in both the presence and absence of FN, with an EAEC 042aafA mutant strain transformed with a plasmid harboring the native aafA gene with a site-directed mutation in Lys72 residue (K72A and K72R strains). No changes in IL-8 secretion were observed. Finally, SEM immunogold assay of cells incubated with FN and infected with EAEC revealed that AAF fimbriae can bind to cells either directly or mediated by FN. Our data suggests that FN participates in AAF/II fimbriae-mediated adherence of EAEC to epithelial cells, but not in the inflammatory response of cells infected by this pathogen. PMID:28008386

  4. Progesterone metabolism in cultured amniotic fluid cells.

    PubMed

    Beling, C G; Cederqvist, L L

    1978-01-01

    Amniotic fluid cells obtained by amnicentesis at 16-20 weeks' gestation were grown in culture until a confluent monolayer of cell had been formed. Radiolabeled pregnenolone, progesterone and 20 alpha-dihydroprogesterone were added to the cell cultures; steroid metabolites which formed after 24 and 48 hours of incubation were identified. Incubation of the cell cultures with pregnenolone-3H resulted in the formation of progesterone, 17alpha-progesterone and 20 alpha-dihydroprogesterone. A significant amount of progesterone was identified after incubating the cell cultures with 20 alpha-dihydroprogesterone. The results indicate that 3 beta-ol-dehydrogenase, 17 alpha-hydroxylase and 20 alpha-hydroxysteroid dehydrogenase enzymes are present in cultured amniotic fluid cells obtained at 16-20 weeks' gestation.

  5. CD271 as a marker to identify mesenchymal stem cells from diverse sources before culture

    PubMed Central

    Álvarez-Viejo, María; Menéndez-Menéndez, Yolanda; Otero-Hernández, Jesús

    2015-01-01

    Mesenchymal stem cells, due to their characteristics are ideal candidates for cellular therapy. Currently, in culture these cells are defined by their adherence to plastic, specific surface antigen expression and multipotent differentiation potential. However, the in vivo identification of mesenchymal stem cells, before culture, is not so well established. Pre-culture identification markers would ensure higher purity than that obtained with selection based on adherence to plastic. Up until now, CD271 has been described as the most specific marker for the characterization and purification of human bone marrow mesenchymal stem cells. This marker has been shown to be specifically expressed by these cells. Thus, CD271 has been proposed as a versatile marker to selectively isolated and expand multipotent mesenchymal stem cells with both immunosuppressive and lymphohematopoietic engraftment-promoting properties. This review focuses on this marker, specifically on identification of mesenchymal stem cells from different tissues. Literature revision suggests that CD271 should not be defined as a universal marker to identify mesenchymal stem cells before culture from different sources. In the case of bone marrow or adipose tissue, CD271 could be considered a quite suitable marker; however this marker seems to be inadequate for the isolation of mesenchymal stem cells from other tissues such as umbilical cord blood or wharton’s jelly among others. PMID:25815130

  6. Passive control of cell locomotion using micropatterns: the effect of micropattern geometry on the migratory behavior of adherent cells.

    PubMed

    Yoon, Sang-Hee; Kim, Young Kyun; Han, Eui Don; Seo, Young-Ho; Kim, Byeong Hee; Mofrad, Mohammad R K

    2012-07-07

    Directed cell migration is critical to a variety of biological and physiological processes. Although simple topographical patterns such as parallel grooves and three-dimensional post arrays have been studied to guide cell migration, the effect of the dimensions and shape of micropatterns, which respectively represent the amount and gradient of physical spatial cues, on cell migration has not yet been fully explored. This motivates a quantitative characterization of cell migration in response to micropatterns having different widths and divergence angles. The changes in the migratory (and even locational) behavior of adherent cells, when the cells are exposed to physical spatial cues imposed by the micropatterns, are explored here using a microfabricated biological platform, nicknamed the "Rome platform". The Rome platform, made of a biocompatible, ultraviolet (UV) curable polymer (ORMOCOMP), consists of 3 μm thick micropatterns with different widths of 3 to 75 μm and different divergence angles of 0.5 to 5.0°. The migration paths through which NIH 3T3 fibroblasts move on the micropatterns are analyzed with a persistent random walk model, thus quantifying the effect of the divergence angle of micropatterns on cell migratory characteristics such as cell migration speed, directional persistence time, and random motility coefficient. The effect of the width of micropatterns on cell migratory characteristics is also extensively investigated. Cell migration direction is manipulated by creating the gradient of physical spatial cues (that is, divergence angle of micropatterns), while cell migration speed is controlled by modulating the amount of them (namely, width of micropatterns). In short, the amount and gradient of physical spatial cues imposed by changing the width and divergence angle of micropatterns make it possible to control the rate and direction of cell migration in a passive way. These results offer a potential for reducing the healing time of open wounds

  7. Embryonic Stem Cells: Isolation, Characterization and Culture

    NASA Astrophysics Data System (ADS)

    Amit, Michal; Itskovitz-Eldor, Joseph

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

  8. Degradation of heparin proteoglycan in cultured mouse mastocytoma cells.

    PubMed Central

    Jacobsson, K G; Lindahl, U

    1987-01-01

    Pulse-labelling of mouse mastocytoma cell cultures, established from ascites fluid, with inorganic [35S]sulphate for 1 h yielded labelled heparin proteoglycan containing polysaccharide chains of Mr 60,000-100,000. After chase incubation for 24 h most of the 35S appeared in intracellular polysaccharide fragments similar in size to commercially available heparin, Mr 5000-25,000, as indicated by gel chromatography. Products isolated from cultures after 6 h of chase incubation consisted of partially degraded free polysaccharide chains and, in addition, residual proteoglycans that were of smaller size than the proteoglycans initially pulse-labelled. The polysaccharide chains released by alkali treatment from the residual chase-incubated proteoglycans were of the same size as the chains derived from proteoglycans after 1 h of pulse labelling. These results suggest that the intracellular degradation of heparin proteoglycan to polysaccharide fragments is initiated by release of intact polysaccharide chains, probably by action of a peptidase, and is pursued through cleavage of these chains by an endoglycosidase. An endoglucuronidase with stringent substrate specificity [Thunberg, Bäckström, Wasteson, Ogren & Lindahl (1982) J. Biol. Chem. 257, 10278-10282] has previously been implicated in the latter step. Cultures of more purified mastocytoma cells (essentially devoid of macrophages) did not metabolize [35S]heparin proteoglycan to polysaccharide fragments, but instead accumulated free intact polysaccharide chains, i.e. the postulated intermediate of the complete degradation pathway. When such purified cells were co-cultured with adherent mouse peritoneal cells, presumably macrophages, formation of polysaccharide fragments was observed. It is tentatively proposed that the expression of endoglucuronidase activity by the mast cells depends on collaboration between these cells and macrophages. PMID:3120695

  9. Comparative adherence of Candida albicans and Candida dubliniensis to human buccal epithelial cells and extracellular matrix proteins.

    PubMed

    Jordan, Rachael P C; Williams, David W; Moran, Gary P; Coleman, David C; Sullivan, Derek J

    2014-04-01

    Candida albicans and Candida dubliniensis are very closely related pathogenic yeast species. Despite their close relationship, C. albicans is a far more successful colonizer and pathogen of humans. The purpose of this study was to determine if the disparity in the virulence of the two species is attributed to differences in their ability to adhere to human buccal epithelial cells (BECs) and/or extracellular matrix proteins. When grown overnight at 30°C in yeast extract peptone dextrose, genotype 1 C. dubliniensis isolates were found to be significantly more adherent to human BECs than C. albicans or C. dubliniensis genotypes 2-4 (P < 0.001). However, when the yeast cells were grown at 37°C, no significant difference between the adhesion of C. dubliniensis genotype 1 and C. albicans to human BECs was observed, and C. dubliniensis genotype 1 and C. albicans adhered to BECs in significantly greater numbers than the other C. dubliniensis genotypes (P < 0.001). Using surface plasmon resonance analysis, C. dubliniensis isolates were found to adhere in significantly greater numbers than C. albicans to type I and IV collagen, fibronectin, laminin, vitronectin, and proline-rich peptides. These data suggest that C. albicans is not more adherent to epithelial cells or matrix proteins than C. dubliniensis and therefore other factors must contribute to the greater levels of virulence exhibited by C. albicans.

  10. Correlative VIS-fluorescence and soft X-ray cryo-microscopy/tomography of adherent cells

    PubMed Central

    Hagen, Christoph; Guttmann, Peter; Klupp, Barbara; Werner, Stephan; Rehbein, Stefan; Mettenleiter, Thomas C.; Schneider, Gerd; Grünewald, Kay

    2012-01-01

    Soft X-ray cryo-microscopy/tomography of vitreous samples is becoming a valuable tool in structural cell biology. Within the ‘water-window’ wavelength region (2.34–4.37 nm), it provides absorption contrast images with high signal to noise ratio and resolution of a few tens of nanometer. Soft X-rays with wavelengths close to the K-absorption edge of oxygen penetrate biological samples with thicknesses in the micrometer range. Here, we report on the application of a recently established extension of the transmission soft X-ray cryo-microscope (HZB TXM) at the beamline U41-XM of the BESSY II electron storage ring by an in-column epi-fluorescence and reflected light cryo-microscope. We demonstrate the new capability for correlative fluorescence and soft X-ray cryo-microscopy/tomography of this instrument along a typical life science experimental approach – the correlation of a fluorophore-tagged protein (pUL34-GFP of pseudorabies virus, PrV, the nuclear membrane-anchored component of the nuclear egress complex of the Herpesviridae which interacts with viral pUL31) in PrV pUL34-GFP/pUL31 coexpressing mammalian cells, with virus-induced vesicular structures in the nucleus, expanding the nucleoplasmic reticulum. Taken together, our results demonstrate new possibilities to study the role of specific proteins in substructures of adherent cells, especially of the nucleus in toto, accessible to electron microscopy in thinned samples only. PMID:22210307

  11. Culture medium refinement by dialysis for the expansion of human induced pluripotent stem cells in suspension culture.

    PubMed

    Nath, Suman Chandra; Nagamori, Eiji; Horie, Masanobu; Kino-Oka, Masahiro

    2017-01-01

    Human induced pluripotent stem cells (hiPSCs) secrete essential autocrine factors that are removed along with toxic metabolites when the growth medium is exchanged daily. In this study, after determining the minimum inhibitory level of lactic acid for hiPSCs, a medium refining system was constructed by which toxic metabolites were removed from used culture medium and autocrine factors as well as other growth factors were recycled. Specifically, about 87 % of the basic fibroblast growth factor and 80 % of transforming growth factor beta 1 were retained in the refined medium after dialysis. The refined medium efficiently potentiated the proliferation of hiPS cells in adherent culture. When the refining system was used to refresh medium in suspension culture, a final cell density of (1.1 ± 0.1) × 10(6) cells mL(-1) was obtained, with 99.5 ± 0.2 % OCT 3/4 and 78.3 ± 1.1 % TRA-1-60 expression, on day 4 of culture. These levels of expression were similar to those observed in the conventional suspension culture. With this method, culture medium refinement by dialysis was established to remove toxic metabolites, recycle autocrine factors as well as other growth factors, and reduce the use of macromolecules for the expansion of hiPSCs in suspension culture.

  12. Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) assay

    EPA Science Inventory

    The Embryonic Stem Cell Test (EST) is an assay which evaluates xenobiotic-induced effects using three endpoints: mouse embryonic stem cell (mESC) differentiation, mESC viability, and 3T3-cell viability. Our research goal was to develop an improved high-throughput assay by establi...

  13. Scalable Culture and Cryopreservation of Human Embryonic Stem Cells on Microcarriers

    PubMed Central

    Nie, Ying; Bergendahl, Veit; Hei, Derek J.; Jones, Jeffrey M.; Palecek, Sean P.

    2009-01-01

    As a result of their pluripotency and potential for unlimited self-renewal, human embryonic stem cells (hESCs) hold tremendous promise in regenerative medicine. An essential prerequisite for the widespread application of hESCs is the establishment of effective and efficient protocols for large-scale cell culture, storage, and distribution. At laboratory scales hESCs are cultured adherent to tissue culture plates; these culture techniques are labor-intensive and do not scale to high cell numbers. In an effort to facilitate larger scale hESC cultivation, we investigated the feasibility of culturing hESCs adherent to microcarriers. We modified the surface of Cytodex 3 microcarriers with either Matrigel or mouse embryonic fibroblasts (MEFs). hESC colonies were effectively expanded in a pluripotent, undifferentiated state on both Matrigel-coated microcarriers and microcarriers seeded with a MEF monolayer. While the hESC expansion rate on MEF-microcarriers was less than that on MEF-plates, the doubling time of hESCs on Matrigel-microcarriers was indistinguishable from that of hESCs expanded on Matrigel-coated tissue culture plates. Standard hESC cryopreservation methodologies are plagued by poor viability and high differentiation rates upon thawing. Here, we demonstrate that cryopreservation of hESCs adherent to microcarriers in cryovials provides a higher recovery of undifferentiated cells than cryopreservation of cells in suspension. Together, these results suggest that microcarrier-based stabilization and culture may facilitate hESC expansion and storage for research and therapeutic applications. PMID:19197994

  14. Serum-Free Suspension Culture of MDCK Cells for Production of Influenza H1N1 Vaccines.

    PubMed

    Huang, Ding; Peng, Wen-Juan; Ye, Qian; Liu, Xu-Ping; Zhao, Liang; Fan, Li; Xia-Hou, Kang; Jia, Han-Jing; Luo, Jian; Zhou, Lin-Ting; Li, Bei-Bei; Wang, Shi-Lei; Xu, Wen-Ting; Chen, Ze; Tan, Wen-Song

    2015-01-01

    Development of serum-free suspension cell culture processes is very important for influenza vaccine production. Previously, we developed a MDCK suspension cell line in a serum-free medium. In the present study, the growth kinetics of suspension MDCK cells and influenza virus production in the serum-free medium were investigated, in comparison with those of adherent MDCK cells in both serum-containing and serum-free medium. It was found that the serum-free medium supported the stable subculture and growth of both adherent and suspension cells. In batch culture, for both cell lines, the growth kinetics in the serum-free medium was comparable with those in the serum-containing medium and a commercialized serum-free medium. In the serum-free medium, peak viable cell density (VCD), haemagglutinin (HA) and median tissue culture infective dose (TCID50) titers of the two cell lines reached 4.51×106 cells/mL, 2.94Log10(HAU/50 μL) and 8.49Log10(virions/mL), and 5.97×106 cells/mL, 3.88Log10(HAU/50 μL), and 10.34Log10(virions/mL), respectively. While virus yield of adherent cells in the serum-free medium was similar to that in the serum-containing medium, suspension culture in the serum-free medium showed a higher virus yield than adherent cells in the serum-containing medium and suspension cells in the commercialized serum-free medium. However, the percentage of infectious viruses was lower for suspension culture in the serum-free medium. These results demonstrate the great potential of this suspension MDCK cell line in serum-free medium for influenza vaccine production and further improvements are warranted.

  15. Analysis of the Adherence of Dental Pulp Stem Cells on Two-Dimensional and Three-Dimensional Silk Fibroin-Based Biomaterials: Applications in Regenerative Dentistry.

    PubMed

    Pecci-Lloret, María Pilar; Vera-Sánchez, Mar; Aznar-Cervantes, Salvador; García-Bernal, David; Sánchez, Ricardo Oñate; Pecci-Lloret, Miguel Ramón; Moraleda, José María; Cenis, José Luis; Rodríguez-Lozano, Francisco Javier

    2017-02-22

    Among various biomaterials used as scaffolds in tissue engineering, silk fibroin is a highly attractive material. A scaffold should be biocompatible and nontoxic, with optimal physical features and mechanical properties. For this reason, tissue-engineering approaches in regenerative medicine have focused on investigating the biocompatibility of possible biomaterials by analyzing cell-scaffold interaction properties. The aim of the present study was to examine the biocompatibility of silk fibroin as a film (two-dimensional [2D]) and a scaffold (three-dimensional [3D]) after being cellularized with human dental pulp stem cells (hDPSCs). Human dental pulp stem cells were isolated from healthy patients aged between 18 and 31 years. Further, silk fibroin-based 2D films and 3D scaffolds were prepared. Human dental pulp stem cells were directly seeded onto the biomaterial surfaces and their proliferation, adherence, and cell morphology were analyzed after 24, 120, and 168 hours. Additionally, the characteristics of the silk fibroin 2D films and 3D scaffolds before and after cell seeding were analyzed by scanning electron microscopy. After the initial 24 hours, silk fibroin-based 3D scaffolds displayed more adhered cells with a suitable fibroblastic morphology than those displayed on the 2D films. After longer culture times, hDPSCs proliferated sufficiently to cover the entire surface of the 3D silk fibroin scaffold, whereas the 2D films were only partially covered. Our results indicate the good in vitro biocompatibility of silk fibroin-based biomaterials, especially when 3D scaffolds rather than 2D films are used.

  16. Effects of enamel matrix proteins on adherence, proliferation and migration of epithelial cells: A real-time in vitro study

    PubMed Central

    Wyganowska-Swiatkowska, Marzena; Urbaniak, Paulina; Lipinski, Daniel; Szalata, Marlena; Borysiak, Karolina; Jakun, Jerzy; Kotwicka, Malgorzata

    2017-01-01

    Enamel matrix derivative (EMD) can mimic odontogenic effects by inducing the proliferation and differentiation of connective tissue progenitor cells, stimulating bone growth and arresting epithelial cells migration. To the best of our knowledge, there is no data indicating that any active component of EMD reduces epithelial cell viability. The present study examines the impact of commercial lyophilized EMD, porcine recombinant amelogenin (prAMEL; 21.3 kDa) and tyrosine-rich amelogenin peptide (TRAP) on the adherence, proliferation and migration of human epithelial cells in real-time. The tongue carcinoma cell line SCC-25 was stimulated with EMD, porcine recombinant AMEL and TRAP, at concentrations of 12.5, 25 and 50 µg/ml. Cell adherence, migration and proliferation were monitored in real-time using the xCELLigence system. No significant effects of EMD on the morphology, adhesion, proliferation and migration of SCC-25 cells were observed. However, porcine recombinant AMEL had a dose-dependent inhibitory effect on SCC-25 cell proliferation and migration. Predominantly, no notable differences were found between control and TRAP-treated cells in terms of cell adhesion and migration, a decrease in proliferation was observed, but this was not statistically significant. EMD and its active components do not increase the tongue cancer cell viability. PMID:28123485

  17. Effects of enamel matrix proteins on adherence, proliferation and migration of epithelial cells: A real-time in vitro study.

    PubMed

    Wyganowska-Swiatkowska, Marzena; Urbaniak, Paulina; Lipinski, Daniel; Szalata, Marlena; Borysiak, Karolina; Jakun, Jerzy; Kotwicka, Malgorzata

    2017-01-01

    Enamel matrix derivative (EMD) can mimic odontogenic effects by inducing the proliferation and differentiation of connective tissue progenitor cells, stimulating bone growth and arresting epithelial cells migration. To the best of our knowledge, there is no data indicating that any active component of EMD reduces epithelial cell viability. The present study examines the impact of commercial lyophilized EMD, porcine recombinant amelogenin (prAMEL; 21.3 kDa) and tyrosine-rich amelogenin peptide (TRAP) on the adherence, proliferation and migration of human epithelial cells in real-time. The tongue carcinoma cell line SCC-25 was stimulated with EMD, porcine recombinant AMEL and TRAP, at concentrations of 12.5, 25 and 50 µg/ml. Cell adherence, migration and proliferation were monitored in real-time using the xCELLigence system. No significant effects of EMD on the morphology, adhesion, proliferation and migration of SCC-25 cells were observed. However, porcine recombinant AMEL had a dose-dependent inhibitory effect on SCC-25 cell proliferation and migration. Predominantly, no notable differences were found between control and TRAP-treated cells in terms of cell adhesion and migration, a decrease in proliferation was observed, but this was not statistically significant. EMD and its active components do not increase the tongue cancer cell viability.

  18. Identification of Cell Surface-Exposed Proteins Involved in the Fimbria-Mediated Adherence of Enteroaggregative Escherichia coli to Intestinal Cells

    PubMed Central

    Izquierdo, Mariana; Navarro-Garcia, Fernando; Nava-Acosta, Raul; Nataro, James P.; Ruiz-Perez, Fernando

    2014-01-01

    Fimbria-mediated adherence to the intestinal epithelia is a key step in enteroaggregative Escherichia coli (EAEC) pathogenesis. To date, four fimbriae have been described for EAEC; aggregative adherence fimbria II (AAF/II) is the most important adherence factor for EAEC prototype strain 042. Previously, we described results showing that extracellular matrix (ECM) components might be involved in the recognition of AAF/II fimbriae by intestinal cells. In this study, we sought to identify novel potential receptors on intestinal epithelial cells recognized by the AAF/II fimbriae. Purified AafA-dsc protein, the major subunit of AAF/II fimbriae, was incubated with a monolayer of T84 cells, cross-linked to the surface-exposed T84 cell proteins, and immunoprecipitated by using anti-AafA antibodies. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of cellular proteins bound to AafA-dsc protein identified laminin (previously recognized as a potential receptor for AAF/II) and cytokeratin 8 (CK8). Involvement of the major subunit of AAF/II fimbriae (AafA protein) in the binding to recombinant CK8 was confirmed by adherence assays with purified AAF/II fimbriae, AafA-dsc protein, and strain 042. Moreover, HEp-2 cells transfected with CK8 small interfering RNA (siRNA) showed reduced 042 adherence compared with cells transfected with scrambled siRNA as a control. Adherence of 042 to HEp-2 cells preincubated with antibodies against ECM proteins or CK8 was substantially reduced. Altogether, our results supported the idea of a role of CK8 as a potential receptor for EAEC. PMID:24516112

  19. Primary Human Uterine Leiomyoma Cell Culture Quality Control: Some Properties of Myometrial Cells Cultured under Serum Deprivation Conditions in the Presence of Ovarian Steroids

    PubMed Central

    Sumikawa, Joana Tomomi; Batista, Fabrício Pereira; Paredes-Gamero, Edgar J.; Girão, Manoel J. B. C.; Oliva, Maria Luiza V.

    2016-01-01

    Cell culture is considered the standard media used in research to emulate the in vivo cell environment. Crucial in vivo experiments cannot be conducted in humans and depend on in vitro methodologies such as cell culture systems. However, some procedures involving the quality control of cells in culture have been gradually neglected by failing to acknowledge that primary cells and cell lines change over time in culture. Thus, we report methods based on our experience for monitoring primary cell culture of human myometrial cells derived from uterine leiomyoma. We standardized the best procedure of tissue dissociation required for the study of multiple genetic marker systems that include species-specific antigens, expression of myofibroblast or myoblast markers, growth curve, serum deprivation, starvation by cell cycle synchronization, culture on collagen coated plates, and 17 β-estradiol (E2) and progesterone (P4) effects. The results showed that primary myometrial cells from patients with uterine leiomyoma displayed myoblast phenotypes before and after in vitro cultivation, and leiomyoma cells differentiated into mature myocyte cells under the appropriate differentiation-inducing conditions (serum deprivation). These cells grew well on collagen coated plates and responded to E2 and P4, which may drive myometrial and leiomyoma cells to proliferate and adhere into a focal adhesion complex involvement in a paracrine manner. The establishment of these techniques as routine procedures will improve the understanding of the myometrial physiology and pathogenesis of myometrium-derived diseases such as leiomyoma. Mimicking the in vivo environment of fibrotic conditions can prevent false results and enhance results that are based on cell culture integrity. PMID:27391384

  20. Cryopreservation of adherent neuronal networks.

    PubMed

    Ma, Wu; O'Shaughnessy, Thomas; Chang, Eddie

    2006-07-31

    Neuronal networks have been widely used for neurophysiology, drug discovery and toxicity testing. An essential prerequisite for future widespread application of neuronal networks is the development of efficient cryopreservation protocols to facilitate their storage and transportation. Here is the first report on cryopreservation of mammalian adherent neuronal networks. Dissociated spinal cord cells were attached to a poly-d-lysine/laminin surface and allowed to form neuronal networks. Adherent neuronal networks were embedded in a thin film of collagen gel and loaded with trehalose prior to transfer to a freezing medium containing DMSO, FBS and culture medium. This was followed by a slow rate of cooling to -80 degrees C for 24 h and then storage for up to 2 months in liquid nitrogen at -196 degrees C. The three components: DMSO, collagen gel entrapment and trehalose loading combined provided the highest post-thaw viability, relative to individual or two component protocols. The post-thaw cells with this protocol demonstrated similar neuronal and astrocytic markers and morphological structure as those detected in unfrozen cells. Fluorescent dye FM1-43 staining revealed active recycling of synaptic vesicles upon depolarizing stimulation in the post-thaw neuronal networks. These results suggest that a combination of DMSO, collagen gel entrapment and trehalose loading can significantly improve conventional slow-cooling methods in cryopreservation of adherent neuronal networks.

  1. HUMAN VASCULAR ENDOTHELIAL CELLS IN CULTURE

    PubMed Central

    Gimbrone, Michael A.; Cotran, Ramzi S.; Folkman, Judah

    1974-01-01

    Human endothelial cells, obtained by collagenase treatment of term umbilical cord veins, were cultured using Medium 199 supplemented with 20% fetal calf serum. Small clusters of cells initially spread on plastic or glass, coalesced and grew to form confluent monolayers of polygonal cells by 7 days. Cells in primary and subcultures were identified as endothelium by the presence of Weibel-Palade bodies by electron microscopy. A morphologically distinct subpopulation of cells contaminating some primary endothelial cultures was selectively subcultured, and identified by ultrastructural criteria as vascular smooth muscle. Autoradiography of endothelial cells after exposure to [3H]thymidine showed progressive increases in labeling in growing cultures beginning at 24 h. In recently confluent cultures, labeling indices were 2.4% in central closely packed regions, and 53.2% in peripheral growing regions. 3 days after confluence, labeling was uniform, being 3.5 and 3.9% in central and peripheral areas, respectively. When small areas of confluent cultures were experimentally "denuded," there were localized increases in [3H]thymidine labeling and eventual reconstitution of the monolayer. Liquid scintillation measurements of [3H]thymidine incorporation in primary and secondary endothelial cultures in microwell trays showed a similar correlation of DNA synthesis with cell density. These data indicate that endothelial cell cultures may provide a useful in vitro model for studying pathophysiologic factors in endothelial regeneration. PMID:4363161

  2. Emulsions Containing Perfluorocarbon Support Cell Cultures

    NASA Technical Reports Server (NTRS)

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

    1990-01-01

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

  3. Tocopherol production in plant cell cultures.

    PubMed

    Caretto, Sofia; Nisi, Rossella; Paradiso, Annalisa; De Gara, Laura

    2010-05-01

    Tocopherols, collectively known as vitamin E, are lipophilic antioxidants, essential dietary components for mammals and exclusively synthesized by photosynthetic organisms. Of the four forms (alpha, beta, gamma and delta), alpha-tocopherol is the major vitamin E form present in green plant tissues, and has the highest vitamin E activity. Synthetic alpha-tocopherol, being a racemic mixture of eight different stereoisomers, always results less effective than the natural form (R,R,R) alpha-tocopherol. This raises interest in obtaining this molecule from natural sources, such as plant cell cultures. Plant cell and tissue cultures are able to produce and accumulate valuable metabolites that can be used as food additives, nutraceuticals and pharmaceuticals. Sunflower cell cultures, growing under heterotrophic conditions, were exploited to establish a suitable in vitro production system of natural alpha-tocopherol. Optimization of culture conditions, precursor feeding and elicitor application were used to improve the tocopherol yields of these cultures. Furthermore, these cell cultures were useful to investigate the relationship between alpha-tocopherol biosynthesis and photomixotrophic culture conditions, revealing the possibility to enhance tocopherol production by favouring sunflower cell photosynthetic properties. The modulation of alpha-tocopherol levels in plant cell cultures can provide useful hints for a regulatory impact on tocopherol metabolism.

  4. Constructing a High Density Cell Culture System

    NASA Technical Reports Server (NTRS)

    Spaulding, Glenn F. (Inventor)

    1996-01-01

    An annular culture vessel for growing mammalian cells is constructed in a one piece integral and annular configuration with an open end which is closed by an endcap. The culture vessel is rotatable about a horizontal axis by use of conventional roller systems commonly used in culture laboratories. The end wall of the endcap has tapered access ports to frictionally and sealingly receive the ends of hypodermic syringes. The syringes permit the introduction of fresh nutrient and withdrawal of spent nutrients. The walls are made of conventional polymeric cell culture material and are subjected to neutron bombardment to form minute gas permeable perforations in the walls.

  5. 3D Cell Culture in Alginate Hydrogels

    PubMed Central

    Andersen, Therese; Auk-Emblem, Pia; Dornish, Michael

    2015-01-01

    This review compiles information regarding the use of alginate, and in particular alginate hydrogels, in culturing cells in 3D. Knowledge of alginate chemical structure and functionality are shown to be important parameters in design of alginate-based matrices for cell culture. Gel elasticity as well as hydrogel stability can be impacted by the type of alginate used, its concentration, the choice of gelation technique (ionic or covalent), and divalent cation chosen as the gel inducing ion. The use of peptide-coupled alginate can control cell–matrix interactions. Gelation of alginate with concomitant immobilization of cells can take various forms. Droplets or beads have been utilized since the 1980s for immobilizing cells. Newer matrices such as macroporous scaffolds are now entering the 3D cell culture product market. Finally, delayed gelling, injectable, alginate systems show utility in the translation of in vitro cell culture to in vivo tissue engineering applications. Alginate has a history and a future in 3D cell culture. Historically, cells were encapsulated in alginate droplets cross-linked with calcium for the development of artificial organs. Now, several commercial products based on alginate are being used as 3D cell culture systems that also demonstrate the possibility of replacing or regenerating tissue. PMID:27600217

  6. Insect cell culture in reagent bottles.

    PubMed

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

    2014-01-01

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

  7. Cell receptor and surface ligand density effects on dynamic states of adhering circulating tumor cells.

    PubMed

    Zheng, Xiangjun; Cheung, Luthur Siu-Lun; Schroeder, Joyce A; Jiang, Linan; Zohar, Yitshak

    2011-10-21

    Dynamic states of cancer cells moving under shear flow in an antibody-functionalized microchannel are investigated experimentally and theoretically. The cell motion is analyzed with the aid of a simplified physical model featuring a receptor-coated rigid sphere moving above a solid surface with immobilized ligands. The motion of the sphere is described by the Langevin equation accounting for the hydrodynamic loadings, gravitational force, receptor-ligand bindings, and thermal fluctuations; the receptor-ligand bonds are modeled as linear springs. Depending on the applied shear flow rate, three dynamic states of cell motion have been identified: (i) free motion, (ii) rolling adhesion, and (iii) firm adhesion. Of particular interest is the fraction of captured circulating tumor cells, defined as the capture ratio, via specific receptor-ligand bonds. The cell capture ratio decreases with increasing shear flow rate with a characteristic rate. Based on both experimental and theoretical results, the characteristic flow rate increases monotonically with increasing either cell-receptor or surface-ligand density within certain ranges. Utilizing it as a scaling parameter, flow-rate dependent capture ratios for various cell-surface combinations collapse onto a single curve described by an exponential formula.

  8. Primary cell cultures from sea urchin ovaries: a new experimental tool.

    PubMed

    Mercurio, Silvia; Di Benedetto, Cristiano; Sugni, Michela; Candia Carnevali, M Daniela

    2014-02-01

    In the present work, primary cell cultures from ovaries of the edible sea urchin Paracentrotus lividus were developed in order to provide a simple and versatile experimental tool for researches in echinoderm reproductive biology. Ovary cell phenotypes were identified and characterized by different microscopic techniques. Although cell cultures could be produced from ovaries at all stages of maturation, the cells appeared healthier and viable, displaying a higher survival rate, when ovaries at early stages of gametogenesis were used. In terms of culture medium, ovarian cells were successfully cultured in modified Leibovitz-15 medium, whereas poor results were obtained in minimum essential medium Eagle and medium 199. Different substrates were tested, but ovarian cells completely adhered only on poly-L-lysine. To improve in vitro conditions and stimulate cell proliferation, different serum-supplements were tested. Fetal calf serum and an originally developed pluteus extract were detrimental to cell survival, apparently accelerating processes of cell death. In contrast, cells cultured with sea urchin egg extract appeared larger and healthier, displaying an increased longevity that allowed maintaining them for up to 1 month. Overall, our study provides new experimental bases and procedures for producing successfully long-term primary cell cultures from sea urchin ovaries offering a good potential to study echinoid oogenesis in a controlled system and to investigate different aspects of echinoderm endocrinology and reproductive biology.

  9. Rethinking adherence.

    PubMed

    Steiner, John F

    2012-10-16

    In 2012, the Centers for Medicare & Medicaid Services (CMS) will introduce measures of adherence to oral hypoglycemic, antihypertensive, and cholesterol-lowering drugs into its Medicare Advantage quality program. To meet these quality goals, delivery systems will need to develop and disseminate strategies to improve adherence. The design of adherence interventions has too often been guided by the mistaken assumptions that adherence is a single behavior that can be predicted from readily available patient characteristics and that individual clinicians alone can improve adherence at the population level.Effective interventions require recognition that adherence is a set of interacting behaviors influenced by individual, social, and environmental forces; adherence interventions must be broadly based, rather than targeted to specific population subgroups; and counseling with a trusted clinician needs to be complemented by outreach interventions and removal of structural and organizational barriers. To achieve the adherence goals set by CMS, front-line clinicians, interdisciplinary teams, organizational leaders, and policymakers will need to coordinate efforts in ways that exemplify the underlying principles of health care reform.

  10. Development of a Modular Automated System for Maintenance and Differentiation of Adherent Human Pluripotent Stem Cells.

    PubMed

    Crombie, Duncan E; Daniszewski, Maciej; Liang, Helena H; Kulkarni, Tejal; Li, Fan; Lidgerwood, Grace E; Conquest, Alison; Hernández, Damian; Hung, Sandy S; Gill, Katherine P; De Smit, Elisabeth; Kearns, Lisa S; Clarke, Linda; Sluch, Valentin M; Chamling, Xitiz; Zack, Donald J; Wong, Raymond C B; Hewitt, Alex W; Pébay, Alice

    2017-03-01

    Patient-specific induced pluripotent stem cells (iPSCs) have tremendous potential for development of regenerative medicine, disease modeling, and drug discovery. However, the processes of reprogramming, maintenance, and differentiation are labor intensive and subject to intertechnician variability. To address these issues, we established and optimized protocols to allow for the automated maintenance of reprogrammed somatic cells into iPSCs to enable the large-scale culture and passaging of human pluripotent stem cells (PSCs) using a customized TECAN Freedom EVO. Generation of iPSCs was performed offline by nucleofection followed by selection of TRA-1-60-positive cells using a Miltenyi MultiMACS24 Separator. Pluripotency markers were assessed to confirm pluripotency of the generated iPSCs. Passaging was performed using an enzyme-free dissociation method. Proof of concept of differentiation was obtained by differentiating human PSCs into cells of the retinal lineage. Key advantages of this automated approach are the ability to increase sample size, reduce variability during reprogramming or differentiation, and enable medium- to high-throughput analysis of human PSCs and derivatives. These techniques will become increasingly important with the emergence of clinical trials using stem cells.

  11. Cell culture from sponges: pluripotency and immortality.

    PubMed

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

    2007-10-01

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

  12. Effect of growth phase on the adherence to and invasion of Caco-2 epithelial cells by Campylobacter.

    PubMed

    Ganan, M; Campos, G; Muñoz, R; Carrascosa, A V; de Pascual-Teresa, S; Martinez-Rodriguez, A J

    2010-05-30

    The effect of growth phase on the adherence to and invasion of Caco-2 epithelial cells by five strains of Campylobacter was studied. No significant differences were observed between the behaviors in the exponential or stationary phases for the most stationary-phase tolerant strains (C. jejuni 118 and C. coli LP2), while the strains that produced a greater reduction in the viability in the stationary phase (C. jejuni 11351, C. jejuni 11168 and C. jejuni LP1), also presented reduced adherence to and invasion of Caco-2 cells. In order to find a possible explanation for the observed differences, the presence of putative virulence factors was studied in the analyzed strains. In spite of the fact that C. jejuni 118 and C. jejuni 11168 strains showed a different adherence to and invasion of Caco-2 cells behavior, they posses identical alleles for ciaB, cadF, and pldA loci. From the virulence factors analyzed, only the flaA locus was different among both strains.

  13. Expansion of CD133+ colon cancer cultures retaining stem cell properties to enable cancer stem cell target discovery

    PubMed Central

    Fang, D D; Kim, Y J; Lee, C N; Aggarwal, S; McKinnon, K; Mesmer, D; Norton, J; Birse, C E; He, T; Ruben, S M; Moore, P A

    2010-01-01

    Background: Despite earlier studies demonstrating in vitro propagation of solid tumour cancer stem cells (CSCs) as non-adherent tumour spheres, it remains controversial as to whether CSCs can be maintained in vitro. Additional validation of the CSC properties of tumour spheres would support their use as CSC models and provide an opportunity to discover additional CSC cell surface markers to aid in CSC detection and potential elimination. Methods: Primary tumour cells isolated from 13 surgically resected colon tumour specimens were propagated using serum-free CSC-selective conditions. The CSC properties of long-term cultured tumour spheres were established and mass spectrometry-based proteomics performed. Results: Freshly isolated CD133+ colorectal cancer cells gave rise to long-term tumour sphere (or spheroids) cultures maintaining CD133 expression. These spheroid cells were able to self-renew and differentiate into adherent epithelial lineages and recapitulate the phenotype of the original tumour. Relative to their differentiated progeny, tumour spheroid cells were more resistant to the chemotherapeutic irinotecan. Finally, CD44, CD166, CD29, CEACAM5, cadherin 17, and biglycan were identified by mass spectrometry to be enriched in CD133+ tumour spheroid cells. Conclusion: Our data suggest that ex vivo-expanded colon CSCs isolated from clinical specimens can be maintained in culture enabling the identification of CSC cell surface-associated proteins. PMID:20332776

  14. Suppression of unprimed T and B cells in antibody responses by irradiation-resistant and plastic-adherent suppressor cells in Toxoplasma gondii-infected mice

    SciTech Connect

    Suzuki, Y.; Kobayashi, A.

    1983-04-01

    In the acute phase of Toxoplasma infection, the function of both helper T and B cells was suppressed in primary antibody responses to dinitrophenol (DNP)-conjugated protein antigens. During the course of infection, the suppressive effect on T cells seems to continue longer than that on B cells, since suppression in responses to sheep erythrocytes, a T-dependent antigen, persisted longer than those to DNP-Ficoll, a T-independent antigen. Plastic-adherent cells from the spleens of Toxoplasma-infected and X-irradiated (400 rads) mice had strong suppressor activity in primary anti-sheep erythrocyte antibody responses of normal mouse spleen cells in vitro. These data suggest that the activation of irradiation-resistant and plastic-adherent suppressor cells causes the suppression of both T and B cells in Toxoplasma-infected mice.

  15. Porcine mitral valve interstitial cells in culture.

    PubMed

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

    1988-11-01

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

  16. Preserving elemental content in adherent mammalian cells for analysis by synchrotron‐based x‐ray fluorescence microscopy

    PubMed Central

    JIN, QIAOLING; PAUNESKU, TATJANA; LAI, BARRY; GLEBER, SOPHIE‐CHARLOTTE; CHEN, SI; FINNEY, LYDIA; VINE, DAVID; VOGT, STEFAN; WOLOSCHAK, GAYLE

    2016-01-01

    Summary Trace metals play important roles in biological function, and x‐ray fluorescence microscopy (XFM) provides a way to quantitatively image their distribution within cells. The faithfulness of these measurements is dependent on proper sample preparation. Using mouse embryonic fibroblast NIH/3T3 cells as an example, we compare various approaches to the preparation of adherent mammalian cells for XFM imaging under ambient temperature. Direct side‐by‐side comparison shows that plunge‐freezing‐based cryoimmobilization provides more faithful preservation than conventional chemical fixation for most biologically important elements including P, S, Cl, K, Fe, Cu, Zn and possibly Ca in adherent mammalian cells. Although cells rinsed with fresh media had a great deal of extracellular background signal for Cl and Ca, this approach maintained cells at the best possible physiological status before rapid freezing and it does not interfere with XFM analysis of other elements. If chemical fixation has to be chosen, the combination of 3% paraformaldehyde and 1.5 % glutaraldehyde preserves S, Fe, Cu and Zn better than either fixative alone. When chemically fixed cells were subjected to a variety of dehydration processes, air drying was proved to be more suitable than other drying methods such as graded ethanol dehydration and freeze drying. This first detailed comparison for x‐ray fluorescence microscopy shows how detailed quantitative conclusions can be affected by the choice of cell preparation method. PMID:27580164

  17. CD66 nonspecific cross-reacting antigens are involved in neutrophil adherence to cytokine-activated endothelial cells

    PubMed Central

    1992-01-01

    Neutrophil adherence to cytokine-activated endothelial cell (EC) monolayers depends on the expression of the endothelial leukocyte adhesion molecule-1 (ELAM-1). The ligand for ELAM-1 is the sialylated Lewis-x antigen (SLe(x)) structure. The selectin LAM-1 (or LECAM-1) has been described as one of the SLe(x)-presenting glycoproteins involved in neutrophil binding to ELAM-1. Other presenter molecules have not yet been described. Our data demonstrate that the carcinoembryonic antigen (CEA)-like surface molecules on neutrophils--known as the nonspecific cross-reacting antigens (NCAs)--are involved in neutrophil adherence to monolayers of IL-1-beta-activated EC. The NCAs are recognized by CD66 (NCA-160 and NCA-90) and CD67 (NCA-95). Because NCA-95 and NCA-90 have previously been found to be phosphatidylinositol (PI)-linked, paroxysmal nocturnal hemoglobinuria (PNH) neutrophils (which lack PI- linked surface proteins) were tested as well. PNH neutrophils showed a diminished binding to activated EC. CD66 (on PNH cells still recognizing the transmembrane NCA-160 form) still inhibited the adherence of PNH cells to IL-1-beta-activated EC, but to a limited extent. Soluble CEA(-related) antigens inhibited normal neutrophil adherence as well, whereas neutrophil transmigration was unaffected. Sialidase-treatment as well as CD66 preclearing abolished the inhibitory capacity of the CEA(-related) antigens. The binding of soluble CEA antigens to IL-1-beta-pretreated EC was blocked by anti- ELAM-1. These soluble antigens, as well as the neutrophil NCA-160 and NCA-90, both recognized by CD66 antibodies, presented the SLe(x) determinant. Together, these findings indicate that the CD66 antigens (i.e., NCA-160/NCA-90) function as presenter molecules of the SLe(x) oligosaccharide structures on neutrophils that bind to ELAM-1 on EC. PMID:1378450

  18. Culture and Manipulation of Embryonic Cells

    PubMed Central

    Edgar, Lois G.; Goldstein, Bob

    2012-01-01

    The direct manipulation of embryonic cells is an important tool for addressing key questions in cell and developmental biology. C. elegans is relatively unique among genetic model systems in being amenable to manipulation of embryonic cells. Embryonic cell manipulation has allowed the identification of cell interactions by direct means, and it has been an important technique for dissecting mechanisms by which cell fates are specified, cell divisions are oriented, and morphogenesis is accomplished. Here, we present detailed methods for isolating, manipulating and culturing embryonic cells of C. elegans. PMID:22226523

  19. Sustaining a "culture of silence" in the neonatal intensive care unit during nonemergency situations: a grounded theory on ensuring adherence to behavioral modification to reduce noise levels.

    PubMed

    Swathi, S; Ramesh, A; Nagapoornima, M; Fernandes, Lavina M; Jisina, C; Rao, P N Suman; Swarnarekha, A

    2014-01-01

    The aim of this study was to generate a substantive theory explaining how the staff in a resource-limited neonatal intensive care unit (NICU) of a developing nation manage to ensure adherence to behavioral modification components of a noise reduction protocol (NsRP) during nonemergency situations. The study was conducted after implementation of an NsRP in a level III NICU of south India. The normal routine of the NICU is highly dynamic because of various categories of staff conducting clinical rounds followed by care-giving activities. This is unpredictably interspersed with very noisy emergency management of neonates who suddenly fall sick. In-depth interviews were conducted with 36 staff members of the NICU (20 staff nurses, six nursing aides, and 10 physicians). Group discussions were conducted with 20 staff nurses and six nursing aides. Data analysis was done in line with the reformulated grounded theory approach, which was based on inductive examination of textual information. The results of the analysis showed that the main concern was to ensure adherence to behavioral modification components of the NsRP. This was addressed by using strategies to "sustain a culture of silence in NICU during nonemergency situations" (core category). The main strategies employed were building awareness momentum, causing awareness percolation, developing a sense of ownership, expansion of caring practices, evolution of adherence, and displaying performance indicators. The "culture of silence" reconditions the existing staff and conditions new staff members joining the NICU. During emergency situations, a "noisy culture" prevailed because of pragmatic neglect of behavioral modification when life support overrode all other concerns. In addition to this, the process of operant conditioning should be formally conducted once every 18 months. The results of this study may be adapted to create similar strategies and establish context specific NsRPs in NICUs with resource constraints.

  20. Adherence of Enterohemorrhagic Escherichia coli to Human Epithelial Cells: The Role of Intimin

    DTIC Science & Technology

    1995-04-28

    Typhlocolltlsd genotype· adherence" LAlFAS + NA LAlFAS NAIweak DAIweak FAS· 118 Intimate bacterial adherence and NE lesions, as described by Staley...Additionally, two independent TnphoA mutants of EHEC strain CL-8 (0157:H7) were isolated and found deficient in bacterial factors necessary for NE lesion...intestinal NE lesions in gnotobiotic piglets. In vitro attachment and in vivo lesion formation by 86-24eaeMO was fully restored by a clone of EHEC 86-24

  1. Myosin types in cultured muscle cells

    PubMed Central

    1980-01-01

    Fluorescent antibodies against fast skeletal, slow skeletal, and ventricular myosins were applied to muscle cultures from embryonic pectoralis and ventricular myocadium of the chicken. A number of spindle-shaped mononucleated cells, presumably myoblasts, and all myotubes present in skeletal muscle cultures were labeled by all three antimyosin antisera. In contrast, in cultures from ventricular myocardium all muscle cells were labeled by anti-ventricular myosin, whereas only part of them were stained by anti-slow skeletal myosin and rare cells reacted with anti-fast skeletal myosin. The findings indicate that myosin(s) present in cultured embryonic skeletal muscle cells contains antigenic determinants similar to those present in adult fast skeletal, slow skeletal, and ventricular myosins. PMID:6156177

  2. Serum Proteins Enhance Dispersion Stability and Influence the Cytotoxicity and Dosimetry of ZnO Nanoparticles in Suspension and Adherent Cancer Cell Models

    NASA Astrophysics Data System (ADS)

    Anders, Catherine B.; Chess, Jordan J.; Wingett, Denise G.; Punnoose, Alex

    2015-11-01

    Agglomeration and sedimentation of nanoparticles (NPs) within biological solutions is a major limitation in their use in many downstream applications. It has been proposed that serum proteins associate with the NP surface to form a protein corona that limits agglomeration and sedimentation. Here, we investigate the effect of fetal bovine serum (FBS) proteins on the dispersion stability, dosimetry, and NP-induced cytotoxicity of cationic zinc oxide nanoparticles (nZnO) synthesized via forced hydrolysis with a core size of 10 nm. Two different in vitro cell culture models, suspension and adherent, were evaluated by comparing a phosphate buffered saline (PBS) nZnO dispersion (nZnO/PBS) and an FBS-stabilized PBS nZnO dispersion (nZnO - FBS/PBS). Surface interactions of FBS on nZnO were analyzed via spectroscopic and optical techniques. Fourier transformed infrared spectroscopy (FTIR) confirmed the adsorption of negatively charged protein components on the cationic nZnO surface through the disappearance of surfaced-adsorbed carboxyl functional groups and the subsequent detection of vibrational modes associated with the protein backbone of FBS-associated proteins. Further confirmation of these interactions was noted in the isoelectric point shift of the nZnO from the characteristic pH of 9.5 to a pH of 6.1. In nZnO - FBS/PBS dispersions, the FBS reduced agglomeration and sedimentation behaviors to impart long-term improvements (>24 h) to the nZnO dispersion stability. Furthermore, mathematical dosimetry models indicate that nZnO - FBS/PBS dispersions had consistent NP deposition patterns over time unlike unstable nZnO/PBS dispersions. In suspension cell models, the stable nZnO - FBS/PBS dispersion resulted in a ~33 % increase in the NP-induced cytotoxicity for both Jurkat leukemic and Hut-78 lymphoma cancer cells. In contrast, the nZnO - FBS/PBS dispersion resulted in 49 and 71 % reductions in the cytotoxicity observed towards the adherent breast (T-47D) and prostate

  3. Serum Proteins Enhance Dispersion Stability and Influence the Cytotoxicity and Dosimetry of ZnO Nanoparticles in Suspension and Adherent Cancer Cell Models.

    PubMed

    Anders, Catherine B; Chess, Jordan J; Wingett, Denise G; Punnoose, Alex

    2015-12-01

    Agglomeration and sedimentation of nanoparticles (NPs) within biological solutions is a major limitation in their use in many downstream applications. It has been proposed that serum proteins associate with the NP surface to form a protein corona that limits agglomeration and sedimentation. Here, we investigate the effect of fetal bovine serum (FBS) proteins on the dispersion stability, dosimetry, and NP-induced cytotoxicity of cationic zinc oxide nanoparticles (nZnO) synthesized via forced hydrolysis with a core size of 10 nm. Two different in vitro cell culture models, suspension and adherent, were evaluated by comparing a phosphate buffered saline (PBS) nZnO dispersion (nZnO/PBS) and an FBS-stabilized PBS nZnO dispersion (nZnO - FBS/PBS). Surface interactions of FBS on nZnO were analyzed via spectroscopic and optical techniques. Fourier transformed infrared spectroscopy (FTIR) confirmed the adsorption of negatively charged protein components on the cationic nZnO surface through the disappearance of surfaced-adsorbed carboxyl functional groups and the subsequent detection of vibrational modes associated with the protein backbone of FBS-associated proteins. Further confirmation of these interactions was noted in the isoelectric point shift of the nZnO from the characteristic pH of 9.5 to a pH of 6.1. In nZnO - FBS/PBS dispersions, the FBS reduced agglomeration and sedimentation behaviors to impart long-term improvements (>24 h) to the nZnO dispersion stability. Furthermore, mathematical dosimetry models indicate that nZnO - FBS/PBS dispersions had consistent NP deposition patterns over time unlike unstable nZnO/PBS dispersions. In suspension cell models, the stable nZnO - FBS/PBS dispersion resulted in a ~33 % increase in the NP-induced cytotoxicity for both Jurkat leukemic and Hut-78 lymphoma cancer cells. In contrast, the nZnO - FBS/PBS dispersion resulted in 49 and 71 % reductions in the cytotoxicity observed towards the adherent breast (T-47D) and prostate

  4. The extracellular adherence protein (Eap) of Staphylococcus aureus acts as a proliferation and migration repressing factor that alters the cell morphology of keratinocytes.

    PubMed

    Eisenbeis, Janina; Peisker, Henrik; Backes, Christian S; Bur, Stephanie; Hölters, Sebastian; Thewes, Nicolas; Greiner, Markus; Junker, Christian; Schwarz, Eva C; Hoth, Markus; Junker, Kerstin; Preissner, Klaus T; Jacobs, Karin; Herrmann, Mathias; Bischoff, Markus

    2017-02-01

    Staphyloccocus aureus is a major human pathogen and a common cause for superficial and deep seated wound infections. The pathogen is equipped with a large arsenal of virulence factors, which facilitate attachment to various eukaryotic cell structures and modulate the host immune response. One of these factors is the extracellular adherence protein Eap, a member of the "secretable expanded repertoire adhesive molecules" (SERAM) protein family that possesses adhesive and immune modulatory properties. The secreted protein was previously shown to impair wound healing by interfering with host defense and neovascularization. However, its impact on keratinocyte proliferation and migration, two major steps in the re-epithelialization process of wounds, is not known. Here, we report that Eap affects the proliferation and migration capacities of keratinocytes by altering their morphology and adhesive properties. In particular, treatment of non-confluent HaCaT cell cultures with Eap resulted in cell morphology changes as well as a significant reduction in cell proliferation and migration. Eap-treated HaCaT cells changed their appearance from an oblong via a trapezoid to an astral-like shape, accompanied by decreases in cell volume and cell stiffness, and exhibited significantly increased cell adhesion. Eap had a similar influence on endothelial and cancer cells, indicative for a general effect of Eap on eukaryotic cell morphology and functions. Specifically, Eap was found to interfere with growth factor-stimulated activation of the mitogen-activated protein kinase (MAPK) pathway that is known to be responsible for cell shape modulation, induction of proliferation and migration of epithelial cells. Western blot analyses revealed that Eap blocked the phosphorylation of extracellular signal-regulated kinase 1 and 2 (Erk1/2) in keratinocyte growth factor (KGF)-stimulated HaCaT cells. Together, these data add another antagonistic mechanism of Eap in wound healing, whereby the

  5. Genome Wide assessment of Early Osseointegration in Implant-Adherent Cells

    NASA Astrophysics Data System (ADS)

    Thalji, Ghadeer N.

    Objectives: To determine the molecular processes involved in osseointegration. Materials and methods: A structured literature review concerning in vitro and in vivo molecular assessment of osseointegration was performed. A rat and a human model were then used to identify the early molecular processes involved in osseointegration associated with a micro roughened and nanosurface superimposed featured implants. In the rat model, 32 titanium implants with surface topographies exhibiting a micro roughened (AT-II) and nanosurface superimposed featured implants (AT-I) were placed in the tibiae of 8 rats and subsequently harvested at 2 and 4 days after placement. Whereas in the human model, four titanium mini-implants with either a moderately roughened surface (TiOblast) or super-imposed nanoscale topography (Osseospeed) were placed in edentulous sites of eleven systemically healthy subjects and subsequently removed after 3 and 7 days. Total RNA was isolated from cells adherent to retrieved implants. A whole genome microarray using the Affymetrix 1.1 ST Array platform was used to describe the gene expression profiles that were differentially regulated by the implant surfaces. Results: The literature review provided evidence that particular topographic cues can be specifically integrated among the many extracellular signals received by the cell in its signal transduction network. In the rat model, functionally relevant categories related to ossification, skeletal system development, osteoblast differentiation, bone development and biomineral tissue development were upregulated and more prominent at AT-I compared to AT-II. In the human model, there were no significant differences when comparing the two-implant surfaces at each time point. However, the microarray identified several genes that were differentially regulated at day 7 vs. day 3 for both implant surfaces. Functionally relevant categories related to the extracellular matrix, collagen fibril organization and

  6. The Escherichia coli O157:H7 cattle immunoproteome includes outer membrane protein A (OmpA), a modulator of adherence to bovine rectoanal junction squamous epithelial (RSE) cells.

    PubMed

    Kudva, Indira T; Krastins, Bryan; Torres, Alfredo G; Griffin, Robert W; Sheng, Haiqing; Sarracino, David A; Hovde, Carolyn J; Calderwood, Stephen B; John, Manohar

    2015-06-01

    Building on previous studies, we defined the repertoire of proteins comprising the immunoproteome (IP) of Escherichia coli O157:H7 (O157) cultured in DMEM supplemented with norepinephrine (O157 IP), a β-adrenergic hormone that regulates E. coli O157 gene expression in the gastrointestinal tract, using a variation of a novel proteomics-based platform proteome mining tool for antigen discovery, called "proteomics-based expression library screening" (PELS; Kudva et al., 2006). The E. coli O157 IP (O157-IP) comprised 91 proteins, and included those identified previously using proteomics-based expression library screening, and also proteins comprising DMEM and bovine rumen fluid proteomes. Outer membrane protein A (OmpA), a common component of the above proteomes, and reportedly a contributor to E. coli O157 adherence to cultured HEp-2 epithelial cells, was interestingly found to be a modulator rather than a contributor to E. coli O157 adherence to bovine rectoanal junction squamous epithelial cells. Our results point to a role for yet to be identified members of the O157-IP in E. coli O157 adherence to rectoanal junction squamous epithelial cells, and additionally implicate a possible role for the outer membrane protein A regulator, TdcA, in the expression of such adhesins. Our observations have implications for the development of efficacious vaccines for preventing E. coli O157 colonization of the bovine gastrointestinal tract.

  7. Banks of cell cultures for biotechnology.

    PubMed

    Radaeva, I F; Bogryantseva, M P; Nechaeva, E A

    2012-08-01

    Seeding and working cell banks were created and stored in cell culture collection. The banks were certified in accordance with international and national requirements. Cultures of 293, MT-4, L-68, FECH-16-1, FECH-16-2, 4647, MDCK, CHO TK(-), and CHO pE cells were recommended by Medical Immunobiological Preparation Committee for the use in the production of medical immunobiological preparations. The stock is sufficient enough for supplying standard cell material for the production of medical immunobiological preparations over few decades.

  8. Automated microraft platform to identify and collect non-adherent cells successfully gene-edited with CRISPR-Cas9.

    PubMed

    Attayek, Peter J; Waugh, Jennifer P; Hunsucker, Sally A; Grayeski, Philip J; Sims, Christopher E; Armistead, Paul M; Allbritton, Nancy L

    2017-05-15

    Microraft arrays have been used to screen and then isolate adherent and non-adherent cells with very high efficiency and excellent viability; however, manual screening and isolation limits the throughput and utility of the technology. In this work, novel hardware and software were developed to automate the microraft array platform. The developed analysis software identified microrafts on the array with greater than 99% sensitivity and cells on the microrafts with 100% sensitivity. The software enabled time-lapse imaging and the use of temporally varying characteristics as sort criteria. The automated hardware released microrafts with 98% efficiency and collected released microrafts with 100% efficiency. The automated system was used to examine the temporal variation in EGFP expression in cells transfected with CRISPR-Cas9 components for gene editing. Of 11,499 microrafts possessing a single cell, 220 microrafts were identified as possessing temporally varying EGFP-expression. Candidate cells (n=172) were released and collected from the microraft array and screened for the targeted gene mutation. Two cell colonies were successfully gene edited demonstrating the desired mutation.

  9. Effect of mannoproteins on the growth, gastrointestinal viability, and adherence to Caco-2 cells of lactic acid bacteria.

    PubMed

    Ganan, M; Carrascosa, A V; de Pascual-Teresa, S; Martinez-Rodriguez, A J

    2012-03-01

    Yeast cell wall (YCW) preparations and yeast mannoprotein extracts have been effective against some enteropathogenic bacteria as Campylobacter jejuni, Escherichia coli, and Salmonella, and they can affect the population of beneficial lactic acid bacteria (LAB). In this work, we studied the effect of a mannoprotein extract on five strains of LAB. This extract was metabolised by the bacteria, enhancing their survival in simulated gastrointestinal juice, and increasing the adherence of Lactobacillus plantarum, L. salivarius, and Enterococcus faecium to Caco-2 cells. Yeast mannoproteins are promising naturally occurring compounds that could be used to enhance LAB intestinal populations and control pathogens.

  10. Increased mechanosensitivity of cells cultured on nanotopographies

    PubMed Central

    Salvi, Joshua D.; Lim, Jung Yul; Donahue, Henry J.

    2012-01-01

    Enhancing cellular mechanosensitivity is recognized as a novel tool for successful musculoskeletal tissue engineering. We examined the hypothesis that mechanosensitivity of human mesenchymal stem cells (hMSCs) is enhanced on nanotopographic substrates relative to flat surfaces. hMSCs were cultured on polymer-demixed, randomly distributed nanoisland surfaces with varying island heights and changes in intracellular calcium concentration, [Ca2+]i, in response to fluid flow induced shear stress were quantifide. Stem cells cultured on specific scale nanotopographies displayed greater intracellular calcium responses to fluid flow. hMSCs cultured on 10-20 nm high nanoislands displayed a greater percentage of cells responding in calcium relative to cells cultured on flat control, and showed greater average [Ca2+]i increase relative to cells cultured on other nanoislands (45-80 nm high nanoislands). As [Ca2+]i is an important regulator of downstream signaling, as well as proliferation and differentiation of hMSCs, this observation suggests that specific scale nanotopographies provide an optimal milieu for promoting stem cell mechanotransduction activity. That mechanical signals and substrate nanotopography may synergistically regulate cell behavior is of significant interest in the development of regenerative medicine protocols. PMID:20851397

  11. Monitoring changes in proteome during stepwise adaptation of a MDCK cell line from adherence to growth in suspension.

    PubMed

    Kluge, Sabine; Benndorf, Dirk; Genzel, Yvonne; Scharfenberg, Klaus; Rapp, Erdmann; Reichl, Udo

    2015-08-20

    Adaptation of continuous cell lines to growth in suspension in a chemically defined medium has significant advantages for design and optimization in manufacturing of biologicals. In this work, changes in the protein expression level during a step-wise adaptation of an adherent Madin Darby canine kidney (MDCK) cell line to suspension growth were analyzed. Therefore, three cell line adaptations were performed independently. Two adaptations were monitored closely to characterize short term changes in protein expression levels after serum deprivation. In addition, initial stages of suspension growth were analyzed for both adaptations. The third adaptation involved MDCK suspension cells (MDCKSUS2) grown over an extended time period to achieve robust growth characteristics. Here, cells of the final stage of adaptation were compared with their parental cell line (MDCKADH). A combination of two dimensional differential gel electrophoresis for relative protein quantification and tandem mass spectrometry for protein identification enabled insights into cellular physiology. The two closely monitored cell line adaptations followed different routes regarding specific changes in protein expression but resulted in similar proteome profiles at the initial stages of suspension growth analyzed. Compared to the MDCKADH cells more than 90% of all changes in the protein expression level were identified after serum deprivation and were related to cytoskeletal structure, genetic information processing and cellular metabolism. Myosin proteins, involved in cellular detachment by actin-myosin contractile mechanisms were also differentially expressed. Interestingly, for both of the two adaptations, proteins linked for tumorigenicity, like lactoylglutathione lyase and sulfotransferase 1A1 were differentially expressed. In contrast, none of these proteins were differentially expressed for the MDCKSUS2 cell line. Overall, proteomic monitoring allowed identification of key proteins involved in

  12. Characterization of a distinct population of circulating human non-adherent endothelial forming cells and their recruitment via intercellular adhesion molecule-3.

    PubMed

    Appleby, Sarah L; Cockshell, Michaelia P; Pippal, Jyotsna B; Thompson, Emma J; Barrett, Jeffrey M; Tooley, Katie; Sen, Shaundeep; Sun, Wai Yan; Grose, Randall; Nicholson, Ian; Levina, Vitalina; Cooke, Ira; Talbo, Gert; Lopez, Angel F; Bonder, Claudine S

    2012-01-01

    Circulating vascular progenitor cells contribute to the pathological vasculogenesis of cancer whilst on the other hand offer much promise in therapeutic revascularization in post-occlusion intervention in cardiovascular disease. However, their characterization has been hampered by the many variables to produce them as well as their described phenotypic and functional heterogeneity. Herein we have isolated, enriched for and then characterized a human umbilical cord blood derived CD133(+) population of non-adherent endothelial forming cells (naEFCs) which expressed the hematopoietic progenitor cell markers (CD133, CD34, CD117, CD90 and CD38) together with mature endothelial cell markers (VEGFR2, CD144 and CD31). These cells also expressed low levels of CD45 but did not express the lymphoid markers (CD3, CD4, CD8) or myeloid markers (CD11b and CD14) which distinguishes them from 'early' endothelial progenitor cells (EPCs). Functional studies demonstrated that these naEFCs (i) bound Ulex europaeus lectin, (ii) demonstrated acetylated-low density lipoprotein uptake, (iii) increased vascular cell adhesion molecule (VCAM-1) surface expression in response to tumor necrosis factor and (iv) in co-culture with mature endothelial cells increased the number of tubes, tubule branching and loops in a 3-dimensional in vitro matrix. More importantly, naEFCs placed in vivo generated new lumen containing vasculature lined by CD144 expressing human endothelial cells (ECs). Extensive genomic and proteomic analyses of the naEFCs showed that intercellular adhesion molecule (ICAM)-3 is expressed on their cell surface but not on mature endothelial cells. Furthermore, functional analysis demonstrated that ICAM-3 mediated the rolling and adhesive events of the naEFCs under shear stress. We suggest that the distinct population of naEFCs identified and characterized here represents a new valuable therapeutic target to control aberrant vasculogenesis.

  13. Characterization of a Distinct Population of Circulating Human Non-Adherent Endothelial Forming Cells and Their Recruitment via Intercellular Adhesion Molecule-3

    PubMed Central

    Thompson, Emma J.; Barrett, Jeffrey M.; Tooley, Katie; Sen, Shaundeep; Sun, Wai Yan; Grose, Randall; Nicholson, Ian; Levina, Vitalina; Cooke, Ira; Talbo, Gert; Lopez, Angel F.; Bonder, Claudine S.

    2012-01-01

    Circulating vascular progenitor cells contribute to the pathological vasculogenesis of cancer whilst on the other hand offer much promise in therapeutic revascularization in post-occlusion intervention in cardiovascular disease. However, their characterization has been hampered by the many variables to produce them as well as their described phenotypic and functional heterogeneity. Herein we have isolated, enriched for and then characterized a human umbilical cord blood derived CD133+ population of non-adherent endothelial forming cells (naEFCs) which expressed the hematopoietic progenitor cell markers (CD133, CD34, CD117, CD90 and CD38) together with mature endothelial cell markers (VEGFR2, CD144 and CD31). These cells also expressed low levels of CD45 but did not express the lymphoid markers (CD3, CD4, CD8) or myeloid markers (CD11b and CD14) which distinguishes them from ‘early’ endothelial progenitor cells (EPCs). Functional studies demonstrated that these naEFCs (i) bound Ulex europaeus lectin, (ii) demonstrated acetylated-low density lipoprotein uptake, (iii) increased vascular cell adhesion molecule (VCAM-1) surface expression in response to tumor necrosis factor and (iv) in co-culture with mature endothelial cells increased the number of tubes, tubule branching and loops in a 3-dimensional in vitro matrix. More importantly, naEFCs placed in vivo generated new lumen containing vasculature lined by CD144 expressing human endothelial cells (ECs). Extensive genomic and proteomic analyses of the naEFCs showed that intercellular adhesion molecule (ICAM)-3 is expressed on their cell surface but not on mature endothelial cells. Furthermore, functional analysis demonstrated that ICAM-3 mediated the rolling and adhesive events of the naEFCs under shear stress. We suggest that the distinct population of naEFCs identified and characterized here represents a new valuable therapeutic target to control aberrant vasculogenesis. PMID:23144795

  14. Computational modeling of adherent cell growth in a hollow-fiber membrane bioreactor for large-scale 3-D bone tissue engineering.

    PubMed

    Mohebbi-Kalhori, Davod; Behzadmehr, Amin; Doillon, Charles J; Hadjizadeh, Afra

    2012-09-01

    The use of hollow-fiber membrane bioreactors (HFMBs) has been proposed for three-dimensional bone tissue growth at the clinical scale. However, to achieve an efficient HFMB design, the relationship between cell growth and environmental conditions must be determined. Therefore, in this work, a dynamic double-porous media model was developed to determine nutrient-dependent cell growth for bone tissue formation in a HFMB. The whole hollow-fiber scaffold within the bioreactor was treated as a porous domain in this model. The domain consisted of two interpenetrating porous regions, including a porous lumen region available for fluid flow and a porous extracapillary space filled with a collagen gel that contained adherent cells for promoting long-term growth into tissue-like mass. The governing equations were solved numerically and the model was validated using previously published experimental results. The contributions of several bioreactor design and process parameters to the performance of the bioreactor were studied. The results demonstrated that the process and design parameters of the HFMB significantly affect nutrient transport and thus cell behavior over a long period of culture. The approach presented here can be applied to any cell type and used to develop tissue engineering hollow-fiber scaffolds.

  15. Exploring the cultural context of HIV stigma on antiretroviral therapy adherence among people living with HIV/AIDS in southwest Nigeria.

    PubMed

    Okoror, Titilayo A; Falade, Catherine O; Olorunlana, Adetayo; Walker, Ebunlomo M; Okareh, Oladapo T

    2013-01-01

    This study explored the cultural context of HIV stigma on antiretroviral therapy adherence among people living with HIV/AIDS (PLWHA) in southwest Nigeria. Using purposive sampling, participants were recruited through a community-based organization. Consenting PLWHA participated in in-depth interviews and focus group discussions that were audio-taped. Using Deacon's conceptual framework of stigma, four opinion guides facilitated the interviews and discussions. Interviews and discussions were conducted in three languages, and lasted from 45 min to 2 h. A total of 35 women and men participated in the study. Participants ranged in age from 22 to 58 years, with an average of 4 years since clinical diagnosis of HIV/AIDS. All participants were receiving ART, and self-reported high adherence level. Using thematic analysis, three themes emerged: life before ART, life after ART, and strategies used in ART adherence. In describing their lives before ART, participants reported experiencing self, anticipated and enacted stigmas due to their sickly appearance from HIV-related complications. After initiating ART, participants talked about friends and families "returning to them" and "apologizing for abandoning" them once they started "looking well." In response to anticipated stigma, many reported sticking to their medications. Drawing from the cultural milieu as part of their strategies, participants discussed the use of plastic bags for medications and àkònpó, as ways of diverting attention from their use of many medications. Implications for ART program policies and stigma interventions were discussed, along with limitation of a short-term ART study on stigma since long-term use of ART can contribute to stigma by way of lipoatrophy as PLWHA age.

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

    PubMed

    Föllmann, W; Weber, S; Birkner, S

    2000-10-01

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

  17. Human cell culture in a space bioreactor

    NASA Technical Reports Server (NTRS)

    Morrison, Dennis R.

    1988-01-01

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

  18. Cell culture models of transmissible spongiform encephalopathies.

    PubMed

    Béranger, F; Mangé, A; Solassol, J; Lehmann, S

    2001-11-30

    In this review, we describe the generation and use of cell culture models of transmissible spongiform encephalopathies, also known as prion diseases. These models include chronically prion-infected cell lines, as well as cultures expressing variable amounts of wild-type, mutated, or chimeric prion proteins. These cell lines have been widely used to investigate the biology of both the normal and the pathological isoform of the prion protein. They have also contributed to the comprehension of the pathogenic processes occurring in transmissible spongiform encephalopathies and in the development of new therapeutic approaches of these diseases.

  19. Glycosylation of Fluorophenols by Plant Cell Cultures

    PubMed Central

    Shimoda, Kei; Kubota, Naoji; Kondo, Yoko; Sato, Daisuke; Hamada, Hiroki

    2009-01-01

    Fluoroaromatic compounds are used as agrochemicals and released into environment as pollutants. Glycosylation of 2-, 3-, and 4-fluorophenols using plant cell cultures of Nicotiana tabacum was investigated to elucidate their potential to metabolize these compounds. Cultured N. tabacum cells converted 2-fluorophenol into its β-glucoside (60%) and β-gentiobioside (10%). 4-Fluorophenol was also glycosylated to its β-glucoside (32%) and β-gentiobioside (6%) by N. tabacum cells. On the other hand, N. tabacum glycosylated 3-fluorophenol to β-glucoside (17%). PMID:19564930

  20. Pitfalls in cell culture work with xanthohumol.

    PubMed

    Motyl, M; Kraus, B; Heilmann, J

    2012-01-01

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

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

    DOEpatents

    Stampfer, Martha R; Garbe, James C

    2015-02-24

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

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

    SciTech Connect

    Stampfer, Martha R.; Garbe, James C.

    2016-06-28

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

  3. Microfabricated surface designs for cell culture and diagnosis.

    PubMed

    Matsuda, T; Chung, D J

    1994-01-01

    Grooved and holed surfaces with a well fabricated design may serve as microsubstrates for cell culture and microreactors for diagnosis. In this study, the authors prepared chemically treated, micrometer scale grooved and holed glass surfaces by combined surface modification and ultraviolet (UV) excimer laser ablation techniques, as follows. 1) Microcell-culture substrate: Amino group attached glass surfaces, prepared by the treatment with an aminopropylsilane, were condensed with a carboxylated radical initiator. Subsequently, polyacrylamide was grafted by surface initiated radical polymerization to create a very hydrophilic surface layer. Ultraviolet excimer laser beams (KrF: 248 nm) were irradiated through a microscope onto surfaces to create grooves or holes that were 10 and 50 microns in width or diameter, respectively. The depth, depending on the irradiation light strength, ranged from a few to several tenths of a micrometer. On endothelial cell (EC) seeding, ECs adhered and grew on the bottoms of the grooved or holed surface where glass was exposed on ablation. Little cell adhesion was observed on non ablated, grafted surfaces. Endothelial cells aligned along the groove, resulting in very narrow tube like tissue formation, whereas ECs tended to form a multilayered spherical aggregate in a hole. A single cell resided in a 10 microns square hole. 2) Microreactor for diagnosis: The glass surface, treated with a fluorinated silane, was ablated to create round holes. On addition of a few microliters of water, water could be quantitatively transferred into a hole because of the water repellent characteristics of non ablated, fluorinated glass. As a model of a microreactor, enzyme reactions to affect different levels of glucose were carried out in tiny holed surfaces.

  4. Local pulsatile contractions are an intrinsic property of the myosin 2A motor in the cortical cytoskeleton of adherent cells

    PubMed Central

    Baird, Michelle A.; Billington, Neil; Wang, Aibing; Adelstein, Robert S.; Sellers, James R.; Fischer, Robert S.; Waterman, Clare M.

    2017-01-01

    The role of nonmuscle myosin 2 (NM2) pulsatile dynamics in generating contractile forces required for developmental morphogenesis has been characterized, but whether these pulsatile contractions are an intrinsic property of all actomyosin networks is not known. Here we used live-cell fluorescence imaging to show that transient, local assembly of NM2A “pulses” occurs in the cortical cytoskeleton of single adherent cells of mesenchymal, epithelial, and sarcoma origin, independent of developmental signaling cues and cell–cell or cell–ECM interactions. We show that pulses in the cortical cytoskeleton require Rho-associated kinase– or myosin light chain kinase (MLCK) activity, increases in cytosolic calcium, and NM2 ATPase activity. Surprisingly, we find that cortical cytoskeleton pulses specifically require the head domain of NM2A, as they do not occur with either NM2B or a 2B-head-2A-tail chimera. Our results thus suggest that pulsatile contractions in the cortical cytoskeleton are an intrinsic property of the NM2A motor that may mediate its role in homeostatic maintenance of tension in the cortical cytoskeleton of adherent cells. PMID:27881665

  5. Adhesion to and invasion of cultured human cells by Bartonella bacilliformis.

    PubMed Central

    Hill, E M; Raji, A; Valenzuela, M S; Garcia, F; Hoover, R

    1992-01-01

    Bartonella bacilliformis was tested for its ability to adhere to and invade tissue culture cell monolayers. The parasite was able to efficiently bind and penetrate human dermal fibroblasts, human laryngeal epithelium, and human umbilical vein endothelial cells. Exposure of the organism to immune serum prepared against a crude Bartonella extract containing cell wall and membranous material resulted in decreased ability of the parasite to invade host cells. There was also an overall reduction in the invasiveness of bartonellae and total host cell association when human laryngeal epithelial cells and human umbilical vein endothelial cells were preexposed to cytochalasin D, indicating an active involvement of host cells in the uptake of bartonellae. Transmission electron microscopy revealed the presence of bartonellae inside and outside intracellular vacuoles. These data suggest that a surface-associated factor is involved in the invasion process and that internalization of the parasite by host cells involves a microfilament-dependent process similar to phagocytosis. Images PMID:1398917

  6. Eradication of Mycoplasma contaminations from cell cultures.

    PubMed

    Uphoff, Cord C; Drexler, Hans G

    2014-04-14

    Mycoplasma contaminations have a multitude of effects on cultured cell lines that may influence the results of experiments or pollute bioactive substances isolated from the eukaryotic cells. The elimination of mycoplasma contaminations from cell cultures with antibiotics has been proven to be a practical alternative to discarding and re-establishing important or irreplaceable cell lines. Different fluoroquinolones, tetracyclins, pleuromutilins, and macrolides shown to have strong anti-mycoplasma properties are employed for the decontamination. These antibiotics are applied as single treatments, as combination treatment of two antibiotics in parallel or successively, or in combination with a surface-active peptide to enhance the action of the antibiotic. The protocols in this unit allow eradication of mycoplasmas, prevention of the development of resistant mycoplasma strains, and potential cure of heavily contaminated and damaged cells. Consistent and permanent alterations to eukaryotic cells attributable to the treatment have not been demonstrated.

  7. Impact of release dynamics of laser-irradiated polymer micropallets on the viability of selected adherent cells

    PubMed Central

    Ma, Huan; Mismar, Wael; Wang, Yuli; Small, Donald W.; Ras, Mat; Allbritton, Nancy L.; Sims, Christopher E.; Venugopalan, Vasan

    2012-01-01

    We use time-resolved interferometry, fluorescence assays and computational fluid dynamics (CFD) simulations to examine the viability of confluent adherent cell monolayers to selection via laser microbeam release of photoresist polymer micropallets. We demonstrate the importance of laser microbeam pulse energy and focal volume position relative to the glass–pallet interface in governing the threshold energies for pallet release as well as the pallet release dynamics. Measurements using time-resolved interferometry show that increases in laser pulse energy result in increasing pallet release velocities that can approach 10 m s−1 through aqueous media. CFD simulations reveal that the pallet motion results in cellular exposure to transient hydrodynamic shear stress amplitudes that can exceed 100 kPa on microsecond timescales, and which produces reduced cell viability. Moreover, CFD simulation results show that the maximum shear stress on the pallet surface varies spatially, with the largest shear stresses occurring on the pallet periphery. Cell viability of confluent cell monolayers on the pallet surface confirms that the use of larger pulse energies results in increased rates of necrosis for those cells situated away from the pallet centre, while cells situated at the pallet centre remain viable. Nevertheless, experiments that examine the viability of these cell monolayers following pallet release show that proper choices for laser microbeam pulse energy and focal volume position lead to the routine achievement of cell viability in excess of 90 per cent. These laser microbeam parameters result in maximum pallet release velocities below 6 m s−1 and cellular exposure of transient hydrodynamic shear stresses below 20 kPa. Collectively, these results provide a mechanistic understanding that relates pallet release dynamics and associated transient shear stresses with subsequent cellular viability. This provides a quantitative, mechanistic basis for determining

  8. Production of the Escherichia coli common pilus by uropathogenic E. coli is associated with adherence to HeLa and HTB-4 cells and invasion of mouse bladder urothelium.

    PubMed

    Saldaña, Zeus; De la Cruz, Miguel A; Carrillo-Casas, Erika Margarita; Durán, Laura; Zhang, Yushan; Hernández-Castro, Rigoberto; Puente, José L; Daaka, Yehia; Girón, Jorge A

    2014-01-01

    Uropathogenic Escherichia coli (UPEC) strains cause urinary tract infections and employ type 1 and P pili in colonization of the bladder and kidney, respectively. Most intestinal and extra-intestinal E. coli strains produce a pilus called E. coli common pilus (ECP) involved in cell adherence and biofilm formation. However, the contribution of ECP to the interaction of UPEC with uroepithelial cells remains to be elucidated. Here, we report that prototypic UPEC strains CFT073 and F11 mutated in the major pilin structural gene ecpA are significantly deficient in adherence to cultured HeLa (cervix) and HTB-4 (bladder) epithelial cells in vitro as compared to their parental strains. Complementation of the ecpA mutant restored adherence to wild-type levels. UPEC strains produce ECP upon growth in Luria-Bertani broth or DMEM tissue culture medium preferentially at 26°C, during incubation with cultured epithelial cells in vitro at 37°C, and upon colonization of mouse bladder urothelium ex vivo. ECP was demonstrated on and inside exfoliated bladder epithelial cells present in the urine of urinary tract infection patients. The ability of the CFT073 ecpA mutant to invade the mouse tissue was significantly reduced. The presence of ECP correlated with the architecture of the biofilms produced by UPEC strains on inert surfaces. These data suggest that ECP can potentially be produced in the bladder environment and contribute to the adhesive and invasive capabilities of UPEC during its interaction with the host bladder. We propose that along with other known adhesins, ECP plays a synergistic role in the multi-step infection of the urinary tract.

  9. Production of the Escherichia coli Common Pilus by Uropathogenic E. coli Is Associated with Adherence to HeLa and HTB-4 Cells and Invasion of Mouse Bladder Urothelium

    PubMed Central

    Carrillo-Casas, Erika Margarita; Durán, Laura; Zhang, Yushan; Hernández-Castro, Rigoberto; Puente, José L.; Daaka, Yehia; Girón, Jorge A.

    2014-01-01

    Uropathogenic Escherichia coli (UPEC) strains cause urinary tract infections and employ type 1 and P pili in colonization of the bladder and kidney, respectively. Most intestinal and extra-intestinal E. coli strains produce a pilus called E. coli common pilus (ECP) involved in cell adherence and biofilm formation. However, the contribution of ECP to the interaction of UPEC with uroepithelial cells remains to be elucidated. Here, we report that prototypic UPEC strains CFT073 and F11 mutated in the major pilin structural gene ecpA are significantly deficient in adherence to cultured HeLa (cervix) and HTB-4 (bladder) epithelial cells in vitro as compared to their parental strains. Complementation of the ecpA mutant restored adherence to wild-type levels. UPEC strains produce ECP upon growth in Luria-Bertani broth or DMEM tissue culture medium preferentially at 26°C, during incubation with cultured epithelial cells in vitro at 37°C, and upon colonization of mouse bladder urothelium ex vivo. ECP was demonstrated on and inside exfoliated bladder epithelial cells present in the urine of urinary tract infection patients. The ability of the CFT073 ecpA mutant to invade the mouse tissue was significantly reduced. The presence of ECP correlated with the architecture of the biofilms produced by UPEC strains on inert surfaces. These data suggest that ECP can potentially be produced in the bladder environment and contribute to the adhesive and invasive capabilities of UPEC during its interaction with the host bladder. We propose that along with other known adhesins, ECP plays a synergistic role in the multi-step infection of the urinary tract. PMID:25036370

  10. Interventions for improving adherence to iron chelation therapy in people with sickle cell disease or thalassaemia

    PubMed Central

    Fortin, Patricia M; Madgwick, Karen V; Trivella, Marialena; Hopewell, Sally; Doree, Carolyn; Estcourt, Lise J

    2016-01-01

    This is the protocol for a review and there is no abstract. The objectives are as follows: To identify and assess the effectiveness of interventions to improve adherence to iron chelation therapy compared to standard care in people with SCD or thalassaemia including: identifying and assessing the effectiveness of different types of interventions (psychological and psychosocial, educational, medication interventions, or multi-component interventions);identifying and assessing the effectiveness of interventions specific to different age groups (children, adolescents, adults). PMID:27713668

  11. HIV pill reminder device shows some adherence improvement. Technology now switched to cell phone.

    PubMed

    2005-12-01

    Researchers studying a population of HIV patients found that a pill reminder improved adherence for those who were memory impaired. "One reason patients don't take medications is because they simply forget it, and it's more of an issue in the population we studied because some started with mild cognitive impairment," says Adriana Andrade, MD, MPH, an assistant professor at Johns Hopkins University Division of Infectious Diseases in Baltimore, MD.

  12. Quantitative Proteomic and Phosphoproteomic Comparison of 2D and 3D Colon Cancer Cell Culture Models.

    PubMed

    Yue, Xiaoshan; Lukowski, Jessica K; Weaver, Eric M; Skube, Susan B; Hummon, Amanda B

    2016-12-02

    Cell cultures are widely used model systems. Some immortalized cell lines can be grown in either two-dimensional (2D) adherent monolayers or in three-dimensional (3D) multicellular aggregates, or spheroids. Here, the quantitative proteome and phosphoproteome of colon carcinoma HT29 cells cultures in 2D monolayers and 3D spheroids were compared with a stable isotope labeling of amino acids (SILAC) labeling strategy. Two biological replicates from each sample were examined, and notable differences in both the proteome and the phosphoproteome were determined by nanoliquid chromatography tandem mass spectrometry (LC-MS/MS) to assess how growth configuration affects molecular expression. A total of 5867 protein groups, including 2523 phosphoprotein groups and 8733 phosphopeptides were identified in the samples. The Gene Ontology analysis revealed enriched GO terms in the 3D samples for RNA binding, nucleic acid binding, enzyme binding, cytoskeletal protein binding, and histone binding for their molecular functions (MF) and in the process of cell cycle, cytoskeleton organization, and DNA metabolic process for the biological process (BP). The KEGG pathway analysis indicated that 3D cultures are enriched for oxidative phosphorylation pathways, metabolic pathways, peroxisome pathways, and biosynthesis of amino acids. In contrast, analysis of the phosphoproteomes indicated that 3D cultures have decreased phosphorylation correlating with slower growth rates and lower cell-to-extracellular matrix interactions. In sum, these results provide quantitative assessments of the effects on the proteome and phosphoproteome of culturing cells in 2D versus 3D cell culture configurations.

  13. Platelet adhesion to human umbilical vein endothelial cells cultured on anionic hydrogel scaffolds.

    PubMed

    Chen, Yong Mei; Tanaka, Masaru; Gong, Jian Ping; Yasuda, Kazunori; Yamamoto, Sadaaki; Shimomura, Masatsugu; Osada, Yoshihito

    2007-04-01

    In this work we describe experiments designed to understand the human platelet adhesion to human umbilical vein endothelial cells (HUVECs) cultured on various kinds of chemically cross-linked anionic hydrogels, which were synthesized by radical polymerization. HUVECs could proliferate to sub-confluent or confluent on poly(acrylic acid) (PAA), poly(2-acrylamido-2-methyl-propane sulfonic acid sodium salt) (PNaAMPS), and poly(sodium p-styrene sulfonate) (PNaSS) gels. The proliferation behavior was not sensitive to the cross-linker concentration of the gels. However, the platelet adhesion on the HUVECs cultured on these gels showed different behavior, as revealed by human platelet adhesion test in static conditions. Only a few platelets adhered on the HUVEC sheets cultured on PNaAMPS gels with 4 and 10mol% cross-linker concentrations, and completely no platelet adhered on the HUVEC sheets cultured on PNaSS gels with 4 and 10mol% cross-linker concentrations. On the other hand, a large number of platelets adhered on the HUVECs cultured on PAA gels with 1, 2mol% cross-linker concentrations and PNaAMPS gel with 2mol% cross-linker concentration. Furthermore, the study showed that promote of the glycocalyx of HUVECs with transforming growth factor-beta(1) (TGF-beta(1)) decreased platelet adhesion, and degrade the glycocalyx with heparinase I increased platelet adhesion. The results suggested that the glycocalyx of cultured HUVECs modulates platelet compatibility, and the amount of glycocalyx secreted by HUVECs dependents on the chemical structure and cross-linker concentration of gel scaffolds. This result should be applied to make the hybrid artificial blood vessel composes of gels and endothelial cells with high platelet compatibility.

  14. Cell culture experiments planned for the space bioreactor

    NASA Technical Reports Server (NTRS)

    Morrison, Dennis R.; Cross, John H.

    1987-01-01

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

  15. Effects of teicoplanin on cell number of cultured cell lines

    PubMed Central

    Kashkolinejad-Koohi, Tahere; Saadat, Iraj

    2015-01-01

    Teicoplanin is a glycopeptide antibiotic with a wide variation in human serum half-life. It is also a valuable alternative of vancomycin. There is however no study on its effect on cultured cells. The aim of the present study was to test the effect of teicoplanin on cultured cell lines CHO, Jurkat E6.1 and MCF-7. The cultured cells were exposed to teicoplanin at final concentrations of 0–11000 μg/ml for 24 hours. To determine cell viability, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test was performed. At low concentrations of teicoplanin the numbers of cultured cells (due to cell proliferation) were increased in the three cell lines examined. The maximum cell proliferation rates were observed at concentrations of 1000, 400, and 200 μg/ml of teicoplanin for CHO, MCF-7 and Jurkat cell lines, respectively. Cell toxicity was observed at final concentrations over 2000, 6000, and 400 μg/ml of teicoplanin for CHO, MCF-7 and Jurkat cell lines, respectively. A dose-dependent manner of cell toxicity was observed. Our present findings indicated that teicoplanin at clinically used concentrations induced cell proliferation. It should therefore be used cautiously, particularly in children, pregnant women and patients with cancer. PMID:27486356

  16. Lipid Accumulation in Hypoxic Tissue Culture Cells

    PubMed Central

    Gordon, Gerald B.; Barcza, Maureen A.; Bush, Marilyn E.

    1977-01-01

    Lipid droplets have long been recognized by light microscopy to accumulate in hypoxic cells both in vivo and in vitro. In the present tissue culture experiments, correlative electron microscopic observations and lipid analyses were performed to determine the nature and significance of lipid accumulation in hypoxia. Strain L mouse fibroblasts were grown in suspension culture, both aerobically and under severe oxygen restriction obtained by gassing cultures daily with an 8% CO2-92% nitrogen mixture. After 48 hours, hypoxic cells showed an increase in total lipid/protein ratio of 42% over control cells. Most of this increase was accounted for by an elevation in the level of cellular triglyceride from 12.3 ± 0.9 μg/mg cell protein in aerobic cultures to 41.9 ± 0.7 in the hypoxic cultures, an increase of 240%. Levels of cellular free fatty acids (FFA) were 96% higher in the hypoxic cultures. No significant changes in the levels of cellular phospholipid or cholesterol were noted. Electron microscopic examination revealed the accumulation of homogeneous cytoplasmic droplets. The hypoxic changes were reversible upon transferring the cultures to aerobic atmospheres with disappearance of the lipid. These experiments indicate that hypoxic injury initially results in triglyceride and FFA accumulation from an inability to oxidize fatty acids taken up from the media and not from autophagic processes, as described in other types of cell injury associated with the sequestration of membranous residues and intracellular cholesterol and phospholipid accumulation. ImagesFigure 3Figure 4Figure 5Figure 6Figure 7Figure 1Figure 2 PMID:196505

  17. Neurofilament expression in cultured rat adenohypophysial cells.

    PubMed

    Quintanar, J L; Salinas, E

    2001-01-01

    The aim of the present work was to investigate in cultured rat adenohypophysial cells: a) the presence of neurofilaments of 200 kDa (NF-H), b) the effect of thyroid hormone (T(3)) and thyrotropin releasing hormone (TRH) on the expression of NF-H and c) the possible role of NF-H on thyrotropin (TSH) secretion. The presence of NF-H was observed by immunocytochemistry in cultured rat adenohypophysial cells. The exposure to T(3) for 12 h produced a significant increase in NF-H expression; whereas incubation with TRH or T(3)+TRH resulted in no change. The cells treated with T(3) or TRH or T(3)+TRH for 24 h showed no alteration. However, incubation for 48 h with TRH or T(3)+TRH caused significant decrease in NF-H expression. Incubation with NF-H antibodies produced a significant inhibition of calcium-induced TSH release in digitonin-permeabilized adenohypophysial cells. These results provide evidence that NF-H is present in cultured rat adenohypophysial cells, and that T(3) and TRH can modify NF-H expression. It can be suggested that in cultured adenohypophysial cells, NF-H may play a role in the secretory process.

  18. Biochemical Assays of Cultured Cells

    NASA Technical Reports Server (NTRS)

    Barlow, G. H.

    1985-01-01

    Subpopulations of human embryonic kidney cells isolated from continuous flow electrophoresis experiments performed at McDonnell Douglas and on STS-8 have been analyzed. These analyses have included plasminogen activator assays involving indirect methodology on fibrin plated and direct methodology using chromogenic substrates. Immunological studies were performed and the conditioned media for erythropoietin activity and human granulocyte colony stimulating (HGCSF) activity was analyzed.

  19. Inhibition of Streptococcus pneumoniae adherence to human epithelial cells in vitro by the probiotic Lactobacillus rhamnosus GG

    PubMed Central

    2013-01-01

    Background Colonization of the nasopharynx by Streptococcus pneumoniae is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. Probiotic bacteria can influence disease outcomes through various mechanisms, including inhibition of pathogen colonization. Here, we examine the effect of the probiotic Lactobacillus rhamnosus GG (LGG) on S. pneumoniae colonization of human epithelial cells using an in vitro model. We investigated the effects of LGG administered before, at the same time as, or after the addition of S. pneumoniae on the adherence of four pneumococcal isolates. Results LGG significantly inhibited the adherence of all the pneumococcal isolates tested. The magnitude of inhibition varied with LGG dose, time of administration, and the pneumococcal isolate used. Inhibition was most effective when a higher dose of LGG was administered prior to establishment of pneumococcal colonization. Mechanistic studies showed that LGG binds to epithelial cells but does not affect pneumococcal growth or viability. Administration of LGG did not lead to any significant changes in host cytokine responses. Conclusions These findings demonstrate that LGG can inhibit pneumococcal colonization of human epithelial cells in vitro and suggest that probiotics could be used clinically to prevent the establishment of pneumococcal carriage. PMID:23561014

  20. Cell Culture on MEMS Platforms: A Review

    PubMed Central

    Ni, Ming; Tong, Wen Hao; Choudhury, Deepak; Rahim, Nur Aida Abdul; Iliescu, Ciprian; Yu, Hanry

    2009-01-01

    Microfabricated systems provide an excellent platform for the culture of cells, and are an extremely useful tool for the investigation of cellular responses to various stimuli. Advantages offered over traditional methods include cost-effectiveness, controllability, low volume, high resolution, and sensitivity. Both biocompatible and bio-incompatible materials have been developed for use in these applications. Biocompatible materials such as PMMA or PLGA can be used directly for cell culture. However, for bio-incompatible materials such as silicon or PDMS, additional steps need to be taken to render these materials more suitable for cell adhesion and maintenance. This review describes multiple surface modification strategies to improve the biocompatibility of MEMS materials. Basic concepts of cell-biomaterial interactions, such as protein adsorption and cell adhesion are covered. Finally, the applications of these MEMS materials in Tissue Engineering are presented. PMID:20054478

  1. Integrated bioprocessing for plant cell cultures.

    PubMed

    Choi, J W; Cho, G H; Byun, S Y; Kim, D I

    2001-01-01

    Plant cell suspension culture has become the focus of much attention as a tool for the production of secondary metabolites including paclitaxel, a well-known anticancer agent. Recently, it has also been regarded as one of the host systems for the production of recombinant proteins. In order to produce phytochemicals using plant cell cultures, efficient processes must be developed with adequate bioreactor design. Most of the plant secondary metabolites are toxic to cells at the high concentrations required during culture. Therefore, if the product could be removed in situ during culture, productivity might be enhanced due to the alleviation of this toxicity. In situ removal or extractive bioconversion of such products can be performed by in situ extraction with various kinds of organic solvents. In situ adsorption using polymeric resins is another possibility. Using the fact that secondary metabolites are generally hydrophobic, various integrated bioprocessing techniques can be designed not only to lower toxicity, but also to enhance productivity. In this article, in situ extraction, in situ adsorption, utilization of cyclodextrins, and the application of aqueous two-phase systems in plant cell cultures are reviewed.

  2. Activation of circulated immune cells and inflammatory immune adherence are involved in the whole process of acute venous thrombosis

    PubMed Central

    Wang, Le-Min; Duan, Qiang-Lin; Yang, Fan; Yi, Xiang-Hua; Zeng, Yu; Tian, Hong-Yan; Lv, Wei; Jin, Yun

    2014-01-01

    Objective: To investigate localization and distribution of integrin subunit β1, β2 and β3 and morphological changes of ligand-recepter binding in thrombi of acute pulmonary embolism (PE) patients and explore activation of circulated immune cells, inflammatory immune adherence and coagulation response in acute venous thrombosis. Methods: Thrombi were collected from patients with acute PE. Immunohistochemistry was done to detect the expression and distribution of integrin β1, β2 and β3 in cells within thrombi, and ligands of integrin subunit β1, β2 and β3 were also determined by immunohistochemistry within the thrombi. Results: 1) Acute venous thrombi were red thrombi composed of skeletons and filamentous mesh containing large amounts of red blood cells and white blood cells; 2) Integrin subunit β1, β2 and β3 were expressed on lymphocytes, neutrophils and platelets; 3) No expression of integrin β1 ligands: Laminin, Fibronectin, Collagen I or Collagen-II on lymphocytes; integrin β2 ligands including ICAM, factor X and iC3b are distributed on neutrophils, and ligand fibrinogen bound to neutrophils; integrin β3 was expressed on platelets which form the skeleton of thrombi and bound to fibrinogen to construct mesh structure; 4) Factor Xa was expressed on the filamentous mesh; 5) Filamentous mesh was fully filled with red blood cell dominant blood cells. Conclusion: Acute venous thrombosis is an activation process of circulated lymphocytes, neutrophils and platelets mainly, and a whole process including integrin subunit β2 and β3 binding with their ligands. Activation of immune cells, inflammatory immune adherence and coagulation response are involved in the acute venous thrombosis. PMID:24753749

  3. Development of a cell culture surface conversion technique using alginate thin film for evaluating effect upon cellular differentiation.

    PubMed

    Nakashima, Y; Tsusu, K; Minami, K; Nakanishi, Y

    2014-06-01

    Here, we sought to develop a cell culture surface conversion technique that would not damage living cells. An alginate thin film, formed on a glass plate by spin coating of sodium alginate solution and dipping into calcium chloride solution, was used to inhibit adhesion of cells. The film could be removed by ethylenediaminetetraacetate (EDTA) at any time during cell culture, permitting observation of cellular responses to conversion of the culture surface in real time. Additionally, we demonstrated the validity of the alginate thin film coating method and the performance of the film. The thickness of the alginate thin film was controlled by varying the rotation speed during spin coating. Moreover, the alginate thin film completely inhibited the adhesion of cultured cells to the culture surface, irrespective of the thickness of the film. When the alginate thin film was removed from the culture surface by EDTA, the cultured cells adhered to the culture surface, and their morphology changed. Finally, we achieved effective differentiation of C2C12 myoblasts into myotube cells by cell culture on the convertible culture surface, demonstrating the utility of our novel technique.

  4. Development of a cell culture surface conversion technique using alginate thin film for evaluating effect upon cellular differentiation

    SciTech Connect

    Nakashima, Y.; Tsusu, K.; Minami, K.; Nakanishi, Y.

    2014-06-15

    Here, we sought to develop a cell culture surface conversion technique that would not damage living cells. An alginate thin film, formed on a glass plate by spin coating of sodium alginate solution and dipping into calcium chloride solution, was used to inhibit adhesion of cells. The film could be removed by ethylenediaminetetraacetate (EDTA) at any time during cell culture, permitting observation of cellular responses to conversion of the culture surface in real time. Additionally, we demonstrated the validity of the alginate thin film coating method and the performance of the film. The thickness of the alginate thin film was controlled by varying the rotation speed during spin coating. Moreover, the alginate thin film completely inhibited the adhesion of cultured cells to the culture surface, irrespective of the thickness of the film. When the alginate thin film was removed from the culture surface by EDTA, the cultured cells adhered to the culture surface, and their morphology changed. Finally, we achieved effective differentiation of C2C12 myoblasts into myotube cells by cell culture on the convertible culture surface, demonstrating the utility of our novel technique.

  5. Replication of human immunodeficiency virus type 1 in primary dendritic cell cultures.

    PubMed Central

    Langhoff, E; Terwilliger, E F; Bos, H J; Kalland, K H; Poznansky, M C; Bacon, O M; Haseltine, W A

    1991-01-01

    The ability of the human immunodeficiency virus type 1 (HIV-1) to replicate in primary blood dendritic cells was investigated. Dendritic cells compose less than 1% of the circulating leukocytes and are nondividing cells. Highly purified preparations of dendritic cells were obtained using recent advances in cell fractionation. The results of these experiments show that dendritic cells, in contrast to monocytes and T cells, support the active replication of all strains of HIV-1 tested, including T-cell tropic and monocyte/macrophage tropic isolates. The dendritic cell cultures supported much more virus production than did cultures of primary unseparated T cells, CD4+ T cells, and adherent as well as nonadherent monocytes. Replication of HIV-1 in dendritic cells produces no noticeable cytopathic effect nor does it decrease total cell number. The ability of the nonreplicating dendritic cells to support high levels of replication of HIV-1 suggests that this antigen-presenting cell population, which is also capable of supporting clonal T-cell growth, may play a central role in HIV pathogenesis, serving as a source of continued infection of CD4+ T cells and as a reservoir of virus infection. Images PMID:1910172

  6. Human placental eXpanded (PLX) mesenchymal-like adherent stromal cells confer neuroprotection to nerve growth factor (NGF)-differentiated PC12 cells exposed to ischemia by secretion of IL-6 and VEGF.

    PubMed

    Lahiani, Adi; Zahavi, Efrat; Netzer, Nir; Ofir, Racheli; Pinzur, Lena; Raveh, Shani; Arien-Zakay, Hadar; Yavin, Ephraim; Lazarovici, Philip

    2015-02-01

    Mesenchymal stem cells are potent candidates in stroke therapy due to their ability to secrete protective anti-inflammatory cytokines and growth factors. We investigated the neuroprotective effects of human placental mesenchymal-like adherent stromal cells (PLX) using an established ischemic model of nerve growth factor (NGF)-differentiated pheochromocytoma PC12 cells exposed to oxygen and glucose deprivation (OGD) followed by reperfusion. Under optimal conditions, 2 × 10⁵ PLX cells, added in a trans-well system, conferred 30-60% neuroprotection to PC12 cells subjected to ischemic insult. PC12 cell death, measured by LDH release, was reduced by PLX cells or by conditioned medium derived from PLX cells exposed to ischemia, suggesting the active release of factorial components. Since neuroprotection is a prominent function of the cytokine IL-6 and the angiogenic factor VEGF165, we measured their secretion using selective ELISA of the cells under ischemic or normoxic conditions. IL-6 and VEGF165 secretion by co-culture of PC12 and PLX cells was significantly higher under ischemic compared to normoxic conditions. Exogenous supplementation of 10 ng/ml each of IL-6 and VEGF165 to insulted PC12 cells conferred neuroprotection, reminiscent of the neuroprotective effect of PLX cells or their conditioned medium. Growth factors as well as co-culture conditioned medium effects were reduced by 70% and 20% upon pretreatment with 240 ng/ml Semaxanib (anti VEGF165) and/or 400 ng/ml neutralizing anti IL-6 antibody, respectively. Therefore, PLX-induced neuroprotection in ischemic PC12 cells may be partially explained by IL-6 and VEGF165 secretion. These findings may also account for the therapeutic effects seen in clinical trials after treatment with these cells.

  7. Tracking in real time the crawling dynamics of adherent living cells with a high resolution surface plasmon microscope

    NASA Astrophysics Data System (ADS)

    Streppa, L.; Berguiga, L.; Boyer Provera, E.; Ratti, F.; Goillot, E.; Martinez Torres, C.; Schaeffer, L.; Elezgaray, Juan; Arneodo, A.; Argoul, F.

    2016-03-01

    We introduce a high resolution scanning surface plasmon microscope for long term imaging of living adherent mouse myoblast cells. The coupling of a high numerical aperture objective lens with a fibered heterodyne interferometer provides both enhanced sensitivity and long term stability. This microscope takes advantage of the plasmon resonance excitation and the amplification of the electromagnetic field in near-field distance to the gold coated coverslip. This plasmon enhanced evanescent wave microscopy is particularly attractive for the study of cell adhesion and motility since it can be operated without staining of the biological sample. We show that this microscope allows very long-term imaging of living samples, and that it can capture and follow the temporal deformation of C2C12 myoblast cell protusions (lamellipodia), during their migration on a at surface.

  8. Cell culture and senescence in uterine fibroids.

    PubMed

    Markowski, Dominique Nadine; Bartnitzke, Sabine; Belge, Gazanfer; Drieschner, Norbert; Helmke, Burkhard Maria; Bullerdiek, Jörn

    2010-10-01

    The in vitro growth of cells from uterine fibroids is characterized by an early onset of senescence. Often, an even lower growth potential than that of matching myometrial cells is noted. Also, the tremendous differences in the expression of the high mobility group protein HMGA2 seen when comparing fibroids of different genetic subtypes are surprisingly not reflected by significant differences in their growth potential in vitro. We aimed to evaluate possible changes of the HMGA2 expression level between the native tissue and cell cultures, so we performed quantitative real-time polymerase chain reaction studies that revealed a marked decrease of the HMGA2 mRNA in culture in those cases with overexpression of HMGA2. In the two cases initially showing the highest expression, it decreased by approximately 97%. Associated with the decrease of HMGA2 was a clearly increased expression of the senescence-associated p19(Arf). Together, these findings explain the similar behavior of cell cultures from fibroids of different genetic subgroups and may also offer an explanation for the early onset of in vitro senescence in these cell cultures.

  9. An Evaluation of Matrix-Containing and Humanised Matrix-Free 3-Dimensional Cell Culture Systems for Studying Breast Cancer

    PubMed Central

    Roberts, Grace C.; Morris, Paul G.; Moss, Marcus A.; Maltby, Sarah L.; Palmer, Chelsea A.; Nash, Claire E.; Smart, Emily; Holliday, Deborah L.; Speirs, Valerie

    2016-01-01

    Background 3D cell cultures are emerging as more physiologically meaningful alternatives to monolayer cultures for many biological applications. They are attractive because they more closely mimic in vivo morphology, especially when co-cultured with stromal fibroblasts. Methodology/Principal Findings We compared the efficacy of 3 different 3D cell culture systems; collagen I, low attachment culture vessels and a modification of Fibrolife®, a specialised humanised cell culture medium devoid of animal-derived components, using breast cancer cell lines representative of the different molecular subtypes of breast cancer, cultured alone or with human mammary fibroblasts with a view to developing matrix-free humanised systems. 3D collagen I culture supported the growth of a range of breast cancer cell lines. By modifying the composition of Fibrolife® to epiFL, matrix-free cell culture was possible. During sequential transfer to epiFL breast cancer cells gradually detached from the flask, growing progressively as spheroids. Phenotype was stable and reversible with cells remaining actively proliferating and easily accessible throughout culture. They could also be revived from frozen stocks. To achieve co-culture with fibroblasts in epiFL required use of low attachment culture vessels instead of standard plastic as fibroblasts remained adherent in epiFL. Here, cancer cell spheroids were allowed to form before adding fibroblasts. Immunohistochemical examination showed fibroblasts scattered throughout the epithelial spheroid, not dissimilar to the relationship of tumour stroma in human breast cancer. Conclusions Because of its ease of handling, matrix-free 3D cell culture may be a useful model to study the influence of fibroblasts on breast cancer epithelial cells with use of epiFL culture medium taking this a step further towards a fully humanised 3D model. This methodology could be applied to other types of cancer cell lines, making this a versatile technique for cancer

  10. Evaluation of 309 Environmental Chemicals Using a Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity Assay

    PubMed Central

    Chandler, Kelly J.; Barrier, Marianne; Jeffay, Susan; Nichols, Harriette P.; Kleinstreuer, Nicole C.; Singh, Amar V.; Reif, David M.; Sipes, Nisha S.; Judson, Richard S.; Dix, David J.; Kavlock, Robert; Hunter, Edward S.; Knudsen, Thomas B.

    2011-01-01

    The vast landscape of environmental chemicals has motivated the need for alternative methods to traditional whole-animal bioassays in toxicity testing. Embryonic stem (ES) cells provide an in vitro model of embryonic development and an alternative method for assessing developmental toxicity. Here, we evaluated 309 environmental chemicals, mostly food-use pesticides, from the ToxCast™ chemical library using a mouse ES cell platform. ES cells were cultured in the absence of pluripotency factors to promote spontaneous differentiation and in the presence of DMSO-solubilized chemicals at different concentrations to test the effects of exposure on differentiation and cytotoxicity. Cardiomyocyte differentiation (α,β myosin heavy chain; MYH6/MYH7) and cytotoxicity (DRAQ5™/Sapphire700™) were measured by In-Cell Western™ analysis. Half-maximal activity concentration (AC50) values for differentiation and cytotoxicity endpoints were determined, with 18% of the chemical library showing significant activity on either endpoint. Mining these effects against the ToxCast Phase I assays (∼500) revealed significant associations for a subset of chemicals (26) that perturbed transcription-based activities and impaired ES cell differentiation. Increased transcriptional activity of several critical developmental genes including BMPR2, PAX6 and OCT1 were strongly associated with decreased ES cell differentiation. Multiple genes involved in reactive oxygen species signaling pathways (NRF2, ABCG2, GSTA2, HIF1A) were strongly associated with decreased ES cell differentiation as well. A multivariate model built from these data revealed alterations in ABCG2 transporter was a strong predictor of impaired ES cell differentiation. Taken together, these results provide an initial characterization of metabolic and regulatory pathways by which some environmental chemicals may act to disrupt ES cell growth and differentiation. PMID:21666745

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

    PubMed

    Runkle, Kristin B; Witze, Eric S

    2016-01-01

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

  12. Establishment of primary bovine intestinal epithelial cell culture and clone method.

    PubMed

    Zhan, Kang; Lin, Miao; Liu, Ming-Mei; Sui, Yang-Nan; Zhao, Guo-Qi

    2017-01-01

    The aim of this study was to establish bovine intestinal epithelial cell (BIEC) line and provide a novel clone cell method. Although various strategies of bovine cell culture and clone techniques have been reported, these methods remain not established. Here, we culture successfully primary BIECs and establish a novel clone cell method. Our result showed that BIECs could be successfully cultured and passaged about generation 5. These cellular aggregates and clusters were adherent loosely at day 2 of culture. Cell aggregates and clusters start to proliferate after approximately 4 d. The BIECs showed positive reaction against cytokeratin 18, E-cadherin, and characteristics of epithelial-like morphology. In addition, the fatty acid-binding proteins (FABPs), villin, and intestinal peptidase (IP) band were positive in BIECs. Our results suggest that the establishment of culturing and clone BIEC methods will apply to isolate and clone other primary cells. These BIECs could therefore contribute to the study of bovine intestinal nutrient absorption and regulation, immune regulation, and the pathogenesis of the bovine intestinal disease, which will provide intestinal cell model in vitro.

  13. Measurement of polyphosphoinositides in cultured mammalian cells.

    PubMed

    Cooke, Frank T

    2009-01-01

    The seven phosphorylated derivatives of phosphatidylinositol (PtdIns), often collectively referred to as polyphosphoinositides (PPIn), are a minor component of eukaryotic cell membranes. Nevertheless, their synthesis is needed for an ever-increasing spectrum of cellular processes, including regulation of the actin cytoskeleton, chemotaxis, membrane trafficking, glucose uptake, and organelle acidification. PPIn metabolism is regulated dynamically by a network of kinases and phosphatases. Furthermore, synthesis of PPIn can be provoked by external stimuli; for example, the second messenger phosphatidylinositol 3,4,5-trisphosphate rapidly and transiently accumulates in cells challenged with agonists such as PDGF that activate receptor tyrosine kinases. The measurement of PPIn levels in in vivo cultured cells has been vital to our understanding of the metabolism and function of these important signaling molecules; methods are described herein that allow measurement of PPIn levels in culture cells in vivo.

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

    NASA Technical Reports Server (NTRS)

    Parks, Kelsey

    2009-01-01

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

  15. Progress Towards Drosophila Epithelial Cell Culture

    PubMed Central

    Simcox, Amanda

    2015-01-01

    Drosophila epithelial research is at the forefront of the field; however, there are no well-characterized epithelial cell lines that could provide a complementary in vitro model for studies conducted in vivo. Here, a protocol is described that produces epithelial cell lines. The method uses genetic manipulation of oncogenes or tumor suppressors to induce embryonic primary culture cells to rapidly progress to permanent cell lines. It is, however, a general method and the type of cells that comprise a given line is not controlled experimentally. Indeed, only a small fraction of the lines produced are epithelial in character. For this reason, additional work needs to be done to develop a more robust epithelial cell-specific protocol. It is expected that Drosophila epithelial cell lines will have great utility for in vitro analysis of epithelial biology, particularly high-throughput analyses such as RNAi screens. PMID:23097097

  16. NF-{kappa}B signaling is activated and confers resistance to apoptosis in three-dimensionally cultured EGFR-mutant lung adenocarcinoma cells

    SciTech Connect

    Sakuma, Yuji; Yamazaki, Yukiko; Nakamura, Yoshiyasu; Yoshihara, Mitsuyo; Matsukuma, Shoichi; Koizume, Shiro; Miyagi, Yohei

    2012-07-13

    Highlights: Black-Right-Pointing-Pointer EGFR-mutant cells in 3D culture resist EGFR inhibition compared with suspended cells. Black-Right-Pointing-Pointer Degradation of I{kappa}B and activation of NF-{kappa}B are observed in 3D-cultured cells. Black-Right-Pointing-Pointer Inhibiting NF-{kappa}B enhances the efficacy of the EGFR inhibitor in 3D-cultured cells. -- Abstract: Epidermal growth factor receptor (EGFR)-mutant lung adenocarcinoma cells in suspension undergo apoptosis to a greater extent than adherent cells in a monolayer when EGFR autophosphorylation is inhibited by EGFR tyrosine kinase inhibitors (TKIs). This suggests that cell adhesion to a culture dish may activate an anti-apoptotic signaling pathway other than the EGFR pathway. Since the microenvironment of cells cultured in a monolayer are substantially different to that of cells existing in three-dimension (3D) in vivo, we assessed whether two EGFR-mutant lung adenocarcinoma cell lines, HCC827 and H1975, were more resistant to EGFR TKI-induced apoptosis when cultured in a 3D extracellular matrix (ECM) as compared with in suspension. The ECM-adherent EGFR-mutant cells in 3D were significantly less sensitive to treatment with WZ4002, an EGFR TKI, than the suspended cells. Further, a marked degradation of I{kappa}B{alpha}, the inhibitor of nuclear factor (NF)-{kappa}B, was observed only in the 3D-cultured cells, leading to an increase in the activation of NF-{kappa}B. Moreover, the inhibition of NF-{kappa}B with pharmacological inhibitors enhanced EGFR TKI-induced apoptosis in 3D-cultured EGFR-mutant cells. These results suggest that inhibition of NF-{kappa}B signaling would render ECM-adherent EGFR-mutant lung adenocarcinoma cells in vivo more susceptible to EGFR TKI-induced cell death.

  17. 3D culture for cardiac cells.

    PubMed

    Zuppinger, Christian

    2016-07-01

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

  18. Adherence to Mediterranean diet is associated with methylation changes in inflammation-related genes in peripheral blood cells.

    PubMed

    Arpón, A; Riezu-Boj, J I; Milagro, F I; Razquin, C; Martínez-González, M A; Corella, D; Estruch, R; Casas, R; Fitó, M; Ros, E; Salas-Salvadó, J; Martínez, J A

    2017-02-08

    Epigenetic processes, including DNA methylation, might be modulated by environmental factors such as the diet, which in turn have been associated with the onset of several diseases such as obesity or cardiovascular events. Meanwhile, Mediterranean diet (MedDiet) has demonstrated favourable effects on cardiovascular risk, blood pressure, inflammation and other complications related to excessive adiposity. Some of these effects could be mediated by epigenetic modifications. Therefore, the objective of this study was to investigate whether the adherence to MedDiet is associated with changes in the methylation status from peripheral blood cells. A subset of 36 individuals was selected within the Prevención con Dieta Mediterránea (PREDIMED)-Navarra study, a randomised, controlled, parallel trial with three groups of intervention in high cardiovascular risk volunteers, two with a MedDiet and one low-fat control group. Changes in methylation between baseline and 5 years were studied. DNA methylation arrays were analysed by several robust statistical tests and functional classifications. Eight genes related to inflammation and immunocompetence (EEF2, COL18A1, IL4I1, LEPR, PLAGL1, IFRD1, MAPKAPK2, PPARGC1B) were finally selected as changes in their methylation levels correlated with adherence to MedDiet and because they presented sensitivity related to a high variability in methylation changes. Additionally, EEF2 methylation levels positively correlated with concentrations of TNF-α and CRP. This report is apparently the first showing that adherence to MedDiet is associated with the methylation of the reported genes related to inflammation with a potential regulatory impact.

  19. Prevention and Detection of Mycoplasma Contamination in Cell Culture

    PubMed Central

    Nikfarjam, Laleh; Farzaneh, Parvaneh

    2012-01-01

    One of the main problems in cell culture is mycoplasma infection. It can extensively affect cell physiology and metabolism. As the applications of cell culture increase in research, industrial production and cell therapy, more concerns about mycoplasma contamination and detection will arise. This review will provide valuable information about: 1. the ways in which cells are contaminated and the frequency and source of mycoplasma species in cell culture; 2. the ways to prevent mycoplasma contamination in cell culture; 3. the importance of mycoplasma tests in cell culture; 4. different methods to identify mycoplasma contamination; 5. the consequences of mycoplasma contamination in cell culture and 6. available methods to eliminate mycoplasma contamination. Awareness about the sources of mycoplasma and pursuing aseptic techniques in cell culture along with reliable detection methods of mycoplasma contamination can provide an appropriate situation to prevent mycoplasma contamination in cell culture. PMID:23508237

  20. Cell Culture Assay for Human Noroviruses [response

    SciTech Connect

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

    2007-07-01

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

  1. A simple method to obtain pure cultures of multiciliated ependymal cells from adult rodents.

    PubMed

    Grondona, J M; Granados-Durán, P; Fernández-Llebrez, P; López-Ávalos, M D

    2013-01-01

    Ependymal cells form an epithelium lining the ventricular cavities of the vertebrate brain. Numerous methods to obtain primary culture ependymal cells have been developed. Most of them use foetal or neonatal rat brain and the few that utilize adult brain hardly achieve purity. Here, we describe a simple and novel method to obtain a pure non-adherent ependymal cell culture from explants of the striatal and septal walls of the lateral ventricles. The combination of a low incubation temperature followed by a gentle enzymatic digestion allows the detachment of most of the ependymal cells from the ventricular wall in a period of 6 h. Along with ependymal cells, a low percentage (less than 6 %) of non-ependymal cells also detaches. However, they do not survive under two restrictive culture conditions: (1) a simple medium (alpha-MEM with glucose) without any supplement; and (2) a low density of 1 cell/µl. This purification method strategy does not require cell labelling with antibodies and cell sorting, which makes it a simpler and cheaper procedure than other methods previously described. After a period of 48 h, only ependymal cells survive such conditions, revealing the remarkable survival capacity of ependymal cells. Ependymal cells can be maintained in culture for up to 7-10 days, with the best survival rates obtained in Neurobasal supplemented with B27 among the tested media. After 7 days in culture, ependymal cells lose most of the cilia and therefore the mobility, while acquiring radial glial cell markers (GFAP, BLBP, GLAST). This interesting fact might indicate a reprogramming of the cell identity.

  2. Probing nanoparticle interactions in cell culture media.

    PubMed

    Sabuncu, Ahmet C; Grubbs, Janna; Qian, Shizhi; Abdel-Fattah, Tarek M; Stacey, Michael W; Beskok, Ali

    2012-06-15

    Nanoparticle research is often performed in vitro with little emphasis on the potential role of cell culture medium. In this study, gold nanoparticle interactions with cell culture medium and two cancer cell lines (human T-cell leukemia Jurkat and human pancreatic carcinoma PANC1) were investigated. Gold nanoparticles of 10, 25, 50, and 100 nm in diameter at fixed mass concentration were tested. Size distributions and zeta potentials of gold nanoparticles suspended in deionized (DI) water and Dulbecco's Modified Eagle's Media (DMEM) supplemented with fetal calf serum (FCS) were measured using dynamic light scattering (DLS) technique. In DI water, particle size distributions exhibited peaks around their nominal diameters. However, the gold nanoparticles suspended in DMEM supplemented with FCS formed complexes around 100 nm, regardless of their nominal sizes. The DLS and UV-vis spectroscopy results indicate gold nanoparticle agglomeration in DMEM that is not supplemented by FCS. The zeta potential results indicate that protein rich FCS increases the dispersion quality of gold nanoparticle suspensions through steric effects. Cellular uptake of 25 and 50 nm gold nanoparticles by Jurkat and PANC1 cell lines were investigated using inductively coupled plasma-mass spectroscopy. The intracellular gold level of PANC1 cells was higher than that of Jurkat cells, where 50 nm particles enter cells at faster rates than the 25 nm particles.

  3. Cell division modulates prion accumulation in cultured cells.

    PubMed

    Ghaemmaghami, Sina; Phuan, Puay-Wah; Perkins, Beth; Ullman, Julie; May, Barnaby C H; Cohen, Fred E; Prusiner, Stanley B

    2007-11-13

    The phenotypic effect of prions on host cells is influenced by the physical properties of the prion strain and its level of accumulation. In mammalian cell cultures, prion accumulation is determined by the interplay between de novo prion formation, catabolism, cell division, and horizontal cell-to-cell transmission. Understanding this dynamic enables the analytical modeling of protein-based heritability and infectivity. Here, we quantitatively measured these competing effects in a subline of neuroblastoma (N2a) cells and propose a concordant reaction mechanism to explain the kinetics of prion propagation. Our results show that cell division leads to a predictable reduction in steady-state prion levels but not to complete clearance. Scrapie-infected N2a cells were capable of accumulating different steady-state levels of prions, dictated partly by the rate of cell division. We also show that prions in this subline of N2a cells are transmitted primarily from mother to daughter cells, rather than horizontal cell-to-cell transmission. We quantitatively modeled our kinetic results based on a mechanism that assumes a subpopulation of prions is capable of self-catalysis, and the levels of this subpopulation reach saturation in fully infected cells. Our results suggest that the apparent effectiveness of antiprion compounds in culture may be strongly influenced by the growth phase of the target cells.

  4. LINE-1 Cultured Cell Retrotransposition Assay.

    PubMed

    Kopera, Huira C; Larson, Peter A; Moldovan, John B; Richardson, Sandra R; Liu, Ying; Moran, John V

    2016-01-01

    The Long INterspersed Element-1 (LINE-1 or L1) retrotransposition assay has facilitated the discovery and characterization of active (i.e., retrotransposition-competent) LINE-1 sequences from mammalian genomes. In this assay, an engineered LINE-1 containing a retrotransposition reporter cassette is transiently transfected into a cultured cell line. Expression of the reporter cassette, which occurs only after a successful round of retrotransposition, allows the detection and quantification of the LINE-1 retrotransposition efficiency. This assay has yielded insight into the mechanism of LINE-1 retrotransposition. It also has provided a greater understanding of how the cell regulates LINE-1 retrotransposition and how LINE-1 retrotransposition impacts the structure of mammalian genomes. Below, we provide a brief introduction to LINE-1 biology and then detail how the LINE-1 retrotransposition assay is performed in cultured mammalian cells.

  5. [Good cell culture practice--implementation of a relational cell culture database].

    PubMed

    Philipp, Marcel O; Falkner, Erwin; Kapeller, Barbara; Eberl, Heidrun; Frick, Wolfram; Macfelda, Karin; Losert, Udo M

    2002-01-01

    The claim for cell culture to provide validable in vitro models for biomedical research postulates evasion of possible fatal record keeping errors. A prototype of a relational computer database for IBM-compatible personal computers using Microsoft(r) Windows 95/98/2000 and NT for administration of cell culture data has been developed using Microsoft(r) Access 98 (Microsoft Corporation, Redmond, USA), -Access Basic, -Visual Basic and Structured Query Language (SQL) (IBM Corporation, Armonk, USA), and was tested successfully. The modular software application manages the many aspects of cell culture laboratory record keeping like detailed information on tissue donor, primary cell isolation/cell line origin, immunohistochemical/molecular biological characterisation, cell countings at passaging/subcultivation/cell aliquotation and cryopreservation. One main feature is a collection of all methods performed at our cell culture laboratory, where linked tables and files store specific informations. Entries into the database are checked via validation rules for correctness to avoid mistakes. The developed prototype has been demonstrated to be an adaptable, reliable tool for improving quality of information storage according to Good Scientific Practice (GSP), Good Cell Culture Practice (GCCP) and general ISO certification trends.

  6. Modification of adherence to plastic and to human buccal cells of Candida albicans and Candida dubliniensis by a subinhibitory concentration of itraconazole.

    PubMed

    Blanco, M T; Morales, J J; Lucio, L; Pérez-Giraldo, C; Hurtado, C; Gómez-García, A C

    2006-02-01

    Exposure to subinhibitory concentrations of antifungal agents can influence the adherence of Candida spp. to the host cell. In this study the adherence of Candida albicans ATCC 10231 and Candida dubliniensis CECT 11455 to plastic and to human buccal epithelial cells was evaluated following pre-exposure to 0.5 x minimum inhibitory capacity (MIC) of itraconazole and compared with the corresponding cellular surface hydrophobicity. The yeasts were grown in Sabouraud broth or RPMI-1640 with itraconazole (0.5 x MIC) for 24-26 h at 37 degrees C and the drug was then removed. The adhesion capacity to plastic was studied by turbidimetry in a polystyrene microtiter plate. The adhesion of the yeast to buccal epithelial cells was determined using microscopy techniques. The cellular surface hydrophobicity levels were determined by the microbial adhesion hydrocarbons test. Pre-exposure to itraconazole decreased plastic adherence and cellular surface hydrophobicity in both species when grown in RPMI. When C. albicans was grown in Sabouraud broth, it was nonhydrophobic and did not adhere and therefore no change was detected with the antibiotic. Itraconazole increased adherence to buccal epithelial cells in both species and media studied, as compared to controls without antifungal agents. To study the effects of these antifungal agents on pathogenicity mechanisms, it will be necessary to standardize the methodology for evaluation to determine their in vivo therapeutic efficacy.

  7. Non-specific pinocytosis by human endothelial cells cultured as multicellular aggregates: uptake of lucifer yellow and horse radish peroxidase.

    PubMed

    Catizone, A; Chiantore, M V; Andreola, F; Coletti, D; Medolago Albani, L; Alescio, T

    1996-12-01

    We have analyzed the pattern of time-dependent and concentration-dependent incorporation of Lucifer Yellow CH (LY) and Horseradish Peroxidase (HRP) by human umbilical vein endothelial cells cultured on a non-adhesive substratum, where they they become organized into stable, multicellular aggregates. The data were compared with those previously obtained from low-density cultures of non-growing endothelial cells adherent to plastic. While the linear trend of the incorporation kinetics is preserved, the rate of uptake with both time and concentrations is highly dependent on the culture conditions, namely typology of cell-cell and cell-substrate interactions. An at least two-fold increase of the rate of uptake was observed with both markers in the aggregated cells. The extracellular concentration of LY required to saturate the binding capacity of the cell surface shifts from approximately 0.25 mg/ml, with the adherent cells, to approximately 0.5 mg/ml in the aggregated cells; the rate of uptake of three different forms of HRP shows, besides a sharp quantitative increase, also qualitative variations, testified by differential changes of their incorporation rates. These results are entirely consistent with the assumption that the association of the endothelial cells into multicellular aggregates increases the rate of pinocytic uptake by modifying the physicochemical properties of the cell surface, thereby increasing its differential affinity for the extracellular markers.

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

    NASA Technical Reports Server (NTRS)

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

    1998-01-01

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

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

    PubMed

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

    1998-08-01

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

  10. Plant cell cultures: bioreactors for industrial production.

    PubMed

    Ruffoni, Barbara; Pistelli, Laura; Bertoli, Alessandra; Pistelli, Luisa

    2010-01-01

    The recent biotechnology boom has triggered increased interest in plant cell cultures, since a number of firms and academic institutions investigated intensively to rise the production of very promising bioactive compounds. In alternative to wild collection or plant cultivation, the production of useful and valuable secondary metabolites in large bioreactors is an attractive proposal; it should contribute significantly to future attempts to preserve global biodiversity and alleviate associated ecological problems. The advantages of such processes include the controlled production according to demand and a reduced man work requirement. Plant cells have been grown in different shape bioreactors, however, there are a variety of problems to be solved before this technology can be adopted on a wide scale for the production of useful plant secondary metabolites. There are different factors affecting the culture growth and secondary metabolite production in bioreactors: the gaseous atmosphere, oxygen supply and CO2 exchange, pH, minerals, carbohydrates, growth regulators, the liquid medium rheology and cell density. Moreover agitation systems and sterilization conditions may negatively influence the whole process. Many types ofbioreactors have been successfully used for cultivating transformed root cultures, depending on both different aeration system and nutrient supply. Several examples of medicinal and aromatic plant cultures were here summarized for the scale up cultivation in bioreactors.

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

    NASA Technical Reports Server (NTRS)

    Cogoli, Augusto

    1992-01-01

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

  12. Functional Genomic Analysis of Candida albicans Adherence Reveals a Key Role for the Arp2/3 Complex in Cell Wall Remodelling and Biofilm Formation

    PubMed Central

    Ketela, Troy; Cowen, Leah E.

    2016-01-01

    Fungal biofilms are complex, structured communities that can form on surfaces such as catheters and other indwelling medical devices. Biofilms are of particular concern with Candida albicans, one of the leading opportunistic fungal pathogens of humans. C. albicans biofilms include yeast and filamentous cells that are surrounded by an extracellular matrix, and they are intrinsically resistant to antifungal drugs such that resolving biofilm infections often requires surgery to remove the contaminated device. C. albicans biofilms form through a regulated process of adhesion to surfaces, filamentation, maturation, and ultimately dispersion. To uncover new strategies to block the initial stages of biofilm formation, we utilized a functional genomic approach to identify genes that modulate C. albicans adherence. We screened a library of 1,481 double barcoded doxycycline-repressible conditional gene expression strains covering ~25% of the C. albicans genome. We identified five genes for which transcriptional repression impaired adherence, including: ARC18, PMT1, MNN9, SPT7, and orf19.831. The most severe adherence defect was observed upon transcriptional repression of ARC18, which encodes a member of the Arp2/3 complex that is involved in regulation of the actin cytoskeleton and endocytosis. Depletion of components of the Arp2/3 complex not only impaired adherence, but also caused reduced biofilm formation, increased cell surface hydrophobicity, and increased exposure of cell wall chitin and β-glucans. Reduced function of the Arp2/3 complex led to impaired cell wall integrity and activation of Rho1-mediated cell wall stress responses, thereby causing cell wall remodelling and reduced adherence. Thus, we identify important functional relationships between cell wall stress responses and a novel mechanism that controls adherence and biofilm formation, thereby illuminating novel strategies to cripple a leading fungal pathogen of humans. PMID:27870871

  13. The effect of prebiotics on adherence of probiotics.

    PubMed

    Kadlec, Robert; Jakubec, Martin

    2014-01-01

    Prebiotics are generally considered to promote the function or viability of probiotics via their fermentation, but their effect on the adherence of probiotics is still unclear. In this study, we examined the effect of 4 commercially available prebiotics [Orafti GR, Orafti P95, and Orafti Synergy (Beneo GmbH, Mannheim, Germany), and Vivinal (Friesland Foods Domo, Amersfoort, the Netherlands)] and 3 simple saccharides (glucose, galactose, and lactose) on the adherence of 5 probiotic type strains, 2 lactococci starter cultures, and 5 potential dairy probiotic strains from the Culture Collection of Dairy Microorganisms (Tábor, Czech Republic). Adherence was tested in microtiter plates on the following types of substrate: polystyrene alone and polystyrene coated with either porcine mucus or cocultures of the human colon cell lines Caco2 and HT29-MXT (1:9 ratio of HT29-MXT:Caco2). Adherence was evaluated as a change in fluorescence in the well of a microtiter plate. The most commonly observed effect (with a few exceptions) of prebiotics was decreased adherence of the tested strains observed on all types of substrate. The tested saccharides, which are part of the residual compounds of the used prebiotics, had a very similar effect-eliciting a decrease in adherence ability in the majority of the probiotic strains.

  14. An in vitro adherence assay reveals that Helicobacter pylori exhibits cell lineage-specific tropism in the human gastric epithelium.

    PubMed Central

    Falk, P; Roth, K A; Borén, T; Westblom, T U; Gordon, J I; Normark, S

    1993-01-01

    Helicobacter pylori is a microaerophilic bacterium found in the stomach of asymptomatic humans as well as patients with acid peptic disease and gastric adenocarcinoma. We have developed an in situ adherence assay to examine the cell lineage-specific nature of binding of this organism and to characterize the nature of cell surface receptors that recognize its adhesin. Fluorescein isothiocyanate-labeled H. pylori strains were bound to surface mucous cells present in the pit region of human and rat gastric units but not to mucous neck, parietal, or chief cell lineages present in the glandular domains of these units. Binding was abolished by proteinase K treatment of tissue sections and by pretreatment of the bacteria with bovine submaxillary gland mucin, a rich source of fucosylated and sialylated carbohydrates. Several lines of evidence suggest that binding to surface mucous cells is not dependent upon terminal nonsubstituted alpha 2,3- and alpha 2,6-linked sialic acids in the adhesin receptor: (i) binding was not inhibited by incubating H. pylori strains with sialylated glycoconjugates such as fetuin and free sialyllactose; (ii) immunohistochemical stainings using the sialic acid-specific Sambucus nigra and Maackia amurensis lectins and the cholera toxin B subunit did not detect any sialylated glycoconjugates in these epithelial cells; and (iii) binding was not sensitive to metaperiodate under conditions that selectively cleaved carbons 8 and 9 of terminal nonmodified sialic acids. A role for fucosylated epitopes in the glycoprotein(s) that mediate binding of H. pylori to surface mucous cells was suggested by the facts that this lineage coexpresses the adhesin receptor and major fucosylated histo-blood group antigens, that monoclonal antibodies specific for histo-blood group antigens H, B, and Leb block binding, and that the lectin Ulex europaeus type 1 agglutinin, which is specific for alpha-L-fucose, also bound to the same cells that bound the bacteria

  15. Stability of cultured dental follicle cells.

    PubMed

    Yao, Shaomian; Norton, Jolanna; Wise, Gary E

    2004-06-01

    Because the dental follicle is required for tooth eruption, establishment of dental follicle cell (DFC) lines is needed for experimentation to determine how the cells regulate eruption. Thus, it is critical that the follicle cells in culture remain stable and neither become transformed nor differentiate. To determine the stability of rat DFC cultures in terms of exhibiting contact inhibition of growth when confluent (no transformation), DFC at different passages were analysed using flow cytometry. Gene expression of cyclin E was determined by reverse transcription polymerase chain reaction as a further method to determine if growth was occurring when the cells were confluent. Alkaline phosphatase and von Kossa staining were also performed as a means of determining stability in terms of differentiation; that is, are the DFC maintaining their phenotype or are they differentiating into osteoblasts and osteocytes? After plating cells of a given passage, they initially underwent a rapid phase of growth with 30-40% of the cells in S, G(2) and M (dividing track) as determined by flow cytometry. The number of such cells declined to only 7-15% at preconfluency. At late confluency, only 2 and 5% of the cells were in the dividing track in passages 6 and 9, respectively, but in passage 12 this had risen to 15%. For a given passage of cells, cyclin E gene expression significantly declined in late confluency as compared to the early growth phase. However, in passage 12, the gene expression of cyclin E at late confluency was higher than the expression at late confluency in passage 6. Thus, the DFC were remarkably stable through passage 9, but by passage 12 it appeared that a small percentage of the cells had become transformed and had lost their contact inhibition growth properties. Alkaline phosphatase and von Kossa staining were negative for all passages, suggesting that the cells remained stable in terms of differentiation and did not differentiate into either osteoblasts or

  16. Human melanoma cells derived from lymphatic metastases use integrin alpha v beta 3 to adhere to lymph node vitronectin.

    PubMed Central

    Nip, J; Shibata, H; Loskutoff, D J; Cheresh, D A; Brodt, P

    1992-01-01

    Human melanoma is a highly metastatic cancer and the regional lymph nodes are generally the first site of metastasis. Adhesion to cryostat sections of human lymph nodes was therefore studied using two human melanoma models established from lymph node metastases, namely, MeWo cell lines of diverse metastatic potentials and a highly metastatic cell line of recent origin designated MIM/8. We found a good correlation between the metastatic potentials of the melanoma cells as measured in nude mice and their ability to adhere to cryostat sections of human lymph nodes. When adhesion to immobilized extracellular matrix proteins was measured, a significant increase in adhesion, which correlated with increased metastasis, was seen mainly on vitronectin and to a lesser extent on fibronectin. The adhesion to vitronectin and to the frozen sections were specifically blocked by an RGD-containing peptide, mAb 661 to vitronectin and mAb LM609 to integrin alpha v beta 3. FACS analysis revealed a significant and specific increase in cell surface expression of alpha v beta 3 on the metastatic cells as compared to the parent line. Together these results suggest that the adhesion of melanoma cells to lymph node vitronectin via the alpha v beta 3 receptor plays a role in the process of lymphatic dissemination. Images PMID:1383272

  17. Surface modification of cell culture carriers: routes to anhydride functionalization of polystyrene.

    PubMed

    Nitschke, Mirko; Ricciardi, Serena; Gramm, Stefan; Zschoche, Stefan; Herklotz, Manuela; Rivolo, Paola; Werner, Carsten

    2012-02-01

    Physico-chemical and topographical cues allow to control the behavior of adherent cells. Towards this goal, commercially available cell culture carriers can be finished with a laterally microstructured biomolecular functionalization. As shown in a previous study [Biomacromolecules 4 (2003) 1072], the anhydride moiety facilitates a simple and versatile way to protein binding. The present work addresses the technical issue of anhydride surface functionalization of polystyrene, the most common material for cell culture ware. Different approaches based on low pressure plasma, electron beam and ultraviolet light techniques (i.e. maleic anhydride plasma reactions; plasma, electron beam and UV immobilization of functional polymer thin films; grafting of functional polymers to plasma activated surfaces) are introduced and briefly illustrated with examples. Results are characterized by Fourier transform infrared (FTIR) spectroscopy, X-ray photoelectron spectroscopy (XPS) and ellipsometry. The different routes are compared in terms of technical feasibility and achievable surface properties.

  18. Adherence to Asian and European American Cultural Values and Attitudes toward Seeking Professional Psychological Help among Asian American College Students

    ERIC Educational Resources Information Center

    Kim, Bryan S. K.

    2007-01-01

    Possible relations among enculturation and acculturation to cultural values and attitudes toward seeking professional psychological help were examined among 146 Asian American college students. In addition, possible relations between various dimensions of Asian values and attitudes toward seeking professional psychological help were examined. As…

  19. Mouse bone marrow-derived mast cells (BMMC) change their phenotype when cultured with fibroblasts

    SciTech Connect

    Levi-Schaffer, F.; Austen, K.F.; Stevens, R.L.

    1986-03-05

    The heparin-containing mast cells (HP-MC) that reside in the connective tissues of the mouse, but not the chondroitin sulfate containing mast cells in the gastrointestinal mucosa, stain with safranin when exposed to alcian blue/safranin. Mouse BMMC (the presumptive in vitro counterpart of the in vivo differentiated mucosal mast cell) were cultured for 2-14 days with confluent skin-derived 3T3 fibroblasts in RPMI-1640 containing 10% fetal calf serum and 50% WEHI-3 conditioned medium. Although the BMMC adhered to the fibroblast monolayer, they continued to divide, probably due to the presence of interleukin-3 in the conditioned medium. The mast cells remained viable throughout the period of co-culture, since they failed to release LDG and because they increased their histamine content per cell approx.15-fold. After 8-9 days of co-culture, >50% of the BMMC changed histochemically becoming safranin positive. At this time, 30-50% of the (/sup 35/S)glycosaminoglycans on the proteoglycans synthesized by these co-cultured mass cells were heparin, whereas the initial BMMC synthesized proteoglycans containing only chondroitin sulfate E. That interleukin 3-dependent mouse BMMC can be induced to undergo a phenotypic change so as to express characteristics of a HP-MC suggests that the tissue microenvironment determines the differentiated characteristics of these cells.

  20. Tubulin dynamics in cultured mammalian cells

    PubMed Central

    1984-01-01

    Bovine neurotubulin has been labeled with dichlorotriazinyl- aminofluorescein (DTAF-tubulin) and microinjected into cultured mammalian cells strains PTK1 and BSC. The fibrous, fluorescence patterns that developed in the microinjected cells were almost indistinguishable from the pattern of microtubules seen in the same cells by indirect immunofluorescence. DTAF-tubulin participated in the formation of all visible, microtubule-related structures at all cell cycle stages for at least 48 h after injection. Treatments of injected cells with Nocodazole or Taxol showed that DTAF-tubulin closely mimicked the behavior of endogenous tubulin. The rate at which microtubules incorporated DTAF-tubulin depended on the cell-cycle stage of the injected cell. Mitotic microtubules became fluorescent within seconds while interphase microtubules required minutes. Studies using fluorescence redistribution after photobleaching confirmed this apparent difference in tubulin dynamics between mitotic and interphase cells. The temporal patterns of redistribution included a rapid phase (approximately 3 s) that we attribute to diffusion of free DTAF-tubulin and a second, slower phase that seems to represent the exchange of bleached DTAF-tubulin in microtubules with free, unbleached DTAF- tubulin. Mean half times of redistribution were 18-fold shorter in mitotic cells than they were in interphase cells. PMID:6501419

  1. Side Effects of Culture Media Antibiotics on Cell Differentiation.

    PubMed

    Llobet, Laura; Montoya, Julio; López-Gallardo, Ester; Ruiz-Pesini, Eduardo

    2015-11-01

    Besides the advance in scientific knowledge and the production of different compounds, cell culture can now be used to obtain cells for regenerative medicine. To avoid microbial contamination, antibiotics were usually incorporated into culture media. However, these compounds affect cell biochemistry and may modify the differentiation potential of cultured cells. To check this possibility, we grew human adipose tissue-derived stem cells and differentiated them to adipocyte with or without antibiotics commonly used in these culture protocols, such as a penicillin-streptomycin-amphotericin mix or gentamicin. We show that these antibiotics affect cell differentiation. Therefore, antibiotics should not be used in cell culture because aseptic techniques make these compounds unnecessary.

  2. Intrathecal Transplantation of Autologous Adherent Bone Marrow Cells Induces Functional Neurological Recovery in a Canine Model of Spinal Cord Injury.

    PubMed

    Gabr, Hala; El-Kheir, Wael Abo; Farghali, Haithem A M A; Ismail, Zeinab M K; Zickri, Maha B; El Maadawi, Zeinab M; Kishk, Nirmeen A; Sabaawy, Hatem E

    2015-01-01

    Spinal cord injury (SCI) results in demyelination of surviving axons, loss of oligodendrocytes, and impairment of motor and sensory functions. We have developed a clinical strategy of cell therapy for SCI through the use of autologous bone marrow cells for transplantation to augment remyelination and enhance neurological repair. In a preclinical large mammalian model of SCI, experimental dogs were subjected to a clipping contusion of the spinal cord. Two weeks after the injury, GFP-labeled autologous minimally manipulated adherent bone marrow cells (ABMCs) were transplanted intrathecally to investigate the safety and efficacy of autologous ABMC therapy. The effects of ABMC transplantation in dogs with SCI were determined using functional neurological scoring, and the integration of ABMCs into the injured cords was determined using histopathological and immunohistochemical investigations and electron microscopic analyses of sections from control and transplanted spinal cords. Our data demonstrate the presence of GFP-labeled cells in the injured spinal cord for up to 16 weeks after transplantation in the subacute SCI stage. GFP-labeled cells homed to the site of injury and were detected around white matter tracts and surviving axons. ABMC therapy in the canine SCI model enhanced remyelination and augmented neural regeneration, resulting in improved neurological functions. Therefore, autologous ABMC therapy appears to be a safe and promising therapy for spinal cord injuries.

  3. Functional and Molecular Characterization of Ex Vivo Cultured Epiretinal Membrane Cells from Human Proliferative Diabetic Retinopathy

    PubMed Central

    Veréb, Zoltán; Lumi, Xhevat; Andjelic, Sofija; Globocnik-Petrovic, Mojca; Urbancic, Mojca; Hawlina, Marko; Facskó, Andrea; Petrovski, Goran

    2013-01-01

    Characterization of the cell surface marker phenotype of ex vivo cultured cells growing out of human fibrovascular epiretinal membranes (fvERMs) from proliferative diabetic retinopathy (PDR) can give insight into their function in immunity, angiogenesis, and retinal detachment. FvERMs from uneventful vitrectomies due to PDR were cultured adherently ex vivo. Surface marker analysis, release of immunity- and angiogenesis-pathway-related factors upon TNFα activation and measurement of the intracellular calcium dynamics upon mechano-stimulation using fluorescent dye Fura-2 were all performed. FvERMs formed proliferating cell monolayers when cultured ex vivo, which were negative for endothelial cell markers (CD31, VEGFR2), partially positive for hematopoietic- (CD34, CD47) and mesenchymal stem cell markers (CD73, CD90/Thy-1, and PDGFRβ), and negative for CD105. CD146/MCAM and CD166/ALCAM, previously unreported in cells from fvERMs, were also expressed. Secretion of 11 angiogenesis-related factors (DPPIV/CD26, EG-VEGF/PK1, ET-1, IGFBP-2 and 3, IL-8/CXCL8, MCP-1/CCL2, MMP-9, PTX3/TSG-14, Serpin E1/PAI-1, Serpin F1/PEDF, TIMP-1, and TSP-1) were detected upon TNFα activation of fvERM cells. Mechano-stimulation of these cells induced intracellular calcium propagation representing functional viability and role of these cells in tractional retinal detachment, thus serving as a model for studying tractional forces present in fvERMs in PDR ex vivo. PMID:24195074

  4. Functional and molecular characterization of ex vivo cultured epiretinal membrane cells from human proliferative diabetic retinopathy.

    PubMed

    Veréb, Zoltán; Lumi, Xhevat; Andjelic, Sofija; Globocnik-Petrovic, Mojca; Urbancic, Mojca; Hawlina, Marko; Facskó, Andrea; Petrovski, Goran

    2013-01-01

    Characterization of the cell surface marker phenotype of ex vivo cultured cells growing out of human fibrovascular epiretinal membranes (fvERMs) from proliferative diabetic retinopathy (PDR) can give insight into their function in immunity, angiogenesis, and retinal detachment. FvERMs from uneventful vitrectomies due to PDR were cultured adherently ex vivo. Surface marker analysis, release of immunity- and angiogenesis-pathway-related factors upon TNF α activation and measurement of the intracellular calcium dynamics upon mechano-stimulation using fluorescent dye Fura-2 were all performed. FvERMs formed proliferating cell monolayers when cultured ex vivo, which were negative for endothelial cell markers (CD31, VEGFR2), partially positive for hematopoietic- (CD34, CD47) and mesenchymal stem cell markers (CD73, CD90/Thy-1, and PDGFR β ), and negative for CD105. CD146/MCAM and CD166/ALCAM, previously unreported in cells from fvERMs, were also expressed. Secretion of 11 angiogenesis-related factors (DPPIV/CD26, EG-VEGF/PK1, ET-1, IGFBP-2 and 3, IL-8/CXCL8, MCP-1/CCL2, MMP-9, PTX3/TSG-14, Serpin E1/PAI-1, Serpin F1/PEDF, TIMP-1, and TSP-1) were detected upon TNF α activation of fvERM cells. Mechano-stimulation of these cells induced intracellular calcium propagation representing functional viability and role of these cells in tractional retinal detachment, thus serving as a model for studying tractional forces present in fvERMs in PDR ex vivo.

  5. The induction of suppressor cells in mixed leucocyte cultures and in mixed leucocyte-non-lymphoid cell cultures.

    PubMed Central

    Pawelec, G

    1980-01-01

    X-ray resistant porcine suppressor T cells expressing Ia-like antigens were obtained from mixed cultures of leucocytes and tissue cells (cultured kidney cells, liver cells, endothelial cells, fibroblasts or X-irradiated leucocytes), and were assayed by their ability to suppress lymphocyte proliferation in a second mixed culture. All tissues tested induced suppressor cells although quantitative differences existed between them. Suppressor cell induction was under genetic control by at least two loci, one of which was within the major histocompatibility (MHC) complex. Suppressor cell function was restricted by the MHC type of the responding cell but not the stimulating cell in the second culture. PMID:6445866

  6. Modeling the deformation of a migrating cell adhering to a rigid ligand-coated substrate in the presence of a shear flow

    NASA Astrophysics Data System (ADS)

    Chiam, Keng-Hwee; Lei Lai, Tan; Quek, Raymond

    2007-11-01

    We have developed a computational model for the process in metastasis where tumor cells that have intravasated into the vasculature are carried by the circulation to a distant part of the body. Using a two-dimensional model of a cell as a homogeneous viscoelastic drop that is parametrized by its cytoplasmic viscosity and membrane surface tension, we have shown that the length of the cell membrane that is adhered to the substrate can be expressed in a very simple relation involving only the product of the inverse of the cell's capillary number and the distance that the cell has migrated. We have also shown that this relation may be exploited in determining a cell's cytoplasmic viscosity in terms of mechanical quantities such as adhered length and distance migrated. This may aid in the development of microfluidic devices that may one day serve as a diagnostic tool to screen for tumor cells that have a different stiffness from normal cells. Finally, we have also shown that, when the cell is sufficiently close to the rigid substrate, adhesive forces mediated by receptors on the cell and ligands on the substrate is negligible. We provide evidence for this by showing that the length of the cell membrane adhered to the substrate is independent of the density of adhesion receptors on the cell's membrane.

  7. Advantages and challenges of microfluidic cell culture in polydimethylsiloxane devices.

    PubMed

    Halldorsson, Skarphedinn; Lucumi, Edinson; Gómez-Sjöberg, Rafael; Fleming, Ronan M T

    2015-01-15

    Culture of cells using various microfluidic devices is becoming more common within experimental cell biology. At the same time, a technological radiation of microfluidic cell culture device designs is currently in progress. Ultimately, the utility of microfluidic cell culture will be determined by its capacity to permit new insights into cellular function. Especially insights that would otherwise be difficult or impossible to obtain with macroscopic cell culture in traditional polystyrene dishes, flasks or well-plates. Many decades of heuristic optimization have gone into perfecting conventional cell culture devices and protocols. In comparison, even for the most commonly used microfluidic cell culture devices, such as those fabricated from polydimethylsiloxane (PDMS), collective understanding of the differences in cellular behavior between microfluidic and macroscopic culture is still developing. Moving in vitro culture from macroscopic culture to PDMS based devices can come with unforeseen challenges. Changes in device material, surface coating, cell number per unit surface area or per unit media volume may all affect the outcome of otherwise standard protocols. In this review, we outline some of the advantages and challenges that may accompany a transition from macroscopic to microfluidic cell culture. We focus on decisive factors that distinguish macroscopic from microfluidic cell culture to encourage a reconsideration of how macroscopic cell culture principles might apply to microfluidic cell culture.

  8. Differentiation of mammalian skeletal muscle cells cultured on microcarrier beads in a rotating cell culture system

    NASA Technical Reports Server (NTRS)

    Torgan, C. E.; Burge, S. S.; Collinsworth, A. M.; Truskey, G. A.; Kraus, W. E.

    2000-01-01

    The growth and repair of adult skeletal muscle are due in part to activation of muscle precursor cells, commonly known as satellite cells or myoblasts. These cells are responsive to a variety of environmental cues, including mechanical stimuli. The overall goal of the research is to examine the role of mechanical signalling mechanisms in muscle growth and plasticity through utilisation of cell culture systems where other potential signalling pathways (i.e. chemical and electrical stimuli) are controlled. To explore the effects of decreased mechanical loading on muscle differentiation, mammalian myoblasts are cultured in a bioreactor (rotating cell culture system), a model that has been utilised to simulate microgravity. C2C12 murine myoblasts are cultured on microcarrier beads in a bioreactor and followed throughout differentiation as they form a network of multinucleated myotubes. In comparison with three-dimensional control cultures that consist of myoblasts cultured on microcarrier beads in teflon bags, myoblasts cultured in the bioreactor exhibit an attenuation in differentiation. This is demonstrated by reduced immunohistochemical staining for myogenin and alpha-actinin. Western analysis shows a decrease, in bioreactor cultures compared with control cultures, in levels of the contractile proteins myosin (47% decrease, p < 0.01) and tropomyosin (63% decrease, p < 0.01). Hydrodynamic measurements indicate that the decrease in differentiation may be due, at least in part, to fluid stresses acting on the myotubes. In addition, constraints on aggregate size imposed by the action of fluid forces in the bioreactor affect differentiation. These results may have implications for muscle growth and repair during spaceflight.

  9. The α-helical regions of KERP1 are important in Entamoeba histolytica adherence to human cells

    PubMed Central

    Perdomo, Doranda; Baron, Bruno; Rojo-Domínguez, Arturo; Raynal, Bertrand; England, Patrick; Guillén, Nancy

    2013-01-01

    The lysine and glutamic acid rich protein KERP1 is a unique surface adhesion factor associated with virulence in the human pathogen Entamoeba histolytica. Both the function and structure of this protein remain unknown to this date. Here, we used circular dichroism, analytical ultracentrifugation and bioinformatics modeling to characterize the structure of KERP1. Our findings revealed that it is an α-helical rich protein organized as a trimer, endowed with a very high thermal stability (Tm = 89.6°C). Bioinformatics sequence analyses and 3D-structural modeling indicates that KERP1 central segments could account for protein trimerization. Relevantly, expressing the central region of KERP1 in living parasites, impair their capacity to adhere to human cells. Our observations suggest a link between the inhibitory effect of the isolated central region and the structural features of KERP1. PMID:23378906

  10. High-Throughput Cancer Cell Sphere Formation for Characterizing the Efficacy of Photo Dynamic Therapy in 3D Cell Cultures

    NASA Astrophysics Data System (ADS)

    Chen, Yu-Chih; Lou, Xia; Zhang, Zhixiong; Ingram, Patrick; Yoon, Euisik

    2015-07-01

    Photodynamic therapy (PDT), wherein light sensitive non-toxic agents are locally and selectively activated using light, has emerged as an appealing alternative to traditional cancer chemotherapy. Yet to date, PDT efficacy has been mostly characterized using 2D cultures. Compared to 2D cultures, 3D sphere culture generates unique spatial distributions of nutrients and oxygen for the cells that better mimics the in-vivo conditions. Using a novel polyHEMA (non-adherent polymer) fabrication process, we developed a microfluidic sphere formation platform that can (1) generate 1,024 uniform (size variation <10%) cancer spheres within a 2 cm by 2 cm core area, (2) culture spheres for more than 2 weeks, and (3) allow the retrieval of spheres. Using the presented platform, we have successfully characterized the different responses in 2D and 3D cell culture to PDT. Furthermore, we investigated the treatment resistance effect in cancer cells induced by tumor associated fibroblasts (CAF). Although the CAFs can enhance the resistance to traditional chemotherapy agents, no significant difference in PDT was observed. The preliminary results suggest that the PDT can be an attractive alternative cancer therapy, which is less affected by the therapeutic resistance induced by cancer associated cells.

  11. High-Throughput Cancer Cell Sphere Formation for Characterizing the Efficacy of Photo Dynamic Therapy in 3D Cell Cultures

    PubMed Central

    Chen, Yu-Chih; Lou, Xia; Zhang, Zhixiong; Ingram, Patrick; Yoon, Euisik

    2015-01-01

    Photodynamic therapy (PDT), wherein light sensitive non-toxic agents are locally and selectively activated using light, has emerged as an appealing alternative to traditional cancer chemotherapy. Yet to date, PDT efficacy has been mostly characterized using 2D cultures. Compared to 2D cultures, 3D sphere culture generates unique spatial distributions of nutrients and oxygen for the cells that better mimics the in-vivo conditions. Using a novel polyHEMA (non-adherent polymer) fabrication process, we developed a microfluidic sphere formation platform that can (1) generate 1,024 uniform (size variation <10%) cancer spheres within a 2 cm by 2 cm core area, (2) culture spheres for more than 2 weeks, and (3) allow the retrieval of spheres. Using the presented platform, we have successfully characterized the different responses in 2D and 3D cell culture to PDT. Furthermore, we investigated the treatment resistance effect in cancer cells induced by tumor associated fibroblasts (CAF). Although the CAFs can enhance the resistance to traditional chemotherapy agents, no significant difference in PDT was observed. The preliminary results suggest that the PDT can be an attractive alternative cancer therapy, which is less affected by the therapeutic resistance induced by cancer associated cells. PMID:26153550

  12. Measurement of spatiotemporal intracellular deformation of cells adhered to collagen matrix during freezing of biomaterials.

    PubMed

    Ghosh, Soham; Craig Dutton, J; Han, Bumsoo

    2014-02-01

    Preservation of structural integrity inside cells and at cell-extracellular matrix (ECM) interfaces is a key challenge during freezing of biomaterials. Since the post-thaw functionality of cells depends on the extent of change in the cytoskeletal structure caused by complex cell-ECM adhesion, spatiotemporal deformation inside the cell was measured using a newly developed microbead-mediated particle tracking deformetry (PTD) technique using fibroblast-seeded dermal equivalents as a model tissue. Fibronectin-coated 500 nm diameter microbeads were internalized in cells, and the microbead-labeled cells were used to prepare engineered tissue with type I collagen matrices. After a 24 h incubation the engineered tissues were directionally frozen, and the cells were imaged during the process. The microbeads were tracked, and spatiotemporal deformation inside the cells was computed from the tracking data using the PTD method. Effects of particle size on the deformation measurement method were tested, and it was found that microbeads represent cell deformation to acceptable accuracy. The results showed complex spatiotemporal deformation patterns in the cells. Large deformation in the cells and detachments of cells from the ECM were observed. At the cellular scale, variable directionality of the deformation was found in contrast to the one-dimensional deformation pattern observed at the tissue scale, as found from earlier studies. In summary, this method can quantify the spatiotemporal deformation in cells and can be correlated to the freezing-induced change in the structure of cytosplasm and of the cell-ECM interface. As a broader application, this method may be used to compute deformation of cells in the ECM environment for physiological processes, namely cell migration, stem cell differentiation, vasculogenesis, and cancer metastasis, which have relevance to quantify mechanotransduction.

  13. GLUCOCORTICOID-INDUCED ALTERATION OF THE SURFACE MEMBRANE OF CULTURED HEPATOMA CELLS

    PubMed Central

    Ballard, Philip L.; Tomkins, Gordon M.

    1970-01-01

    Glucocorticoids induce an alteration of the surface of hepatoma tissue culture (HTC) cells as expressed by changes in cell electrophoretic, antigenic, and adhesive properties. The alteration is assayed by the increased adhesiveness of induced cells for a glass surface. The induction process has a lag period of about 3 hr and attains a plateau level after 24–30 hr when 50–80% of the steroid-treated cells are firmly adhered. Less than 10% of untreated cells adhere under the same conditions. Induction is inhibited by actinomycin D and cycloheximide, demonstrates both pH and temperature dependence, and responds to changes in steroid concentration and structure. By contrast, the attachment per se of preinduced cells is not affected by inhibitors of RNA and protein synthesis, fluctuations of temperature and pH, and the presence or absence of the hormone. When the induction process is reversed by removal of steroid or addition of actinomycin D, preinduced adhesiveness is lost with a half-life of 13–24 hr, but in the presence of cycloheximide the loss is accelerated (t1/2 3–5.5 hr). These results suggest that glucocorticoids induce the biosynthesis of a protein which either modifies the cell surface (an enzyme) or is incorporated into surface structures (structural protein). PMID:4327515

  14. Macro to Nano: A Simple Method for Transporting Cultured Cells from Milliliter Scale to Nanoliter Scale

    PubMed Central

    Seale, Kevin T.; Faley, Shannon L.; Chamberlain, Jeff; Wikswo, John P.

    2013-01-01

    Microfluidic devices are well suited for the study of metabolism and paracrine and autocrine signaling because they allow steady or intermittent perfusion of biological cells at cell densities that approach those in living tissue. They also enable the study of small populations of rare cells. However, it can be difficult to introduce the cells into a microfluidic device to achieve and control such densities without damaging or clumping the cells. We describe simple procedures that address the problem of efficient introduction of cells and cell culture media into microfluidic devices using small bore polyetheretherketone (PEEK) tubing and Hamilton gastight syringes. Suspension or adherent cells grown in cell culture flasks are centrifuged and extracted directly from the centrifuge pellet into the end of the PEEK tubing by aspiration. The tube end is then coupled to pre-punched channels in the polydimethylsiloxane (PDMS) microfluidic device by friction fitting. Controlled depression of the syringe plunger expels the cells into the microfluidic device only seconds following aspiration. The gastight syringes and PEEK tubing with PEEK fittings provide a noncompliant source of pressure and suction with a rapid response time that is well suited for short-term intra-microfluidic cellular studies. The benefits of this method are its simplicity, modest expense, the short preparation time required for loading appropriate numbers of cells, and the applicability of the technique to small quantities of rare or expensive cells. This should in turn lead to new applications of microfludic devices to biology and medicine. PMID:20511682

  15. Adhesion of human enterotoxigenic Escherichia coli to human mucus secreting HT-29 cell subpopulations in culture.

    PubMed Central

    Kerneis, S; Bernet, M F; Coconnier, M H; Servin, A L

    1994-01-01

    Enterotoxigenic Escherichia coli (ETEC) bearing the fimbrial colonisation factor antigens CFA/I, CFA/II, CFA/III, and the non-fimbrial antigen 2230 were tested for their ability to adhere to two cultured human intestinal HT-29 mucus secreting cell subpopulations. These populations are referred to as HT29-MTX and HT29-FU, which differ in the amount of secreted mucins and in their gastric or colonic mucin immunoreactivity respectively. Adherence of radiolabelled bacteria to cell monolayers infected apically was assessed. All ETEC strains adhered to the mucus secreting HT29-FU subpopulation, which secretes mucins of colonic immunoreactivity. Visualisation of bacteria by scanning electron microscopy showed that ETEC bound to the HT29-FU cells possessing a brush border, but not to the mucus and that ETEC binding developed as a function of cell differentiation. The adhesion of ETEC to cells possessing a brush border and to mucus secreting cells was also analysed by indirect immunofluorescence in HT29-MTX cells, which secrete mucins of gastric immunoreactivity. Fluorescein isothiocyanate labelling using specific anti-CFA/I antibody was used to show ETEC; rhodamine isothiocyanate labelling using a monoclonal antibody (designated M1) against purified human gastric mucus was used to detect secreted mucins, and rhodamine isothiocyanate labelling using a monoclonal antibody (designated 4H3) against human dipeptidylpeptidase IV was used to show cells possessing a brush border. Binding of bacteria colocalised with dipeptidylpeptidase IV of enterocytes and not with mucins. Images Figure 1 Figure 2 Figure 3 Figure 5 PMID:7959203

  16. Gonococcal and meningococcal pathogenesis as defined by human cell, cell culture, and organ culture assays.

    PubMed Central

    Stephens, D S

    1989-01-01

    Human cells, cell cultures, and organ cultures have been extremely useful for studying the events that occur when gonococci and meningococci encounter human mucosal surfaces. The specificity and selectivity of these events for human cells are striking and correlate with the adaptation of these pathogens for survival on human mucous membranes. To colonize these sites, meningococci and gonococci have developed mechanisms to damage local host defenses such as the mucociliary blanket, to attach to epithelial cells, and to invade these cells. Attachment to epithelial cells mediated by pili, and to some types of cells mediated by PIIs, serves to anchor the organism close to sources of nutrition and allows multiplication. Intracellular invasion, possibly initiated by the major porin protein, may provide additional nutritional support and protection from host defenses. Mucosal invasion may also result in access of gonococci and meningococci to the bloodstream, leading to dissemination. Images PMID:2497953

  17. Process analytical technology (PAT) in insect and mammalian cell culture processes: dielectric spectroscopy and focused beam reflectance measurement (FBRM).

    PubMed

    Druzinec, Damir; Weiss, Katja; Elseberg, Christiane; Salzig, Denise; Kraume, Matthias; Pörtner, Ralf; Czermak, Peter

    2014-01-01

    Modern bioprocesses demand for a careful definition of the critical process parameters (CPPs) already during the early stages of process development in order to ensure high-quality products and satisfactory yields. In this context, online monitoring tools can be applied to recognize unfavorable changes of CPPs during the production processes and to allow for early interventions in order to prevent losses of production batches due to quality issues. Process analytical technologies such as the dielectric spectroscopy or focused beam reflectance measurement (FBRM) are possible online monitoring tools, which can be applied to monitor cell growth as well as morphological changes. Since the dielectric spectroscopy only captures cells with intact cell membranes, even information about dead cells with ruptured or leaking cell membranes can be derived. The following chapter describes the application of dielectric spectroscopy on various virus-infected and non-infected cell lines with respect to adherent as well as suspension cultures in common stirred tank reactors. The adherent mammalian cell lines Vero (African green monkey kidney cells) and hMSC-TERT (telomerase-immortalized human mesenchymal stem cells) are thereby cultured on microcarrier, which provide the required growth surface and allow the cultivation of these cells even in dynamic culture systems. In turn, the insect-derived cell lines S2 and Sf21 are used as examples for cells typically cultured in suspension. Moreover, the FBRM technology as a further monitoring tool for cell culture applications has been included in this chapter using the example of Drosophila S2 insect cells.

  18. Response of adherent cells to mechanical perturbations of the surrounding matrix.

    PubMed

    Ben-Yaakov, Dan; Golkov, Roman; Shokef, Yair; Safran, Samuel A

    2015-02-04

    We present a generic and unified theory to explain how cells respond to perturbations of their mechanical environment such as the presence of neighboring cells, slowly applied stretch, or gradients of matrix rigidity. Motivated by experiments, we calculate the local balance of forces that give rise to a tendency for the cell to locally move or reorient, with a focus on the contribution of feedback and homeostasis to cell contractility (manifested by a fixed displacement, strain or stress) that acts on the adhesions at the cell boundary. These forces can be either reinforced or diminished by elastic stresses due to mechanical perturbations of the matrix. Our model predicts these changes and how their balance with local protrusive forces that act on the cell's leading edge either increase or decrease the tendency of the cell to locally move (toward neighboring cells or rigidity gradients) or reorient (in the direction of slowly applied stretch or rigidity gradients).

  19. Aragonite crystallization in primary cell cultures of multicellular isolates from a hard coral, Pocillopora damicornis.

    PubMed

    Domart-Coulon, I J; Elbert, D C; Scully, E P; Calimlim, P S; Ostrander, G K

    2001-10-09

    The foundation of marine coral reef ecosystems is calcium carbonate accumulated primarily by the action of hard corals (Coelenterata: Anthozoa: Scleractinia). Colonial hard coral polyps cover the surface of the reef and deposit calcium carbonate as the aragonite polymorph, stabilized into a continuous calcareous skeleton. Scleractinian coral skeleton composition and architecture are well documented; however, the cellular mechanisms of calcification are poorly understood. There is little information on the nature of the coral cell types involved or their cooperation in biocalcification. We report aragonite crystallization in primary cell cultures of a hard coral, Pocillopora damicornis. Cells of apical coral colony fragments were isolated by spontaneous in vitro dissociation. Single dissociated cell types were separated by density in a discontinuous Percoll gradient. Primary cell cultures displayed a transient increase in alkaline phosphatase (ALP) activity, to the level observed in intact corals. In adherent multicellular isolate cultures, enzyme activation was followed by precipitation of aragonite. Modification of the ionic formulation of the medium prolonged maintenance of isolates, delayed ALP activation, and delayed aragonite precipitation. These results demonstrate that in vitro crystallization of aragonite in coral cell cultures is possible, and provides an innovative approach to investigate reef-building coral calcification at the cellular level.

  20. The effect of neurosphere culture conditions on the cellular metabolism of glioma cells.

    PubMed

    Kahlert, Ulf Dietrich; Koch, Katharina; Suwala, Abigail Kora; Hartmann, Rudolf; Cheng, Menglin; Maciaczyk, Donata; Willbold, Dieter; Eberhart, Charles G; Glunde, Kristine; Maciaczyk, Jarek

    2015-01-01

    Malignant gliomas, with an average survival time of 16-19 months after initial diagnosis, account for one of the most lethal tumours overall. Current standards in patient care provide only unsatisfying strategies in diagnostic and treatment for high-grade gliomas. Here we describe metabolic phenomena in the choline and glycine network associated with stem cell culture conditions in the classical glioma cell line U87. Using high-resolution proton magnetic resonance spectroscopy of cell culture metabolic extracts we compare the metabolic composition of U87 chronically propagated as adherent culture in medium supplemented with serum to serum-free neurosphere growth. We found that the switch to neurosphere growth, besides the increase of cells expressing the putative glioma stem cell marker CD133, modulated a number of intracellular metabolites including choline, creatine, glycine, and myo-inositol that have been previously reported as potential diagnostic markers in various tumours. These findings highlight the critical influence of culture conditions on glioma cell metabolism, and therefore particular caution should be drawn to the use of in vitro system research in order to investigate cancer metabolism.

  1. Ultra-soft PDMS-based magnetoactive elastomers as dynamic cell culture substrata.

    PubMed

    Mayer, Matthias; Rabindranath, Raman; Börner, Juliane; Hörner, Eva; Bentz, Alexander; Salgado, Josefina; Han, Hong; Böse, Holger; Probst, Jörn; Shamonin, Mikhail; Monkman, Gareth J; Schlunck, Günther

    2013-01-01

    Mechanical cues such as extracellular matrix stiffness and movement have a major impact on cell differentiation and function. To replicate these biological features in vitro, soft substrata with tunable elasticity and the possibility for controlled surface translocation are desirable. Here we report on the use of ultra-soft (Young's modulus <100 kPa) PDMS-based magnetoactive elastomers (MAE) as suitable cell culture substrata. Soft non-viscous PDMS (<18 kPa) is produced using a modified extended crosslinker. MAEs are generated by embedding magnetic microparticles into a soft PDMS matrix. Both substrata yield an elasticity-dependent (14 vs. 100 kPa) modulation of α-smooth muscle actin expression in primary human fibroblasts. To allow for static or dynamic control of MAE material properties, we devise low magnetic field (≈40 mT) stimulation systems compatible with cell-culture environments. Magnetic field-instigated stiffening (14 to 200 kPa) of soft MAE enhances the spreading of primary human fibroblasts and decreases PAX-7 transcription in human mesenchymal stem cells. Pulsatile MAE movements are generated using oscillating magnetic fields and are well tolerated by adherent human fibroblasts. This MAE system provides spatial and temporal control of substratum material characteristics and permits novel designs when used as dynamic cell culture substrata or cell culture-coated actuator in tissue engineering applications or biomedical devices.

  2. Ultra-Soft PDMS-Based Magnetoactive Elastomers as Dynamic Cell Culture Substrata

    PubMed Central

    Mayer, Matthias; Rabindranath, Raman; Börner, Juliane; Hörner, Eva; Bentz, Alexander; Salgado, Josefina; Han, Hong; Böse, Holger; Probst, Jörn; Shamonin, Mikhail; Monkman, Gareth J.; Schlunck, Günther

    2013-01-01

    Mechanical cues such as extracellular matrix stiffness and movement have a major impact on cell differentiation and function. To replicate these biological features in vitro, soft substrata with tunable elasticity and the possibility for controlled surface translocation are desirable. Here we report on the use of ultra-soft (Young’s modulus <100 kPa) PDMS-based magnetoactive elastomers (MAE) as suitable cell culture substrata. Soft non-viscous PDMS (<18 kPa) is produced using a modified extended crosslinker. MAEs are generated by embedding magnetic microparticles into a soft PDMS matrix. Both substrata yield an elasticity-dependent (14 vs. 100 kPa) modulation of α-smooth muscle actin expression in primary human fibroblasts. To allow for static or dynamic control of MAE material properties, we devise low magnetic field (≈40 mT) stimulation systems compatible with cell-culture environments. Magnetic field-instigated stiffening (14 to 200 kPa) of soft MAE enhances the spreading of primary human fibroblasts and decreases PAX-7 transcription in human mesenchymal stem cells. Pulsatile MAE movements are generated using oscillating magnetic fields and are well tolerated by adherent human fibroblasts. This MAE system provides spatial and temporal control of substratum material characteristics and permits novel designs when used as dynamic cell culture substrata or cell culture-coated actuator in tissue engineering applications or biomedical devices. PMID:24204603

  3. Aragonite crystallization in primary cell cultures of multicellular isolates from a hard coral, Pocillopora damicornis

    PubMed Central

    Domart-Coulon, Isabelle J.; Elbert, David C.; Scully, Erik P.; Calimlim, Precilia S.; Ostrander, Gary K.

    2001-01-01

    The foundation of marine coral reef ecosystems is calcium carbonate accumulated primarily by the action of hard corals (Coelenterata: Anthozoa: Scleractinia). Colonial hard coral polyps cover the surface of the reef and deposit calcium carbonate as the aragonite polymorph, stabilized into a continuous calcareous skeleton. Scleractinian coral skeleton composition and architecture are well documented; however, the cellular mechanisms of calcification are poorly understood. There is little information on the nature of the coral cell types involved or their cooperation in biocalcification. We report aragonite crystallization in primary cell cultures of a hard coral, Pocillopora damicornis. Cells of apical coral colony fragments were isolated by spontaneous in vitro dissociation. Single dissociated cell types were separated by density in a discontinuous Percoll gradient. Primary cell cultures displayed a transient increase in alkaline phosphatase (ALP) activity, to the level observed in intact corals. In adherent multicellular isolate cultures, enzyme activation was followed by precipitation of aragonite. Modification of the ionic formulation of the medium prolonged maintenance of isolates, delayed ALP activation, and delayed aragonite precipitation. These results demonstrate that in vitro crystallization of aragonite in coral cell cultures is possible, and provides an innovative approach to investigate reef-building coral calcification at the cellular level. PMID:11593000

  4. Structural characterization and primary in vitro cell culture of locust male germline stem cells and their niche.

    PubMed

    Dorn, David C; Dorn, August

    2011-03-01

    The establishment of in vitro culture systems to expand stem cells and to elucidate the niche/stem cell interaction is among the most sought-after culture systems of our time. To further investigate niche/stem cell interactions, we evaluated in vitro cultures of isolated intact male germline-niche complexes (i.e., apical complexes), complexes with empty niche spaces, and completely empty niches (i.e., isolated apical cells) from the testes of Locusta migratoria and the interaction of these complexes with isolated germline stem cells, spermatogonia (of transit-amplifying stages), cyst progenitor cells, cyst progenitor cell-like cells, cyst cells, and follicle envelope cells. The structural characteristics of these cell types allow the identification of the different cell types in primary cultures, which we studied in detail by light and electron microscopy. In intact testes germline stem cells strongly adhere to their niche (the apical cell), but emigrate from their niche and form filopodia if the apical complex is put into culture with "standard media." The lively movements of the long filopodia of isolated germline stem cells and spermatogonia may be indicative of their search for specific signals to home to their niche. All other incubated cell types (except for follicle envelope cells) expressed rhizopodia and lobopodia. Nevertheless isolated germline stem cells in culture do not migrate to empty niche spaces of nearby apical cells. This could indicate that apical cells lose their germline stem cell attracting ability in vitro, although apical cells devoid of germline stem cells either by emigration of germline stem cells or by mechanical removal of germline stem cells are capable of surviving in vitro up to 56 days, forming many small lobopodia and performing amoeboid movements. We hypothesize that the breakdown of the apical complex in vitro with standard media interrupts the signaling between the germline stem cells and the niche (and conceivably the cyst

  5. Recombinant protein production and insect cell culture and process

    NASA Technical Reports Server (NTRS)

    Spaulding, Glenn (Inventor); Prewett, Tacey (Inventor); Goodwin, Thomas (Inventor); Francis, Karen (Inventor); Andrews, Angela (Inventor); Oconnor, Kim (Inventor)

    1993-01-01

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

  6. The secreted autotransporter toxin, Sat, functions as a virulence factor in Afa/Dr diffusely adhering Escherichia coli by promoting lesions in tight junction of polarized epithelial cells.

    PubMed

    Guignot, Julie; Chaplais, Cécile; Coconnier-Polter, Marie-Hélène; Servin, Alain L

    2007-01-01

    Afa/Dr diffusely adhering Escherichia coli (DAEC) strains are responsible for urinary tract and intestinal infections. Both in intestine and kidney, the epithelial cells forming epithelium are sealed by junctional domains. We provide evidence that the Secreted autotransporter toxin, Sat, belonging to the subfamily of serine protease autotransporters of Enterobacteriaceae (SPATEs), acts as a virulence factor in Afa/Dr DAEC by promoting lesions in the tight junctions (TJs) of polarized epithelial Caco-2/TC7 cells. Southern blot analysis reveals that the prototype strains of the subclass-1 and subclass-2 typical Afa/Dr DAEC strains, hybridize with a sat probe. Using the wild-type IH11128 strain, the recombinant E. coli AAEC185 strain that expresses Sat, the recombinant E. coli that expresses both Dr adhesin and Sat, we report that Sat in monolayers of cultured enterocyte-like Caco-2/TC7 cells, induces rearrangements of the TJs-associated proteins ZO-1, ZO-3 and occludin, and increases the formation of domes as the result of an increase in the paracellular permeability without affecting the transepithelial electrical resistance of the cell monolayers. Moreover, we observe that Sat-induced disassembly of TJs-associated proteins is dependent on the serine protease motif. Finally, an analysis of the prevalence of the sat gene in three collections of Afa/Dr DAEC strains collected from the stools of children with and without diarrhoea, and from the urine of patients with urinary tract infection (UTI) shows that: (i) the sat gene is highly prevalent in UTI-associated Afa/Dr DAEC strains (88% positive), (ii) the sat gene is generally absent from Afa/Dr DAEC strains collected from the stools of children without diarrhoea (16% positive); whereas (iii) it is present in about half of the strains collected from the stools of children with diarrhoea (46% positive).

  7. Adherence of Non-O157 Shiga Toxin–Producing Escherichia coli to Bovine Recto-anal Junction Squamous Epithelial Cells Appears to Be Mediated by Mechanisms Distinct from Those Used by O157

    PubMed Central

    Hovde, Carolyn J.; John, Manohar

    2013-01-01

    Abstract This study presents evidence that the pattern (diffuse or aggregative) of adherence of clinically relevant non-O157 Shiga toxin–producing Escherichia coli (STEC) to bovine recto-anal junction squamous epithelial cells is similar to that of E. coli O157, although the mechanisms of adherence appear to be distinct. Our results further suggest that novel adhesins, and not Intimin, are likely involved in non-O157 STEC adherence to bovine recto-anal junction squamous epithelial cells. These findings have important implications for the development of efficacious modalities for blocking adherence of non-O157 STEC to bovine gastrointestinal epithelial cells. PMID:23510495

  8. Comparison The Effects of Two Monocyte Isolation Methods, Plastic Adherence and Magnetic Activated Cell Sorting Methods, on Phagocytic Activity of Generated Dendritic Cells

    PubMed Central

    Delirezh, Nowruz; Shojaeefar, Ehsan; Parvin, Parva; Asadi, Behnaz

    2013-01-01

    Objective: It is believed that monocyte isolation methods and maturation factors affect the phenotypic and functional characteristics of resultant dendritic cells (DC). In the present study, we compared two monocyte isolation methods, including plastic adherence-dendritic cells (Adh-DC) and magnetic activated cell sorting- dendritic cells (MACS-DC), and their effects on phagocytic activity of differentiated immature DCs (immDCs). Materials and Methods: : In this experimental study, immDCs were generated from plastic adherence and MACS isolated monocytes in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4) in five days. The phagocytic activity of immDCs was analyzed by fluorescein isothiocyanate (FITC)-conjugated latex bead using flow cytometry. One way ANOVA test was used for statistical analysis of differences among experimental groups, including Adh-DC and MACS-DC groups. Results: We found that phagocytic activity of Adh-DC was higher than MACS-DC, whereas the mean fluorescence intensity (MFI) of phagocytic cells was higher in MACS-DC (p<0.05). Conclusion: : We concluded that it would be important to consider phagocytosis parameters of generated DCs before making any decision about monocyte isolation methods to have fully functional DCs. PMID:24027662

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

    PubMed Central

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

    2016-01-01

    A versatile bioreactor suitable for dynamic suspension cell culture under tunable shear stress conditions has been developed and preliminarily tested culturing cancer cell spheroids. By adopting simple technological solutions and avoiding rotating components, the bioreactor exploits the laminar hydrodynamics establishing within the culture chamber enabling dynamic cell suspension in an environment favourable to mass transport, under a wide range of tunable shear stress conditions. The design phase of the device has been supported by multiphysics modelling and has provided a comprehensive analysis of the operating principles of the bioreactor. Moreover, an explanatory example is herein presented with multiphysics simulations used to set the proper bioreactor operating conditions for preliminary in vitro biological tests on a human lung carcinoma cell line. The biological results demonstrate that the ultralow shear dynamic suspension provided by the device is beneficial for culturing cancer cell spheroids. In comparison to the static suspension control, dynamic cell suspension preserves morphological features, promotes intercellular connection, increases spheroid size (2.4-fold increase) and number of cycling cells (1.58-fold increase), and reduces double strand DNA damage (1.5-fold reduction). It is envisioned that the versatility of this bioreactor could allow investigation and expansion of different cell types in the future. PMID:27144306

  10. Human Antibodies to PhtD, PcpA, and Ply Reduce Adherence to Human Lung Epithelial Cells and Murine Nasopharyngeal Colonization by Streptococcus pneumoniae

    PubMed Central

    Kaur, Ravinder; Surendran, Naveen; Ochs, Martina

    2014-01-01

    Streptococcus pneumoniae adherence to human epithelial cells (HECs) is the first step in pathogenesis leading to infections. We sought to determine the role of human antibodies against S. pneumoniae protein vaccine candidates PhtD, PcpA, and Ply in preventing adherence to lung HECs in vitro and mouse nasopharyngeal (NP) colonization in vivo. Human anti-PhtD, -PcpA, and -Ply antibodies were purified and Fab fragments generated. Fabs were used to test inhibition of adherence of TIGR4 and nonencapsulated strain RX1 to A549 lung HECs. The roles of individual proteins in adherence were tested using isogenic mutants of strain TIGR4. Anti-PhtD, -PcpA, and -Ply human antibodies were assessed for their ability to inhibit NP colonization in vivo by passive transfer of human antibody in a murine model. Human antibodies generated against PhtD and PcpA caused a decrease in adherence to A549 cells (P < 0.05). Anti-PhtD but not anti-PcpA antibodies showed a protective role against mouse NP colonization. To our surprise, anti-Ply antibodies also caused a significant (P < 0.05) reduction in S. pneumoniae colonization. Our results support the potential of PhtD, PcpA, and Ply protein vaccine candidates as alternatives to conjugate vaccines to prevent non-serotype-specific S. pneumoniae colonization and invasive infection. PMID:25245804

  11. Ascorbic acid transport into cultured pituitary cells

    SciTech Connect

    Cullen, E.I.; May, V.; Eipper, R.A.

    1986-05-01

    An amidating enzyme designated peptidyl-glycine ..cap alpha..-amidating monooxygenase (PAM) has been studied in a variety of tissues and is dependent on molecular oxygen and stimulated by copper and ascorbic acid. To continue investigating the relationship among cellular ascorbic acid concentrations, amidating ability, and PAM activity, the authors studied ascorbic acid transport in three cell preparations that contain PAM and produce amidated peptides: primary cultures of rat anterior and intermediate pituitary and mouse AtT-20 tumor cells. When incubated in 50 ..mu..M (/sup 14/C)ascorbic acid all three cell preparations concentrated ascorbic acid 20- to 40-fold, producing intracellular ascorbate concentrations of 1 to 2 mM, based on experimentally determined cell volumes. All three cell preparations displayed saturable ascorbic acid uptake with half-maximal initial rates occurring between 9 and 18 ..mu..M ascorbate. Replacing NaCl in the uptake buffer with choline chloride significantly diminished ascorbate uptake in all three preparations. Ascorbic acid efflux from these cells was slow, displaying half-lives of 7 hours. Unlike systems that transport dehydroascorbic acid, the transport system for ascorbic acid in these cells was not inhibited by glucose. Thus, ascorbate is transported into pituitary cells by a sodium-dependent, active transport system.

  12. Transportation of transplantable cell sheets fabricated with temperature-responsive culture surfaces for regenerative medicine.

    PubMed

    Nozaki, Takayuki; Yamato, Masayuki; Inuma, Toshiaki; Nishida, Kohji; Okano, Teruo

    2008-06-01

    Here we report transportation of cell sheets fabricated on temperature-responsive culture surfaces for regenerative medicine. On the surfaces cells adhere, spread and proliferate at 37 degrees C, but upon temperature reduction below 32 degrees C all the cells are spontaneously detached. When cells on the surfaces are challenged by long distance transportation, maintaining the temperature is critical. Therefore, we developed a portable homothermal container to keep the inner temperature at 36 degrees C for > 30 h without any need for batteries or energy supply. We transported and compared fibroblast sheets cultured on temperature-responsive surfaces in the container, at room temperature in a car, or on ice. After 8 h transportation by car, all cells at room temperature and on ice were detached from the surfaces and some were folded and broken into tiny pieces. On the other hand, fibroblast sheets transported in the container retained their adhesion to the dish surfaces and intact cell sheets were successfully harvested by temperature reduction. During the transportation, cell viability and histology were not impaired. This unique transportation device would be useful for cell sheet-based regenerative medicine utilizing temperature-responsive culture surfaces.

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

    DOEpatents

    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.

  14. Use of long-term human marrow cultures to demonstrate progenitor cell precursors in marrow treated with 4-hydroperoxycyclophosphamide

    SciTech Connect

    Winton, E.F.; Colenda, K.W.

    1987-07-01

    The continued retrieval of progenitor cells (CFU-GEMM, BFU-E, CFU-E, CFU-GM) from human long-term marrow cultures (LTMC) is not uncommonly used as evidence that proliferation and differentiation are occurring in more primitive hematopoietic stem cells (HSC) in these cultures. Alternatively, the continued presence of progenitors in LTMC could be the result of survival and/or limited self-renewal of progenitor cells present when the culture was initiated, and such progenitors would have little relevance to the parent HSC. The following studies were designed to determine the relative contributions of precursors of progenitor cells to the total progenitor cells present in LTMC using a two-stage regeneration model. The adherent layer in LTMC was established over 3 weeks, irradiated (875 rad) to permanently eliminate resident hematopoietic cells, and recharged with autologous cryo-preserved marrow that was either treated or not treated (control) with 4-hydroperoxycyclophosphamide (4-HC, 100 micrograms/ml for 30 min). The 4-HC-treated marrow contained no progenitor cells, yet based on clinical autologous bone marrow transplant experience, has intact HSC. Within 1-3 weeks, progenitor cells reappeared in the irradiated LTMC recharged with 4-HC-treated marrow, and were preferentially located in the adherent layer. By 2-6 weeks, the number of progenitor cells in the adherent layer of LTMC recharged with 4-HC marrow was equivalent to control LTMC. The progenitors regenerating in the irradiated LTMC recharged with 4-HC-treated marrow appear to originate from precursors of progenitor cells, perhaps HSC. We propose this model may be useful in elucidating cellular and molecular correlates of progenitor cell regeneration from precursors.

  15. Monolayer and three-dimensional cell culture and living tissue culture of gallbladder epithelium.

    PubMed

    Nakanuma, Y; Katayanagi, K; Kawamura, Y; Yoshida, K

    1997-10-01

    Several models for preparing and isolating human and animal gallbladder epithelial cells, including low-grade gallbladder carcinoma cells, as well as proposed systems for culturing these isolated epithelial cells are reviewed here. Several reports concerning tissue culture of the gallbladder are also reviewed. The cell culture systems are divided into monolayer cell culture on collagen-coated or uncoated culture dishes or other culture substrate and three-dimensional cell culture in collagen gel. To prepare and isolate gallbladder epithelial cells, digestion of the gallbladder mucosa, abrasion of the mucosal epithelial cells, and excision of epithelial outgrowth of mucosal explants are applied. In monolayer cell culture, most of the specific biological features of isolated and cultured cells characteristic to the gallbladder are gradually lost after several passages, though quantitative and objective analyses of the pathophysiology of cultured cells and their secretory substances can be performed. Tissue culture using explants of the gallbladder has mainly been used for physiological studies of the gallbladder, such as investigating the transport of water and electrolytes. In this tissue culture system, quantitative assessment is difficult, though the original and specific biological and histological characteristics of the gallbladder are retained. Three-dimensional collagen gel culture could be an ideal model combining monolayer cell culture and tissue culture systems, and create controllable conditions or environments when several biologically active substances, such as growth factors, proinflammatory cytokines and adhesion molecules, are added to the culture medium. Advantages and shortcomings of individual cultivation models are discussed, and selecting the culture model most appropriate to the purpose of the study will facilitate investigations of the biology and pathogenetic mechanisms of gallbladder diseases such as cholelithiasis.

  16. Stably transfected adherent cancer cell models with decreased expression of 5'-nucleotidase cN-II.

    PubMed

    Bricard, Gabriel; Cros-Perrial, Emeline; Machon, Christelle; Dumontet, Charles; Jordheim, Lars Petter

    2016-12-01

    The 5'-nucleotidase cN-II has been shown to be associated with the sensitivity to nucleoside analogues, the survival of cytarabine treated leukemia patients and to cell proliferation. Due to the lack of relevant cell models for solid tumors, we developed four cell lines with low cN-II expression and characterized them concerning their in vitro sensitivity to cancer drugs and their intracellular nucleotide pools. All four cell models had an important decrease of cN-II expression but did not show modified sensitivity, cell proliferation or nucleotide pools. Our cell models will be important for the study of the role of cN-II in human cancer cells.

  17. An Introductory Undergraduate Course Covering Animal Cell Culture Techniques

    ERIC Educational Resources Information Center

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

    2004-01-01

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

  18. 21 CFR 864.2280 - Cultured animal and human cells.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Cultured animal and human cells. 864.2280 Section 864.2280 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES... Cultured animal and human cells. (a) Identification. Cultured animal and human cells are in...

  19. 21 CFR 864.2280 - Cultured animal and human cells.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Cultured animal and human cells. 864.2280 Section 864.2280 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES... Cultured animal and human cells. (a) Identification. Cultured animal and human cells are in...

  20. Transmission of mechanical stresses within the cytoskeleton of adherent cells: a theoretical analysis based on a multi-component cell model.

    PubMed

    Tracqui, Philippe; Ohayon, Jacques

    2004-01-01

    How environmental mechanical forces affect cellular functions is a central problem in cell biology. Theoretical models of cellular biomechanics provide relevant tools for understanding how the contributions of deformable intracellular components and specific adhesion conditions at the cell interface are integrated for determining the overall balance of mechanical forces within the cell. We investigate here the spatial distributions of intracellular stresses when adherent cells are probed by magnetic twisting cytometry. The influence of the cell nucleus stiffness on the simulated nonlinear torque-bead rotation response is analyzed by considering a finite element multi-component cell model in which the cell and its nucleus are considered as different hyperelastic materials. We additionally take into account the mechanical properties of the basal cell cortex, which can be affected by the interaction of the basal cell membrane with the extracellular substrate. In agreement with data obtained on epithelial cells, the simulated behaviour of the cell model relates the hyperelastic response observed at the entire cell scale to the distribution of stresses and strains within the nucleus and the cytoskeleton, up to cell adhesion areas. These results, which indicate how mechanical forces are transmitted at distant points through the cytoskeleton, are compared to recent data imaging the highly localized distribution of intracellular stresses.

  1. Vertical nanopillars for in situ probing of nuclear mechanics in adherent cells

    NASA Astrophysics Data System (ADS)

    Hanson, Lindsey; Zhao, Wenting; Lou, Hsin-Ya; Lin, Ziliang Carter; Lee, Seok Woo; Chowdary, Praveen; Cui, Yi; Cui, Bianxiao

    2015-06-01

    The mechanical stability and deformability of the cell nucleus are crucial to many biological processes, including migration, proliferation and polarization. In vivo, the cell nucleus is frequently subjected to deformation on a variety of length and time scales, but current techniques for studying nuclear mechanics do not provide access to subnuclear deformation in live functioning cells. Here we introduce arrays of vertical nanopillars as a new method for the in situ study of nuclear deformability and the mechanical coupling between the cell membrane and the nucleus in live cells. Our measurements show that nanopillar-induced nuclear deformation is determined by nuclear stiffness, as well as opposing effects from actin and intermediate filaments. Furthermore, the depth, width and curvature of nuclear deformation can be controlled by varying the geometry of the nanopillar array. Overall, vertical nanopillar arrays constitute a novel approach for non-invasive, subcellular perturbation of nuclear mechanics and mechanotransduction in live cells.

  2. Vertical nanopillars for in situ probing of nuclear mechanics in adherent cells

    PubMed Central

    Hanson, Lindsey; Zhao, Wenting; Lou, Hsin-Ya; Lin, Ziliang Carter; Lee, Seok Woo; Chowdary, Praveen; Cui, Yi; Cui, Bianxiao

    2016-01-01

    The mechanical stability and deformability of the cell nucleus are crucial to many biological processes, including migration, proliferation and polarization. In vivo, the cell nucleus is frequently subjected to deformation on a variety of length and time scales, but current techniques for studying nuclear mechanics do not provide access to subnuclear deformation in live functioning cells. Here we introduce arrays of vertical nanopillars as a new method for the in situ study of nuclear deformability and the mechanical coupling between the cell membrane and the nucleus in live cells. Our measurements show that nanopillar-induced nuclear deformation is determined by nuclear stiffness, as well as opposing effects from actin and intermediate filaments. Furthermore, the depth, width and curvature of nuclear deformation can be controlled by varying the geometry of the nanopillar array. Overall, vertical nanopillar arrays constitute a novel approach for non-invasive, subcellular perturbation of nuclear mechanics and mechanotransduction in live cells. PMID:25984833

  3. Vertical nanopillars for in situ probing of nuclear mechanics in adherent cells.

    PubMed

    Hanson, Lindsey; Zhao, Wenting; Lou, Hsin-Ya; Lin, Ziliang Carter; Lee, Seok Woo; Chowdary, Praveen; Cui, Yi; Cui, Bianxiao

    2015-06-01

    The mechanical stability and deformability of the cell nucleus are crucial to many biological processes, including migration, proliferation and polarization. In vivo, the cell nucleus is frequently subjected to deformation on a variety of length and time scales, but current techniques for studying nuclear mechanics do not provide access to subnuclear deformation in live functioning cells. Here we introduce arrays of vertical nanopillars as a new method for the in situ study of nuclear deformability and the mechanical coupling between the cell membrane and the nucleus in live cells. Our measurements show that nanopillar-induced nuclear deformation is determined by nuclear stiffness, as well as opposing effects from actin and intermediate filaments. Furthermore, the depth, width and curvature of nuclear deformation can be controlled by varying the geometry of the nanopillar array. Overall, vertical nanopillar arrays constitute a novel approach for non-invasive, subcellular perturbation of nuclear mechanics and mechanotransduction in live cells.

  4. A family of cell-adhering peptides homologous to fibrinogen C-termini

    SciTech Connect

    Levy-Beladev, Liron; Levdansky, Lilia; Gaberman, Elena; Friedler, Assaf; Gorodetsky, Raphael

    2010-10-08

    Research highlights: {yields} Cell-adhesive sequences homologous to fibrinogen C-termini exist in other proteins. {yields} The extended homologous cell-adhesive C-termini peptides family is termed Haptides. {yields} In membrane-like environment random coiled Haptides adopt a helical conformation. {yields} Replacing positively charged residues with alanine reduces Haptides activity. -- Abstract: A family of cell-adhesive peptides homologous to sequences on different chains of fibrinogen was investigated. These homologous peptides, termed Haptides, include the peptides C{beta}, preC{gamma}, and C{alpha}E, corresponding to sequences on the C-termini of fibrinogen chains {beta}, {gamma}, and {alpha}E, respectively. Haptides do not affect cell survival and rate of proliferation of the normal cell types tested. The use of new sensitive assays of cell adhesion clearly demonstrated the ability of Haptides, bound to inert matrices, to mediate attachment of different matrix-dependent cell types including normal fibroblasts, endothelial, and smooth muscle cells. Here we present new active Haptides bearing homologous sequences derived from the C-termini of other proteins, such as angiopoietin 1 and 2, tenascins C and X, and microfibril-associated glycoprotein-4. The cell adhesion properties of all the Haptides were found to be associated mainly with their 11 N-terminal residues. Mutated preC{gamma} peptides revealed that positively charged residues account for their attachment effect. These results suggest a mechanism of direct electrostatic interaction of Haptides with the cell membrane. The extended Haptides family may be applied in modulating adhesion of cells to scaffolds for tissue regeneration and for enhancement of nanoparticulate transfection into cells.

  5. Amino acid pools in cultured muscle cells.

    PubMed

    Low, R B; Stirewalt, W S; Rittling, S R; Woodworth, R C

    1984-01-01

    Compartmentalization of cellular amino acid pools occurs in cultures of cardiac and skeletal muscle cells, but the factors involved in this are not clear. We have further defined this problem by analyzing the intracellular free leucine and the transfer-RNA-(tRNA)-bound leucine pool in cultures of skeletal and cardiac muscle incubated with 3H-leucine in the presence and absence of serum and amino acids. Withdrawal of nitrogen substrates caused substantial changes in leucine pool relationships--in particular, a change in the degree to which intracellular free leucine and tRNA-leucine were derived from the culture medium. In separate experiments, the validity of our tRNA measurements was confirmed by measurements of the specific activity of newly synthesized ferritin after iron induction. We discuss the implications of these findings with regard to factors involved in the control of amino acid flux through the cell, as well as with regard to design of experiments using isotopic amino acids to measure rates of amino acid utilization.

  6. Chromosomal mosaicism in amniotic fluid cell cultures.

    PubMed Central

    Peakman, D C; Moreton, M F; Corn, B J; Robinson, A

    1979-01-01

    Over the past 6 years, using in situ processing methods, we have identified 32 cases of mosaicism in amniotic fluid cell cultures prepared from 1,100 samples. Two of these (45,X/46,XX and 46,XX/47,XX, + 21) were called true mosaics because multiple colonies demonstrated the same abnormal chromosome complement, and on subsequent evaluation of the newborn blood or fetal tissues, mosaicism was confirmed. Of the remaining cases, 29 were designated as pseudomosaics because only single or partial colonies exhibited an aberrant chromosome complement, 12 having a trisomy 2 line. In the final case, a double trisomy was demonstrated in only one of eight colonies in the first culture, but in the culture from a repeat sample an additional two colonies showed the same double trisomy. Since no abnormal cells were observed in infant blood, it was postulated that the mosaicism may only have been present in the extraembryonic tissues. It is our conviction that the use of these cloning methods should diminish the danger of misdiagnosis in genetic amniocentesis. PMID:453199

  7. Analytical methodology for metabolomics study of adherent mammalian cells using NMR, GC-MS and LC-HRMS.

    PubMed

    Madji Hounoum, Blandine; Blasco, Hélène; Nadal-Desbarats, Lydie; Diémé, Binta; Montigny, Frédéric; Andres, Christian R; Emond, Patrick; Mavel, Sylvie

    2015-11-01

    We developed a methodology for the analysis of intracellular metabolites using nuclear magnetic resonance spectrometry (NMR), gas-chromatography coupled with mass spectrometry (GC-MS), and liquid chromatography coupled with high resolution mass spectrometry (LC-HRMS). The main steps for analysis of adherent cells in order to recover the widest possible range of intracellular compounds are blocking metabolic activity by quenching and extraction of intracellular metabolites. We explored three protocols to quench NSC-34 cell metabolism and four different extraction methods, analyzed by NMR. On the basis of the number of metabolites extracted and their relative standard deviation (RSD) analyzed by NMR, the most reproducible protocol [quenching by MeOH at -40 °C and extraction with CH2Cl2/MeOH/H2O (3:3:2)] was used to obtain intracellular media to be analyzed by GC-MS and LC-HRMS. GC-MS analysis was optimized by three oximation procedures followed by silylation derivatization and these were compared to silylation alone. Using reversed-phase liquid chromatography (C18), four different gradients for LC-MS were compared. The analytical protocols were determined to establish the reliability and suitability of sample treatments required to achieve the correct biological analysis of untargeted mammalian cell metabolomics.

  8. Intestinal Mucus Gel and Secretory Antibody are Barriers to Campylobacter jejuni Adherence to INT 407 Cells

    DTIC Science & Technology

    1987-06-01

    An in vitro mucus assay was developed to study the role of mucus gel and secretory immunoglobulin A (sIgA) in preventing attachment of Campylobacter ... jejuni to INT 407 cells. An overlay of rabbit small intestinal mucus was found to impede the attachment of C. jejuni to a monolayer of INT 407 cells

  9. Adherence to Mediterranean-style dietary pattern and risk of esophageal squamous cell carcinoma: a case-control study in Iran

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The benefit of adherence to a Mediterranean-style dietary pattern in relation to the risk of esophageal squamous cell carcinoma (ESCC) has not been investigated among non-Mediterranean high-risk populations. The objective of the present study was to examine the association of compliance with the Med...

  10. Increased crystal-cell interaction in vitro under co-culture of renal tubular cells and adipocytes by in vitro co-culture paracrine systems simulating metabolic syndrome.

    PubMed

    Ichikawa, Jun; Okada, Atsushi; Taguchi, Kazumi; Fujii, Yasuhiro; Zuo, Li; Niimi, Kazuhiro; Hamamoto, Shuzo; Kubota, Yasue; Umemoto, Yukihiro; Itoh, Yasunori; Yasui, Takahiro; Kawai, Noriyasu; Tozawa, Keiichi; Kohri, Kenjiro

    2014-02-01

    We established an experimental co-culture system for renal tubular cells and adipocytes to investigate kidney stone formation mechanisms under metabolic syndrome (MetS) conditions and examined the interaction between these cells morphologically and genetically. M-1s and 3T3-L1s were cultured individually (control, CON), with 24-h culture media from each cell type added to the other cell type (replacement, RP) in 2-layer co-culture dishes for 24 h (transwell, TW). M-1s wer