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Sample records for algal cell cultures

  1. Air pollutant production by algal cell cultures

    NASA Technical Reports Server (NTRS)

    Fong, F.; Funkhouser, E. A.

    1982-01-01

    The production of phytotoxic air pollutants by cultures of Chlorella vulgaris and Euglena gracilis is considered. Algal and plant culture systems, a fumigation system, and ethylene, ethane, cyanide, and nitrogen oxides assays are discussed. Bean, tobacco, mustard green, cantaloupe and wheat plants all showed injury when fumigated with algal gases for 4 hours. Only coleus plants showed any resistance to the gases. It is found that a closed or recycled air effluent system does not produce plant injury from algal air pollutants.

  2. Algal culture studies for CELSS

    NASA Technical Reports Server (NTRS)

    Radmer, R.; Behrens, P.; Arnett, K.; Gladue, R.; Cox, J.; Lieberman, D.

    1987-01-01

    Microalgae are well-suited as a component of a Closed Environmental Life Support System (CELSS), since they can couple the closely related functions of food production and atmospheric regeneration. The objective was to provide a basis for predicting the response of CELSS algal cultures, and thus the food supply and air regeneration system, to changes in the culture parameters. Scenedesmus growth was measured as a function of light intensity, and the spectral dependence of light absorption by the algae as well as algal respiration in the light were determined as a function of cell concentration. These results were used to test and confirm a mathematical model that describes the productivity of an algal culture in terms of the competing processes of photosynthesis and respiration. The relationship of algal productivity to cell concentration was determined at different carbon dioxide concentrations, temperatures, and light intensities. The maximum productivity achieved by an air-grown culture was found to be within 10% of the computed maximum productivity, indicating that CO2 was very efficiently removed from the gas stream by the algal culture. Measurements of biomass productivity as a function of cell concentration at different light intensities indicated that both the productivity and efficiency of light utilization were greater at higher light intensities.

  3. A colorimetric assay for determination of cell viability in algal cultures.

    PubMed

    Capasso, Juan M; Cossío, Belén R; Berl, Tomás; Rivard, Christopher J; Jiménez, Carlos

    2003-07-01

    In this work, we propose the determination of cell viability in algal cultures by using a colorimetric assay widely used for estimation of cell proliferation in animal cell cultures. The method is based on in vivo reduction by metabolically active cells of a tetrazolium compound (MTS=3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenil)-2H-tetrazolium, inner salt) to a colored formazan, with maximal absorbance at 490 nm, that is released to the culture medium. For this purpose, we have tested two microalgae with high commercial value (Dunaliella and Spirulina) and two seaweeds with different morphology (Ulva and Gracilaria). Color development in this assay is directly proportional to the number of viable cells, to the incubation time in the presence of the assay solution, and to the incubation temperature. A direct significant correlation was found between algal photosynthesis rate and color development in all species used through this work. Moreover, the intensity of absorbance at 490 nm was significantly lower in stressed cells (e.g. in nutrient-limited cultures, in the presence of toxic substances, and in osmotically-stressed cultures). We conclude that cell viability of algal cultures can be rapidly and easily estimated through colorimetric determination of the reduction of MTS to formazan.

  4. Mass algal culture system

    DOEpatents

    Raymond, Lawrence P.

    1981-01-01

    An apparatus and process for the culture of algae in a liquid medium is disclosed. The medium circulates through an open trough and is exposed to an atmosphere which is temperature regulated. The nutrient content of the liquid medium is regulated to control the chemical composition growth and reproduction characteristics of the cultured algae. Before it is allowed to strike the medium, sunlight is passed through a filter to remove wavelengths which are not photosynthetically active. Heat energy can be recovered from the filter.

  5. Mass algal culture system

    DOEpatents

    Raymond, Lawrence P.

    1982-01-01

    An apparatus and process for the culture of algae in a liquid medium is disclosed. The medium circulates through an open trough and is exposed to an atmosphere which is temperature regulated. The nutrient content of the liquid medium is regulated to control the chemical composition growth and reproduction characteristics of the cultured algae. Before it is allowed to strike the medium, sunlight is passed through a filter to remove wavelengths which are not photosynthetically active. Heat energy can be recovered from the filter.

  6. Method and system of culturing an algal mat

    DOEpatents

    Das, Keshav C; Cannon, Benjamin R; Bhatnagar, Ashish; Chinnasamy, Senthil

    2014-05-13

    A system and method for culturing algae are presented. The system and method utilize a fog of growth medium that is delivered to an algal mat generator along with a stream of CO.sub.2 to promote growth of algal cells contained in the generator.

  7. Algal recycling enhances algal productivity and settleability in Pediastrum boryanum pure cultures.

    PubMed

    Park, Jason B K; Craggs, Rupert J; Shilton, Andy N

    2015-12-15

    Recycling a portion of gravity harvested algae (i.e. algae and associated bacteria biomass) has been shown to improve both algal biomass productivity and harvest efficiency by maintaining the dominance of a rapidly-settleable colonial alga, Pediastrum boryanum in both pilot-scale wastewater treatment High Rate Algal Ponds (HRAP) and outdoor mesocosms. While algal recycling did not change the relative proportions of algae and bacteria in the HRAP culture, the contribution of the wastewater bacteria to the improved algal biomass productivity and settleability with the recycling was not certain and still required investigation. P. boryanum was therefore isolated from the HRAP and grown in pure culture on synthetic wastewater growth media under laboratory conditions. The influence of recycling on the productivity and settleability of the pure P. boryanum culture was then determined without wastewater bacteria present. Six 1 L P. boryanum cultures were grown over 30 days in a laboratory growth chamber simulating New Zealand summer conditions either with (Pr) or without (Pc) recycling of 10% of gravity harvested algae. The cultures with recycling (Pr) had higher algal productivity than the controls (Pc) when the cultures were operated at both 4 and 3 d hydraulic retention times by 11% and 38% respectively. Furthermore, algal recycling also improved 1 h settleability from ∼60% to ∼85% by increasing the average P. boryanum colony size due to the extended mean cell residence time and promoted formation of large algal bio-flocs (>500 μm diameter). These results demonstrate that the presence of wastewater bacteria was not necessary to improve algal productivity and settleability with algal recycling.

  8. Carbon and light limitation in mass algal culture

    SciTech Connect

    Brune, D.E.

    1980-01-01

    The carbon limited kinetic responses of various fast growing algal species have been summarized. These results suggest that the growth responses of many algae used in mass culture may best be represented as a Monod fit of the specific growth rate (..mu..) to the free carbon dioxide concentration (CO/sub 2//sub f/). The environmental modifiers of primary importance appear to be light levels, temperature and the ionic strength of the growth media. The various mathematical models describing the algal biological response to limitng CO/sub 2//sub f/ concentration, the carbonate equilibrium chemistry and the physical configration of a flow-through microbial culture are combined to yield equations which predict the pH, total carbon concentration (C/sub T/) and algal cell concentration of a continuous alga culture, given a ..mu../sub max/ and K/sub SCO2/ for the alga of interest. This model is further used to illustrate the under-utilization of inorganic carbon in mass algal cultures in which the pH is uncontrolled. One method of pH control in such cultures involves the utilization of CO/sub 2/ supply from bacterial degradation of waste organics in the influent culture medium. In such a situation both the culture pH and algal cell production will often be governed by either carbon or light limitation depending primarily on the influent BOD loading, detention time and culture depth. In spite of the obvious over-simplification of considering only light and carbon limits in describing the behavior of mass algal culture, comparisons to actual field data suggest that these two parameters will be of paramount importance in controlling net algal cell production rates.

  9. Algal culture studies related to a Closed Ecological Life Support System (CELSS)

    NASA Technical Reports Server (NTRS)

    Radmer, R. O.; Ollinger, O.; Venables, A.; Fernandez, E.

    1982-01-01

    Studies with algal cultures which relate to closed ecological life support systems (CELSS) are discussed. A description of a constant cell density apparatus for continuous culture of algae is included. Excretion of algal by-products, and nitrogen utilization and excretion are discussed.

  10. Regulation of the pigment optical density of an algal cell: filling the gap between photosynthetic productivity in the laboratory and in mass culture.

    PubMed

    Formighieri, Cinzia; Franck, Fabrice; Bassi, Roberto

    2012-11-30

    An increasing number of investors is looking at algae as a viable source of biofuels, beside cultivation for human/animal feeding or to extract high-value chemicals and pharmaceuticals. However, present biomass productivities are far below theoretical estimations implying that a large part of the available photosynthetically active radiation is not used in photosynthesis. Light utilisation inefficiency and rapid light attenuation within a mass culture due to high pigment optical density of wild type strains have been proposed as major limiting factors reducing solar-to-biomass conversion efficiency. Analysis of growth yields of mutants with reduced light-harvesting antennae and/or reduced overall pigment concentration per cell, generated by either mutagenesis or genetic engineering, could help understanding limiting factors for biomass accumulation in photobioreactor. Meanwhile, studies on photo-acclimation can provide additional information on the average status of algal cells in a photobioreactor to be used in modelling-based predictions. Identifying limiting factors in solar-to-biomass conversion efficiency is the first step for planning strategies of genetic improvement and domestication of algae to finally fill the gap between theoretical and industrial photosynthetic productivity. PMID:22426090

  11. An analysis of the productivity of a CELSS continuous algal culture system

    NASA Technical Reports Server (NTRS)

    Radmer, R.; Behrens, P.; Fernandez, E.; Arnett, K.

    1986-01-01

    One of the most attractive aspects of using algal cultures as plant components for a Closed Ecological Life Support Systems (CELSS) is the efficiency with which they can be grown. Although algae are not necessarily intrinsically more efficient than higher plants, the ease which they can be handled and manipulated (more like chemical reagents than plants), and the culturing techniques available, result in much higher growth rates than are usually attainable with higher plants. Furthermore, preliminary experiments have demonstrated that algal growth and physiology is not detectable altered in a microgravity environment, (1) whereas the response of higher plants to zero gravity is unknown. In order to rationally design and operate culture systems, it is necessary to understand how the macroparameters of a culture system, e.g., productivity, are related to the physiological aspects of the algal culture. A first principles analysis of culture system is discussed, and a mathematical model that describes the relationship of culture productivity to the cell concentration of light-limited culture is derived. The predicted productivity vs cell concentration curve agrees well with the experimental data obtained to test this model, indicating that this model permits an accurate prediction of culture productivity given the growth parameters of the system.

  12. Stability of alginate-immobilized algal cells

    SciTech Connect

    Dainty, A.L.; Goulding, K.H.; Robinson, P.K.; Simpkins, I; Trevan, M.D.

    1986-01-01

    Investigations were carried out using immobilized Chlorella cells to determine the diameter, compressibility, tolerance to phosphate chelation, and ability to retain algal cells during incubation of various alginate beads. These physical bead-characteristics were affected by a variety of interactive factors, including multivalent cation type (hardening agent) and cell, cation, and alginate concentration, the latter exhibiting a predominant influence. The susceptibility of alginate beads to phosphate chelation involved a complex interaction of cation type, concentration, and pH of phosphate solution. A scale of response ranging from gel swelling to gel shrinking was observed for a range of conditions. However, stable Ca alginate beads were maintained in incubation media with a pH of 5.5 and a phosphate concentration of 5 micro M. A preliminary investigation into cell leakage from the beads illustrated the importance of maintaining a stable gel structure and limiting cell growth to reduce leakage.

  13. Sludge-grown algae for culturing aquatic organisms: Part I. Algal growth in sludge extracts

    NASA Astrophysics Data System (ADS)

    Hung, K. M.; Chiu, S. T.; Wong, M. H.

    1996-05-01

    This project is aimed at studying the feasibility of using sewage sludge to prepare culture media for microalgae ( Chlorella-HKBU) and the use of the sludge-grown algae as a feed for some aquatic organisms. Part I of the project included results on preparing sludge extracts and their use on algal culture. By comparing two culturing techniques, “aeration” and “shaking,” it was noted that both lag and log phases were shortened in the aeration system. A subsequent experiment noted that algal growth subject to aeration rates of 1.0 and 1.5 liters/min had similar lag and log phases. In addition, both aeration rates had a significantly higher ( P < 0.05) final cell density than that of 0.5 liters/min. A detailed study on the variation of growth conditions on the algal growth was done. The results indicated that pH values of all the cultures declined below 5 at day 12. The removal rates of ammonia N ranged from 62% to 70%. The sludge-grown algae contained a rather substantial amount of heavy metals (µg/g): Zn 289 581, Cu 443 682, Ni 310 963, Mn 96 126, Cr 25 118, and Fe 438 653. This implied that the rather high levels of heavy metals may impose adverse effects on higher trophic organisms.

  14. Sodium efflux from perfused giant algal cells.

    PubMed

    Clint, G M; Macrobbie, E A

    1987-06-01

    Internodal cells of the giant alga Chara corallina were perfused internally to replace the native cytoplasm, tonoplast and vacuole with artificial cytoplasm. Sodium efflux from perfused cells, measured by including (22)Na in the perfusion media, was increased by increasing the internal sodium concentration and by decreasing the external pH, and was inhibited by external application of the renal diuretic amiloride. The sodium efflux was markedly ATP-dependent, with a 50-fold decrease in efflux observed after perfusion with media lacking ATP. Efflux in the presence of ATP was reduced by 33% by inclusion of 10 μM N,N'-dicyclohexylcarbodiimide in the perfusion medium. The membrane potential of the perfused cells approximated that of intact cells from the same culture. It is suggested that sodium efflux in perfused Chara cells proceeds via a secondary antiporter with protons, regulated by ATP in a catalytic role and with the proton motive force acting as the energy source.

  15. Algal cell disruption using microbubbles to localize ultrasonic energy

    PubMed Central

    Krehbiel, Joel D.; Schideman, Lance C.; King, Daniel A.; Freund, Jonathan B.

    2015-01-01

    Microbubbles were added to an algal solution with the goal of improving cell disruption efficiency and the net energy balance for algal biofuel production. Experimental results showed that disruption increases with increasing peak rarefaction ultrasound pressure over the range studied: 1.90 to 3.07 MPa. Additionally, ultrasound cell disruption increased by up to 58% by adding microbubbles, with peak disruption occurring in the range of 108 microbubbles/ml. The localization of energy in space and time provided by the bubbles improve efficiency: energy requirements for such a process were estimated to be one-fourth of the available heat of combustion of algal biomass and one-fifth of currently used cell disruption methods. This increase in energy efficiency could make microbubble enhanced ultrasound viable for bioenergy applications and is expected to integrate well with current cell harvesting methods based upon dissolved air flotation. PMID:25311188

  16. Algal omics: unlocking bioproduct diversity in algae cell factories.

    PubMed

    Guarnieri, Michael T; Pienkos, Philip T

    2015-03-01

    Rapid advances in "omic" technologies are helping to unlock the full potential of microalgae as multi-use feedstocks, with utility in an array of industrial biotechnology, biofuel, and biomedical applications. In turn, algae are emerging as highly attractive candidates for development as microbial cell factories. In this review, we examine the wide array of potential algal bioproducts, with a focus upon the role of omic technologies in driving bioproduct discovery and optimization in microalgal systems.

  17. [Temporal-spatial distribution of algal cells during drought period in Daning River of Three Gorges].

    PubMed

    Zhang, Yong-Sheng; Zheng, Bing-Hui; Wang, Kun; Jiang, Xia; Zheng, Hao

    2013-06-01

    In order to provide basic data for algal bloom warning system, the study on temporal-spatial distribution of algal cells was carried out in Daning River of Three Gorges form April to September, 2011. The results of temporal distribution were as follows: the dominant algal species were blue algal, green algal and diatom. During the test, the density proportion of blue algae increased continuously, the density proportion of diatom decreased, while the density proportion of green algae did not change significantly. The results of spatial distribution were as follows: algal density was extremely significantly correlated with water temperature and chlorophyll a (Chl a), the correlation coefficient were 0.97 and 0.95, respectively; algal density was significantly correlated with light intensity (LI), dissolved oxygen (DO), pH and dissoluble total phosphorus (DTP), the correlation coefficient were 0.87, 0.83, 082 and 0.82, respectively; the algal density in 0 m of Caziba was higher than those in other water depths, and in Baishuihe the highest algal density occurred at 2.0 m water depth in June and July, in Shuanglong most algal cells were found in 0 m and 2.0 m in July, August and September, in Dachang algal density in different water depth did not change significantly during the test; the proportion of different algal species in vertical direction was different in the test, probably because different algal species fitted different environments.

  18. Flagellar waveform dynamics of freely swimming algal cells

    NASA Astrophysics Data System (ADS)

    Kurtuldu, H.; Tam, D.; Hosoi, A. E.; Johnson, K. A.; Gollub, J. P.

    2013-07-01

    We present quantitative measurements of time-dependent flagellar waveforms for freely swimming biflagellated algal cells, for both synchronous and asynchronous beating. We use the waveforms in conjunction with resistive force theory as well as a singularity method to predict a cell's time-dependent velocity for comparison with experiments. While net propulsion is thought to arise from asymmetry between the power and recovery strokes, we show that hydrodynamic interactions between the flagella and cell body on the return stroke make an important contribution to enhance net forward motion.

  19. Flagellar waveform analysis of swimming algal cells

    NASA Astrophysics Data System (ADS)

    Kurtuldu, Huseyin; Johnson, Karl; Gollub, Jerry

    2011-11-01

    The twin flagella of the green alga Chlamydomas reinhardtii are driven by dynein molecular motors to oscillate at about 50-60 Hz in a breaststroke motion. For decades, Chlamydomas has been used as a model organism for studies of flagellar motility, and of genetic disorders of ciliary motion. However, little is known experimentally about the flagellar waveforms, and the resulting time-dependent force distribution along the 250 nm diameter flagella. Here, we study flagellar dynamics experimentally by confining cells in quasi-2D liquid films. From simultaneous measurements of the cell body velocity and the time-dependent velocities along the center lines of the two flagella, we determine the drag coefficients, and estimate the power expended by the body and the flagella, comparing our findings with measurements based on the induced fluid flow field. We contrast the results for the quite different beating patterns of synchronous and asynchronous flagella, respectively. Supported by NSF Grant DMR-0803153.

  20. Separation of algal cells from water by column flotation

    SciTech Connect

    Liu, J.C.; Chen, Y.M.; Ju, Y.H.

    1999-08-01

    The dispersed air flotation (DiAF) process was utilized to separate algal cells (Chlorella sp.) from water. Two types of collector, cationic N-cetyl-N,N,N-trimethylammonium bromide (CTAB) and anionic sodium dodecylsulfate (SDS), were used. It was observed that 20% of cell removal was achieved in the presence of 40 mg/L of SDS, and ca. 86% of the cells were removed at 40 mg/L of CTAB. Upon the addition of 10 mg/L of chitosan, over 90% of the cells were removed when SDS (20 mg/L) was used as the collector. Air flow rate affected cell flotation slightly. Optimum pH values for cell flotation were from 4.0 to 5.0. Flotation efficiency decreased at high ionic strength. The electrostatic interaction between collector and cell surface plays a critical role in the separation processes.

  1. [Cell cultures].

    PubMed

    Cipro, Simon; Groh, Tomáš

    2014-01-01

    Cell or tissue cultures (both terms are interchangeable) represent a complex process by which eukaryotic cells are maintained in vitro outside their natural environment. They have a broad usage covering not only scientific field but also diagnostic one since they represent the most important way of monoclonal antibodies production which are used for both diagnostic and therapeutic purposes. Cell cultures are also used as a "cultivation medium" in virology and for establishing proliferating cells in cytodiagnostics. They are well-established and easy-to-handle models in the area of research, e.g. as a precious source of nucleic acids or proteins. This paper briefly summarizes their importance and methods as well as the pitfalls of the cultivation and new trends in this field. PMID:24624984

  2. Plant and algal cell walls: diversity and functionality

    PubMed Central

    Popper, Zoë A.; Ralet, Marie-Christine; Domozych, David S.

    2014-01-01

    Background Although plants and many algae (e.g. the Phaeophyceae, brown, and Rhodophyceae, red) are only very distantly related they are united in their possession of carbohydrate-rich cell walls, which are of integral importance being involved in many physiological processes. Furthermore, wall components have applications within food, fuel, pharmaceuticals, fibres (e.g. for textiles and paper) and building materials and have long been an active topic of research. As shown in the 27 papers in this Special Issue, as the major deposit of photosynthetically fixed carbon, and therefore energy investment, cell walls are of undisputed importance to the organisms that possess them, the photosynthetic eukaryotes (plants and algae). The complexities of cell wall components along with their interactions with the biotic and abiotic environment are becoming increasingly revealed. Scope The importance of plant and algal cell walls and their individual components to the function and survival of the organism, and for a number of industrial applications, are illustrated by the breadth of topics covered in this issue, which includes papers concentrating on various plants and algae, developmental stages, organs, cell wall components, and techniques. Although we acknowledge that there are many alternative ways in which the papers could be categorized (and many would fit within several topics), we have organized them as follows: (1) cell wall biosynthesis and remodelling, (2) cell wall diversity, and (3) application of new technologies to cell walls. Finally, we will consider future directions within plant cell wall research. Expansion of the industrial uses of cell walls and potentially novel uses of cell wall components are both avenues likely to direct future research activities. Fundamentally, it is the continued progression from characterization (structure, metabolism, properties and localization) of individual cell wall components through to defining their roles in almost every

  3. Flagellar Waveform Dynamics of Freely Swimming Algal Cells

    NASA Astrophysics Data System (ADS)

    Gollub, Jerry; Kurtuldu, Huseyin; Johnson, Karl

    2012-02-01

    We study the time-dependent conformation patterns of flagella driving freely swimming algal cells, and use this information to determine the time-dependent forces acting on the cells, the induced cell velocities, and the power injected into the surrounding fluid. Conformational waves are evident along the flagella, as are synchronization transitions. The observed dynamics relate directly to the behavior of the dynein molecular motors ``walking'' along the microtubules of the flagella. We find that the irreversibility giving rise to net propulsion is due to the hydrodynamic interactions between the flagella and cell body. We determine the time-dependent power injected into the fluid by the cell body and flagella, and show that the propulsion efficiency is only about 3%.

  4. Landfill leachate treatment using bacto-algal co-culture: An integrated approach using chemical analyses and toxicological assessment.

    PubMed

    Kumari, Moni; Ghosh, Pooja; Thakur, Indu Shekhar

    2016-06-01

    The present study aims to evaluate the feasibility of leachate treatment using a synergistic approach by microalgae and bacteria. Leachate from one of the landfill of Northern India showed the presence of various toxic organic contaminants like naphthalene, benzene, phenol and their derivatives, napthols, pesticides, epoxides, phthalates and halogenated organic compounds. ICP-AES analysis revealed high concentrations of Zn, Cr, Fe, Ni, and Pb beyond the maximum permissible limit of discharge. Bacto-algal co-culture was found to be the most efficient in removal of toxic organic contaminants and heavy metals. Further, detoxification efficiency of bacto-algal treatment was evaluated by Methyl tetrazolium (MTT) assay for cytotoxicity and alkaline comet assay for genotoxicity using hepatoma HepG2 cells. Reduction in toxicity was confirmed by an increase in LC50 by 1.9 fold and reduction in Olive Tail Moment by 40.6 fold after 10 days of treatment. Results of the study indicate bioremediation and detoxification potency of bacto-algal co-culture for leachate treatment.

  5. Landfill leachate treatment using bacto-algal co-culture: An integrated approach using chemical analyses and toxicological assessment.

    PubMed

    Kumari, Moni; Ghosh, Pooja; Thakur, Indu Shekhar

    2016-06-01

    The present study aims to evaluate the feasibility of leachate treatment using a synergistic approach by microalgae and bacteria. Leachate from one of the landfill of Northern India showed the presence of various toxic organic contaminants like naphthalene, benzene, phenol and their derivatives, napthols, pesticides, epoxides, phthalates and halogenated organic compounds. ICP-AES analysis revealed high concentrations of Zn, Cr, Fe, Ni, and Pb beyond the maximum permissible limit of discharge. Bacto-algal co-culture was found to be the most efficient in removal of toxic organic contaminants and heavy metals. Further, detoxification efficiency of bacto-algal treatment was evaluated by Methyl tetrazolium (MTT) assay for cytotoxicity and alkaline comet assay for genotoxicity using hepatoma HepG2 cells. Reduction in toxicity was confirmed by an increase in LC50 by 1.9 fold and reduction in Olive Tail Moment by 40.6 fold after 10 days of treatment. Results of the study indicate bioremediation and detoxification potency of bacto-algal co-culture for leachate treatment. PMID:26890189

  6. Isolation of AHL-degrading bacteria from micro-algal cultures and their impact on algal growth and on virulence of Vibrio campbellii to prawn larvae.

    PubMed

    Pande, Gde Sasmita Julyantoro; Natrah, Fatin Mohd Ikhsan; Flandez, Ace Vincent Bravo; Kumar, Uday; Niu, Yufeng; Bossier, Peter; Defoirdt, Tom

    2015-12-01

    Inactivation of quorum sensing (QS) signal molecules, such as acylhomoserine lactones (AHLs) of pathogenic bacteria, has been proposed as a novel method to combat bacterial diseases in aquaculture. Despite the importance of micro-algae for aquaculture, AHL degradation by bacteria associated with micro-algal cultures has thus far not been investigated. In this study, we isolated Pseudomonas sp. NFMI-T and Bacillus sp. NFMI-C from open cultures of the micro-algae Tetraselmis suecica and Chaetoceros muelleri, respectively. An AHL degradation assay showed that either monocultures or co-cultures of the isolates were able to degrade the AHL N-hexanoyl-L-homoserine lactone. In contrast, only Bacillus sp. NFMI-C was able to inactivate N-hydroxybutanoyl-L-homoserine lactone, the AHL produced by Vibrio campbellii. The isolated bacteria were able to persist for up to 3 weeks in conventionalized micro-algal cultures, indicating that they were able to establish and maintain themselves within open algal cultures. Using gnotobiotic algal cultures, we found that the isolates did not affect growth of the micro-algae from which they were isolated, whereas a mixture of both isolates increased the growth of Tetraselmis and decreased the growth of Chaetoceros. Finally, addition of Bacillus sp. NFMI-C to the rearing water of giant river prawn (Macrobrachium rosenbergii) larvae significantly improved survival of the larvae when challenged with pathogenic V. campbellii, whereas it had no effect on larval growth. PMID:26344339

  7. Raman spectroscopy for the characterization of algal cells

    NASA Astrophysics Data System (ADS)

    Samek, Ota; Jonáš, Alexandr; Pilát, Zdeněk; Zemánek, Pavel; Nedbal, Ladislav; Tříska, Jan; Kotas, Petr; Trtílek, Martin

    2010-12-01

    Raman spectroscopy can elucidate fundamental questions about intercellular variability and what governs it. Moreover, knowing the metabolic response on single cell level this can significantly contribute to the study and use of microalgae in systems biology and biofuel technology. Raman spectroscopy is capable to measure nutrient dynamics and metabolism in vivo, in real-time, label free making it possible to monitor/evaluate population variability. Also, degree of unsaturation of the algae oil (iodine value) can be measured using Raman spectra obtained from single microalgae. The iodine value is the determination of the amount of unsaturation contained in fatty acids (in the form of double bonds). Here we demonstrate the capacity of the spatially resolved Raman microspectroscopy to determine the effective iodine value in lipid storage bodies of individual living algal cells. We employed the characteristic peaks in the Raman scattering spectra at 1,656 cm-1 (cis C=C stretching mode) and 1,445 cm-1 (CH2 scissoring mode) as the markers defining the ratio of unsaturated-to-saturated carbon-carbon bonds of the fatty acids in the algal lipids.

  8. Nitrogen recycling from fuel-extracted algal biomass: residuals as the sole nitrogen source for culturing Scenedesmus acutus.

    PubMed

    Gu, Huiya; Nagle, Nick; Pienkos, Philip T; Posewitz, Matthew C

    2015-05-01

    In this study, the reuse of nitrogen from fuel-extracted algal residues was investigated. The alga Scenedesmus acutus was found to be able to assimilate nitrogen contained in amino acids, yeast extracts, and proteinaceous alga residuals. Moreover, these alternative nitrogen resources could replace nitrate in culturing media. The ability of S. acutus to utilize the nitrogen remaining in processed algal biomass was unique among the promising biofuel strains tested. This alga was leveraged in a recycling approach where nitrogen is recovered from algal biomass residuals that remain after lipids are extracted and carbohydrates are fermented to ethanol. The protein-rich residuals not only provided an effective nitrogen resource, but also contributed to a carbon "heterotrophic boost" in subsequent culturing, improving overall biomass and lipid yields relative to the control medium with only nitrate. Prior treatment of the algal residues with Diaion HP20 resin was required to remove compounds inhibitory to algal growth.

  9. Bioengineering aspects of inorganic carbon supply to mass algal cultures. Final report

    SciTech Connect

    Goldman, J.C.

    1980-06-01

    The work included in this report is part of an ongoing study (currently funded by the Solar Energy Research Institute - Subcontract No. XR-9-8144-1) on the inorganic carbon requirements of microalgae under mass culture conditions and covers the period June 1, 1978 through May 31, 1979. It is divided into two parts appended herein. The first part is a literature review on the inorganic carbon chemical system in relation to algal growth requirements, and the second part deals with the kinetics of inorganic carbon-limited growth of two freshwater chlorophytes including the effect of carbon limitation on cellular chemical composition. Additional experiment research covered under this contract was reported in the Proceedings of the 3rd Annual Biomass Energy Systems Conferences, pp. 25-32, Bioengineering aspects of inorganic carbon supply to mass algal cultures. Report No. SERI/TP-33-285.

  10. Coral host cells acidify symbiotic algal microenvironment to promote photosynthesis

    PubMed Central

    Barott, Katie L.; Venn, Alexander A.; Perez, Sidney O.; Tambutté, Sylvie; Tresguerres, Martin

    2015-01-01

    Symbiotic dinoflagellate algae residing inside coral tissues supply the host with the majority of their energy requirements through the translocation of photosynthetically fixed carbon. The algae, in turn, rely on the host for the supply of inorganic carbon. Carbon must be concentrated as CO2 in order for photosynthesis to proceed, and here we show that the coral host plays an active role in this process. The host-derived symbiosome membrane surrounding the algae abundantly expresses vacuolar H+-ATPase (VHA), which acidifies the symbiosome space down to pH ∼4. Inhibition of VHA results in a significant decrease in average H+ activity in the symbiosome of up to 75% and a significant reduction in O2 production rate, a measure of photosynthetic activity. These results suggest that host VHA is part of a previously unidentified carbon concentrating mechanism for algal photosynthesis and provide mechanistic evidence that coral host cells can actively modulate the physiology of their symbionts. PMID:25548188

  11. Rapid algal culture diagnostics for open ponds using multispectral image analysis.

    PubMed

    Murphy, Thomas E; Macon, Keith; Berberoglu, Halil

    2014-01-01

    This article presents a multispectral image analysis approach for probing the spectral backscattered irradiance from algal cultures. It was demonstrated how this spectral information can be used to measure algal biomass concentration, detect invasive species, and monitor culture health in real time. To accomplish this, a conventional RGB camera was used as a three band photodetector for imaging cultures of the green alga Chlorella sp. and the cyanobacterium Anabaena variabilis. A novel floating reference platform was placed in the culture, which enhanced the sensitivity of image color intensity to biomass concentration. Correlations were generated between the RGB color vector of culture images and the biomass concentrations for monocultures of each strain. These correlations predicted the biomass concentrations of independently prepared cultures with average errors of 22 and 14%, respectively. Moreover, the difference in spectral signatures between the two strains was exploited to detect the invasion of Chlorella sp. cultures by A. variabilis. Invasion was successfully detected for A. variabilis to Chlorella sp. mass ratios as small as 0.08. Finally, a method was presented for using multispectral imaging to detect thermal stress in A. variabilis. These methods can be extended to field applications to provide delay free process control feedback for efficient operation of large scale algae cultivation systems.

  12. Rapid algal culture diagnostics for open ponds using multispectral image analysis.

    PubMed

    Murphy, Thomas E; Macon, Keith; Berberoglu, Halil

    2014-01-01

    This article presents a multispectral image analysis approach for probing the spectral backscattered irradiance from algal cultures. It was demonstrated how this spectral information can be used to measure algal biomass concentration, detect invasive species, and monitor culture health in real time. To accomplish this, a conventional RGB camera was used as a three band photodetector for imaging cultures of the green alga Chlorella sp. and the cyanobacterium Anabaena variabilis. A novel floating reference platform was placed in the culture, which enhanced the sensitivity of image color intensity to biomass concentration. Correlations were generated between the RGB color vector of culture images and the biomass concentrations for monocultures of each strain. These correlations predicted the biomass concentrations of independently prepared cultures with average errors of 22 and 14%, respectively. Moreover, the difference in spectral signatures between the two strains was exploited to detect the invasion of Chlorella sp. cultures by A. variabilis. Invasion was successfully detected for A. variabilis to Chlorella sp. mass ratios as small as 0.08. Finally, a method was presented for using multispectral imaging to detect thermal stress in A. variabilis. These methods can be extended to field applications to provide delay free process control feedback for efficient operation of large scale algae cultivation systems. PMID:24265121

  13. The effect of light direction and suspended cell concentrations on algal biofilm growth rates.

    PubMed

    Schnurr, Peter J; Espie, George S; Allen, D Grant

    2014-10-01

    Algae biofilms were grown in a semicontinuous flat plate biofilm photobioreactor to study the effects of light direction and suspended algal cell populations on algal biofilm growth. It was determined that, under the growth conditions and biofilm thicknesses studied, light direction had no effect on long-term algal biofilm growth (26 days); however, light direction did affect the concentration of suspended algal cells by influencing the photon flux density in the growth medium in the photobioreactors. This suspended algal cell population affected short-term (7 days) algae cell recruitment and algal biofilm growth, but additional studies showed that enhanced suspended algal cell populations did not affect biofilm growth rates over the long term (26 days). Studying profiles of light transmittance through biofilms as they grew showed that most of the light became attenuated by the biomass after just a few days of growth (88 % after 3 days). The estimated biofilm thicknesses after these few days of growth were approximately 150 μm. The light attenuation data suggests that, although the biofilms grew to 700-900 μm, under these light intensities, only the first few hundred micrometers of the biofilm is receiving enough light to be photosynthetically active. We postulate that this photosynthetically active layer of the biofilm grows adjacent to the light source, while the rest of the biofilm is in a stationary growth phase. The results of this study have implications for algal biofilm photobioreactor design and operation.

  14. The effect of light direction and suspended cell concentrations on algal biofilm growth rates.

    PubMed

    Schnurr, Peter J; Espie, George S; Allen, D Grant

    2014-10-01

    Algae biofilms were grown in a semicontinuous flat plate biofilm photobioreactor to study the effects of light direction and suspended algal cell populations on algal biofilm growth. It was determined that, under the growth conditions and biofilm thicknesses studied, light direction had no effect on long-term algal biofilm growth (26 days); however, light direction did affect the concentration of suspended algal cells by influencing the photon flux density in the growth medium in the photobioreactors. This suspended algal cell population affected short-term (7 days) algae cell recruitment and algal biofilm growth, but additional studies showed that enhanced suspended algal cell populations did not affect biofilm growth rates over the long term (26 days). Studying profiles of light transmittance through biofilms as they grew showed that most of the light became attenuated by the biomass after just a few days of growth (88 % after 3 days). The estimated biofilm thicknesses after these few days of growth were approximately 150 μm. The light attenuation data suggests that, although the biofilms grew to 700-900 μm, under these light intensities, only the first few hundred micrometers of the biofilm is receiving enough light to be photosynthetically active. We postulate that this photosynthetically active layer of the biofilm grows adjacent to the light source, while the rest of the biofilm is in a stationary growth phase. The results of this study have implications for algal biofilm photobioreactor design and operation. PMID:25149444

  15. 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.

  16. Culturing Selenastrum capricornutum (Chlorophyta) in a synthetic algal nutrient medium with defined mineral particulates

    USGS Publications Warehouse

    Kuwabara, J.S.; Davis, J.A.; Chang, Cecily C.Y.

    1985-01-01

    Algal nutrient studies in chemically-defined media typically employ a synthetic chelator to prevent iron hydroxide precipitation. Micronutrient-particulate interactions may, however, significantly affect chemical speciation and hence biovailability of these nutrients in natural waters. A technique is described by which Selenastrum capricornutum Printz (Chlorophyta) may be cultured in a medium where trace metal speciation (except iron) is controlled, not by organic chelation, but by sorption onto titanium dioxide. Application of this culturing protocol in conjunction with results from sorption studies of nutrient ions on mineral particles provides a means of studying biological impacts of sorptive processes in aquatic environments. ?? 1985 Dr W. Junk Publishers.

  17. Production of biofuel using molluscan pseudofeces derived from algal cells

    DOEpatents

    Das, Keshav C.; Chinnasamy, Senthil; Shelton, James; Wilde, Susan B.; Haynie, Rebecca S.; Herrin, James A.

    2012-08-28

    Embodiments of the present disclosure provide for novel strategies to harvest algal lipids using mollusks which after feeding algae from the growth medium can convert algal lipids into their biomass or excrete lipids in their pseudofeces which makes algae harvesting energy efficient and cost effective. The bioconverter, filter-feeding mollusks and their pseudofeces can be harvested and converted to biocrude using an advanced thermochemical liquefaction technology. Methods, systems, and materials are disclosed for the harvest and isolation of algal lipids from the mollusks, molluscan feces and molluscan pseudofeces.

  18. Host-microbe interactions as a driver of acclimation to salinity gradients in brown algal cultures.

    PubMed

    Dittami, Simon M; Duboscq-Bidot, Laëtitia; Perennou, Morgan; Gobet, Angélique; Corre, Erwan; Boyen, Catherine; Tonon, Thierry

    2016-01-01

    Like most eukaryotes, brown algae live in association with bacterial communities that frequently have beneficial effects on their development. Ectocarpus is a genus of small filamentous brown algae, which comprises a strain that has recently colonized freshwater, a rare transition in this lineage. We generated an inventory of bacteria in Ectocarpus cultures and examined the effect they have on acclimation to an environmental change, that is, the transition from seawater to freshwater medium. Our results demonstrate that Ectocarpus depends on bacteria for this transition: cultures that have been deprived of their associated microbiome do not survive a transfer to freshwater, but restoring their microflora also restores the capacity to acclimate to this change. Furthermore, the transition between the two culture media strongly affects the bacterial community composition. Examining a range of other closely related algal strains, we observed that the presence of two bacterial operational taxonomic units correlated significantly with an increase in low salinity tolerance of the algal culture. Despite differences in the community composition, no indications were found for functional differences in the bacterial metagenomes predicted to be associated with algae in the salinities tested, suggesting functional redundancy in the associated bacterial community. Our study provides an example of how microbial communities may impact the acclimation and physiological response of algae to different environments, and thus possibly act as facilitators of speciation. It paves the way for functional examinations of the underlying host-microbe interactions, both in controlled laboratory and natural conditions.

  19. Coupled nutrient removal and biomass production with mixed algal culture: impact of biotic and abiotic factors.

    PubMed

    Su, Yanyan; Mennerich, Artur; Urban, Brigitte

    2012-08-01

    The influence of biotic (algal inoculum concentration) and abiotic factors (illumination cycle, mixing velocity and nutrient strength) on the treatment efficiency, biomass generation and settleability were investigated with selected mixed algal culture. Dark condition led to poor nutrient removal efficiency. No significant difference in the N, P removal and biomass settleability between continuous and alternating illumination was observed, but a higher biomass generation capability for the continuous illumination was obtained. Different mixing velocity led to similar phosphorus removal efficiencies (above 98%) with different retention times. The reactor with 300 rpm mixing velocity had the best N removal capability. For the low strength wastewater, the N rates were 5.4±0.2, 9.1±0.3 and 10.8±0.3 mg/l/d and P removal rates were 0.57±0.03, 0.56±0.03 and 0.72±0.05 mg/l/d for reactors with the algal inoculum concentration of 0.2, 0.5 and 0.8 g/l, respectively. Low nutrient removal efficiency and poor biomass settleability were obtained for high strength wastewater.

  20. Effects of four rice paddy herbicides on algal cell viability and the relationship with population recovery.

    PubMed

    Nagai, Takashi; Ishihara, Satoru; Yokoyama, Atsushi; Iwafune, Takashi

    2011-08-01

    Paddy herbicides are a high-risk concern for aquatic plants, including algae, because they easily flow out from paddy fields into rivers, with toxic effects. The effect on algal population dynamics, including population recovery after timed exposure, must be assessed. Therefore, we demonstrated concentration-response relationships of four paddy herbicides for algal growth inhibition and mortality, and the relationship between the effect on algal cell viability and population recovery following exposure. We used SYTOX Green dye assay and flow cytometry to assess cell viability of the alga Pseudokirchneriella subcapitata. Live cells could be clearly distinguished from dead cells during herbicide exposure. Our results showed that pretilachlor and quinoclamine had both algicidal and algistatic effects, whereas bensulfuron-methyl only had an algistatic effect, and pentoxazone only had an algicidal effect. Then, a population recovery test following a 72-h exposure was conducted. The algal population recovered in all tests, but the periods required for recovery differed among exposure concentrations and herbicides. The periods required for recovery were inconsistent with the dead cell ratio at the beginning of the recovery test; that is, population recovery could not be described only by cell viability. Consequently, the temporal effect of herbicides and subsequent recovery of the algal population could be described not only by the toxicity characteristics but also by toxicokinetics, such as rate of uptake, transport to the target site, and elimination of the substance from algal cells. PMID:21590715

  1. Biomass recycle as a means to improve the energy efficiency of CELSS algal culture systems

    NASA Astrophysics Data System (ADS)

    Radmer, R.; Cox, J.; Lieberman, D.; Behrens, P.; Arnett, K.

    Algal cultures can be very rapid and efficient means to generate biomass and regenerate the atmosphere for closed environmental life support systems. However, as in the case of most higher plants, a significant fraction of the biomass produced by most algae cannot be directly converted to a useful food product by standard food technology procedures. This waste biomass will serve as an energy drain on the overall system unless it can be efficiently recycled without a significant loss of its energy content. We report experiments in which cultures of the algae Scenedesmus obliquus were grown in the light and at the expense of an added carbon source, which either replaced or supplemented the actinic light. As part of these experiments we tested hydrolyzed waste biomass from these same algae to determine whether the algae themselves could be made part of the biological recycling process. Results indicate that hydrolyzed algal (and plant) biomass can serve as carbon and energy sources for the growth of these algae, suggesting that the efficiency of the closed system could be significantly improved using this recycling process.

  2. Biomass recycle as a means to improve the energy efficiency of CELSS algal culture systems.

    PubMed

    Radmer, R; Cox, J; Lieberman, D; Behrens, P; Arnett, K

    1987-01-01

    Algal cultures can be very rapid and efficient means to generate biomass and regenerate the atmosphere for closed environmental life support systems. However, as in the case of most higher plants, a significant fraction of the biomass produced by most algae cannot be directly converted to a useful food product by standard food technology procedures. This waste biomass will serve as an energy drain on the overall system unless it can be efficiently recycled without a significant loss of its energy content. We report experiments in which cultures of the algae Scenedesmus obliquus were grown in the light and at the expense of an added carbon source, which either replaced or supplemented the actinic light. As part of these experiments we tested hydrolyzed waste biomass from these same algae to determine whether the algae themselves could be made part of the biological recycling process. Results indicate that hydrolyzed algal (and plant) biomass can serve as carbon and energy sources for the growth of these algae, suggesting that the efficiency of the closed system could be significantly improved using this recycling process.

  3. Biomass recycle as a means to improve the energy efficiency of CELSS algal culture systems

    NASA Technical Reports Server (NTRS)

    Radmer, R.; Cox, J.; Lieberman, D.; Behrens, P.; Arnett, K.

    1987-01-01

    Algal cultures can be very rapid and efficient means to generate biomass and regenerate the atmosphere for closed environmental life support systems. However, as in the case of most higher plants, a significant fraction of the biomass produced by most algae cannot be directly converted to a useful food product by standard food technology procedures. This waste biomass will serve as an energy drain on the overall system unless it can be efficiently recycled without a significant loss of its energy content. Experiments are reported in which cultures of the alga Scenedesmus obliquus were grown in the light and at the expense of an added carbon source, which either replaced or supplemented the actinic light. As part of these experiments, hydrolyzed waste biomass from these same algae were tested to determine whether the algae themselves could be made part of the biological recycling process. Results indicate that hydrolyzed algal (and plant) biomass can serve as carbon and energy sources for the growth of these algae, suggesting that the efficiency of the closed system could be significantly improved using this recycling process.

  4. Impact of harmful algal blooms on wild and cultured animals in the Gulf of California.

    PubMed

    Núñez Vázquez, Erick J; Lizarraga, Ismael Gárate; Schmidt, Christine J Band; Tapia, Amaury Cordero; Cortes, David J Lopez; Sandoval, Francisco E Hernandez; Tapia, Alejandra Heredia; Guzman, Jose J Bustillos

    2011-07-01

    Historical documents and classic works together with recent specialized literature have described Harmful Algal Blooms (HABs) in the Gulf of California. This is a review of HABs impact (qualitative and quantitative) during the last decades in the Gulf of California on wild (mammals, birds, fishes, and invertebrates) and cultured animals (shrimps and fishes). Microalgal species responsible of noxious effects are Noctiluca scintillans, Cochlodinium polykrikoides, Gymnodinium catenatum, Prorocentrum minimum, Akashiwo sanguinea, Chattonella subsalsa Ch. marina, Chattonella sp., Heterocapsa sp., Dinophysis sp., Fibrocapsa japonica, Heterosigma akashiwo, Thalassiosira sp., Chaetoceros spp., Pseudo-nitzschia australis, P fraudulenta, Pseudo-nitzschia sp., Trichodesmium erythraeum and ScSchizotrix calcicola. Emphasis is given to the necessity to continue with interdisciplinary studies in oceanography, ecology, toxicology and toxinology interrelated with biomedical sciences such as physiology, pathology, epidemiology and animal health.

  5. Optimizing algal cultivation & productivity : an innovative, multidiscipline, and multiscale approach.

    SciTech Connect

    Murton, Jaclyn K.; Hanson, David T.; Turner, Tom; Powell, Amy Jo; James, Scott Carlton; Timlin, Jerilyn Ann; Scholle, Steven; August, Andrew; Dwyer, Brian P.; Ruffing, Anne; Jones, Howland D. T.; Ricken, James Bryce; Reichardt, Thomas A.

    2010-04-01

    Progress in algal biofuels has been limited by significant knowledge gaps in algal biology, particularly as they relate to scale-up. To address this we are investigating how culture composition dynamics (light as well as biotic and abiotic stressors) describe key biochemical indicators of algal health: growth rate, photosynthetic electron transport, and lipid production. Our approach combines traditional algal physiology with genomics, bioanalytical spectroscopy, chemical imaging, remote sensing, and computational modeling to provide an improved fundamental understanding of algal cell biology across multiple cultures scales. This work spans investigations from the single-cell level to ensemble measurements of algal cell cultures at the laboratory benchtop to large greenhouse scale (175 gal). We will discuss the advantages of this novel, multidisciplinary strategy and emphasize the importance of developing an integrated toolkit to provide sensitive, selective methods for detecting early fluctuations in algal health, productivity, and population diversity. Progress in several areas will be summarized including identification of spectroscopic signatures for algal culture composition, stress level, and lipid production enabled by non-invasive spectroscopic monitoring of the photosynthetic and photoprotective pigments at the single-cell and bulk-culture scales. Early experiments compare and contrast the well-studied green algae chlamydomonas with two potential production strains of microalgae, nannochloropsis and dunnaliella, under optimal and stressed conditions. This integrated approach has the potential for broad impact on algal biofuels and bioenergy and several of these opportunities will be discussed.

  6. The influence of protozoa with a filtered and non-filtered seawater culture of Tetraselmis sp., and effects to the bacterial and algal communities over 10 days.

    PubMed

    Erkelens, Mason; Ball, Andrew S; Lewis, David M

    2014-12-01

    In this study a filter was used to remove protozoa and its effects on a Tetraselmis sp. culture were evaluated in terms of final total lipid, final total dry weight, cell counts, and both the bacterial and algal communities. The protozoa species observed within this study was identified as Cohnilembus reniformis. It was observed that on the final day no C. reniformis were present in filtered cultures compared to the non-filtered culture which contained 40±3 C. reniformis/mL. The presence of C. reniformis within the culture did not affect the total lipid or the total dry weight recovered, suggesting that Tetraselmis sp. was capable of surviving and growing in the presence of C. reniformis. Overall it is suggested that an 11 μm filter was effective at removing protozoa, though growing a microalgae culture without filtration did not show any significant effect.

  7. Digestion of algal biomass for electricity generation in microbial fuel cells.

    PubMed

    Nishio, Koichi; Hashimoto, Kazuhito; Watanabe, Kazuya

    2013-01-01

    Algal biomass serves as a fuel for electricity generation in microbial fuel cells. This study constructed a model consortium comprised of an alga-digesting Lactobacillus and an iron-reducing Geobacter for electricity generation from photo-grown Clamydomonas cells. Total power-conversion efficiency (from Light to electricity) was estimated to be 0.47%.

  8. Flocculation of algal cells by amphoteric chitosan-based flocculant.

    PubMed

    Dong, Changlong; Chen, Wei; Liu, Cheng

    2014-10-01

    A kind of amphoteric chitosan-based flocculant (quaternized carboxymethyl chitosan, denoted as QCMC) has been prepared. QCMC presented significant improvement of water solubility in the whole pH range. The effects of pH, dosage, temperature and original turbidity of algal water on the flocculation performance were investigated. The optimal dosages of QCMC at pH 5, 9 and 12 with original turbidity of 20NTU at 20°C were 0.1, 0.6 and 2.0mg/L, respectively, which were much less than that of chitosan, PAM, Al2(SO4)3 and FeCl3. The floc properties during grow, breakage and regrow period were also evaluated at different pH values in terms of floc size, strength and density. It was demonstrated that QCMC produced larger, stronger and denser flocs than Al2(SO4)3. There is every indication that QCMC is more suitable for algal harvesting than other traditional coagulants or flocculants.

  9. Plasticity of Total and Intracellular Phosphorus Quotas in Microcystis aeruginosa Cultures and Lake Erie Algal Assemblages.

    PubMed

    Saxton, Matthew A; Arnold, Robert J; Bourbonniere, Richard A; McKay, Robert Michael L; Wilhelm, Steven W

    2012-01-01

    Blooms of the potentially toxic cyanobacterium Microcystis are common events globally, and as a result significant resources continue to be dedicated to monitoring and controlling these events. Recent studies have shown that a significant proportion of total cell-associated phosphorus (P) in marine phytoplankton can be surface adsorbed; as a result studies completed to date do not accurately report the P demands of these organisms. In this study we measure the total cell-associated and intracellular P as well as growth rates of two toxic strains of Microcystis aeruginosa Kütz grown under a range of P concentrations. The results show that the intracellular P pool in Microcystis represents a percentage of total cell-associated P (50-90%) similar to what has been reported for actively growing algae in marine systems. Intracellular P concentrations (39-147 fg cell(-1)) generally increased with increasing P concentrations in the growth medium, but growth rate and the ratio of total cell-associated to intracellular P remained generally stable. Intracellular P quotas and growth rates in cells grown under the different P treatments illustrate the ability of this organism to successfully respond to changes in ambient P loads, and thus have implications for ecosystem scale productivity models employing P concentrations to predict algal bloom events. PMID:22279445

  10. Influence of the Cell Wall on Intracellular Delivery to Algal Cells by Electroporation and Sonication

    PubMed Central

    Azencott, Harold R.; Peter, Gary F.; Prausnitz, Mark R.

    2007-01-01

    To assess the cell wall’s role as a barrier to intracellular delivery, wild-type Chlamydomonas reinhardtii algal cells and mutant cells lacking a cell wall were exposed to electroporation or sonication. Flow cytometry determined intracellular uptake of calcein and bovine serum albumin (BSA) and loss of cell viability as functions of electroporation transmembrane potential and acoustic energy. Electroporation of wild-type cells increased calcein uptake with increasing transmembrane potential, but delivered much less BSA. Electroporation of wall-deficient cells had similar effects on calcein uptake, but increased BSA uptake as much as 7.5-fold relative to wild-type cells, which indicated that the cell wall was a significant barrier to BSA delivery during electroporation. Sonication of wild-type cells caused calcein and BSA uptake at similar levels. This suggests that the cell wall barrier to BSA delivery can be overcome by sonication. Increased electroporation transmembrane potential or acoustic energy also caused increased loss of cell viability, where wall-deficient cells were especially susceptible to lysis. Overall, we believe this is the first study to compare the effects of electroporation and sonication in a direct fashion in any cell type. Specifically, these findings suggest that electroporation primarily transports molecules across the plasma membrane, because its mechanism is specific to lipid bilayer disruption, whereas sonication transports molecules across both the plasma membrane and cell wall, because it non-specifically disrupts cell-surface barriers. PMID:17602827

  11. Climatic forcings on algal cell size through the Cenozoic

    NASA Astrophysics Data System (ADS)

    Hannisdal, B.; Henderiks, J.

    2009-04-01

    Marine phytoplankton constitute the base of the marine food chain and are fundamental components in biogeochemical cycles. A better understanding of both the mechanisms and the rates of climatic adaptation by marine phytoplankton remains a critical challenge in predicting future impacts of climate change. Using coccolithophores (a prominent group of calcifying unicellular algae) as an example, we explore why different species respond differently to environmental change, and determine what climatic parameters drive changes in cell size over geological time scales. We use comprehensive time series of coccolith size - a proxy for cell size - over the Cenozoic, targeting the ancestry of extant taxa that have revealed distinct physiological responses in culture experiments. We employ a novel, information-theoretic method to quantify the relative influence of different environmental variables on coccolith size. This model-free technique detects the significance and relative strength of the causal influence of multiple climatic forcing variables (including non-linear relationships), while correcting for possible common causal interactions among the forcings. Our analyses suggest that the macroevolutionary size decrease within the Reticulofenestra lineage (the ancestry of today's most prolific bloomer Emiliania huxleyi) primarily reflects a physiological adaptation to CO2 limitation. In contrast, macroevolutionary size variability within the Coccolithus lineage (the ancestry of Coccolithus pelagicus and C. braarudii), while influenced by long-term trends in ocean temperature, is not affected by changes in CO2. Improved understanding of such heterogeneity within ecosystem functional groups, including taxon-specific climatic adaptation strategies, will be of fundamental importance to the assessment of future climate change impact and mitigation.

  12. Effects of anodic oxidation of a substoichiometric titanium dioxide reactive electrochemical membrane on algal cell destabilization and lipid extraction.

    PubMed

    Hua, Likun; Guo, Lun; Thakkar, Megha; Wei, Dequan; Agbakpe, Michael; Kuang, Liyuan; Magpile, Maraha; Chaplin, Brian P; Tao, Yi; Shuai, Danmeng; Zhang, Xihui; Mitra, Somenath; Zhang, Wen

    2016-03-01

    Efficient algal harvesting, cell pretreatment and lipid extraction are the major steps challenging the algal biofuel industrialization. To develop sustainable solutions for economically viable algal biofuels, our research aims at devising innovative reactive electrochemical membrane (REM) filtration systems for simultaneous algal harvesting and pretreatment for lipid extraction. The results in this work particularly demonstrated the use of the Ti4O7-based REM in algal pretreatment and the positive impacts on lipid extraction. After REM treatment, algal cells exhibited significant disruption in morphology and photosynthetic activity due to the anodic oxidation. Cell lysis was evidenced by the changes of fluorescent patterns of dissolved organic matter (DOM) in the treated algal suspension. The lipid extraction efficiency increased from 15.2 ± 0.6 g-lipidg-algae(-1) for untreated algae to 23.4 ± 0.7 g-lipidg-algae(-1) for treated algae (p<0.05), which highlights the potential to couple algal harvesting with cell pretreatment in an integrated REM filtration process. PMID:26722810

  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. A Simple and Rapid Protocol for Measuring Neutral Lipids in Algal Cells Using Fluorescence

    PubMed Central

    Storms, Zachary J.; Cameron, Elliot; de la Hoz Siegler, Hector; McCaffrey, William C.

    2014-01-01

    Algae are considered excellent candidates for renewable fuel sources due to their natural lipid storage capabilities. Robust monitoring of algal fermentation processes and screening for new oil-rich strains requires a fast and reliable protocol for determination of intracellular lipid content. Current practices rely largely on gravimetric methods to determine oil content, techniques developed decades ago that are time consuming and require large sample volumes. In this paper, Nile Red, a fluorescent dye that has been used to identify the presence of lipid bodies in numerous types of organisms, is incorporated into a simple, fast, and reliable protocol for measuring the neutral lipid content of Auxenochlorella protothecoides, a green alga. The method uses ethanol, a relatively mild solvent, to permeabilize the cell membrane before staining and a 96 well micro-plate to increase sample capacity during fluorescence intensity measurements. It has been designed with the specific application of monitoring bioprocess performance. Previously dried samples or live samples from a growing culture can be used in the assay. PMID:24961928

  15. Time-response of cultured deep-sea benthic foraminifera to different algal diets

    NASA Astrophysics Data System (ADS)

    Heinz, P.; Hemleben, Ch; Kitazato, H.

    2002-03-01

    The vertical distribution of benthic foraminifera in the surface sediment is influenced by environmental factors, mainly by food and oxygen supply. An experiment of three different time series was performed to investigate the response of deep-sea benthic foraminifera to simulated phytodetritus pulses under stable oxygen concentrations. Each series was fed constantly with one distinct algal species in equivalent amounts. The temporal reactions of the benthic foraminifera with regard to the vertical distribution in the sediment, the total number, and the species composition were observed and compared within the three series. Additionally, oxygen contents and bacterial cell numbers were measured to ensure that these factors were invariable and did not influence foraminiferal communities. The addition of algae leads to higher population densities 21 days after food was added. Higher numbers of individuals were probably caused by higher organic levels, which in turn induced reproduction. A stronger response is found after feeding with Amphiprora sp. and Pyramimonas sp., compared to Dunaliella tertiolecta. At a constant high oxygen supply, no migration to upper layers was observed after food addition, and more individuals were found in deeper layers. The laboratory results thus agree with the predictions of the TROX-model. An epifaunal microhabitat preference was shown for Adercotryma glomerata. Hippocrepina sp. was spread over the entire sediment depth with a shallow infaunal maximum. Melonis barleeanum preferred a deeper infaunal habitat. Bacterial cell concentrations were stable during the laboratory experiments and showed no significant response to higher organic fluxes.

  16. Algal autolysate medium to label proteins for NMR in mammalian cells.

    PubMed

    Fuccio, Carmelo; Luchinat, Enrico; Barbieri, Letizia; Neri, Sara; Fragai, Marco

    2016-04-01

    In-cell NMR provides structural and functional information on proteins directly inside living cells. At present, the high costs of the labeled media for mammalian cells represent a limiting factor for the development of this methodology. Here we report a protocol to prepare a homemade growth medium from Spirulina platensis autolysate, suitable to express uniformly labeled proteins inside mammalian cells at a reduced cost-per-sample. The human proteins SOD1 and Mia40 were overexpressed in human cells grown in (15)N-enriched S. platensis algal-derived medium, and high quality in-cell NMR spectra were obtained. PMID:27106902

  17. Unicellular Algal Growth: A Biomechanical Approach to Cell Wall Dynamics

    NASA Astrophysics Data System (ADS)

    Kam, Royce; Levine, Herbert

    1997-11-01

    We model a growing cell in a calcium solution as an elastic shell on short time scales. The turgor pressure and elastic properties (Young's modulus, thickness) of the cell wall determine a stressed cell shape. Enzyme-mediated relaxation of the unstressed toward the stressed configuration results in a slow (plastic) deformation of the cell. The cell wall thickness is then modulated by calcium-mediated fusion of material and elongation. We analyze small perturbations to a circular cell and find an instability related to modulations of the wall thickness, leading to growth rates which peak at a finite wave number.

  18. Role of initial cell density of algal bioassay of toxic chemicals.

    PubMed

    Singh, Prashant Kumar; Shrivastava, Alok Kumar

    2016-07-01

    A variety of toxicants such as, metal ions, pesticides, dyes, etc. are continuously being introduced anthropogenically in the environment and adversely affect to the biotic component of the ecosystem. Therefore, the assessment of negative effects of these toxicants is required. However, toxicity assessment anticipated by chemical analysis are extremely poor, therefore the application of the living systems for the same is an excellent approach. Concentration of toxicant as well as cell density both influenced the result of the algal toxicity assay. Here, Scenedesmus sp, a very fast growing green microalgae was selected for study the effects of initial cell densities on the toxicity of Cu(II), Cd(II), Zn(II), paraquat and 2,4-D. Results demonstrated concentration dependent decrease in biomass and specific growth rate of Scenedesmus sp. on exposure of abovesaid toxicants. Paraquat and 2,4-D emerged as extremely toxic to the test alga which reflected from the lowest EC value and very steep decline in biomass was evident with increasing concentration of paraquat and 2,4-D in the medium. Result also demonstrated that initial cell density is a very important parameter than specific growth rate for algal bioassay of various toxicants. Present study clearly illustrated that the use of smaller cell density is always recommended for assaying toxicity of chemicals in algal assays. PMID:26593761

  19. 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)

  20. Fish stem cell cultures.

    PubMed

    Hong, Ni; Li, Zhendong; Hong, Yunhan

    2011-04-13

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

  1. Direct extraction of photosynthetic electrons from single algal cells by nanoprobing system.

    PubMed

    Ryu, WonHyoung; Bai, Seoung-Jai; Park, Joong Sun; Huang, Zubin; Moseley, Jeffrey; Fabian, Tibor; Fasching, Rainer J; Grossman, Arthur R; Prinz, Fritz B

    2010-04-14

    There are numerous sources of bioenergy that are generated by photosynthetic processes, for example, lipids, alcohols, hydrogen, and polysaccharides. However, generally only a small fraction of solar energy absorbed by photosynthetic organisms is converted to a form of energy that can be readily exploited. To more efficiently use the solar energy harvested by photosynthetic organisms, we evaluated the feasibility of generating bioelectricity by directly extracting electrons from the photosynthetic electron transport chain before they are used to fix CO(2) into sugars and polysaccharides. From a living algal cell, Chlamydomonas reinhardtii, photosynthetic electrons (1.2 pA at 6000 mA/m(2)) were directly extracted without a mediator electron carrier by inserting a nanoelectrode into the algal chloroplast and applying an overvoltage. This result may represent an initial step in generating "high efficiency" bioelectricity by directly harvesting high energy photosynthetic electrons. PMID:20201533

  2. Improved photobiological H2 production in engineered green algal cells.

    PubMed

    Kruse, Olaf; Rupprecht, Jens; Bader, Klaus-Peter; Thomas-Hall, Skye; Schenk, Peer Martin; Finazzi, Giovanni; Hankamer, Ben

    2005-10-01

    Oxygenic photosynthetic organisms use solar energy to split water (H2O) into protons (H+), electrons (e-), and oxygen. A select group of photosynthetic microorganisms, including the green alga Chlamydomonas reinhardtii, has evolved the additional ability to redirect the derived H+ and e- to drive hydrogen (H2) production via the chloroplast hydrogenases HydA1 and A2 (H2 ase). This process occurs under anaerobic conditions and provides a biological basis for solar-driven H2 production. However, its relatively poor yield is a major limitation for the economic viability of this process. To improve H2 production in Chlamydomonas, we have developed a new approach to increase H+ and e- supply to the hydrogenases. In a first step, mutants blocked in the state 1 transition were selected. These mutants are inhibited in cyclic e- transfer around photosystem I, eliminating possible competition for e- with H2ase. Selected strains were further screened for increased H2 production rates, leading to the isolation of Stm6. This strain has a modified respiratory metabolism, providing it with two additional important properties as follows: large starch reserves (i.e. enhanced substrate availability), and a low dissolved O2 concentration (40% of the wild type (WT)), resulting in reduced inhibition of H2ase activation. The H2 production rates of Stm6 were 5-13 times that of the control WT strain over a range of conditions (light intensity, culture time, +/- uncoupler). Typically, approximately 540 ml of H2 liter(-1) culture (up to 98% pure) were produced over a 10-14-day period at a maximal rate of 4 ml h(-1) (efficiency = approximately 5 times the WT). Stm6 therefore represents an important step toward the development of future solar-powered H2 production systems. PMID:16100118

  3. Flow cytometric measurement of pollutant stresses on algal cells

    SciTech Connect

    Berglund, D.L.; Eversman, S.

    1988-03-01

    The lichen Usnea fulvoreagens (Raes). Raes. was treated with four pH levels (5.5, 4.5, 3.5, and 2.5) of simulated acid rain (sulfuric acid, nitric acid, and a 1:1 combination of both) and automobile exhaust. The samples were dissociated and analyzed by a Becton-Dickinson FACS 440 flow cytometer. Analyses included measurement of chlorophyll autofluorescence and fluorescence due to uptake of fluorescein diacetate (FDA) and calcofluor white M2R (CFW). Cell parameters measured were esterase activity (FDA), membrane permeability (FDA, CFW), and intracellular pH (FDA). Mean fluorescence intensity from FDA staining and numbers of events were incorporated with autofluorescence information to produce a stress index of relative cell stress. Results indicated that highly stressed samples (lower pH treatments and greater exposure to exhaust) exhibited a low stress index of FDA fluorescence.Au

  4. Flow cytometric measurement of pollutant stresses on algal cells.

    PubMed

    Berglund, D L; Eversman, S

    1988-03-01

    The lichen Usnea fulvoreagens (Räs). Räs. was treated with four pH levels (5.5, 4.5, 3.5, and 2.5) of simulated acid rain (sulfuric acid, nitric acid, and a 1:1 combination of both) and automobile exhaust. The samples were dissociated and analyzed by a Becton-Dickinson FACS 440 flow cytometer. Analyses included measurement of chlorophyll autofluorescence and fluorescence due to uptake of fluorescein diacetate (FDA) and calcofluor white M2R (CFW). Cell parameters measured were esterase activity (FDA), membrane permeability (FDA, CFW), and intracellular pH (FDA). Mean fluorescence intensity from FDA staining and numbers of events were incorporated with autofluorescence information to produce a "stress index" of relative cell stress. Results indicated that highly stressed samples (lower pH treatments and greater exposure to exhaust) exhibited a low "stress index" of FDA fluorescence.

  5. Digital Microfluidic Cell Culture.

    PubMed

    Ng, Alphonsus H C; Li, Bingyu Betty; Chamberlain, M Dean; Wheeler, Aaron R

    2015-01-01

    Digital microfluidics (DMF) is a droplet-based liquid-handling technology that has recently become popular for cell culture and analysis. In DMF, picoliter- to microliter-sized droplets are manipulated on a planar surface using electric fields, thus enabling software-reconfigurable operations on individual droplets, such as move, merge, split, and dispense from reservoirs. Using this technique, multistep cell-based processes can be carried out using simple and compact instrumentation, making DMF an attractive platform for eventual integration into routine biology workflows. In this review, we summarize the state-of-the-art in DMF cell culture, and describe design considerations, types of DMF cell culture, and cell-based applications of DMF. PMID:26643019

  6. Liver Cell Culture Devices

    PubMed Central

    Andria, B.; Bracco, A.; Cirino, G.; Chamuleau, R. A. F. M.

    2010-01-01

    In the last 15 years many different liver cell culture devices, consisting of functional liver cells and artificial materials, have been developed. They have been devised for numerous different applications, such as temporary organ replacement (a bridge to liver transplantation or native liver regeneration) and as in vitro screening systems in the early stages of the drug development process, like assessing hepatotoxicity, hepatic drug metabolism, and induction/inhibition studies. Relevant literature is summarized about artificial human liver cell culture systems by scrutinizing PubMed from 2003 to 2009. Existing devices are divided in 2D configurations (e.g., static monolayer, sandwich, perfused cells, and flat plate) and 3D configurations (e.g., liver slices, spheroids, and different types of bioreactors). The essential features of an ideal liver cell culture system are discussed: different types of scaffolds, oxygenation systems, extracellular matrixes (natural and artificial), cocultures with nonparenchymal cells, and the role of shear stress problems. Finally, miniaturization and high-throughput systems are discussed. All these factors contribute in their own way to the viability and functionality of liver cells in culture. Depending on the aim for which they are designed, several good systems are available for predicting hepatotoxicity and hepatic metabolism within the general population. To predict hepatotoxicity in individual cases genomic analysis might be essential as well. PMID:26998397

  7. 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

  8. Culturing Uveal Melanoma Cells.

    PubMed

    Angi, Martina; Versluis, Mieke; Kalirai, Helen

    2015-04-01

    A major challenge in cancer research is the use of appropriate models with which to study a specific biological question. Cell lines have long been used to study cellular processes and the effects of individual molecules because they are easy to use, grow rapidly, produce reproducible results and have a strong track record in research. In uveal melanoma in particular, the absence of animal models that faithfully replicate the behavior of the human disease has propagated the generation and use of numerous cell lines by individual research groups. This in itself, however, can be viewed as a problem due to the lack of standardization when characterizing these entities to determine how closely they reflect the genetic and phenotypic characteristics of this disease. The alternative is to use in vitro primary cultures of cells obtained directly from uveal melanoma patient samples, but this too has its difficulties. Primary cell cultures are difficult to use, hard to obtain and can show considerable heterogeneity. In this article, we review the following: (1) the uveal melanoma cell lines that are currently available, discussing the importance of establishing a bank of those that represent the molecular heterogeneity of uveal melanoma; (2) the methods used to isolate and perform short-term cultures of primary uveal melanoma cells, and (3) the establishment of 3D tissue culture models that bridge the gap between 2D in vitro systems and in vivo models with which to dissect cancer biology and perform therapeutic screens. PMID:27171555

  9. 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.

  10. 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.

  11. 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

  12. Cell death in a harmful algal bloom causing species Alexandrium tamarense upon an algicidal bacterium induction.

    PubMed

    Zhang, Huajun; Lv, Jinglin; Peng, Yun; Zhang, Su; An, Xinli; Xu, Hong; Zhang, Jun; Tian, Yun; Zheng, Wei; Zheng, Tianling

    2014-09-01

    Harmful algal blooms occur throughout the world, destroying aquatic ecosystems and threatening human health. The culture supernatant of the marine algicidal bacteria DHQ25 was able to lysis dinoflagellate Alexandrium tamarense. Loss of photosynthetic pigments, accompanied by a decline in Photosystem II (PSII) photochemical efficiency (Fv/Fm), in A. tamarense was detected under bacterial supernatant stress. Transmission electron microscope analysis showed obvious morphological modifications of chloroplast dismantling as a part of the algicidal process. The PSII electron transport chain was seriously blocked, with its reaction center damaged. This damage was detected in a relative transcriptional level of psbA and psbD genes, which encode the D1 and D2 proteins in the PSII reaction center. And the block in the electron transport chain of PSII might generate excessive reactive oxygen species (ROS) which could destroy the membrane system and pigment synthesis and activated enzymic antioxidant systems including superoxide dismutase (SOD) and catalase (CAT). This study indicated that marine bacteria with indirect algicidal activity played an important role in the changes of photosynthetic process in a harmful algal bloom species.

  13. Algal cell disruption using microbubbles to localize ultrasonic energy for biofuel extraction

    NASA Astrophysics Data System (ADS)

    Krehbiel, Joel; Sch, Lance; King, Daniel; Freund, Jonathan

    2014-11-01

    Cell disruption is a critical step in the production of algal-based biofuels, but current mechanical disruption methods require significant energy, typically more than actually available in the cell's oil. We propose and investigate an ultrasound disruption process using ultrasound contrast agents to localize the delivered energy. Experiments in a flow cell with focused ultrasound show a significant benefit. The degree of disruption increases with increasing peak rarefactional ultrasound pressure for pressures between 1.90 and 3.07 MPa and increasing microbubble concentration up to 12 . 5 ×107 bubbles/ml. Estimates suggest the energy of this method is less than one fourth of the energy of other industrial mechanical disruption techniques and comparable with theoretical disruption estimates. The increase in efficiency would make this technique viable for bioenergy applications.

  14. Rescue of heavy metal effects on cell physiology of the algal model system Micrasterias by divalent ions.

    PubMed

    Volland, Stefanie; Bayer, Elisabeth; Baumgartner, Verena; Andosch, Ancuela; Lütz, Cornelius; Sima, Evelyn; Lütz-Meindl, Ursula

    2014-01-15

    Recent studies have shown that metals such as copper, zinc, aluminum, cadmium, chromium, iron and lead cause severe dose-dependent disturbances in growth, morphogenesis, photosynthetic and respiratory activity as well as on ultrastructure and function of organelles in the algal model system Micrasterias denticulata (Volland et al., 2011, 2012; Andosch et al., 2012). In the present investigation we focus on amelioration of these adverse effects of cadmium, chromium and lead by supplying the cells with different antioxidants and essential micronutrients to obtain insight into metal uptake mechanisms and subcellular metal targets. This seems particularly interesting as Micrasterias is adapted to extremely low-concentrated, oligotrophic conditions in its natural bog environment. The divalent ions of iron, zinc and calcium were able to diminish the effects of the metals cadmium, chromium and lead on Micrasterias. Iron showed most ameliorating effects on cadmium and chromium in short- and long-term treatments and improved cell morphogenesis, ultrastructure, cell division rates and photosynthesis. Analytical transmission electron microscopic (TEM) methods (electron energy loss spectroscopy (EELS) and electron spectroscopic imaging (ESI)) revealed that chromium uptake was decreased when Micrasterias cells were pre-treated with iron, which resulted in no longer detectable intracellular chromium accumulations. Zinc rescued the detrimental effects of chromium on net-photosynthesis, respiration rates and electron transport in PS II. Calcium and gadolinium were able to almost completely compensate the inhibiting effects of lead and cadmium on cell morphogenesis after mitosis, respectively. These results indicate that cadmium is taken up by calcium and iron transporters, whereas chromium appears to enter the algae cells via iron and zinc carriers. It was shown that lead is not taken up into Micrasterias at all but exerts its adverse effects on cell growth by substituting cell

  15. Rescue of heavy metal effects on cell physiology of the algal model system Micrasterias by divalent ions.

    PubMed

    Volland, Stefanie; Bayer, Elisabeth; Baumgartner, Verena; Andosch, Ancuela; Lütz, Cornelius; Sima, Evelyn; Lütz-Meindl, Ursula

    2014-01-15

    Recent studies have shown that metals such as copper, zinc, aluminum, cadmium, chromium, iron and lead cause severe dose-dependent disturbances in growth, morphogenesis, photosynthetic and respiratory activity as well as on ultrastructure and function of organelles in the algal model system Micrasterias denticulata (Volland et al., 2011, 2012; Andosch et al., 2012). In the present investigation we focus on amelioration of these adverse effects of cadmium, chromium and lead by supplying the cells with different antioxidants and essential micronutrients to obtain insight into metal uptake mechanisms and subcellular metal targets. This seems particularly interesting as Micrasterias is adapted to extremely low-concentrated, oligotrophic conditions in its natural bog environment. The divalent ions of iron, zinc and calcium were able to diminish the effects of the metals cadmium, chromium and lead on Micrasterias. Iron showed most ameliorating effects on cadmium and chromium in short- and long-term treatments and improved cell morphogenesis, ultrastructure, cell division rates and photosynthesis. Analytical transmission electron microscopic (TEM) methods (electron energy loss spectroscopy (EELS) and electron spectroscopic imaging (ESI)) revealed that chromium uptake was decreased when Micrasterias cells were pre-treated with iron, which resulted in no longer detectable intracellular chromium accumulations. Zinc rescued the detrimental effects of chromium on net-photosynthesis, respiration rates and electron transport in PS II. Calcium and gadolinium were able to almost completely compensate the inhibiting effects of lead and cadmium on cell morphogenesis after mitosis, respectively. These results indicate that cadmium is taken up by calcium and iron transporters, whereas chromium appears to enter the algae cells via iron and zinc carriers. It was shown that lead is not taken up into Micrasterias at all but exerts its adverse effects on cell growth by substituting cell

  16. Rescue of heavy metal effects on cell physiology of the algal model system Micrasterias by divalent ions

    PubMed Central

    Volland, Stefanie; Bayer, Elisabeth; Baumgartner, Verena; Andosch, Ancuela; Lütz, Cornelius; Sima, Evelyn; Lütz-Meindl, Ursula

    2014-01-01

    Recent studies have shown that metals such as copper, zinc, aluminum, cadmium, chromium, iron and lead cause severe dose-dependent disturbances in growth, morphogenesis, photosynthetic and respiratory activity as well as on ultrastructure and function of organelles in the algal model system Micrasterias denticulata (Volland et al., 2011, 2012; Andosch et al., 2012). In the present investigation we focus on amelioration of these adverse effects of cadmium, chromium and lead by supplying the cells with different antioxidants and essential micronutrients to obtain insight into metal uptake mechanisms and subcellular metal targets. This seems particularly interesting as Micrasterias is adapted to extremely low-concentrated, oligotrophic conditions in its natural bog environment. The divalent ions of iron, zinc and calcium were able to diminish the effects of the metals cadmium, chromium and lead on Micrasterias. Iron showed most ameliorating effects on cadmium and chromium in short- and long-term treatments and improved cell morphogenesis, ultrastructure, cell division rates and photosynthesis. Analytical transmission electron microscopic (TEM) methods (electron energy loss spectroscopy (EELS) and electron spectroscopic imaging (ESI)) revealed that chromium uptake was decreased when Micrasterias cells were pre-treated with iron, which resulted in no longer detectable intracellular chromium accumulations. Zinc rescued the detrimental effects of chromium on net-photosynthesis, respiration rates and electron transport in PS II. Calcium and gadolinium were able to almost completely compensate the inhibiting effects of lead and cadmium on cell morphogenesis after mitosis, respectively. These results indicate that cadmium is taken up by calcium and iron transporters, whereas chromium appears to enter the algae cells via iron and zinc carriers. It was shown that lead is not taken up into Micrasterias at all but exerts its adverse effects on cell growth by substituting cell

  17. Degradation of algal organic matter using microbial fuel cells and its association with trihalomethane precursor removal.

    PubMed

    Wang, Huan; Liu, Dongmei; Lu, Lu; Zhao, Zhiwei; Xu, Yongpeng; Cui, Fuyi

    2012-07-01

    In order to provide an alternative for removal of algal organic matter (AOM) produced during algal blooms in aquatic environment, microbial fuel cell (MFC) was used to study AOM degradation and its association with THM precursor removal. The chemical oxygen demand (COD) removals in MFCs were 81 ± 6% and 73 ± 3% for AOM from Microcystis aeruginosa (AOM(M)) and Chlorella vulgaris (AOM(C)), respectively. THM precursor was also effectively degraded (AOM(M) 85 ± 2%, AOM(C) 72 ± 4%). The major AOM components (proteins, lipids, and carbohydrates) were obviously removed in MFCs. The contribution of each component to the THM formation potential (THMFP) was obtained based on calculation. The THMFP produced from soluble microbial products was very low. If the energy input during operation process was not considered, MFCs treatment could recover electrical energy of 0.29 ± 0.02 kWh/kg COD (AOM(M)) and 0.35 ± 0.06 kWh/kg COD (AOM(C)).

  18. Characteristic changes in algal organic matter derived from Microcystis aeruginosa in microbial fuel cells.

    PubMed

    Wang, Huan; Lu, Lu; Liu, Dongmei; Cui, Fuyi; Wang, Peng

    2015-11-01

    The objective of this study was to investigate behavior of algal organic matter (AOM) during bioelectrochemical oxidation in microbial fuel cell in terms of compositions and structures. Study revealed that the AOM derived from blue-green algae Microcystis aeruginosa could be degraded more completely (82% COD removal) in microbial fuel cells (MFCs) than by anaerobic fermentation (24% COD removal) in a control reactor without closed-circuit electrode and electricity was produced simultaneously. A variety of techniques were used to characterize the changes in AOM compositions and structures during bioelectrochemical oxidation. The presence of syntrophic interactions between electrochemical active bacteria and fermentative bacteria to degrade large molecular organics into small molecular substances, which could be oxidized by electrode but not by fermentation. The dominant tryptophan protein-like substances, humic acid-like substances and Chlorophyll a in AOM were highly degraded during MFC treatment.

  19. 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.

  20. Mapping of nutrient-induced biochemical changes in living algal cells using synchrotron infrared microspectroscopy.

    PubMed

    Heraud, Philip; Wood, Bayden R; Tobin, Mark J; Beardall, John; McNaughton, Don

    2005-08-15

    High quality Fourier transform infrared (FTIR) spectra were acquired from living Micrasterias hardyi cells maintained in an IR transparent flow-through cell using a FTIR microscope coupled to a synchrotron light source. Spectral maps of living, nutrient-replete cells showed band intensities consistent with the known location of the nucleus and the chloroplasts. These were very similar to maps acquired from fixed, air-dried cells. Bands due to lipids were lowest in absorbance in the region of the nucleus and highest in the chloroplast region and this trend was reversed for the absorbance of bands attributed to protein. Spectra acquired in 10 microm steps across living phosphorus-starved (P-starved) cells, repeated approximately every 30 min, were consistent over time, and bands correlated well with the known position of the nucleus and the observed chloroplasts, corroborating the observations with replete cells. Experiments in which missing nutrients were re-supplied to starved cells showed that cells could be maintained in a functional state in the flow-through cell for up to one day. Nitrogen-starved cells re-supplied with N showed an increase in lipid in all positions measured across the cell over a 23 h period of re-supply, with the largest increases occurring in positions where the chloroplasts were observed. Re-supply of phosphorus to P-starved cells produced no changes in bands attributable to lipid or protein. Due to their thin cell body ( approximately 12 microm) and large diameter ( approximately 300 microm) Micrasterias sp. make an ideal spectroscopic model to study nutrient kinetics in algal cells.

  1. Validation of algal viability treated with total residual oxidant and organic matter by flow cytometry.

    PubMed

    Lee, Junghyun; Choi, Eun Joo; Rhie, Kitae

    2015-08-15

    Algal cell growth after starch and oxidant treatments in seawater species (Isochrysis galbana and Phaeodactylum tricornutum) and freshwater species (Selenastrum capricornutum and Scenedesmus obliquus) were evaluated by flow cytometry with fluorescein diacetate (FDA) staining to determine algal viability. Growth of algal cell was found to be significantly different among groups treated with NaOCl, starch and/or sodium thiosulfate, which are active substance (Total Residual Oxidant; TRO as Cl2), organic compound to meet efficacy testing standard and neutralizer of TRO by Ballast Water Management Convention of International Maritime Organization, respectively. The viability of algal cell treated with TRO in starch-add culture of 5days after treatment and neutralization was decreased significantly. ATP contents of the treated algal cells corresponded to the FL1 fluorescent signal of flow cytometry with FDA staining. I. galbana was the most sensitive to TRO-neutralized cultures during viability analysis.

  2. Algal culture studies related to a Closed Ecological Life Support System (CELSS)

    NASA Technical Reports Server (NTRS)

    Radmer, R.; Behrens, P.; Fernandez, E.; Ollinger, O.; Howell, C.; Venables, A.; Huggins, D.; Gladue, R.

    1984-01-01

    In many respects, algae would be the ideal plant component for a biologically based controlled life support system, since they are eminently suited to the closely coupled functions of atmosphere regeneration and food production. Scenedesmus obliquus and Spirulina platensis were grown in three continuous culture apparatuses. Culture vessels their operation and relative merits are described. Both light and nitrogen utilization efficiency are examined. Long term culture issues are detailed and a discussion of a plasmid search in Spirulina is included.

  3. Chlorophyll fluorescence imaging of individual algal cells: effects of herbicide on Spirogyra distenta at different growth stages.

    PubMed

    Endo, Ryosuke; Omasa, Kenji

    2004-08-01

    Serious environmental degradation of aquatic ecosystems has been caused by eutrophication and by pollutants such as herbicides. Therefore, measurement of in situ algal photosynthetic activity is important for environmental monitoring. With ordinary nonimaging fluorometers, algal chlorophyll fluorescence can be measured easily, but heterogeneity within samples cannot be detected. Effects of a herbicide preparation containing 3-(3,4-dichlorophenyl)-1,1 -dimethylurea (DCMU) on photosynthetic activity at different growth stages of Spirogyra distenta were investigated by using a computer-aided microscopic imaging system for chlorophyll afluorescence. Photosystem II photochemical yield (phiPSII) images were used to diagnose photosynthetic activity of spiral filate chloroplasts in algal cells. The herbicide treatment caused a stronger decline in phiPSII values in younger than in mature algae cells. This result indicated that heterogeneity within algal samples should be considered when algae are used for environmental monitoring. Thus, measurement of chlorophyll fluorescence from young and mature chloroplasts with a microscopic imaging system makes it possible to improve the sensitivity for monitoring the environmental degradation of aquatic ecosystems.

  4. 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.

  5. 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. PMID:24924388

  6. Accelerating Commercialization of Algal Biofuels Through Partnerships (Brochure)

    SciTech Connect

    Not Available

    2011-10-01

    This brochure describes National Renewable Energy Laboratory's (NREL's) algal biofuels research capabilities and partnership opportunities. NREL is accelerating algal biofuels commercialization through: (1) Advances in applied biology; (2) Algal strain development; (3) Development of fuel conversion pathways; (4) Techno-economic analysis; and (5) Development of high-throughput lipid analysis methodologies. NREL scientists and engineers are addressing challenges across the algal biofuels value chain, including algal biology, cultivation, harvesting and extraction, and fuel conversion. Through partnerships, NREL can share knowledge and capabilities in the following areas: (1) Algal Biology - A fundamental understanding of algal biology is key to developing cost-effective algal biofuels processes. NREL scientists are experts in the isolation and characterization of microalgal species. They are identifying genes and pathways involved in biofuel production. In addition, they have developed a high-throughput, non-destructive technique for assessing lipid production in microalgae. (2) Cultivation - NREL researchers study algal growth capabilities and perform compositional analysis of algal biomass. Laboratory-scale photobioreactors and 1-m2 open raceway ponds in an on-site greenhouse allow for year-round cultivation of algae under a variety of conditions. A bioenergy-focused algal strain collection is being established at NREL, and our laboratory houses a cryopreservation system for long-term maintenance of algal cultures and preservation of intellectual property. (3) Harvesting and Extraction - NREL is investigating cost-effective harvesting and extraction methods suitable for a variety of species and conditions. Areas of expertise include cell wall analysis and deconstruction and identification and utilization of co-products. (4) Fuel Conversion - NREL's excellent capabilities and facilities for biochemical and thermochemical conversion of biomass to biofuels are being

  7. Simultaneous wastewater treatment, electricity generation and biomass production by an immobilized photosynthetic algal microbial fuel cell.

    PubMed

    He, Huanhuan; Zhou, Minghua; Yang, Jie; Hu, Youshuang; Zhao, Yingying

    2014-05-01

    A photosynthetic algal microbial fuel cell (PAMFC) was constructed by the introduction of immobilized microalgae (Chlorella vulgaris) into the cathode chamber of microbial fuel cells to fulfill electricity generation, biomass production and wastewater treatment. The immobilization conditions, including the concentration of immobilized matrix, initial inoculation concentration and cross-linking time, were investigated both for the growth of C. vulgaris and power generation. It performed the best at 5 % sodium alginate and 2 % calcium chloride as immobilization matrix, initial inoculation concentration of 10(6) cell/mL and cross-linking time of 4 h. Our findings indicated that C. vulgaris immobilization was an effective and promising approach to improve the performance of PAMFC, and after optimization the power density and Coulombic efficiency improved by 258 and 88.4 %, respectively. Important parameters such as temperature and light intensity were optimized on the performance. PAMFC could achieve a COD removal efficiency of 92.1 %, and simultaneously the maximum power density reached 2,572.8 mW/m(3) and the Coulombic efficiency was 14.1 %, under the light intensity of 5,000 lux and temperature at 25 °C.

  8. Phylogenomic Analyses Indicate that Early Fungi Evolved Digesting Cell Walls of Algal Ancestors of Land Plants.

    PubMed

    Chang, Ying; Wang, Sishuo; Sekimoto, Satoshi; Aerts, Andrea L; Choi, Cindy; Clum, Alicia; LaButti, Kurt M; Lindquist, Erika A; Yee Ngan, Chew; Ohm, Robin A; Salamov, Asaf A; Grigoriev, Igor V; Spatafora, Joseph W; Berbee, Mary L

    2015-05-14

    As decomposers, fungi are key players in recycling plant material in global carbon cycles. We hypothesized that genomes of early diverging fungi may have inherited pectinases from an ancestral species that had been able to extract nutrients from pectin-containing land plants and their algal allies (Streptophytes). We aimed to infer, based on pectinase gene expansions and on the organismal phylogeny, the geological timing of the plant-fungus association. We analyzed 40 fungal genomes, three of which, including Gonapodya prolifera, were sequenced for this study. In the organismal phylogeny from 136 housekeeping loci, Rozella diverged first from all other fungi. Gonapodya prolifera was included among the flagellated, predominantly aquatic fungal species in Chytridiomycota. Sister to Chytridiomycota were the predominantly terrestrial fungi including zygomycota I and zygomycota II, along with the ascomycetes and basidiomycetes that comprise Dikarya. The Gonapodya genome has 27 genes representing five of the seven classes of pectin-specific enzymes known from fungi. Most of these share a common ancestry with pectinases from Dikarya. Indicating functional and sequence similarity, Gonapodya, like many Dikarya, can use pectin as a carbon source for growth in pure culture. Shared pectinases of Dikarya and Gonapodya provide evidence that even ancient aquatic fungi had adapted to extract nutrients from the plants in the green lineage. This implies that 750 million years, the estimated maximum age of origin of the pectin-containing streptophytes represents a maximum age for the divergence of Chytridiomycota from the lineage including Dikarya.

  9. Disruption of cell-to-cell signaling does not abolish the antagonism of Phaeobacter gallaeciensis toward the fish pathogen Vibrio anguillarum in algal systems.

    PubMed

    Prol García, M J; D'Alvise, P W; Gram, L

    2013-09-01

    Quorum sensing (QS) regulates Phaeobacter gallaeciensis antagonism in broth systems; however, we demonstrate here that QS is not important for antagonism in algal cultures. QS mutants reduced Vibrio anguillarum to the same extent as the wild type. Consequently, a combination of probiotic Phaeobacter and QS inhibitors is a feasible strategy for aquaculture disease control.

  10. Bioengineering Aspects of Inorganic Carbon Supply to Mass Algal Cultures: Final Report

    SciTech Connect

    Goldman, J. C.

    1981-04-01

    Regardless of the application, the basic biotechnology of large-scale outdoor cultures involves many common features, particularly in the requirement for adequate nutrients such as carbon, nitrogen, and phosphorus to ensure that light is the sole limiting yield determinant. Whereas the required quantities of nitrogen and phosphorus are fairly simple, to estimate, those for inorganic carbon are far more complex.

  11. Hydrothermal liquefaction of mixed-culture algal biomass from wastewater treatment system into bio-crude oil.

    PubMed

    Chen, Wan-Ting; Zhang, Yuanhui; Zhang, Jixiang; Yu, Guo; Schideman, Lance C; Zhang, Peng; Minarick, Mitchell

    2014-01-01

    In this study, a mixed-culture algal biomass harvested from a functioning wastewater treatment system (AW) was hydrothermally converted into bio-crude oils. The highest bio-crude oil yield (49% of volatile matter) and the highest energy recovery were obtained at 300 °C with 1 h retention time. The highest heating value of the bio-crude oil was 33.3 MJ/kg, produced at 320 °C and 1h retention time. Thermogravimetric analysis showed approximately 60% of the bio-crude oils were distilled in the range of 200-550 °C; and the solid residue might be suitable for use in asphalt. GC-MS results indicated that the bio-crude oil contained hydrocarbons and fatty acids, while the aqueous product was rich in organic acids and cyclic amines. The nitrogen recovery (NR) in the bio-crude oil ranged from 8.41% to 16.8%, which was lower than the typical range of 25%-53% from previous studies. PMID:24287452

  12. Research and development of shallow algal mass culture systems for the production of oils

    SciTech Connect

    Laws, E.A.

    1984-10-01

    The major accomplishment of the past nine months' work was the identification of a microalgal species which can be grown in the system on a 12-month basis without temperature control. The most promising species identified to date is a strain of platymonas sp. This strain grows rapidly at temperatures from 20/sup 0/ to 34/sup 0/C, and at salinities from 1.5 to 3.5%. Neither the lower temperature limit nor the lower salinity limit of the strain are known at this time. A factorial experiment designed to determine optimum growth conditions indicated that the optimum culture depth was 10 cm, the optimum pH about 7.5, and the optimum flow rate about 30 cm/s. A major discovery was that diluting the culture every third day greatly enhanced production. In this dilution mode daily yields averaged 46 g/m/sup 2/ ash-free dry weight (AFDW) over a one-month period, and photosynthetic efficiencies averaged 11% (based on visible light energy). The former figure is over twice the best long-term yields achieved in microalgal mass culture systems grown exclusively on inorganic nutrients.

  13. The use of physical and virtual infrastructures for the validation of algal cryopreservation methods in international culture collections.

    PubMed

    Day, John G; Iorenz, Maike; Wilding, Thomas A; Friedl, Thomas; Harding, Keith; Pröschold, Thomas; Brennan, Debra; Müller, Julia; Santos, Lília M A; Santos, M Fátima; Osório, Hugo C; Amaral, Raquel; Lukesova, Alena; Hrouzek, Pavel; Lukes, Martin; Elster, Josef; Lukavsky, Jaromír; Probert, Ian; Ryan, Matthew J; Benson, Erica E

    2007-01-01

    Two cryopreservation methods, colligative cryoprotection coupled with controlled cooling and vitrification-based, encapsulation-dehydration were validated by five members of the EU research infrastructure consortium, COBRA, and two independent external validators. The test strain Chlorella vulgaris SAG 211-11b was successfully cryopreserved using two-step cooling employing passive (Mr Frosty) and Controlled Rate Freezers (CRF) attaining the desired recovery target within 15% of the median viability level (94%). Significant differences (p < 0.05) between cooling regimes were observed where Mr Frosty was more variable (Inter-Quartile Range being 21.5%, versus 13.0% for CRF samples). Viability assessment using fluorescein diacetate gave significantly (P < 0.0001) higher survival than growth in agar with median values being 96% and 89%, respectively. On employing encapsulation-dehydration, greater variability between some validators was observed, with six labs observing recovery in 100% of the beads (84-95% of cells surviving) and one lab observing survival in 80% of the treated beads. Bead disruption followed by algal growth in agar was considered the most reliable and accurate method of assessing cell survival for encapsulation-dehydration. PMID:18075705

  14. Comparison of cell rupturing by ozonation and ultrasonication for algal lipid extraction from Chlorella vulgaris.

    PubMed

    Huang, Yuanxing; Hong, Andy; Zhang, Daofang; Li, Liang

    2014-01-01

    Cell disruption is essential for lipid collection from cultivated microalgae. This study examines the performance of ultrasonication (US), conventional bubbling ozonation (CBO), and pressure-assisted ozonation (PAO) as a cell rupturing technique to obtain algal lipid from a freshwater unicellular microalgae Chlorella vulgaris, which was grown in BG11 medium at a temperature of 25 degrees C and illuminated by artificial lighting with light/dark cycle of 12 h/12 h. Changes in total organic carbon, total nitrogen, total phosphorous, and chlorophyll contents in the algae suspension after ozonation and US treatments were measured to evaluate the effectiveness of cell rupture by these techniques. Lipid yields of 21 and 27 g/100 g biomass were obtained using US and PAO, respectively. Lipid yields of about 5 g/100 g biomass were obtained using CBO. In all rupturing treatments, C16 and C18 compounds were found to be predominant accounting for 90% of the fatty acids. Using US for rupturing, fatty acids of C 16:0, C18:1, and C18:2 were predominant, accounting for 76 +/- 4.2% of all the fatty acids. Using CBO and PAO involving ozone, fatty acids of C16:0 and C18:0 were predominant, accounting for 63-94% of the products. The results suggest that saturated fatty acid methyl ester (FAME) products are predominant with oxidative ozonation rupturing while unsaturated FAME products of lower-melting points predominant with physical ultrasonic rupturing means. PAO was an effective cell rupture method for biodiesel production with high lipid yield and more saturated hydrocarbon products.

  15. Ureaplasma infection of cell cultures.

    PubMed Central

    Kotani, H; McGarrity, G J

    1986-01-01

    Studies were performed to characterize the effects of ureaplasmas in HeLa, 3T6, and CV-1 cell cultures. The ureaplasmas studied were human Ureaplasma urealyticum T960 (serotype VIII), bovine U. diversum T95, simian strain T167-2, ovine strain 1202, canine strain D1M-C, and feline strains 382 and FT2-B. FT2-B was the only ureaplasma to grow in the cell free culture medium, Dulbecco modified Eagle-Earle medium containing 10% fetal bovine serum. The growth pattern of the ureaplasmas varied in the different cell cultures, but each strain grew in at least two of the cell cultures, suggesting a requirement for a product of the cell culture and for low concentrations of urea. When growth occurred, organisms grew to concentrations that approached, but did not equal, those observed in 10B broth. Most, but not all, ureaplasmas grew quickly, reaching peak titers 2 days after infection. Canine strain D1M-C did not grow in 3T6, but showed rapid growth in HeLa and CV-1 cells, killing both cultures, In some systems, e.g., U. urealyticum T960 and simian strain T167-2, the infection persisted, and ureaplasmas could be recovered from cell cultures four passages after infection, when studies were terminated. The cell culture ureaplasmas grew on T agar, but not on mycoplasma agar medium. Images PMID:3699891

  16. Vernalophrys algivore gen. nov., sp. nov. (Rhizaria: Cercozoa: Vampyrellida), a New Algal Predator Isolated from Outdoor Mass Culture of Scenedesmus dimorphus.

    PubMed

    Gong, Yingchun; Patterson, David J; Li, Yunguang; Hu, Zixuan; Sommerfeld, Milton; Chen, Yongsheng; Hu, Qiang

    2015-06-15

    Microbial contamination is the main cause of loss of biomass yield in microalgal cultures, especially under outdoor environmental conditions. Little is known about the identities of microbial contaminants in outdoor mass algal cultures. In this study, a new genus and species of vampyrellid amoeba, Vernalophrys algivore, is described from cultures of Scenedesmus dimorphus in open raceway ponds and outdoor flat-panel photobioreactors. This vampyrellid amoeba was a significant grazer of Scenedesmus and was frequently associated with a very rapid decline in algal numbers. We report on the morphology, subcellular structure, feeding behavior, molecular phylogeny, and life cycle. The new amoeba resembles Leptophrys in the shape of trophozoites and pseudopodia and in the mechanism of feeding (mainly by engulfment). It possesses two distinctive regions in helix E10_1 (nucleotides 117 to 119, CAA) and E23_1 (nucleotides 522 and 523, AG) of the 18S rRNA gene. It did not form a monophyletic group with Leptophrys in molecular phylogenetic trees. We establish a new genus, Vernalophrys, with the type species Vernalophrys algivore. The occurrence, impact of the amoeba on mass culture of S. dimorphus, and means to reduce vampyrellid amoeba contamination in Scenedesmus cultures are addressed. The information obtained from this study will be useful for developing an early warning system and control measures for preventing or treating this contaminant in microalgal mass cultures.

  17. Vernalophrys algivore gen. nov., sp. nov. (Rhizaria: Cercozoa: Vampyrellida), a New Algal Predator Isolated from Outdoor Mass Culture of Scenedesmus dimorphus

    PubMed Central

    Patterson, David J.; Li, Yunguang; Hu, Zixuan; Sommerfeld, Milton; Chen, Yongsheng

    2015-01-01

    Microbial contamination is the main cause of loss of biomass yield in microalgal cultures, especially under outdoor environmental conditions. Little is known about the identities of microbial contaminants in outdoor mass algal cultures. In this study, a new genus and species of vampyrellid amoeba, Vernalophrys algivore, is described from cultures of Scenedesmus dimorphus in open raceway ponds and outdoor flat-panel photobioreactors. This vampyrellid amoeba was a significant grazer of Scenedesmus and was frequently associated with a very rapid decline in algal numbers. We report on the morphology, subcellular structure, feeding behavior, molecular phylogeny, and life cycle. The new amoeba resembles Leptophrys in the shape of trophozoites and pseudopodia and in the mechanism of feeding (mainly by engulfment). It possesses two distinctive regions in helix E10_1 (nucleotides 117 to 119, CAA) and E23_1 (nucleotides 522 and 523, AG) of the 18S rRNA gene. It did not form a monophyletic group with Leptophrys in molecular phylogenetic trees. We establish a new genus, Vernalophrys, with the type species Vernalophrys algivore. The occurrence, impact of the amoeba on mass culture of S. dimorphus, and means to reduce vampyrellid amoeba contamination in Scenedesmus cultures are addressed. The information obtained from this study will be useful for developing an early warning system and control measures for preventing or treating this contaminant in microalgal mass cultures. PMID:25819973

  18. 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.

  19. Pitfalls, artefacts and open questions in chlorophyll thermoluminescence of leaves or algal cells.

    PubMed

    Ducruet, Jean-Marc

    2013-07-01

    Thermoluminescence of intact photosynthetic organisms, leaves or algal cells, raises specific problems. The constitutive S2/3Q B (-) B bands constitute major probes of the state of photosystem II in vivo. The presence of a dark-stable acidic lumen causes a temperature downshift of B bands, specially the S3 B band, providing a lumen pH indicator. This is accompanied by a broadening of the S3 B band that becomes an envelope of elementary B bands. The occasional AT, Q and C bands are briefly examined in an in vivo context. It is emphasized that freezing below the nucleation temperature is not necessary for physiological studies, but a source of artefacts, hence should be avoided. In intact photosynthetic structures, a dark-electron transfer from stroma reductants to the quinonic acceptors of photosystem II via the cyclic/chlororespiratory pathways, strongly stimulated by moderate warming, gives rise to the afterglow (AG) luminescence emission that reflects chloroplast energy status. The decomposition of complex TL signals into elementary bands is necessary to determine the maximum temperature T m and the area of each of them. A comparison of TL signals after 1 flash and 2 flashes prevents from confusing the three main bands observed in vivo, i.e. the S2 and S3 B bands and the AG band. Finally, the thermoluminescence bands arising sometimes above 50 °C are mentioned. The basic principles of (thermo)luminescence established on isolated thylakoids should not be applied directly without a careful examination of in vivo conditions.

  20. Suspension culture of mammalian cells.

    PubMed

    Birch, J R; Arathoon, R

    1990-01-01

    Mammalian cell suspension culture systems are being used increasingly in the biotechnology industry. This is due to their many advantages including simplicity and homogeneity of culture. Suspension systems are very adaptable (e.g., for microcarrier, microencapsulation, or other methods of culture). Their engineering is thoroughly understood and standardized at large scale, and automation and cleaning procedures are well established. Suspension systems offer the possibility of quick implementation of production protocols due to their ability to be scaled easily once the basic culture parameters are understood. The only main disadvantage of the suspension culture systems to date is their inapplicability for the production of human vaccines from either primary cell lines or from normal human diploid cell lines (Hayflick et al., 1987 and references therein). One of the great advantages of suspension culture is the opportunity it provides to study interactions of metabolic and production phenomena in chemostat or turbidostat steady-state systems. Furthermore, in suspension culture systems from which cell number and cell mass measurements are easy to obtain, rigorous and quantitative estimations of the effects of growth conditions or perturbations of metabolic homeostasis can be made. Such studies can speed up the development of optimal processes. With our increasing understanding of factors influencing expression in mammalian cells (Cohen and Levinson, 1988; Santoro et al., 1988) and the direct application of new methods in suspension culture (Rhodes and Birch, 1988), its usefulness and importance is likely to increase in the future. In this chapter, we have described some of the potential uses of the various suspension culture systems and have covered most of the established technology and literature. Due to the rapid developments and needs in the biotechnology industry and the versatility of suspension culture systems, it is probable that many more variations on this

  1. 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.

  2. The Influence of Salinity, Growth Rate and Temperature on D/H Fractionation in Algal Lipids from Culture and Field Studies

    NASA Astrophysics Data System (ADS)

    Sachs, J. P.; Schwab, V.; Sachse, D.; Cash, A.; Nelson, D.; Zhang, Z.; Kawka, O.

    2007-12-01

    The use of compound-specific D/H ratios to decipher biochemical, geochemical, oceanographic, and climatic processes is expanding rapidly. The relative success of these efforts depends on an understanding of the environmental conditions that influence the deuterium depletion relative to environmental water observed in all plant, algal and bacterial lipids, and the sensitivity of D/H fractionation responses to changes in those environmental conditions. Presently very little is known about this interplay between the environment and D/H fraction in algal lipids. Here we present results from field studies (in the Chesapeake Bay, Christmas Island, the Great Salt Lake, and saline basins in Alberta and Saskatchewan) and culture studies (both continuous and batch) that indicate that salinity, growth rate and temperature each influence D/H fractionation in algal lipids to varying degrees, depending on the algae and the lipid. Our initial results indicate that D/H fractionation (1) decreases with increasing salinity, (2) increases with increasing growth rate in isoprenoid lipids, (3) is insensitive to growth rate in acetogenic lipids, and (4) increases with increasing temperature.

  3. Algal Culture Material

    ERIC Educational Resources Information Center

    Baldock, R.

    1971-01-01

    Suggests suitable species of microscopic green algae for demonstrating diversity of form, increasing complexity in related species, the animal" and plant" characteristics of protists, and protist behavior. (AL)

  4. Expression of the nucleus-encoded chloroplast division genes and proteins regulated by the algal cell cycle.

    PubMed

    Miyagishima, Shin-Ya; Suzuki, Kenji; Okazaki, Kumiko; Kabeya, Yukihiro

    2012-10-01

    Chloroplasts have evolved from a cyanobacterial endosymbiont and their continuity has been maintained by chloroplast division, which is performed by the constriction of a ring-like division complex at the division site. It is believed that the synchronization of the endosymbiotic and host cell division events was a critical step in establishing a permanent endosymbiotic relationship, such as is commonly seen in existing algae. In the majority of algal species, chloroplasts divide once per specific period of the host cell division cycle. In order to understand both the regulation of the timing of chloroplast division in algal cells and how the system evolved, we examined the expression of chloroplast division genes and proteins in the cell cycle of algae containing chloroplasts of cyanobacterial primary endosymbiotic origin (glaucophyte, red, green, and streptophyte algae). The results show that the nucleus-encoded chloroplast division genes and proteins of both cyanobacterial and eukaryotic host origin are expressed specifically during the S phase, except for FtsZ in one graucophyte alga. In this glaucophyte alga, FtsZ is persistently expressed throughout the cell cycle, whereas the expression of the nucleus-encoded MinD and MinE as well as FtsZ ring formation are regulated by the phases of the cell cycle. In contrast to the nucleus-encoded division genes, it has been shown that the expression of chloroplast-encoded division genes is not regulated by the host cell cycle. The endosymbiotic gene transfer of minE and minD from the chloroplast to the nuclear genome occurred independently on multiple occasions in distinct lineages, whereas the expression of nucleus-encoded MIND and MINE is regulated by the cell cycle in all lineages examined in this study. These results suggest that the timing of chloroplast division in algal cell cycle is restricted by the cell cycle-regulated expression of some but not all of the chloroplast division genes. In addition, it is

  5. Short-term algal toxicity test based on phosphate uptake.

    PubMed

    Kaneko, H Hidehiro; Shimada, Akiko; Hirayama, Kimiaki

    2004-04-01

    In order to develop a short-term algal toxicity test, the growth of and the phosphate uptake by the green alga Selenastrum capricornutum during batch culture were observed. In the control medium, S. capricornutum took up phosphate earlier than it grew. It was also observed that the phosphate uptake was inhibited by the presence of a toxicant. From these results, phosphate uptake was considered as one of the useful effect parameters for a short-term algal toxicity test. As the removal rate of phosphate from the medium is a function of the amount of algal cell initially inoculated, the test period is variable. The relationship between the amount of inoculation and phosphate uptake was examined and the test conditions suitable for a 3-h toxicity test were established as one example. According to this test procedure, the inhibitory effect of some toxicants on the phosphate uptake was determined. For comparison, a conventional algal assay based on algal growth was also performed. The EC50s for both tests were close. This indicated that the algal toxicity test method proposed in this paper would be useful for the uses where rapidity is required. PMID:15087199

  6. Algal-CAMs: isoforms of a cell adhesion molecule in embryos of the alga Volvox with homology to Drosophila fasciclin I.

    PubMed

    Huber, O; Sumper, M

    1994-09-15

    Proof that plants possess homologs of animal adhesion proteins is lacking. In this paper we describe the generation of monoclonal antibodies that interfere with cell-cell contacts in the 4-cell embryo of the multicellular alga Volvox carteri, resulting in a hole between the cells. The number of following cell divisions is reduced and the cell division pattern is altered drastically. Antibodies given at a later stage of embryogenesis specifically inhibit inversion of the embryo, a morphogenetic movement that turns the embryo inside out. Immunofluorescence microscopy localizes the antigen (Algal-CAM) at cell contact sites of the developing embryo. Algal-CAM is a protein with a three-domain structure: an N-terminal extensin-like domain characteristic for plant cell walls and two repeats with homology to fasciclin I, a cell adhesion molecule involved in the neuronal development of Drosophila. Alternatively spliced variants of Algal-CAM mRNA were detected that are produced under developmental control. Thus, Algal-CAM is the first plant homolog of animal adhesion proteins. PMID:7925267

  7. 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.

  8. Black silicon SERS substrate: effect of surface morphology on SERS detection and application of single algal cell analysis.

    PubMed

    Deng, Yu-Luen; Juang, Yi-Je

    2014-03-15

    In this study, we have investigated the effect of the surface morphology of the black silicon substrate on surface enhanced Raman spectroscopy (SERS) and explored its application of single algal cell detection. By adjusting the O2 and SF6 flow rates in the cryogenic plasma etching process, different surface morphologies of the black silicon substrate was produced without performing the lithographic process. It was found the Raman signals were better enhanced as the tip density of the black silicon substrate increased. In addition, as the thickness of the deposited gold layer increased, the SERS effect increased as well, which could be owing to the generation of more hot spots by bridging individual silicon tips through deposition of gold layer. For the black silicon substrate with tip density of 30 tips/μm(2) and covered by 400 nm deposited gold layer, the detection limit of 10 fM R6G solution concentration with uniform SERS effect across the substrate was achieved. Furthermore, detection of individual algal cell (Chlorella vulgaris) was performed at the SERS substrate as fabricated and the Raman signals of carotenoid and lipid were substantially enhanced.

  9. Reminiscences of work with Alex Hope: the movement of water and ions in giant algal cells, 1963-1967.

    PubMed

    Barry, Peter H

    2009-12-01

    This article, from the Tribute to Alex Hope Symposium at the 2008 Australian Society for Biophysics meeting, represents reminiscences of PhD studies done under my former supervisor, Professor Alex Hope. The studies demonstrated and quantified electroosmosis in giant algal cells of Chara and isolated segments of cell wall by measuring instantaneous current-induced volume flows. The studies also uncovered and modelled an unstirred-layer transport number effect that gave rise to an additional transiently increasing current-induced volume flow component, which could be mistaken for electroosmosis. In addition, action potential induced volume flows and pressure changes were measured in these cells and successfully modelled. An appreciation of the influence of Alex Hope and his laboratory environment, together with some of the further studies that resulted from this work, is also mentioned.

  10. Algal biocathode for in situ terminal electron acceptor (TEA) production: synergetic association of bacteria-microalgae metabolism for the functioning of biofuel cell.

    PubMed

    Venkata Mohan, S; Srikanth, S; Chiranjeevi, P; Arora, Somya; Chandra, Rashmi

    2014-08-01

    Replacement of energy intensive mechanical aeration with sustainable oxygenic photosynthesis by microalgae at cathode was studied in dual-chambered microbial fuel cell (MFC). The synergistic association between bacterial fermentation at anode and the oxygenic photosynthesis of microalgae at cathode facilitated good power output as well as treatment efficiency. However, MFC operation during spring showed higher bioelectrogenic activity (57.0 mW/m(2)) over summer (1.1 mW/m(2)) due to the higher oxygenic photosynthetic activity of microalgae and respective dissolved oxygen (DO) levels. This can be attributed to RuBisCO inactivation under high temperatures and light intensity of summer, which prevented rich algal biomass growth as well as their photosynthetic activity. Unlike abiotic cathode, the algal cathode potential increased with operation time due to the algal biomass growth during spring but was negligible during summer. The catalytic currents on voltammetric signatures and the bioprocess parameters also corroborated well with the observed power output.

  11. Red and green algal origin of diatom membrane transporters: insights into environmental adaptation and cell evolution.

    PubMed

    Chan, Cheong Xin; Reyes-Prieto, Adrian; Bhattacharya, Debashish

    2011-01-01

    Membrane transporters (MTs) facilitate the movement of molecules between cellular compartments. The evolutionary history of these key components of eukaryote genomes remains unclear. Many photosynthetic microbial eukaryotes (e.g., diatoms, haptophytes, and dinoflagellates) appear to have undergone serial endosymbiosis and thereby recruited foreign genes through endosymbiotic/horizontal gene transfer (E/HGT). Here we used the diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum as models to examine the evolutionary origin of MTs in this important group of marine primary producers. Using phylogenomics, we used 1,014 diatom MTs as query against a broadly sampled protein sequence database that includes novel genome data from the mesophilic red algae Porphyridium cruentum and Calliarthron tuberculosum, and the stramenopile Ectocarpus siliculosus. Our conservative approach resulted in 879 maximum likelihood trees of which 399 genes show a non-lineal history between diatoms and other eukaryotes and prokaryotes (at the bootstrap value ≥70%). Of the eukaryote-derived MTs, 172 (ca. 25% of 697 examined phylogenies) have members of both red/green algae as sister groups, with 103 putatively arising from green algae, 19 from red algae, and 50 have an unresolved affiliation to red and/or green algae. We used topology tests to analyze the most convincing cases of non-lineal gene history in which red and/or green algae were nested within stramenopiles. This analysis showed that ca. 6% of all trees (our most conservative estimate) support an algal origin of MTs in stramenopiles with the majority derived from green algae. Our findings demonstrate the complex evolutionary history of photosynthetic eukaryotes and indicate a reticulate origin of MT genes in diatoms. We postulate that the algal-derived MTs acquired via E/HGT provided diatoms and other related microbial eukaryotes the ability to persist under conditions of fluctuating ocean chemistry, likely contributing to

  12. Predicting Pb bioavailability to freshwater microalgae in the presence of fulvic acid: algal cell density as a variable.

    PubMed

    Slaveykova, Vera I

    2007-11-01

    In order to better understand the relationship between lead speciation and its bioavailability in natural waters, the interactions between Pb(II), fulvic acid and the freshwater alga, Chlorella kesslerii were studied at various algal cell densities. An increase in cellular lead or fulvic acid adsorbed to algae was observed with decrease of the cell density from ca. 10(7) to 10(5)cells ml(-1). In the presence of fulvic acid, cellular Pb was greater than that expected for the same free lead ion concentrations in the absence of fulvic acid in agreement to our previous study. This effect was found to be more pronounced at lower cell density, in accordance with increased fulvic acid adsorption to algae. Good fit between experimental observations and model predictions of cellular Pb at various cell densities, was observed by assuming that fulvic acid adsorbed to algae give rise to additional binding sites for Pb(II). The findings of this study indicate that a further extension of the biotic ligand model which includes the formation of a ternary complex and cell density (or concentration) as an input parameter is needed to improve its site-specific predictive capacity, especially in the case of dissolved organic matter-rich surface waters. This extension of predictive capacity would allow to reduce the deviations from the BLM model predictions for microalgae in the presence of dissolved organic matter and hence will serve as a mechanistic tool for establishing ambient site-specific water quality criteria. PMID:17555791

  13. Cell Culture, Technology: Enhancing the Culture of Diagnosing Human Diseases.

    PubMed

    Hudu, Shuaibu Abdullahi; Alshrari, Ahmed Subeh; Syahida, Ahmad; Sekawi, Zamberi

    2016-03-01

    Cell culture involves a complex of processes of cell isolation from their natural environment (in vivo) and subsequent growth in a controlled environmental artificial condition (in vitro). Cells from specific tissues or organs are cultured as short term or established cell lines which are widely used for research and diagnosis, most specially in the aspect of viral infection, because pathogenic viral isolation depends on the availability of permissible cell cultures. Cell culture provides the required setting for the detection and identification of numerous pathogens of humans, which is achieved via virus isolation in the cell culture as the "gold standard" for virus discovery. In this review, we summarized the views of researchers on the current role of cell culture technology in the diagnosis of human diseases. The technological advancement of recent years, starting with monoclonal antibody development to molecular techniques, provides an important approach for detecting presence of viral infection. They are also used as a baseline for establishing rapid tests for newly discovered pathogens. A combination of virus isolation in cell culture and molecular methods is still critical in identifying viruses that were previously unrecognized. Therefore, cell culture should be considered as a fundamental procedure in identifying suspected infectious viral agent. PMID:27134874

  14. Cell Culture, Technology: Enhancing the Culture of Diagnosing Human Diseases

    PubMed Central

    Alshrari, Ahmed Subeh; Syahida, Ahmad; Sekawi, Zamberi

    2016-01-01

    Cell culture involves a complex of processes of cell isolation from their natural environment (in vivo) and subsequent growth in a controlled environmental artificial condition (in vitro). Cells from specific tissues or organs are cultured as short term or established cell lines which are widely used for research and diagnosis, most specially in the aspect of viral infection, because pathogenic viral isolation depends on the availability of permissible cell cultures. Cell culture provides the required setting for the detection and identification of numerous pathogens of humans, which is achieved via virus isolation in the cell culture as the “gold standard” for virus discovery. In this review, we summarized the views of researchers on the current role of cell culture technology in the diagnosis of human diseases. The technological advancement of recent years, starting with monoclonal antibody development to molecular techniques, provides an important approach for detecting presence of viral infection. They are also used as a baseline for establishing rapid tests for newly discovered pathogens. A combination of virus isolation in cell culture and molecular methods is still critical in identifying viruses that were previously unrecognized. Therefore, cell culture should be considered as a fundamental procedure in identifying suspected infectious viral agent. PMID:27134874

  15. Cell Culture, Technology: Enhancing the Culture of Diagnosing Human Diseases.

    PubMed

    Hudu, Shuaibu Abdullahi; Alshrari, Ahmed Subeh; Syahida, Ahmad; Sekawi, Zamberi

    2016-03-01

    Cell culture involves a complex of processes of cell isolation from their natural environment (in vivo) and subsequent growth in a controlled environmental artificial condition (in vitro). Cells from specific tissues or organs are cultured as short term or established cell lines which are widely used for research and diagnosis, most specially in the aspect of viral infection, because pathogenic viral isolation depends on the availability of permissible cell cultures. Cell culture provides the required setting for the detection and identification of numerous pathogens of humans, which is achieved via virus isolation in the cell culture as the "gold standard" for virus discovery. In this review, we summarized the views of researchers on the current role of cell culture technology in the diagnosis of human diseases. The technological advancement of recent years, starting with monoclonal antibody development to molecular techniques, provides an important approach for detecting presence of viral infection. They are also used as a baseline for establishing rapid tests for newly discovered pathogens. A combination of virus isolation in cell culture and molecular methods is still critical in identifying viruses that were previously unrecognized. Therefore, cell culture should be considered as a fundamental procedure in identifying suspected infectious viral agent.

  16. 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...

  17. 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...

  18. 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...

  19. 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...

  20. 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...

  1. Techniques for mammalian cell tissue culture.

    PubMed

    Phelan, Mary C

    2006-11-01

    This appendix opens with detailed discussions on the latest principles of sterile technique and preparation of culture media. Step-by-step protocols describe trypsinizing and subculturing monolayer cultures, passaging suspension cultures, freezing and thawing cells, counting cells using a hemacytometer, and preparing cells for transport. PMID:18428384

  2. Techniques for mammalian cell tissue culture.

    PubMed

    Phelan, Mary C

    2006-05-01

    This appendix opens with detailed discussions on the latest principles of sterile technique and preparation of culture media. Step-by-step protocols describe trypsinizing and subculturing monolayer cultures, passaging suspension cultures, freezing and thawing cells, counting cells using a hemacytometer, and preparing cells for transport. PMID:18265370

  3. Techniques for mammalian cell tissue culture.

    PubMed

    Phelan, Mary C

    2006-05-01

    This unit opens with detailed discussions on the latest principles of sterile technique and preparation of culture media. Step-by-step protocols describe trypsinizing and subculturing monolayer cultures, passaging suspension cultures, freezing and thawing cells, counting cells using a hemacytometer, and preparing cells for transport. PMID:18770828

  4. Techniques for mammalian cell tissue culture.

    PubMed

    Phelan, Mary C

    2006-12-01

    This appendix opens with detailed discussions on the latest principles of sterile technique and preparation of culture media. Step-by-step protocols describe trypsinizing and subculturing monolayer cultures, passaging suspension cultures, freezing and thawing cells, counting cells using a hemacytometer, and preparing cells for transport. PMID:18429293

  5. Multistage carcinogenesis in cell culture.

    PubMed

    Rubin, H

    2001-01-01

    Rodent fibroblasts explanted from embryos to culture undergo a period of declining growth rate in serial passages leading to crisis, followed by the appearance of variants which can multiply indefinitely. If the "immortal" cell line was established by low density passage, i.e., 3T3 cells, it has a low saturation density and is non-tumorigenic. If it was established by high density passage, it has a high saturation density and is tumorigenic. The establishment of cells goes through successive stages, including increased capacity to multiply in low serum concentration, growth to high saturation density, growth in suspension, assisted tumour formation in susceptible hosts and unassisted tumour formation. Chromosome aberrations and aneuploidy occur long before the capacity to produce tumours appears. Contrary to conventional belief, human fibroblast populations also undergo a continuous loss of capacity to multiply from the time of explantation, with only the longest surviving clone reaching the Hayflick limit. Neoplastic transformation of rodent cells is strongly favoured by maintaining them in a quiescent state at confluence for prolonged periods, which results in genetic damage to the cells. It also produces a large variety of chromosomal aberrations in human cells and extends their replicative lifespan. Individual clones are more susceptible to spontaneous transformation than their heterogeneous parental cultures. The implications of these results for tumour development in vivo are that oncogenic genetic changes may be common under stressful conditions which restrict replication, and that such changes are maximized when a rogue clone reaches a critical size that reduces stabilizing interactions with neighbouring clones. An alternative explanation, described in the Addendum, which we retrospectively favor is that the easily transformed clones are a minority in the uncloned parental population. The reason they transform before the parental population is that when

  6. Development of a rotating algal biofilm growth system for attached microalgae growth with in situ biomass harvest.

    PubMed

    Gross, Martin; Henry, Wesley; Michael, Clayton; Wen, Zhiyou

    2013-12-01

    This work aimed to develop a rotating algal biofilm (RAB) cultivation system that can be widely adopted by microalgae producers for easy biomass harvest. Algal cells were grown on the surface of a material rotating between nutrient-rich liquid and CO2-rich gaseous phase. Scrapping biomass from the attached surface avoided the expensive harvest operations such as centrifugation. Among various attachment materials, cotton sheet resulted in best algal growth, durability, and cost effectiveness. A lab-scale RAB system was further optimized with harvest frequency, rotation speed, and CO2 levels. The algal biomass from the RAB system had a similar water content as that in centrifuged biomass. An open pond raceway retrofitted with a pilot-scale RAB system resulted in a much higher biomass productivity when compared to a control open pond. Collectively, the research shows that the RAB system is an efficient algal culture system for easy biomass harvest with enhanced biomass productivity.

  7. 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.

  8. Dynamized preparations in cell culture.

    PubMed

    Sunila, Ellanzhiyil Surendran; Kuttan, Ramadasan; Preethi, Korengath Chandran; Kuttan, Girija

    2009-06-01

    Although reports on the efficacy of homeopathic medicines in animal models are limited, there are even fewer reports on the in vitro action of these dynamized preparations. We have evaluated the cytotoxic activity of 30C and 200C potencies of ten dynamized medicines against Dalton's Lymphoma Ascites, Ehrlich's Ascites Carcinoma, lung fibroblast (L929) and Chinese Hamster Ovary (CHO) cell lines and compared activity with their mother tinctures during short-term and long-term cell culture. The effect of dynamized medicines to induce apoptosis was also evaluated and we studied how dynamized medicines affected genes expressed during apoptosis. Mother tinctures as well as some dynamized medicines showed significant cytotoxicity to cells during short and long-term incubation. Potentiated alcohol control did not produce any cytotoxicity at concentrations studied. The dynamized medicines were found to inhibit CHO cell colony formation and thymidine uptake in L929 cells and those of Thuja, Hydrastis and Carcinosinum were found to induce apoptosis in DLA cells. Moreover, dynamized Carcinosinum was found to induce the expression of p53 while dynamized Thuja produced characteristic laddering pattern in agarose gel electrophoresis of DNA. These results indicate that dynamized medicines possess cytotoxic as well as apoptosis-inducing properties. PMID:18955237

  9. Fueling Future with Algal Genomics

    SciTech Connect

    Grigoriev, Igor

    2012-07-05

    Algae constitute a major component of fundamental eukaryotic diversity, play profound roles in the carbon cycle, and are prominent candidates for biofuel production. The US Department of Energy Joint Genome Institute (JGI) is leading the world in algal genome sequencing (http://jgi.doe.gov/Algae) and contributes of the algal genome projects worldwide (GOLD database, 2012). The sequenced algal genomes offer catalogs of genes, networks, and pathways. The sequenced first of its kind genomes of a haptophyte E.huxleyii, chlorarachniophyte B.natans, and cryptophyte G.theta fill the gaps in the eukaryotic tree of life and carry unique genes and pathways as well as molecular fossils of secondary endosymbiosis. Natural adaptation to conditions critical for industrial production is encoded in algal genomes, for example, growth of A.anophagefferens at very high cell densities during the harmful algae blooms or a global distribution across diverse environments of E.huxleyii, able to live on sparse nutrients due to its expanded pan-genome. Communications and signaling pathways can be derived from simple symbiotic systems like lichens or complex marine algae metagenomes. Collectively these datasets derived from algal genomics contribute to building a comprehensive parts list essential for algal biofuel development.

  10. Algal biofuels.

    PubMed

    Razeghifard, Reza

    2013-11-01

    The world is facing energy crisis and environmental issues due to the depletion of fossil fuels and increasing CO2 concentration in the atmosphere. Growing microalgae can contribute to practical solutions for these global problems because they can harvest solar energy and capture CO2 by converting it into biofuel using photosynthesis. Microalgae are robust organisms capable of rapid growth under a variety of conditions including in open ponds or closed photobioreactors. Their reduced biomass compounds can be used as the feedstock for mass production of a variety of biofuels. As another advantage, their ability to accumulate or secrete biofuels can be controlled by changing their growth conditions or metabolic engineering. This review is aimed to highlight different forms of biofuels produced by microalgae and the approaches taken to improve their biofuel productivity. The costs for industrial-scale production of algal biofuels in open ponds or closed photobioreactors are analyzed. Different strategies for photoproduction of hydrogen by the hydrogenase enzyme of green algae are discussed. Algae are also good sources of biodiesel since some species can make large quantities of lipids as their biomass. The lipid contents for some of the best oil-producing strains of algae in optimized growth conditions are reviewed. The potential of microalgae for producing petroleum related chemicals or ready-make fuels such as bioethanol, triterpenic hydrocarbons, isobutyraldehyde, isobutanol, and isoprene from their biomass are also presented.

  11. The color of mass culture: spectral characteristics of a shallow water column through shade-limited algal growth dynamics(1).

    PubMed

    Hewes, Christopher D

    2016-04-01

    It is envisioned that mass algal cultivation for commercial biofuels production will entail the use of large raceway pond systems, which typically have shade-limited photosynthetic growth within depths of 20-30 cm. The attenuation of light and spectral qualities of red, green, and blue wavelengths in a 20-cm water column as a function of Chl-a concentration during exponential and linear phases of growth dynamics for the marine diatom Thalassiosira pseudonana was examined under laboratory conditions. While photosynthetically available radiation (PAR) was in excess throughout the water column during the phase of exponential growth, PAR became rate limiting differently for red, green, and blue wavelengths during the phase of linear growth. The transition from exponential to linear growth occurred at 1-2 mg Chl-a · L-1, whereby a scalar ~5 μmol photons · m-2 · s-1 at 20-cm depth was found to occur as would be anticipated having the compensation point for where rates of photosynthesis and respiration are equal. During the phase of linear growth, red wavelengths became increasingly dominant at depth as Chl-a concentrations increased, being contrary to the optical conditions for those natural bodies of water that forced the evolution of phytoplankton photosynthesis. It is hypothesized this dramatic difference in water column optics between natural and synthetic environments could influence a variety of biological reactions, importantly non-photochemical quenching capacities, which could negatively impact crop yield.

  12. 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)

  13. 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...

  14. 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)

  15. Integrated Bacillus sp. immobilized cell reactor and Synechocystis sp. algal reactor for the treatment of tannery wastewater.

    PubMed

    Sekaran, G; Karthikeyan, S; Nagalakshmi, C; Mandal, A B

    2013-01-01

    The wastewater discharged from leather industries lack biodegradability due to the presence of xenobiotic compounds. The primary clarification and aerobic treatment in Bacillus sp. immobilized Chemo Autotrophic Activated Carbon Oxidation (CAACO) reactor removed considerable amount of pollution parameters. The residual untreated organics in the wastewater was further treated in algal batch reactor inoculated with Synechocystis sp. Sodium nitrate, K(2)HPO(4), MgSO(4).7H(2)O, NH(4)Cl, CaCl(2)·2H(2)O, FeCl(3) (anhydrous), and thiamine hydrochloride, rice husk based activated carbon (RHAC), immobilization of Bacillus sp. in mesoporous activated carbon, sand filter of dimensions diameter, 6 cm and height, 30 cm; and the CAACO reactor of dimensions diameter, 5.5 cm and height, 30 cm with total volume 720 ml, and working volume of 356 ml. In the present investigation, the CAACO treated tannery wastewater was applied to Synechocystis sp. inoculated algal batch reactor of hydraulic residence time 24 h. The BOD(5), COD, and TOC of treated wastewater from algal batch reactor were 20 ± 7, 167 ± 29, and 78 ± 16 mg/l respectively. The integrated CAACO system and Algal batch reactor was operated for 30 days and they accomplished a cumulative removal of BOD(5),COD, TOC, VFA and sulphide as 98 %, 95 %, 93 %, 86 %, and 100 %, respectively. The biokinetic constants for the growth of algae in the batch reactor were specific growth rate, 0.095(day(-1)) and yield coefficient, 3.15 mg of algal biomass/mg of COD destructed. The degradation of xenobiotic compounds in the algal batch reactor was confirmed through HPLC and FT-IR techniques. The integrated CAACO-Algal reactor system established a credible reduction in pollution parameters in the tannery wastewater. The removal mechanism is mainly due to co-metabolism between algae and bacterial species and the organics were completely metabolized rather than by adsorption.

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

    PubMed

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

    1984-05-01

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

  17. Controlled, scalable embryonic stem cell differentiation culture.

    PubMed

    Dang, Stephen M; Gerecht-Nir, Sharon; Chen, Jinny; Itskovitz-Eldor, Joseph; Zandstra, Peter W

    2004-01-01

    Embryonic stem (ES) cells are of significant interest as a renewable source of therapeutically useful cells. ES cell aggregation is important for both human and mouse embryoid body (EB) formation and the subsequent generation of ES cell derivatives. Aggregation between EBs (agglomeration), however, inhibits cell growth and differentiation in stirred or high-cell-density static cultures. We demonstrate that the agglomeration of two EBs is initiated by E-cadherin-mediated cell attachment and followed by active cell migration. We report the development of a technology capable of controlling cell-cell interactions in scalable culture by the mass encapsulation of ES cells in size-specified agarose capsules. When placed in stirred-suspension bioreactors, encapsulated ES cells can be used to produce scalable quantities of hematopoietic progenitor cells in a controlled environment.

  18. 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.

  19. The use of turbidostat culture in investigation of algal heavy-metal toxicity and rotifer population dynamics

    SciTech Connect

    Bennett, W.N.

    1989-01-01

    Using the green alga Chlorella pyrenoidosa Chick, the heavy metals selenium as selenate, cadmium, cadmium + manganese and cadmium + zinc were investigated to assess their toxicity in terms of changes in {mu}{sub max}. It was shown that increases of sublethal concentrations of Se produced a near linear decrease in {mu}{sub max}. A {mu}{sub max}IC{sub 50} was calculated to be 10.1 {mu}M Se. A concentration of 1.8 {mu}M Cd produced a 62% decrease in {mu}{sub max} after 2 generating lag. A recovery of {mu}{sub max} was observed when MnCl{sub 2} or ZnCl{sub 2} was added to the medium in which populations were experiencing sublethal Cd toxicity. The amelioration responses were incomplete with regard to full recovery of {mu}{sub max} and last 20 generations for the Cd-Mn exposure and 7 generations for the Cd-Zn exposure. Measurement of {mu}{sub max} is turbidostat culture was shown to provide a very sensitive measure of toxicity. For the first time, a metazoan, the rotifer Brachionus calyciflorus Pallas, was grown in turbidostat culture and maintained near its {mu}{sub max} for many generations. It was discovered that {mu}{sub max} was subject to selection in this species and increased 51% from 0.053 h{sup {minus}1} to 0.080 h{sup {minus}1} over 8 mo at 25{degree}C.

  20. Replication of cultured lung epithelial cells

    SciTech Connect

    Guzowski, D.; Bienkowski, R.

    1986-03-05

    The authors have investigated the conditions necessary to support replication of lung type 2 epithelial cells in culture. Cells were isolated from mature fetal rabbit lungs (29d gestation) and cultured on feeder layers of mitotically inactivated 3T3 fibroblasts. The epithelial nature of the cells was demonstrated by indirect immunofluorescent staining for keratin and by polyacid dichrome stain. Ultrastructural examination during the first week showed that the cells contained myofilaments, microvilli and lamellar bodies (markers for type 2 cells). The following changes were observed after the first week: increase in cell size; loss of lamellar bodies and appearance of multivesicular bodies; increase in rough endoplasmic reticulum and golgi; increase in tonafilaments and well-defined junctions. General cell morphology was good for up to 10 wk. Cells cultured on plastic surface degenerated after 1 wk. Cell replication was assayed by autoradiography of cultures exposed to (/sup 3/H)-thymidine and by direct cell counts. The cells did not replicate during the first week; however, between 2-10 wk the cells incorporated the label and went through approximately 6 population doublings. They have demonstrated that lung alveolar epithelial cells can replicate in culture if they are maintained on an appropriate substrate. The coincidence of ability to replicate and loss of markers for differentiation may reflect the dichotomy between growth and differentiation commonly observed in developing systems.

  1. Autofluorescence of viable cultured mammalian cells.

    PubMed

    Aubin, J E

    1979-01-01

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

  2. A portable Raman acoustic levitation spectroscopic system for the identification and environmental monitoring of algal cells.

    PubMed

    Wood, Bayden R; Heraud, Philip; Stojkovic, Slobodanka; Morrison, Danielle; Beardall, John; McNaughton, Don

    2005-08-01

    We report the coupling of a portable Raman spectrometer to an acoustic levitation device to enable environmental monitoring and the potential taxonomic identification of microalgae. Spectra of living cells were recorded at 785 nm using a fiber-optic probe coupled to a portable Raman spectrometer. The spectra exhibit an excellent signal-to-noise ratio and clearly show bands from chlorophyll a and beta-carotene. Spectra of levitated photobleached microalgae clearly show a reduction in chlorophyll a concentration relative to beta-carotene after 10 min of exposure to a quartz halogen lamp. Spectra recorded from levitated nitrogen-limited cells also show a significant reduction in bands associated with chlorophyll a, as compared to nitrogen-replete cells. To investigate the diagnostic capability of the technique, four species of microalgae were analyzed. Good quality spectra of all four species were obtained showing varying ratios of beta-carotene to chlorophyll. The combination of an acoustic levitation device and a portable Raman spectrometer shows potential as a taxonomic and environmental monitoring tool with direct application to field studies in remote environments. PMID:16053309

  3. 3D Cell Culture in Alginate Hydrogels.

    PubMed

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

    2015-03-24

    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.

  4. Investigating why recycling gravity harvested algae increases harvestability and productivity in high rate algal ponds.

    PubMed

    Park, J B K; Craggs, R J; Shilton, A N

    2013-09-15

    It has previously been shown that recycling gravity harvested algae promotes Pediastrum boryanum dominance and improves harvestability and biomass production in pilot-scale High Rate Algal Ponds (HRAPs) treating domestic wastewater. In order to confirm the reproducibility of these findings and investigate the mechanisms responsible, this study utilized twelve 20 L outdoor HRAP mesocosms operated with and without algal recycling. It then compared the recycling of separated solid and liquid components of the harvested biomass against un-separated biomass. The work confirmed that algal recycling promoted P. boryanum dominance, improved 1 h-settleability by >20% and increased biomass productivity by >25% compared with controls that had no recycling. With regard to the improved harvestability, of particular interest was that recycling the liquid fraction alone caused a similar improvement in settleability as recycling the solid fraction. This may be due to the presence of extracellular polymeric substances in the liquid fraction. While there are many possible mechanisms that could account for the increased productivity with algal recycling, all but two were systematically eliminated: (i) the mean cell residence time was extended thereby increasing the algal concentration and more fully utilizing the incident sunlight and, (ii) the relative proportions of algal growth stages (which have different specific growth rates) was changed, resulting in a net increase in the overall growth rate of the culture.

  5. Investigating why recycling gravity harvested algae increases harvestability and productivity in high rate algal ponds.

    PubMed

    Park, J B K; Craggs, R J; Shilton, A N

    2013-09-15

    It has previously been shown that recycling gravity harvested algae promotes Pediastrum boryanum dominance and improves harvestability and biomass production in pilot-scale High Rate Algal Ponds (HRAPs) treating domestic wastewater. In order to confirm the reproducibility of these findings and investigate the mechanisms responsible, this study utilized twelve 20 L outdoor HRAP mesocosms operated with and without algal recycling. It then compared the recycling of separated solid and liquid components of the harvested biomass against un-separated biomass. The work confirmed that algal recycling promoted P. boryanum dominance, improved 1 h-settleability by >20% and increased biomass productivity by >25% compared with controls that had no recycling. With regard to the improved harvestability, of particular interest was that recycling the liquid fraction alone caused a similar improvement in settleability as recycling the solid fraction. This may be due to the presence of extracellular polymeric substances in the liquid fraction. While there are many possible mechanisms that could account for the increased productivity with algal recycling, all but two were systematically eliminated: (i) the mean cell residence time was extended thereby increasing the algal concentration and more fully utilizing the incident sunlight and, (ii) the relative proportions of algal growth stages (which have different specific growth rates) was changed, resulting in a net increase in the overall growth rate of the culture. PMID:23866138

  6. Constraints to commercialization of algal fuels.

    PubMed

    Chisti, Yusuf

    2013-09-10

    Production of algal crude oil has been achieved in various pilot scale facilities, but whether algal fuels can be produced in sufficient quantity to meaningfully displace petroleum fuels, has been largely overlooked. Limitations to commercialization of algal fuels need to be understood and addressed for any future commercialization. This review identifies the major constraints to commercialization of transport fuels from microalgae. Algae derived fuels are expensive compared to petroleum derived fuels, but this could change. Unfortunately, improved economics of production are not sufficient for an environmentally sustainable production, or its large scale feasibility. A low-cost point supply of concentrated carbon dioxide colocated with the other essential resources is necessary for producing algal fuels. An insufficiency of concentrated carbon dioxide is actually a major impediment to any substantial production of algal fuels. Sustainability of production requires the development of an ability to almost fully recycle the phosphorous and nitrogen nutrients that are necessary for algae culture. Development of a nitrogen biofixation ability to support production of algal fuels ought to be an important long term objective. At sufficiently large scale, a limited supply of freshwater will pose a significant limitation to production even if marine algae are used. Processes for recovering energy from the algal biomass left after the extraction of oil, are required for achieving a net positive energy balance in the algal fuel oil. The near term outlook for widespread use of algal fuels appears bleak, but fuels for niche applications such as in aviation may be likely in the medium term. Genetic and metabolic engineering of microalgae to boost production of fuel oil and ease its recovery, are essential for commercialization of algal fuels. Algae will need to be genetically modified for improved photosynthetic efficiency in the long term. PMID:23886651

  7. Replication of human endothelial cells in culture.

    PubMed

    Lewis, L J; Hoak, J C; Maca, R D; Fry, G L

    1973-08-01

    Investigative studies dealing with the properties and functions of endothelial cells have been hampered because there has been little or no success in the isolation, growth, and passage of individual cells in large numbers. We have developed a system whereby pure cultures of endothelial cells derived from umbilical veins can be subcultured for at least five serial passages. Many facets of endothelial function and interaction can be evaluated with the use of this new adaptive system of isolation and culture. PMID:4718112

  8. 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.

  9. 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

  10. 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.

  11. Insect cell culture in reagent bottles

    PubMed Central

    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. PMID:26150948

  12. Effects of sodium sulfate on the freshwater microalga Chlamydomonas moewusii: implications for the optimization of algal culture media.

    PubMed

    Mera, Roi; Torres, Enrique; Abalde, Julio

    2016-02-01

    The study of the microalgal growth kinetics is an indispensable tool in all fields of phycology. Knowing the optimal nutrient concentration is an important issue that will help to develop efficient growth systems for these microorganisms. Although nitrogen and phosphorus are well studied for this purpose, sulfur seems to be less investigated. Sulfate is a primary sulfur source used by microalgae; moreover, the concentration of this compound is increasing in freshwater systems due to pollution. The aim of this study was to investigate the effects of different sodium sulfate concentrations in the culture medium on growth and growth kinetics of the freshwater microalga Chlamydomonas moewusii. Production of biomass, chl content, kinetic equations, and a mathematical model that describe the microalgal growth in relation with the concentration of sodium sulfate were obtained. The lowest concentration of sodium sulfate allowing optimal growth was 0.1 mM. Concentrations higher than 3 mM generated a toxic effect. This work demonstrates that this toxic effect was not directly due to the excess of sulfate ion but by the elevation of the ionic strength. An inhibition model was successfully used to simulate the relationship between specific growth rate and sodium sulfate in this microalga.

  13. Effects of sodium sulfate on the freshwater microalga Chlamydomonas moewusii: implications for the optimization of algal culture media.

    PubMed

    Mera, Roi; Torres, Enrique; Abalde, Julio

    2016-02-01

    The study of the microalgal growth kinetics is an indispensable tool in all fields of phycology. Knowing the optimal nutrient concentration is an important issue that will help to develop efficient growth systems for these microorganisms. Although nitrogen and phosphorus are well studied for this purpose, sulfur seems to be less investigated. Sulfate is a primary sulfur source used by microalgae; moreover, the concentration of this compound is increasing in freshwater systems due to pollution. The aim of this study was to investigate the effects of different sodium sulfate concentrations in the culture medium on growth and growth kinetics of the freshwater microalga Chlamydomonas moewusii. Production of biomass, chl content, kinetic equations, and a mathematical model that describe the microalgal growth in relation with the concentration of sodium sulfate were obtained. The lowest concentration of sodium sulfate allowing optimal growth was 0.1 mM. Concentrations higher than 3 mM generated a toxic effect. This work demonstrates that this toxic effect was not directly due to the excess of sulfate ion but by the elevation of the ionic strength. An inhibition model was successfully used to simulate the relationship between specific growth rate and sodium sulfate in this microalga. PMID:26987090

  14. Prechlorination of algae-laden water: The effects of transportation time on cell integrity, algal organic matter release, and chlorinated disinfection byproduct formation.

    PubMed

    Qi, Jing; Lan, Huachun; Liu, Ruiping; Miao, Shiyu; Liu, Huijuan; Qu, Jiuhui

    2016-10-01

    The prechlorination-induced algal organic matter (AOM) released from Microcystis aeruginosa (M. aeruginosa) cells has been reported to serve as a source of precursors for chlorinated disinfection byproducts (DBPs). However, previous studies have mainly focused on the precursors either extracted directly from the cell suspension or derived immediately after algal suspension prechlorination. This study aims to investigate the impacts of water transportation time after algal suspension prechlorination on cell integrity, AOM release, and DBP formation during the dissolved phase chlorination. The damage to cell integrity after prechlorination was indicated to depend not only on chlorine dose but also on transportation time. The highest dissolved organic carbon (DOC) and dissolved organic nitrogen (DON) values were observed at 2 mg/L chlorine preoxidation before transportation, but were obtained at 0.4 mg/L chlorine after 480-min simulated transportation. The variation of DON with transportation time was indicated to be mainly influenced by the small molecular weight nitrogenous organic compounds, such as amino acids. Additionally, formation of the corresponding chlorinated carbonaceous disinfection byproducts (C-DBPs) and nitrogenous disinfection byproducts (N-DBPs) during the dissolved phase chlorination showed the same variation tendency as DOC and DON respectively. The highest C-DBP (98.4 μg/L) and N-DBP (5.5 μg/L) values were obtained at 0.4 mg/L chlorine preoxidation after 480-min simulated transportation. Therefore, when prechlorination is applied for algae-laden water pretreatment, not only chlorine dose but also transportation time needs to be considered with regard to their effects on cell integrity, AOM release, and chlorinated DBP formation.

  15. Prechlorination of algae-laden water: The effects of transportation time on cell integrity, algal organic matter release, and chlorinated disinfection byproduct formation.

    PubMed

    Qi, Jing; Lan, Huachun; Liu, Ruiping; Miao, Shiyu; Liu, Huijuan; Qu, Jiuhui

    2016-10-01

    The prechlorination-induced algal organic matter (AOM) released from Microcystis aeruginosa (M. aeruginosa) cells has been reported to serve as a source of precursors for chlorinated disinfection byproducts (DBPs). However, previous studies have mainly focused on the precursors either extracted directly from the cell suspension or derived immediately after algal suspension prechlorination. This study aims to investigate the impacts of water transportation time after algal suspension prechlorination on cell integrity, AOM release, and DBP formation during the dissolved phase chlorination. The damage to cell integrity after prechlorination was indicated to depend not only on chlorine dose but also on transportation time. The highest dissolved organic carbon (DOC) and dissolved organic nitrogen (DON) values were observed at 2 mg/L chlorine preoxidation before transportation, but were obtained at 0.4 mg/L chlorine after 480-min simulated transportation. The variation of DON with transportation time was indicated to be mainly influenced by the small molecular weight nitrogenous organic compounds, such as amino acids. Additionally, formation of the corresponding chlorinated carbonaceous disinfection byproducts (C-DBPs) and nitrogenous disinfection byproducts (N-DBPs) during the dissolved phase chlorination showed the same variation tendency as DOC and DON respectively. The highest C-DBP (98.4 μg/L) and N-DBP (5.5 μg/L) values were obtained at 0.4 mg/L chlorine preoxidation after 480-min simulated transportation. Therefore, when prechlorination is applied for algae-laden water pretreatment, not only chlorine dose but also transportation time needs to be considered with regard to their effects on cell integrity, AOM release, and chlorinated DBP formation. PMID:27348194

  16. Cyanobacteria Toxin and Cell Propagation through Seven Lake Erie Treatment Plants during the 2013 Algal Bloom Season - abstract

    EPA Science Inventory

    Over the past five years, Lake Erie has been experiencing harmful algal blooms (HABs) of progressively increasing severity. Cognizant of the potential health and economic impacts, the United States Environmental Protection Agency’s (USEPA’s) Water Supply and Water Resources Divis...

  17. Ultraviolet radiation dose calculation for algal suspensions using UVA and UVB extinction coefficients.

    PubMed

    Navarro, Enrique; Muñiz, Selene; Korkaric, Muris; Wagner, Bettina; de Cáceres, Miquel; Behra, Renata

    2014-03-01

    Although the biological importance of ultraviolet light (UVR) attenuation has been recognised in marine and freshwater environments, it is not generally considered in in vitro ecotoxicological studies using algal cell suspensions. In this study, UVA and UVB extinction were determined for cultures of algae with varying cell densities, and the data were used to calculate the corresponding extinction coefficients for both UVA and UVB wavelength ranges. Integrating the Beer-Lambert equation to account for changes in the radiation intensity reaching each depth, from the surface until the bottom of the experimental vessel, we obtained the average UVA and UVB intensity to which the cultured algal cells were exposed. We found that UVR intensity measured at the surface of Chlamydomonas reinhardtii cultures lead to a overestimation of the UVR dose received by the algae by 2-40 times. The approach used in this study allowed for a more accurate estimation of UVA and UVB doses. PMID:24607609

  18. 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

  19. 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

  20. Chronic rabies virus infection of cell cultures.

    PubMed

    Wiktor, T J; Clark, H F

    1972-12-01

    Exposure of both mammalian and reptilian cells in tissue culture to different strains of fixed rabies virus resulted in a carrier type of infection. No cytopathic effect was observed in either type of culture; infected cultures could be maintained by cell transfer for unlimited numbers of passages. A consistent pattern of cyclically rising and falling levels of viral infection was observed by fluorescent-antibody staining techniques and by titration of released infectious virus. Resistance to super-infection by vesicular stomatis virus and the production of an interferon-like substance by infected cells indicated that the maintenance of a carrier type of infection may be interferon-mediated. The degree of susceptibility of rabies-infected cells to immunolysis by antirabies antibody in the presence of complement was found to be correlated with the amount of virus maturation occurring by budding through the cell membrane and not with the presence of immunofluorescent antigen in the cytoplasm of infected cells.

  1. 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

  2. Luminescent Solar Concentrators in the Algal Industry

    NASA Astrophysics Data System (ADS)

    Hellier, Katie; Corrado, Carley; Carter, Sue; Detweiler, Angela; Bebout, Leslie

    2013-03-01

    Today's industry for renewable energy sources and highly efficient energy management systems is rapidly increasing. Development of increased efficiency Luminescent Solar Concentrators (LSCs) has brought about new applications for commercial interests, including greenhouses for agricultural crops. This project is taking first steps to explore the potential of LSCs to enhance production and reduce costs for algae and cyanobacteria used in biofuels and nutraceuticals. This pilot phase uses LSC filtered light for algal growth trials in greenhouses and laboratory experiments, creating specific wavelength combinations to determine effects of discrete solar light regimes on algal growth and the reduction of heating and water loss in the system. Enhancing the optimal spectra for specific algae will not only increase production, but has the potential to lessen contamination of large scale production due to competition from other algae and bacteria. Providing LSC filtered light will reduce evaporation and heating in regions with limited water supply, while the increased energy output from photovoltaic cells will reduce costs of heating and mixing cultures, thus creating a more efficient and cost effective production system.

  3. X-ray microanalysis of calcium in fixed and in shock-frozen hydrated green algal cells: Mougeotia, Spirogyra and Zygnema

    NASA Astrophysics Data System (ADS)

    Rossbacher, Roland; Wagner, Gottfried; Pallaghy, Charles K.

    1984-04-01

    X-ray microanalysis, widely used for ionic detection in cell tissues, was performed on 0.5 μm thick glutaraldehyde-osmiumtetroxide fixed cell sections. Calcium in a water-insoluble state was found in membrane bound vesicles of about 0.5 μm in diameter. No other cell compartment showed a comparable calcium accumulation. In vivo staining by the fluorescent calcium indicator dye chlorotetracycline revealed a vesicular calcium pattern in Mougeotia matching the results by X-ray microanalysis: a dense population of calcium vesicles is located close to the chloroplast edge where the actin filaments are attached. The data find further support from X-ray microanalysis of shock-frozen hydrated Mougeotia cells and of the closely related green algal cells Spirogyra and Zygnema (Zygnemaceae). X-ray microanalysis as performed in this study seems a reliable tool for the detection of water-insoluble calcium stored by membrane-covered cytoplasmic compartments.

  4. Structural Impacts on Thallus and Algal Cell Components of Two Lichen Species in Response to Low-Level Air Pollution in Pacific Northwest Forests

    NASA Astrophysics Data System (ADS)

    Ra, Hyung-Shim Y.; Rubin, Laura; Crang, Richard F. E.

    2004-04-01

    Lichens have long been regarded as bioindicators of air pollution, and structural studies typically have indicated negative impacts in highly polluted areas. In this research, Parmelia sulcata and Platismatia glauca were collected from one clean and two polluted sites in the Pacific Northwest forests of the United States to investigate the anatomical and ultrastructural responses of relatively resistant lichens to moderate air pollution. Light microscopy of polluted materials revealed only slight increases in the algal cell proportions of the thallus, and a decrease in the fungal cells of the medulla. Using transmission electron microscopy, increased lipid droplets in the cytoplasm and an increase in the cell wall thickness of the photobionts were found in the polluted lichens. These results were compared with physiological data in which the net carbon uptake did not show any significant differences; however, the total chlorophyll content was heightened in the polluted samples. The increased total chlorophyll content and the absence of any changes in the algal cell proportions of the polluted samples suggest that the photobionts possessed a higher chlorophyll content per unit volume of the photobiont at polluted sites. The results also indicate that lichens have altered their storage allocation in different cellular compartments. This may be a result of symbiotic readjustment(s) between the photobiont and the mycobiont. In comparison with the physiological results from these two species, these changes do not represent damaging effects by low-level air pollution.

  5. Structural impacts on thallus and algal cell components of two lichen species in response to low-level air pollution in pacific northwest forests.

    PubMed

    Ra, Hyung-Shim Y; Rubin, Laura; Crang, Richard F E

    2004-04-01

    Lichens have long been regarded as bioindicators of air pollution, and structural studies typically have indicated negative impacts in highly polluted areas. In this research, Parmelia sulcata and Platismatia glauca were collected from one clean and two polluted sites in the Pacific Northwest forests of the United States to investigate the anatomical and ultrastructural responses of relatively resistant lichens to moderate air pollution. Light microscopy of polluted materials revealed only slight increases in the algal cell proportions of the thallus, and a decrease in the fungal cells of the medulla. Using transmission electron microscopy, increased lipid droplets in the cytoplasm and an increase in the cell wall thickness of the photobionts were found in the polluted lichens. These results were compared with physiological data in which the net carbon uptake did not show any significant differences; however, the total chlorophyll content was heightened in the polluted samples. The increased total chlorophyll content and the absence of any changes in the algal cell proportions of the polluted samples suggest that the photobionts possessed a higher chlorophyll content per unit volume of the photobiont at polluted sites. The results also indicate that lichens have altered their storage allocation in different cellular compartments. This may be a result of symbiotic readjustment(s) between the photobiont and the mycobiont. In comparison with the physiological results from these two species, these changes do not represent damaging effects by low-level air pollution.

  6. Culture and transfection of axolotl cells.

    PubMed

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

    2015-01-01

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

  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 maintenance of embryonic stem cell cultures.

    PubMed

    Terstegge, Stefanie; Laufenberg, Iris; Pochert, Jörg; Schenk, Sabine; Itskovitz-Eldor, Joseph; Endl, Elmar; Brüstle, Oliver

    2007-01-01

    Embryonic stem cell (ESC) technology provides attractive perspectives for generating unlimited numbers of somatic cells for disease modeling and compound screening. A key prerequisite for these industrial applications are standardized and automated systems suitable for stem cell processing. Here we demonstrate that mouse and human ESC propagated by automated culture maintain their mean specific growth rates, their capacity for multi-germlayer differentiation, and the expression of the pluripotency-associated markers SSEA-1/Oct-4 and Tra-1-60/Tra-1-81/Oct-4, respectively. The feasibility of ESC culture automation may greatly facilitate the use of this versatile cell source for a variety of biomedical applications.

  9. Culture and transfection of axolotl cells.

    PubMed

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

    2015-01-01

    The use of cells grown in vitro has been instrumental for multiple aspects of biomedical research and especially molecular and cellular biology. The ability to grow cells from multicellular organisms like humans, squids, or salamanders is important to simplify the analyses and experimental designs to help understand the biology of these organisms. The advent of the first cell culture has allowed scientists to tease apart the cellular functions, and in many situations these experiments help understand what is happening in the whole organism. In this chapter, we describe techniques for the culture and genetic manipulation of an established cell line from axolotl, a species widely used for studying epimorphic regeneration. PMID:25740487

  10. Automated adherent human cell culture (mesenchymal stem cells).

    PubMed

    Thomas, Robert; Ratcliffe, Elizabeth

    2012-01-01

    Human cell culture processes developed at research laboratory scale need to be translated to large-scale production processes to achieve commercial application to a large market. To allow this transition of scale with consistent process performance and control of costs, it will be necessary to reduce manual processing and increase automation. There are a number of commercially available platforms that will reduce manual process intervention and improve process control for different culture formats. However, in many human cell-based applications, there is currently a need to remain close to the development format, usually adherent culture on cell culture plastic or matrix-coated wells or flasks due to deterioration of cell quality in other environments, such as suspension. This chapter presents an example method for adherent automated human stem cell culture using a specific automated flask handling platform, the CompacT SelecT.

  11. [Variations in the optical absorption and attenuation properties of cultured phytoplankton and their relationships with cell size].

    PubMed

    Zhou, Wen; Sun, Zhao-Hua; Cao, Wen-Xi; Wang, Gui-Fen

    2012-12-01

    The spectral absorption and attenuation coefficients of 16 phytoplankton species were measured in the laboratory using acs instrument. Ancillary measurements included particle size distribution and chlorophyll a concentration (Chl a). The results indicated that both algal cell size and Chl a were the two major factors dominating the magnitudes of the spectral absorption and attenuation coefficients. The spectral behaviors of attenuation spectra were dominated by algal cell size, the relationship of them didn't follow the monotonic function. Both the ratio of absorption in blue and red waveband and the spectral slope of absorption coefficient were influenced by the product of algal cell density and squares of cell size rather than algal cell size alone. The relationship between algal cell size and both absorption and attenuation spectra would be interpreted by Mie theory for homogenous sphere, which imply that the heterogeneity and non-spherical shape in algal cell morphology and internal structure have little effect on the inherent association among them.

  12. Critical evaluation and modeling of algal harvesting using dissolved air flotation. DAF Algal Harvesting Modeling

    SciTech Connect

    Zhang, Xuezhi; Hewson, John C.; Amendola, Pasquale; Reynoso, Monica; Sommerfeld, Milton; Chen, Yongsheng; Hu, Qiang

    2014-07-14

    In our study, Chlorella zofingiensis harvesting by dissolved air flotation (DAF) was critically evaluated with regard to algal concentration, culture conditions, type and dosage of coagulants, and recycle ratio. Harvesting efficiency increased with coagulant dosage and leveled off at 81%, 86%, 91%, and 87% when chitosan, Al3+, Fe3+, and cetyl trimethylammonium bromide (CTAB) were used at dosages of 70, 180, 250, and 500 mg g-1, respectively. The DAF efficiency-coagulant dosage relationship changed with algal culture conditions. In evaluating the influence of the initial algal concentration and recycle ratio revealed that, under conditions typical for algal harvesting, we found that it is possible that the number of bubbles is insufficient. A DAF algal harvesting model was developed to explain this observation by introducing mass-based floc size distributions and a bubble limitation into the white water blanket model. Moreover, the model revealed the importance of coagulation to increase floc-bubble collision and attachment, and the preferential interaction of bubbles with larger flocs, which limited the availability of bubbles to the smaller sized flocs. The harvesting efficiencies predicted by the model agree reasonably with experimental data obtained at different Al3+ dosages, algal concentrations, and recycle ratios. Based on this modeling, critical parameters for efficient algal harvesting were identified.

  13. Critical evaluation and modeling of algal harvesting using dissolved air flotation. DAF Algal Harvesting Modeling

    DOE PAGES

    Zhang, Xuezhi; Hewson, John C.; Amendola, Pasquale; Reynoso, Monica; Sommerfeld, Milton; Chen, Yongsheng; Hu, Qiang

    2014-07-14

    In our study, Chlorella zofingiensis harvesting by dissolved air flotation (DAF) was critically evaluated with regard to algal concentration, culture conditions, type and dosage of coagulants, and recycle ratio. Harvesting efficiency increased with coagulant dosage and leveled off at 81%, 86%, 91%, and 87% when chitosan, Al3+, Fe3+, and cetyl trimethylammonium bromide (CTAB) were used at dosages of 70, 180, 250, and 500 mg g-1, respectively. The DAF efficiency-coagulant dosage relationship changed with algal culture conditions. In evaluating the influence of the initial algal concentration and recycle ratio revealed that, under conditions typical for algal harvesting, we found that itmore » is possible that the number of bubbles is insufficient. A DAF algal harvesting model was developed to explain this observation by introducing mass-based floc size distributions and a bubble limitation into the white water blanket model. Moreover, the model revealed the importance of coagulation to increase floc-bubble collision and attachment, and the preferential interaction of bubbles with larger flocs, which limited the availability of bubbles to the smaller sized flocs. The harvesting efficiencies predicted by the model agree reasonably with experimental data obtained at different Al3+ dosages, algal concentrations, and recycle ratios. Based on this modeling, critical parameters for efficient algal harvesting were identified.« less

  14. Isolation and culture of pulmonary endothelial cells.

    PubMed

    Ryan, U S

    1984-06-01

    Methods for isolation, identification and culture of pulmonary endothelial cells are now routine. In the past, methods of isolation have used proteolytic enzymes to detach cells; thereafter, traditional methods for cell passaging have used trypsin/EDTA mixtures. Cells isolated and passaged using proteolytic enzymes have been useful in establishing the field and in verifying certain endothelial properties. However, there is a growing awareness of the role of endothelial cells in processing vasoactive substances, in responding to hormones and other agonists and in cell-cell interactions with other cell types of the vascular wall, with blood cells and with cellular products. Consequently, a new requirement has arisen for cells in vitro that maintain the differentiated properties of their counterparts in vivo. The deleterious effects of trypsin and other proteolytic enzymes commonly used in cell culture on surface structures of endothelial cells such as enzymes, receptors and junctional proteins, as well as on extracellular layers such as the glycocalyx or "endothelial fuzz," have led to the development of methods that avoid use of proteolytic enzymes at both the isolation step and during subsequent subculture. This chapter describes traditional methods for isolating pulmonary endothelial cells but emphasizes newer approaches using mechanical harvest and scale-up using microcarriers. The new methods allow maintenance of long-term, large-scale cultures of cells that retain the full complement of surface properties and that maintain the cobblestone monolayer morphology and differentiated functional properties. Methods for identification of isolated cells are therefore also considered as methods for validation of cultures during their in vitro lifespan. PMID:6090112

  15. Transferring isolated mitochondria into tissue culture cells

    PubMed Central

    Yang, Yi-Wei; Koob, Michael D.

    2012-01-01

    We have developed a new method for introducing large numbers of isolated mitochondria into tissue culture cells. Direct microinjection of mitochondria into typical mammalian cells has been found to be impractical due to the large size of mitochondria relative to microinjection needles. To circumvent this problem, we inject isolated mitochondria through appropriately sized microinjection needles into rodent oocytes or single-cell embryos, which are much larger than tissue culture cells, and then withdraw a ‘mitocytoplast’ cell fragment containing the injected mitochondria using a modified holding needle. These mitocytoplasts are then fused to recipient cells through viral-mediated membrane fusion and the injected mitochondria are transferred into the cytoplasm of the tissue culture cell. Since mouse oocytes contain large numbers of mouse mitochondria that repopulate recipient mouse cells along with the injected mitochondria, we used either gerbil single-cell embryos or rat oocytes to package injected mouse mitochondria. We found that the gerbil mitochondrial DNA (mtDNA) is not maintained in recipient rho0 mouse cells and that rat mtDNA initially replicated but was soon completely replaced by the injected mouse mtDNA, and so with both procedures mouse cells homoplasmic for the mouse mtDNA in the injected mitochondria were obtained. PMID:22753025

  16. 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.

  17. Algal endosymbionts in European Hydra strains reflect multiple origins of the zoochlorella symbiosis.

    PubMed

    Rajević, Nives; Kovačević, Goran; Kalafatić, Mirjana; Gould, Sven B; Martin, William F; Franjević, Damjan

    2015-12-01

    Symbiotic associations are of broad significance in evolution and biodiversity. Green Hydra is a classic example of endosymbiosis. In its gastrodermal myoepithelial cells it harbors endosymbiotic unicellular green algae, most commonly from the genus Chlorella. We reconstructed the phylogeny of cultured algal endosymbionts isolated and maintained in laboratory conditions for years from green Hydra strains collected from four different geographical sites within Croatia, one from Germany and one from Israel. Nuclear (18S rDNA, ITS region) and chloroplast markers (16S, rbcL) for maximum likelihood phylogenetic analyses were used. We focused on investigating the positions of these algal endosymbiotic strains within the chlorophyte lineage. Molecular analyses established that different genera and species of unicellular green algae are present as endosymbionts in green Hydra, showing that endosymbiotic algae growing within green Hydra sampled from four Croatian localities are not monophyletic. Our results indicate that the intracellular algal endosymbionts of green Hydra have become established several times independently in evolution.

  18. 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

  19. Isolating phagosomes from tissue culture cells.

    PubMed

    Pryor, Paul R; Rofe, Adam P

    2014-12-01

    Phagocytosis is the process by which receptors at the plasma membrane are used to engulf a particle such as a bacterium, parasite, or dead cell. Phagosomes can be isolated from tissue culture cells by various centrifugation methods, including the use of differential density gradients or sucrose step gradients, but these methods are time-consuming or otherwise difficult. We describe here a protocol that avoids centrifugation and relies instead on the uptake of magnetic beads to rapidly isolate the phagosomal compartment from tissue culture cells.

  20. [CO-CULTURE OF BOAR SPERMATOGONIAL CELLS WITH SERTOLI CELLS].

    PubMed

    Savchenkova, I P; Vasil'eva, S A

    2016-01-01

    In the present study, we developed in vitro culture conditions using co-culture of boar spermatogonial cells with Sertoli cells. Testes from 60-day-old crossbred boar were used. A spermatogonia-enriched culture was achieved by enzymatic digestion method and purification by density gradient centrifugation using a discontinuous Percoll gradient and differentiated adherence technique. Lipid drops were detected in isolated Sertoli cells by Oil Red O staining. We have found that the cultivation of boar spermatogonia in the presence of Sertoli cells (up to 35 days) leads to their differentiation as well as in vivo in testis. Association of cells in groups, formation of chains and suspension clusters of the spermatogenic cells were observed on the 10th day. Spermatogonial cellular colonies were noted at the same time. These cellular colonies were analyzed for the expression of genes: Nanog and Plzf in RT PCR. The expression of the Nanog gene in the experimental cellular clones obtained by short-term culture of spermatogonial cells in the presence of Sertoli cells was 200 times higher than the expression of this gene in the freshly isolated spermatogonial cells expression was found in freshly isolated germ cells and in cellular clones derived in vitro. We have found that, in the case of longer cultivation of these cells on Sertoli cells, in vitro process of differentiation of germ cells and formation of single mobile boar spermatozoa occurs at 30-33 days. Cellular population is heterogeneous at this stage. Spermatogenic differentiation in vitro without Sertoli cells stays on the 7th day of cultivation. The results show that co-culture of boar spermatogonia-enriched cells with Sertoli cells can induce their differentiation into spermatozoa in vitro and facilitate obtaining of porcine germ cell culture.

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

    DOEpatents

    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.

  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

    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.

  3. Campylobacter jejuni non-culturable coccoid cells.

    PubMed

    Beumer, R R; de Vries, J; Rombouts, F M

    1992-01-01

    The behaviour of Campylobacter jejuni in the environment is poorly documented. Rapid loss of viability on culture media is reported. This phenomenon is associated with the development of so-called coccoid cells. It has been suggested that these cells can be infective to animals and man. Results obtained with ATP-measurements of coccoid cells and Direct Viable Count (DVC) support this hypothesis. Introduction of coccoid cells into simulated gastric, ileal and colon environments did not result in the presence of culturable cells. Oral administration to laboratory animals and volunteers caused no typical symptoms of campylobacteriosis. Until 30 days after uptake of the cells antibodies against C. jejuni could not be detected in the blood, and the presence of this microorganism in stool samples could not be demonstrated.

  4. Shock Wave Application to Cell Cultures

    PubMed Central

    Holfeld, Johannes; Tepeköylü, Can; Kozaryn, Radoslaw; Mathes, Wolfgang; Grimm, Michael; Paulus, Patrick

    2014-01-01

    Shock waves nowadays are well known for their regenerative effects. Basic research findings showed that shock waves do cause a biological stimulus to target cells or tissue without any subsequent damage. Therefore, in vitro experiments are of increasing interest. Various methods of applying shock waves onto cell cultures have been described. In general, all existing models focus on how to best apply shock waves onto cells. However, this question remains: What happens to the waves after passing the cell culture? The difference of the acoustic impedance of the cell culture medium and the ambient air is that high, that more than 99% of shock waves get reflected! We therefore developed a model that mainly consists of a Plexiglas built container that allows the waves to propagate in water after passing the cell culture. This avoids cavitation effects as well as reflection of the waves that would otherwise disturb upcoming ones. With this model we are able to mimic in vivo conditions and thereby gain more and more knowledge about how the physical stimulus of shock waves gets translated into a biological cell signal (“mechanotransduction"). PMID:24747842

  5. Mechanism and challenges in commercialisation of algal biofuels.

    PubMed

    Singh, Anoop; Nigam, Poonam Singh; Murphy, Jerry D

    2011-01-01

    Biofuels made from algal biomass are being considered as the most suitable alternative energy in current global and economical scenario. Microalgae are known to produce and accumulate lipids within their cell mass which is similar to those found in many vegetable oils. The efficient lipid producer algae cell mass has been reported to contain more than 30% of their cell weight as lipids. According to US DOE microalgae have the potential to produce 100 times more oil per acre land than any terrestrial plants. This article reviews up to date literature on the composition of algae, mechanism of oil droplets, triacylglycerol (TAG) production in algal biomass, research and development made in the cultivation of algal biomass, harvesting strategies, and recovery of lipids from algal mass. The economical challenges in the production of biofuels from algal biomass have been discussed in view of the future prospects in the commercialisation of algal fuels.

  6. 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.

  7. 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... (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture Products § 864.2280 Cultured animal and human cells. (a) Identification. Cultured animal and human cells are in...

  8. 21 CFR 864.2280 - Cultured animal and human cells.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Cultured animal and human cells. 864.2280 Section... (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture Products § 864.2280 Cultured animal and human cells. (a) Identification. Cultured animal and human cells are in...

  9. 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... (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture Products § 864.2280 Cultured animal and human cells. (a) Identification. Cultured animal and human cells are in...

  10. 21 CFR 864.2280 - Cultured animal and human cells.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Cultured animal and human cells. 864.2280 Section... (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture Products § 864.2280 Cultured animal and human cells. (a) Identification. Cultured animal and human cells are in...

  11. 21 CFR 864.2280 - Cultured animal and human cells.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Cultured animal and human cells. 864.2280 Section... (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture Products § 864.2280 Cultured animal and human cells. (a) Identification. Cultured animal and human cells are in...

  12. Pinoresinol from Ipomoea cairica cell cultures.

    PubMed

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

    2002-10-01

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

  13. Culture of human endothelial cells.

    PubMed

    Gallicchio, M A

    2001-01-01

    Endothelial cells line the luminal surface of all blood vessels in the body. The endothelial surface in adult humans is composed of approximately l-6×l0(13) cells and covers an area of 1-7 m(2). Endothelium serves many functions, including fluid and solute exchange through cell contraction, provision of an antithrombogenic surface through tissue plasminogen activator (tPA) and prostacyclin release, synthesis of angiogenic factors such as adenosine, allowance of leukocyte trafficking through adhesion molecule synthesis, presentation of antigens to the immune system, maintenance of vascular tone through nitric oxide and endothelin synthesis, and metabolism of circulating molecules through the release of enzymes such as lipoprotein lipase. PMID:21340938

  14. 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

  15. [Ebola virus reproduction in cell cultures].

    PubMed

    Titenko, A M; Novozhilov, S S; Andaev, E I; Borisova, T I; Kulikova, E V

    1992-01-01

    Ebola-Zaire virus production in Vero and BGM cells was studied. The CPE developed in both cell cultures. The cell monolayer destruction by 80-90% was seen at a low multiplicity of infection in 7-8 days after virus inoculation. An overlay composition was developed for virus titration using plaque assay. The plaque production was shown to be directly proportional to the virus dose. The curve of Ebola virus production in Vero cell culture fluid was determined. At a multiplicity of infection of 0.01 PFU/cell, the maximum virus titer of 10(6.4) PFU/ml was reached in 7 days postinfection. Specific antisera were generated by inoculation of guinea pigs. Indirect immunofluorescent assay was used for testing of virus-specific antigen and antibody.

  16. Growth regulation of cultured human nevus cells.

    PubMed

    Mancianti, M L; Györfi, T; Shih, I M; Valyi-Nagy, I; Levengood, G; Menssen, H D; Halpern, A C; Elder, D E; Herlyn, M

    1993-03-01

    Cells isolated from congenital melanocytic nevi and cultured in vitro have growth characteristics that resemble their premalignant stage in situ. A serum-free, chemically defined medium has been developed that allows continuous growth of established nevus cultures for up to several months. Like primary melanoma cells, nevus cells in high-calcium-containing W489 medium require insulin for growth. In contrast to melanoma cells, nevus cells in serum-free medium require the presence of alpha-melanocyte-stimulating hormone, which enhanced intracellular levels of cyclic adenosine monophosphate. In contrast to the requirements of normal human melanocytes from newborn foreskin, congenital nevus cells grow with less dependency on basic fibroblast growth factor (bFGF). Nevus cultures contain bFGF-like activity, and they express bFGF mRNA. Nevic cells of compound nevi also express bFGF mRNA in situ but only in the junctional areas. These results indicate that bFGF plays an important growth regulatory role for nevus cells in vitro and in vivo. PMID:8440904

  17. Mutagenicity of algal metabolites of benzo(a)pyrene for Salmonella typhimurium

    SciTech Connect

    Schoeny, R.; Cody, T.; Radike, M.; Warshawsky, D.

    1985-01-01

    The metabolism and growth effects of benzo(a)pyrene (BaP) were studied using a freshwater green alga, Selenastrum capricornutum. Algal cultures were incubated under gold light with BaP added at concentrations of 40, 160, 400, and 1200 g/liter for the periods of 1-4 days. The metabolites and BaP were identified and quantified from ethyl acetate extracts of both algal cells and incubation medium. The ethyl acetate extracts were evaluated for genotoxicity using a micro-volume Salmonella typhimurium forward mutation assay with resistance to 8-azaguanine for selection. This assay detected the presence of small quantities of BaP and was particularly sensitive to the mutagenicity of BaP diols. Of those extracts prepared from algae and medium from cultures exposed to 400 g BaP/liter (10 g/25 ml culture), only algal cell extracts from one day's growth were mutagenic. In cultures exposed to 1200 g BaP/liter (30 g/25 ml culture), mutagenic materials were produced or persisted in both algae and media throughout the 4-day incubation. The observed mutagenic response can be attributed in part to the presence of unmetabolized BaP or to BaP diols.

  18. General overview of neuronal cell culture.

    PubMed

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

    2013-01-01

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

  19. 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.

  20. 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. PMID:27590152

  1. 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.

  2. Isolation and Culture of Muscle Stem Cells.

    PubMed

    Mozzetta, Chiara

    2016-01-01

    Polycomb group (PcG) proteins are key epigenetic factors responsible for the proper spatiotemporal repression of defined transcriptional programs along the process of cell differentiation, including myogenesis. The discovery of the pivotal role played by PcG factors during myogenic differentiation relied on the possibility to culture myogenic cells in vitro. We describe here the methods currently used to isolate muscle stem cells (MuSCs) both from single myofibers and from bulk muscles by fluorescence-activated cell sorting (FACS), highlighting experimental details and critical steps. Through these techniques MuSCs can be efficiently isolated and cultured in vitro to recapitulate the different phases of myogenesis: activation, expansion, differentiation, and self-renewal. PMID:27659996

  3. Pretreatment of algae-laden and manganese-containing waters by oxidation-assisted coagulation: Effects of oxidation on algal cell viability and manganese precipitation.

    PubMed

    Lin, Jr-Lin; Hua, Lap-Cuong; Wu, Yuting; Huang, Chihpin

    2016-02-01

    Preoxidation is manipulated to improve performance of algae and soluble manganese (Mn) removal by coagulation-sedimentation for water treatment plants (WTPs) when large amount of soluble Mn presents in algae-laden waters. This study aimed to investigate the effects of preoxidation on the performance of coagulation-sedimentation for the simultaneous removal of algae and soluble Mn, including ionic and complexed Mn. NaOCl, ClO2, and KMnO4 were used to pretreat such algae-laden and Mn containing waters. The variation of algal cell viability, residual cell counts, and concentrations of Mn species prior to and after coagulation-sedimentation step were investigated. Results show that NaOCl dosing was effective in reducing the viability of algae, but precipitated little Mn. ClO2 dosing had a strongest ability to lower algae viability and oxidize ionic and complexed soluble Mn, where KMnO4 dosing oxidized ionic and complexed Mn instead of reducing the viability of cells. Preoxidation by NaOCl only improved the algae removal by sedimentation, whereas most of soluble Mn still remained. On the other hand, ClO2 preoxidation substantially improved the performance of coagulation-sedimentation for simultaneous removal of algae and soluble Mn. Furthermore, KMnO4 preoxidation did improve the removal of algae by sedimentation, but left significant residual Mn in the supernatant. Images from FlowCAM showed changes in aspect ratio (AR) and transparency of algae-Mn flocs during oxidation-assisted coagulation, and indicates that an effective oxidation can improve the removal of most compact algae-Mn flocs by sedimentation. It suggests that an effective preoxidation for reducing algal cell viability and the concentration of soluble Mn is a crucial step for upgrading the performance of coagulation-sedimentation.

  4. Pretreatment of algae-laden and manganese-containing waters by oxidation-assisted coagulation: Effects of oxidation on algal cell viability and manganese precipitation.

    PubMed

    Lin, Jr-Lin; Hua, Lap-Cuong; Wu, Yuting; Huang, Chihpin

    2016-02-01

    Preoxidation is manipulated to improve performance of algae and soluble manganese (Mn) removal by coagulation-sedimentation for water treatment plants (WTPs) when large amount of soluble Mn presents in algae-laden waters. This study aimed to investigate the effects of preoxidation on the performance of coagulation-sedimentation for the simultaneous removal of algae and soluble Mn, including ionic and complexed Mn. NaOCl, ClO2, and KMnO4 were used to pretreat such algae-laden and Mn containing waters. The variation of algal cell viability, residual cell counts, and concentrations of Mn species prior to and after coagulation-sedimentation step were investigated. Results show that NaOCl dosing was effective in reducing the viability of algae, but precipitated little Mn. ClO2 dosing had a strongest ability to lower algae viability and oxidize ionic and complexed soluble Mn, where KMnO4 dosing oxidized ionic and complexed Mn instead of reducing the viability of cells. Preoxidation by NaOCl only improved the algae removal by sedimentation, whereas most of soluble Mn still remained. On the other hand, ClO2 preoxidation substantially improved the performance of coagulation-sedimentation for simultaneous removal of algae and soluble Mn. Furthermore, KMnO4 preoxidation did improve the removal of algae by sedimentation, but left significant residual Mn in the supernatant. Images from FlowCAM showed changes in aspect ratio (AR) and transparency of algae-Mn flocs during oxidation-assisted coagulation, and indicates that an effective oxidation can improve the removal of most compact algae-Mn flocs by sedimentation. It suggests that an effective preoxidation for reducing algal cell viability and the concentration of soluble Mn is a crucial step for upgrading the performance of coagulation-sedimentation. PMID:26689663

  5. 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

  6. Shape memory polymers for active cell culture.

    PubMed

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

    2011-07-04

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

  7. Cell culture models using rat primary alveolar type I cells

    PubMed Central

    Downs, Charles A.; Montgomery, David W.; Merkle, Carrie J.

    2011-01-01

    There is a lack of cell culture models using primary alveolar type I (AT I) cells. The purpose of this study was to develop cell culture models using rat AT I cells and microvascular endothelial cells from the lung (MVECL). Two types of model systems were developed: single and co-culture systems; additionally a 3-dimensional model system was developed. Pure AT I cell (96.3 ±2.7%) and MVECL (97.9 ±1.1 %) preparations were used. AT I cell morphology, mitochondrial number and distribution, actin filament arrangement and number of apoptotic cells at confluence, and telomere attrition were characterized. AT I cells maintained their morphometric characteristics through at least population doubling (PD) 35, while demonstrating telomere attrition through at least PD 100. Furthermore, AT I cells maintained the expression of their specific markers, T1α and AQ-5, through PD 42. For the co-cultures, AT I cells were grown on the top and MVECL were grown on the bottom of fibronectin coated 24 well Transwell Fluroblok™ filter inserts. Neither cell type transmigrated the 1 micron pores. Additionally AT I cells were grown in a thick layer of Matrigel® to create a 3-dimensional model in which primary AT I cells form ring-like structures that resemble an alveolus. The development of these model systems offers the opportunities to investigate AT I cell cells and their interactions with MVECL in response to pharmacological interventions and in the processes of disease, repair and regeneration. PMID:21624488

  8. Development of a floating photobioreactor with internal partitions for efficient utilization of ocean wave into improved mass transfer and algal culture mixing.

    PubMed

    Kim, Z-Hun; Park, Hanwool; Hong, Seong-Joo; Lim, Sang-Min; Lee, Choul-Gyun

    2016-05-01

    Culturing microalgae in the ocean has potentials that may reduce the production cost and provide an option for an economic biofuel production from microalgae. The ocean holds great potentials for mass microalgal cultivation with its high specific heat, mixing energy from waves, and large cultivable area. Suitable photobioreactors (PBRs) that are capable of integrating marine energy into the culture systems need to be developed for the successful ocean cultivation. In this study, prototype floating PBRs were designed and constructed using transparent low-density polyethylene film for microalgal culture in the ocean. To improve the mixing efficiency, various types of internal partitions were introduced within PBRs. Three different types of internal partitions were evaluated for their effects on the mixing efficiency in terms of mass transfer (k(L)a) and mixing time in the PBRs. The partition type with the best mixing efficiency was selected, and the number of partitions was varied from one to three for investigation of its effect on mixing efficiency. When the number of partitions is increased, mass transfer increased in proportion to the number of partitions. However, mixing time was not directly related to the number of partitions. When a green microalga, Tetraselmis sp. was cultivated using PBRs with the selected partition under semi-continuous mode in the ocean, biomass and fatty acid productivities in the PBRs were increased by up to 50 % and 44% at high initial cell density, respectively, compared to non-partitioned ones. The results of internally partitioned PBRs demonstrated potentials for culturing microalgae by efficiently utilizing ocean wave energy into culture mixing in the ocean.

  9. Development of a floating photobioreactor with internal partitions for efficient utilization of ocean wave into improved mass transfer and algal culture mixing.

    PubMed

    Kim, Z-Hun; Park, Hanwool; Hong, Seong-Joo; Lim, Sang-Min; Lee, Choul-Gyun

    2016-05-01

    Culturing microalgae in the ocean has potentials that may reduce the production cost and provide an option for an economic biofuel production from microalgae. The ocean holds great potentials for mass microalgal cultivation with its high specific heat, mixing energy from waves, and large cultivable area. Suitable photobioreactors (PBRs) that are capable of integrating marine energy into the culture systems need to be developed for the successful ocean cultivation. In this study, prototype floating PBRs were designed and constructed using transparent low-density polyethylene film for microalgal culture in the ocean. To improve the mixing efficiency, various types of internal partitions were introduced within PBRs. Three different types of internal partitions were evaluated for their effects on the mixing efficiency in terms of mass transfer (k(L)a) and mixing time in the PBRs. The partition type with the best mixing efficiency was selected, and the number of partitions was varied from one to three for investigation of its effect on mixing efficiency. When the number of partitions is increased, mass transfer increased in proportion to the number of partitions. However, mixing time was not directly related to the number of partitions. When a green microalga, Tetraselmis sp. was cultivated using PBRs with the selected partition under semi-continuous mode in the ocean, biomass and fatty acid productivities in the PBRs were increased by up to 50 % and 44% at high initial cell density, respectively, compared to non-partitioned ones. The results of internally partitioned PBRs demonstrated potentials for culturing microalgae by efficiently utilizing ocean wave energy into culture mixing in the ocean. PMID:26857371

  10. Harmful Algal Blooms (HABs)

    MedlinePlus

    ... Topics Eighth Annual National Conference on Health Communication, Marketing & Media August 19-21, 2014 Atlanta, GA Harmful Algal Blooms Recommend on Facebook Tweet Share Compartir On this Page What's the ...

  11. Harmful Algal Blooms

    USGS Publications Warehouse

    Graham, Jennifer L.

    2007-01-01

    What are Harmful Algal Blooms (HABs)? Freshwater and marine harmful algal blooms (HABs) can occur anytime water use is impaired due to excessive accumulations of algae. HAB occurrence is affected by a complex set of physical, chemical, biological, hydrological, and meteorological conditions making it difficult to isolate specific causative environmental factors. Potential impairments include reduction in water quality, accumulation of malodorous scums in beach areas, algal production of toxins potent enough to poison both aquatic and terrestrial organisms, and algal production of taste-and-odor compounds that cause unpalatable drinking water and fish. HABs are a global problem, and toxic freshwater and (or) marine algae have been implicated in human and animal illness and death in over 45 countries worldwide and in at least 27 U.S. States (Yoo and others, 1995; Chorus and Bartram, 1999; Huisman and others, 2005).

  12. 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.

  13. 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

  14. Electrical excitability of cultured adrenal chromaffin cells.

    PubMed Central

    Biales, B; Dichter, M; Tischler, A

    1976-01-01

    1. Adult human and gerbil adrenal medullary cells were maintained in dissociated cell culture and studied by micro-electrode penetration. 2. In the best recordings, chromaffin cell transmembrane potentials exceeded -50mV. 3. Chromaffin cells were capable of generating all-or-nothing over-shooting action potentials, similar to those generated by sympathetic neurones. 4. The action potentials were blocked by tetrodotoxin (TTX, 10(-6)g/ml.) but were not blocked by removal of Ca or by CoCl2 (10 mM). We conclude that the action potentials are probably generated by a Na mechanism. 5. Chromaffin cells are depolarized by the iontophoretic application of acetylcholine (ACh). This depolarization was accompanied by an increased membrane conductance and could trigger action potentials. 6. Action potentials were also found in cells in fresh slices of gerbil adrenal medullae. Images Plate 1 PMID:1034699

  15. Method of determining the number of cells in cell culture

    SciTech Connect

    Connolly, D.T.

    1990-06-12

    This patent describes a color-sensitivity method for determining the number of cells in in vitro cell culture at a sensitivity as low as about 100 or about 500 cells. It comprises lysing the cells and incubating the lysate with p-nitrophenyl phosphate at acid pH for a predetermined period of time at a temperature of from about 35{degrees} to about 38{degrees}C. and then measuring the color development at 400 to 420 nanometers and correlating the color development with cell number by comparing with a control standard of known cell number.

  16. 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

  17. 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.

  18. Maintenance of algal endosymbionts in Paramecium bursaria: a simple model based on population dynamics.

    PubMed

    Iwai, Sosuke; Fujiwara, Kenji; Tamura, Takuro

    2016-09-01

    Algal endosymbiosis is widely distributed in eukaryotes including many protists and metazoans, and plays important roles in aquatic ecosystems, combining phagotrophy and phototrophy. To maintain a stable symbiotic relationship, endosymbiont population size in the host must be properly regulated and maintained at a constant level; however, the mechanisms underlying the maintenance of algal endosymbionts are still largely unknown. Here we investigate the population dynamics of the unicellular ciliate Paramecium bursaria and its Chlorella-like algal endosymbiont under various experimental conditions in a simple culture system. Our results suggest that endosymbiont population size in P. bursaria was not regulated by active processes such as cell division coupling between the two organisms, or partitioning of the endosymbionts at host cell division. Regardless, endosymbiont population size was eventually adjusted to a nearly constant level once cells were grown with light and nutrients. To explain this apparent regulation of population size, we propose a simple mechanism based on the different growth properties (specifically the nutrient requirements) of the two organisms, and based from this develop a mathematical model to describe the population dynamics of host and endosymbiont. The proposed mechanism and model may provide a basis for understanding the maintenance of algal endosymbionts. PMID:26625979

  19. Maintenance of algal endosymbionts in Paramecium bursaria: a simple model based on population dynamics.

    PubMed

    Iwai, Sosuke; Fujiwara, Kenji; Tamura, Takuro

    2016-09-01

    Algal endosymbiosis is widely distributed in eukaryotes including many protists and metazoans, and plays important roles in aquatic ecosystems, combining phagotrophy and phototrophy. To maintain a stable symbiotic relationship, endosymbiont population size in the host must be properly regulated and maintained at a constant level; however, the mechanisms underlying the maintenance of algal endosymbionts are still largely unknown. Here we investigate the population dynamics of the unicellular ciliate Paramecium bursaria and its Chlorella-like algal endosymbiont under various experimental conditions in a simple culture system. Our results suggest that endosymbiont population size in P. bursaria was not regulated by active processes such as cell division coupling between the two organisms, or partitioning of the endosymbionts at host cell division. Regardless, endosymbiont population size was eventually adjusted to a nearly constant level once cells were grown with light and nutrients. To explain this apparent regulation of population size, we propose a simple mechanism based on the different growth properties (specifically the nutrient requirements) of the two organisms, and based from this develop a mathematical model to describe the population dynamics of host and endosymbiont. The proposed mechanism and model may provide a basis for understanding the maintenance of algal endosymbionts.

  20. Genomics in mammalian cell culture bioprocessing

    PubMed Central

    Wuest, Diane M.; Harcum, Sarah W.; Lee, Kelvin H.

    2013-01-01

    Explicitly identifying the genome of a host organism including sequencing, mapping, and annotating its genetic code has become a priority in the field of biotechnology with aims at improving the efficiency and understanding of cell culture bioprocessing. Recombinant protein therapeutics, primarily produced in mammalian cells, constitute a $108 billion global market. The most common mammalian cell line used in biologic production processes is the Chinese hamster ovary (CHO) cell line, and although great improvements have been made in titer production over the past 25 years, the underlying molecular and physiological factors are not well understood. Confident understanding of CHO bioprocessing elements (e.g. cell line selection, protein production, and reproducibility of process performance and product specifications) would significantly improve with a well understood genome. This review describes mammalian cell culture use in bioprocessing, the importance of obtaining CHO cell line genetic sequences, and the current status of sequencing efforts. Furthermore, transcriptomic techniques and gene expression tools are presented, and case studies exploring genomic techniques and applications aimed to improve mammalian bioprocess performance are reviewed. Finally, future implications of genomic advances are surmised. PMID:22079893

  1. Development in primary cell culture of demosponges.

    PubMed

    De Rosa, Salvatore; De Caro, Salvatore; Iodice, Carmine; Tommonaro, Giuseppina; Stefanov, Kamen; Popov, Simeon

    2003-01-23

    We have established primary cell culture of the marine demosponge Dysidea avara and Suberites domuncula. Microbial contamination was controlled by the use of a pool of antibiotics confirming the goodness of this procedure. Effect of pH, temperature and light was studied to establish the better growth conditions. The comparison of lipid composition of sponge and cells suggested a series of experiments to optimise the medium. A glucose dose-dependent experiment showed that the ideal glucose concentration is 1 g l(-1). Supplementing the medium with unsaturated fatty acid and retinol, no promotion of growth was observed, but the compounds were totally metabolised by cells. Increments from 70 to 160% in the number of cells were observed, supplementing the medium with different concentration of cholesterol. These results suggest that the analysis of the chemical composition of sponge and cells give indication on the composition of the nutrient media.

  2. No-observed-effect concentrations in batch and continuous algal toxicity tests

    SciTech Connect

    Chao, M.R.; Chen, C.Y.

    2000-06-01

    In this study, the authors compare the no-observed-effect concentrations (NOECs) of Cd, Ni, Zn, Cu, and Pb based on different response parameters, using batch and continuous algal toxicity tests. For both batch and continuous tests, parameters based on total cell volume (TCV) were found to be less sensitive than those related to cell densities. The above observation mainly occurred because, under the stresses from metal toxicants evaluated in this and a previous study, the mean cell volume (MCV) of algae increased considerably. The increase of MCV compensates for the effects brought about by the reduction in cell density and eventually results in less variation in TCVs. This study shows that parameters based on cell density are quite sensitive and ideal for the estimation of NOECs. In addition, comparison of the NOEC values derived from different culture techniques shows that the continuous methods generally yields lower NOEC values than that obtained by the batch tests. The results of this study also indicate that the NOEC provides more protection to the test organism than the effective concentration at 10% growth reduction (EC10). For toxicity test methods that produce small variations among replicates, the NOEC is still a good indicator of low toxic effect. Furthermore, for the continuous algal toxicity test, a relatively simple approach is proposed to determine the NOEC values based on the algal culture's control charts. The proposed method produced identical results as those based on conventional hypothesis-testing methods.

  3. 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.

  4. Sixty years in algal physiology and photosynthesis.

    PubMed

    Pirson, A

    1994-06-01

    This personal perspective records research experiences in chemistry and biology at four German universities, two before and two after World War II. The research themes came from cytophysiology of green unicellular algae, in particular their photosynthesis. The function of inorganic ions in photosynthesis and dark respiration was investigated at different degrees of specific mineral stress (deficiencies), and the kinetics of recovery followed after the addition of the missing element. Two types of recovery of photosynthesis were observed: indirect restitution via growth processes and immediate normalisation. From the latter case (K(+), phosphate, Mn(++)) the effect of manganese was emphasized as its role in photosynthetic O2 evolution became established during our research. Other themes of our group, with some bearing on photosynthesis were: synchronization of cell growth by light-dark change and effects of blue (vs. red) light on the composition of green cells. Some experiences in connection with algal mass cultures are included. Discussion of several editorial projects shows how photosynthesis, as an orginally separated field of plant biochemistry and biophysics, became included into general cell physiology and even ecophysiology of green plants. The paper contains an appreciation of the authors' main mentor Kurt Noack (1888-1963) and of Ernst Georg Pringsheim (1881-1970), founder of experimental phycology.

  5. 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.

  6. 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

  7. Iron overload in cultured rat myocardial cells

    NASA Astrophysics Data System (ADS)

    Bauminger, E. R.; Iancu, T. C.; Link, G.; Pinson, A.; Hershko, C.

    1987-03-01

    In order to characterize the nature of iron deposits associated with iron overload in heart cells, Mössbauer spectroscopy and ultrastructural studies were performed in iron loaded heart cell cultures obtained from newborn rats grown in a medium containing 20 μg iron/ml. Maximal uptake of iron after 24 hrs was about 15%. Not more than 20% of the iron in these cells was stored in ferritin and the rest was found in smaller trivalent iron aggregates. With time there was a shift from smaller to larger aggregates. In chase samples there was only a very limited spontaneous release of iron from heart cells. Desferrioxamine, an iron chelating drug, removed a major part of the smaller aggregates, but did not remove ferritin iron.

  8. Measurement of Glucose Uptake in Cultured Cells.

    PubMed

    Yamamoto, Norio; Ueda-Wakagi, Manabu; Sato, Takuya; Kawasaki, Kengo; Sawada, Keisuke; Kawabata, Kyuichi; Akagawa, Mitsugu; Ashida, Hitoshi

    2015-12-08

    Facilitative glucose uptake transport systems are ubiquitous in animal cells and are responsible for transporting glucose across cell surface membranes. Evaluation of glucose uptake is crucial in the study of numerous diseases and metabolic disorders such as myocardial ischemia, diabetes mellitus, and cancer. Detailed in this unit are laboratory methods for assessing glucose uptake into mammalian cells. The unit is divided into five sections: (1) a brief overview of glucose uptake assays in cultured cells; (2) a method for measuring glucose uptake using radiolabeled 3-O-methylglucose; (3) a method for measuring glucose uptake using radiolabeled 2-deoxyglucose (2DG); (4) a microplate method for measuring 2DG-uptake using an enzymatic, fluorometric assay; and (5) a microplate-based method using a fluorescent analog of 2DG.

  9. 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.

  10. 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

  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. Characterisation of algal organic matter produced by bloom-forming marine and freshwater algae.

    PubMed

    Villacorte, L O; Ekowati, Y; Neu, T R; Kleijn, J M; Winters, H; Amy, G; Schippers, J C; Kennedy, M D

    2015-04-15

    Algal blooms can seriously affect the operation of water treatment processes including low pressure (micro- and ultra-filtration) and high pressure (nanofiltration and reverse osmosis) membranes mainly due to accumulation of algal-derived organic matter (AOM). In this study, the different components of AOM extracted from three common species of bloom-forming algae (Alexandrium tamarense, Chaetoceros affinis and Microcystis sp.) were characterised employing various analytical techniques, such as liquid chromatography - organic carbon detection, fluorescence spectroscopy, fourier transform infrared spectroscopy, alcian blue staining and lectin staining coupled with laser scanning microscopy to indentify its composition and force measurement using atomic force microscopy to measure its stickiness. Batch culture monitoring of the three algal species illustrated varying characteristics in terms of growth pattern, cell concentration and AOM release. The AOM produced by the three algal species comprised mainly biopolymers (e.g., polysaccharides and proteins) but some refractory compounds (e.g., humic-like substances) and other low molecular weight acid and neutral compounds were also found. Biopolymers containing fucose and sulphated functional groups were found in all AOM samples while the presence of other functional groups varied between different species. A large majority (>80%) of the acidic polysaccharide components (in terms of transparent exopolymer particles) were found in the colloidal size range (<0.4 μm). The relative stickiness of AOM substantially varied between algal species and that the cohesion between AOM-coated surfaces was much stronger than the adhesion of AOM on AOM-free surfaces. Overall, the composition as well as the physico-chemical characteristics (e.g., stickiness) of AOM will likely dictate the severity of fouling in membrane systems during algal blooms. PMID:25682049

  13. Characterisation of algal organic matter produced by bloom-forming marine and freshwater algae.

    PubMed

    Villacorte, L O; Ekowati, Y; Neu, T R; Kleijn, J M; Winters, H; Amy, G; Schippers, J C; Kennedy, M D

    2015-04-15

    Algal blooms can seriously affect the operation of water treatment processes including low pressure (micro- and ultra-filtration) and high pressure (nanofiltration and reverse osmosis) membranes mainly due to accumulation of algal-derived organic matter (AOM). In this study, the different components of AOM extracted from three common species of bloom-forming algae (Alexandrium tamarense, Chaetoceros affinis and Microcystis sp.) were characterised employing various analytical techniques, such as liquid chromatography - organic carbon detection, fluorescence spectroscopy, fourier transform infrared spectroscopy, alcian blue staining and lectin staining coupled with laser scanning microscopy to indentify its composition and force measurement using atomic force microscopy to measure its stickiness. Batch culture monitoring of the three algal species illustrated varying characteristics in terms of growth pattern, cell concentration and AOM release. The AOM produced by the three algal species comprised mainly biopolymers (e.g., polysaccharides and proteins) but some refractory compounds (e.g., humic-like substances) and other low molecular weight acid and neutral compounds were also found. Biopolymers containing fucose and sulphated functional groups were found in all AOM samples while the presence of other functional groups varied between different species. A large majority (>80%) of the acidic polysaccharide components (in terms of transparent exopolymer particles) were found in the colloidal size range (<0.4 μm). The relative stickiness of AOM substantially varied between algal species and that the cohesion between AOM-coated surfaces was much stronger than the adhesion of AOM on AOM-free surfaces. Overall, the composition as well as the physico-chemical characteristics (e.g., stickiness) of AOM will likely dictate the severity of fouling in membrane systems during algal blooms.

  14. 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…

  15. 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.

  16. Cytotoxicity effects of amiodarone on cultured cells.

    PubMed

    Golli-Bennour, Emna El; Bouslimi, Amel; Zouaoui, Olfa; Nouira, Safa; Achour, Abdellatif; Bacha, Hassen

    2012-07-01

    Amiodarone is a potent anti-arrhythmic drug used for the treatment of cardiac arrhythmias. Although, the effects of amiodarone are well characterized on post-ischemic heart and cardiomyocytes, its toxicity on extra-cardiac tissues is still poorly understood. To this aim, we have monitored the cytotoxicity effects of this drug on three cultured cell lines including hepatocytes (HepG2), epithelial cells (EAhy 926) and renal cells (Vero). We have investigated the effects of amiodarone on (i) cell viabilities, (ii) heat shock protein expressions (Hsp 70) as a parameter of protective and adaptive response and (iii) oxidative damage.Our results clearly showed that amiodarone inhibits cell proliferation, induces an over-expression of Hsp 70 and generates significant amount of reactive oxygen species as measured by lipid peroxidation occurrence. However, toxicity of amiodarone was significantly higher in renal and epithelial cells than in hepatocytes. Vitamin E supplement restores the major part of cell mortalities induced by amiodarone showing that oxidative damage is the predominant toxic effect of the drug.Except its toxicity for the cardiac system, our findings demonstrated that amiodarone can target other tissues. Therefore, kidneys present a high sensibility to this drug which may limit its use with subjects suffering from renal disorders.

  17. Traditional and Modern Cell Culture in Virus Diagnosis.

    PubMed

    Hematian, Ali; Sadeghifard, Nourkhoda; Mohebi, Reza; Taherikalani, Morovat; Nasrolahi, Abbas; Amraei, Mansour; Ghafourian, Sobhan

    2016-04-01

    Cell cultures are developed from tissue samples and then disaggregated by mechanical, chemical, and enzymatic methods to extract cells suitable for isolation of viruses. With the recent advances in technology, cell culture is considered a gold standard for virus isolation. This paper reviews the evolution of cell culture methods and demonstrates why cell culture is a preferred method for identification of viruses. In addition, the advantages and disadvantages of both traditional and modern cell culture methods for diagnosis of each type of virus are discussed. Detection of viruses by the novel cell culture methods is considered more accurate and sensitive. However, there is a need to include some more accurate methods such as molecular methods in cell culture for precise identification of viruses.

  18. Traditional and Modern Cell Culture in Virus Diagnosis

    PubMed Central

    Hematian, Ali; Sadeghifard, Nourkhoda; Mohebi, Reza; Taherikalani, Morovat; Nasrolahi, Abbas; Amraei, Mansour; Ghafourian, Sobhan

    2016-01-01

    Cell cultures are developed from tissue samples and then disaggregated by mechanical, chemical, and enzymatic methods to extract cells suitable for isolation of viruses. With the recent advances in technology, cell culture is considered a gold standard for virus isolation. This paper reviews the evolution of cell culture methods and demonstrates why cell culture is a preferred method for identification of viruses. In addition, the advantages and disadvantages of both traditional and modern cell culture methods for diagnosis of each type of virus are discussed. Detection of viruses by the novel cell culture methods is considered more accurate and sensitive. However, there is a need to include some more accurate methods such as molecular methods in cell culture for precise identification of viruses. PMID:27169004

  19. [Culture of mussel Mytiuls edulis I. mantle cells].

    PubMed

    Daugavet, M A; Blinova, M I

    2015-01-01

    To date, cell lines derived from marine invertebrates have not been available. Hence primary cell cultures serve as model systems for various experiments. In present study we established primary culture of mussel Mytilus edulis L. mantle cells. Cells were isolated by means of explant culture or enzymatic dissociation of mantle tissue. They maintained viability up to 22 months regardless of culture initiation method. In course of culturing, cells, which were transferred onto new plates, successfully attached to a new surface. Physiological activity of cultured cells was also confirmed by formation of crystals, which appeared after 4-6 months. After continuous time of culturing, mantle cells can be cryopreserved using 5 % DMSO with post-freezing survival up to 50%. These results demonstrate that M. edulis mantle cells can maintain viability and physiological activity for exceptionally long time and can be cryopreserved for further examination.

  20. Cell culture device using spatial light modulator

    NASA Astrophysics Data System (ADS)

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

    2009-07-01

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

  1. Rotating bio-reactor cell culture apparatus

    NASA Technical Reports Server (NTRS)

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

    1991-01-01

    A bioreactor system is described in which a tubular housing contains an internal circularly disposed set of blade members and a central tubular filter all mounted for rotation about a common horizontal axis and each having independent rotational support and rotational drive mechanisms. The housing, blade members and filter preferably are driven at a constant slow speed for placing a fluid culture medium with discrete microbeads and cell cultures in a discrete spatial suspension in the housing. Replacement fluid medium is symmetrically input and fluid medium is symmetrically output from the housing where the input and the output are part of a loop providing a constant or intermittent flow of fluid medium in a closed loop.

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

    PubMed

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

    2015-12-01

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

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

    PubMed

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

    2015-12-01

    Perivascular cells are multilineage cells located around the vessel wall and important for wall stabilization. In this study, we evaluated a stem cell media and a perivascular cell-specific media for the culture of primary perivascular cells regarding their cell morphology, doubling time, stem cell properties, and expression of cell type-specific markers. When the two cell culture media were compared to each other, perivascular cells cultured in the stem cell medium had a more elongated morphology and a faster doubling rate and cells cultured in the pericyte medium had a more typical morphology, with several filopodia, and a slower doubling rate. To evaluate stem cell properties, perivascular cells, CD146(-) cells, and mesenchymal stem cells (MSCs) were differentiated into the adipogenic, osteogenic, and chondrogenic lineages. It was seen that perivascular cells, as well as CD146(-) cells and MSCs, cultured in stem cell medium showed greater differentiation than cells cultured in pericyte-specific medium. The expression of pericyte-specific markers CD146, neural/glial antigen 2 (NG2), platelet-derived growth factor receptor-β (PDGFR-β), myosin, and α-smooth muscle actin (α-SMA) could be found in both pericyte cultures, as well as to varying amounts in CD146(-) cells, MSCs, and endothelial cells. The here presented work shows that perivascular cells can adapt to their in vitro environment and cell culture conditions influence cell functionality, such as doubling rate or differentiation behavior. Pericyte-specific markers were shown to be expressed also from cells other than perivascular cells. We can further conclude that CD146(+) perivascular cells are inhomogeneous cell population probably containing stem cell subpopulations, which are located perivascular around capillaries. PMID:26179857

  4. Algal Biofuels Fact Sheet

    SciTech Connect

    2009-10-27

    This fact sheet provides information on algal biofuels, which are generating considerable interest around the world. They may represent a sustainable pathway for helping to meet the U.S. biofuel production targets set by the Energy Independence and Security Act of 2007.

  5. Metabolomic profiling of cultured cancer cells.

    PubMed

    Scoazec, Marie; Durand, Sylvere; Chery, Alexis; Galluzzi, Lorenzo; Kroemer, Guido

    2014-01-01

    Quantitative proteomics approaches have been developed-and now begin to be implemented on a high-throughput basis-to fill-in the large gap between the genomic/transcriptomic setup of (cancer) cells and their phenotypic/behavioral traits, reflecting a significant degree of posttranscriptional regulation in gene expression as well as a robust posttranslational regulation of protein function. However, proteomic profiling assays not only fail to detect labile posttranslational modifications as well as unstable protein-to-protein interactions but also are intrinsically incapable of assessing the enzymatic activity, as opposed to the mere abundance, of a given protein. Thus, determining the abundance of theoretically all the metabolites contained in a cell/tissue/organ/organism may significantly improve the informational value of proteomic approaches. Several techniques have been developed to this aim, including high-performance liquid chromatography (HPLC) coupled to quadrupole time-of-flight (Q-TOF) high-resolution mass spectrometry (HRMS). This approach is particularly advantageous for metabolomic profiling as it offers elevated accuracy and improved sensitivity. Here, we describe a simple procedure to determine the complete complement of intracellular metabolites in cultured malignant cells by HPLC coupled to Q-TOF HRMS. According to this method, (1) cells are collected and processed to minimize contaminations as well as fluctuations in their metabolic profile; (2) samples are separated by HPLC and analyzed on a Q-TOF spectrometer; and (3) data are extracted, normalized, and deconvoluted according to refined mathematical methods. This protocol constitutes a simple approach to determine the intracellular metabolomic profile of cultured cancer cells. With minimal variations (mostly related to sample collection and processing), this method is expected to provide reliable metabolomic data on a variety of cellular samples.

  6. Change in Photosystem II Photochemistry During Algal Growth Phases of Chlorella vulgaris and Scenedesmus obliquus.

    PubMed

    Oukarroum, Abdallah

    2016-06-01

    Sensitivity of photosynthetic processes towards environmental stress is used as a bioanalytical tool to evaluate the responses of aquatic plants to a changing environment. In this paper, change of biomass density, chlorophyll a fluorescence and photosynthetic parameters during growth phases of two microalgae Chlorella vulgaris and Scenedesmus obliquus were studied. The photosynthetic growth behaviour changed significantly with cell age and algae species. During the exponential phase of growth, the photosynthesis capacity reached its maximum and decreased in ageing algal culture during stationary phase. In conclusion, the chlorophyll a fluorescence OJIP method and the derived fluorescence parameters would be an accurate method for obtaining information on maximum photosynthetic capacities and monitoring algal cell growth. This will contribute to more understanding, for example, of toxic actions of pollutants in microalgae test. PMID:26868257

  7. Neutralization of Chlamydia trachomatis in cell culture.

    PubMed Central

    Howard, L V

    1975-01-01

    Neutralization of Chlamydia trachomatis was assayed by the decrease in inclusion-forming units in baby hamster kidney cells grown in culture. Five percent fresh guinea pig sera increased neutralization titers of rabbit antisera 100- to 1,000-fold but had no effect when normal rabbit sera were tested. Neutralization of a type A or B trachoma isolate was strain specific. Neutralization by human eye secretions and sera also was demonstrated when guinea pig sera were included in the test. All of the six human sera tested showed strain specificity against types A or B, in agreement with typing by the fluorescent antibody technique. PMID:1091549

  8. Toward a cell-culture model of cancer

    SciTech Connect

    Kraemer, P.M.

    1984-08-01

    The spontaneous transformation of normal Chinese hamster cells into malignant cells was studied. Hamster cells when grown in culture over an extended series of subcultivations inevitably become malignant even without exposure to a carcinogen. The study of this process using flow cytometry and the implantation of cultured cells into retrievable sponges placed under the skin of a nude mouse is described. (ACR)

  9. Cardiac Cells Beating in Culture: A Laboratory Exercise

    ERIC Educational Resources Information Center

    Weaver, Debora

    2007-01-01

    This article describes how to establish a primary tissue culture, where cells are taken directly from an organ of a living animal. Cardiac cells are taken from chick embryos and transferred to culture dishes. These cells are not transformed and therefore have a limited life span. However, the unique characteristics of cardiac cells are maintained…

  10. Human norovirus culture in B cells.

    PubMed

    Jones, Melissa K; Grau, Katrina R; Costantini, Veronica; Kolawole, Abimbola O; de Graaf, Miranda; Freiden, Pamela; Graves, Christina L; Koopmans, Marion; Wallet, Shannon M; Tibbetts, Scott A; Schultz-Cherry, Stacey; Wobus, Christiane E; Vinjé, Jan; Karst, Stephanie M

    2015-12-01

    Human noroviruses (HuNoVs) are a leading cause of foodborne disease and severe childhood diarrhea, and they cause a majority of the gastroenteritis outbreaks worldwide. However, the development of effective and long-lasting HuNoV vaccines and therapeutics has been greatly hindered by their uncultivability. We recently demonstrated that a HuNoV replicates in human B cells, and that commensal bacteria serve as a cofactor for this infection. In this protocol, we provide detailed methods for culturing the GII.4-Sydney HuNoV strain directly in human B cells, and in a coculture system in which the virus must cross a confluent epithelial barrier to access underlying B cells. We also describe methods for bacterial stimulation of HuNoV B cell infection and for measuring viral attachment to the surface of B cells. Finally, we highlight variables that contribute to the efficiency of viral replication in this system. Infection assays require 3 d and attachment assays require 3 h. Analysis of infection or attachment samples, including RNA extraction and RT-qPCR, requires ∼6 h.

  11. Equipment for large-scale mammalian cell culture.

    PubMed

    Ozturk, Sadettin S

    2014-01-01

    This chapter provides information on commonly used equipment in industrial mammalian cell culture, with an emphasis on bioreactors. The actual equipment used in the cell culture process can vary from one company to another, but the main steps remain the same. The process involves expansion of cells in seed train and inoculation train processes followed by cultivation of cells in a production bioreactor. Process and equipment options for each stage of the cell culture process are introduced and examples are provided. Finally, the use of disposables during seed train and cell culture production is discussed. PMID:24429549

  12. Equipment for large-scale mammalian cell culture.

    PubMed

    Ozturk, Sadettin S

    2014-01-01

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

  13. Metabolic flux rewiring in mammalian cell cultures

    PubMed Central

    Young, Jamey D.

    2013-01-01

    Continuous cell lines (CCLs) engage in “wasteful” glucose and glutamine metabolism that leads to accumulation of inhibitory byproducts, primarily lactate and ammonium. Advances in techniques for mapping intracellular carbon fluxes and profiling global changes in enzyme expression have led to a deeper understanding of the molecular drivers underlying these metabolic alterations. However, recent studies have revealed that CCLs are not necessarily entrenched in a glycolytic or glutaminolytic phenotype, but instead can shift their metabolism toward increased oxidative metabolism as nutrients become depleted and/or growth rate slows. Progress to understand dynamic flux regulation in CCLs has enabled the development of novel strategies to force cultures into desirable metabolic phenotypes, by combining fed-batch feeding strategies with direct metabolic engineering of host cells. PMID:23726154

  14. 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.

  15. 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.

  16. Recombinant Protein Production and Insect Cell Culture and Process

    NASA Technical Reports Server (NTRS)

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

    1997-01-01

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

  17. Algal functional annotation tool

    SciTech Connect

    2012-07-12

    Abstract BACKGROUND: Progress in genome sequencing is proceeding at an exponential pace, and several new algal genomes are becoming available every year. One of the challenges facing the community is the association of protein sequences encoded in the genomes with biological function. While most genome assembly projects generate annotations for predicted protein sequences, they are usually limited and integrate functional terms from a limited number of databases. Another challenge is the use of annotations to interpret large lists of 'interesting' genes generated by genome-scale datasets. Previously, these gene lists had to be analyzed across several independent biological databases, often on a gene-by-gene basis. In contrast, several annotation databases, such as DAVID, integrate data from multiple functional databases and reveal underlying biological themes of large gene lists. While several such databases have been constructed for animals, none is currently available for the study of algae. Due to renewed interest in algae as potential sources of biofuels and the emergence of multiple algal genome sequences, a significant need has arisen for such a database to process the growing compendiums of algal genomic data. DESCRIPTION: The Algal Functional Annotation Tool is a web-based comprehensive analysis suite integrating annotation data from several pathway, ontology, and protein family databases. The current version provides annotation for the model alga Chlamydomonas reinhardtii, and in the future will include additional genomes. The site allows users to interpret large gene lists by identifying associated functional terms, and their enrichment. Additionally, expression data for several experimental conditions were compiled and analyzed to provide an expression-based enrichment search. A tool to search for functionally-related genes based on gene expression across these conditions is also provided. Other features include dynamic visualization of genes on KEGG

  18. Algal functional annotation tool

    2012-07-12

    Abstract BACKGROUND: Progress in genome sequencing is proceeding at an exponential pace, and several new algal genomes are becoming available every year. One of the challenges facing the community is the association of protein sequences encoded in the genomes with biological function. While most genome assembly projects generate annotations for predicted protein sequences, they are usually limited and integrate functional terms from a limited number of databases. Another challenge is the use of annotations tomore » interpret large lists of 'interesting' genes generated by genome-scale datasets. Previously, these gene lists had to be analyzed across several independent biological databases, often on a gene-by-gene basis. In contrast, several annotation databases, such as DAVID, integrate data from multiple functional databases and reveal underlying biological themes of large gene lists. While several such databases have been constructed for animals, none is currently available for the study of algae. Due to renewed interest in algae as potential sources of biofuels and the emergence of multiple algal genome sequences, a significant need has arisen for such a database to process the growing compendiums of algal genomic data. DESCRIPTION: The Algal Functional Annotation Tool is a web-based comprehensive analysis suite integrating annotation data from several pathway, ontology, and protein family databases. The current version provides annotation for the model alga Chlamydomonas reinhardtii, and in the future will include additional genomes. The site allows users to interpret large gene lists by identifying associated functional terms, and their enrichment. Additionally, expression data for several experimental conditions were compiled and analyzed to provide an expression-based enrichment search. A tool to search for functionally-related genes based on gene expression across these conditions is also provided. Other features include dynamic visualization of genes on

  19. Three-dimensional culture may promote cell reprogramming.

    PubMed

    Han, Jin; Chen, Lei; Luo, Guanzheng; Dai, Bin; Wang, Xiujie; Dai, Jianwu

    2013-01-01

    Stem cells reside in stem cells niches, which maintain the balance of self-renewal and differentiation of stem cells. In stem cell niches, cell-cell, cell-extracellular matrix interactions and diffusible signals are important elements. However, another pivotal element is that localized and diffusible signals are all organized as three-dimensional (3-D) structures, which is easily neglected by in vitro cell biology research. Under 3-D culture conditions, the morphology of cells exhibited differently from cultured in traditional two-dimensional (2-D) conditions. Under 3-D culture conditions, the self-renewal and pluripotency of neural stem cells (NSCs) and bone marrow mesenchymal stem cells (BMSCs) were enhanced compared with culturing under 2-D conditions. 3-D cultures could change the transcriptional profile of NSCs compared with 2-D cultures. We hypothesized that 3-D cultures could reprogram mature cells such as fibroblasts to an immature state, like the pluripotent stem cells. The primary results indicated that several ES marker genes were upregulated by 3-D cultures. Though further experiments are needed, this work may provide a method of reprogramming mature cells without gene modifications. PMID:23820263

  20. Three-dimensional cell culturing by magnetic levitation.

    PubMed

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

    2013-10-01

    Recently, biomedical research has moved toward cell culture in three dimensions to better recapitulate native cellular environments. This protocol describes one method for 3D culture, the magnetic levitation method (MLM), in which cells bind with a magnetic nanoparticle assembly overnight to render them magnetic. When resuspended in medium, an external magnetic field levitates and concentrates cells at the air-liquid interface, where they aggregate to form larger 3D cultures. The resulting cultures are dense, can synthesize extracellular matrix (ECM) and can be analyzed similarly to the other culture systems using techniques such as immunohistochemical analysis (IHC), western blotting and other biochemical assays. This protocol details the MLM and other associated techniques (cell culture, imaging and IHC) adapted for the MLM. The MLM requires 45 min of working time over 2 d to create 3D cultures that can be cultured in the long term (>7 d). PMID:24030442

  1. Density gradient electrophoresis of cultured human embryonic kidney cells

    NASA Technical Reports Server (NTRS)

    Plank, L. D.; Kunze, M. E.; Giranda, V.; Todd, P. W.

    1985-01-01

    Ground based confirmation of the electrophoretic heterogeneity of human embryonic kidney cell cultures, the general characterization of their electrophoretic migration, and observations on the general properties of cultures derived from electrophoretic subpopulations were studied. Cell migration in a density gradient electrophoresis column and cell electrophoretic mobility was determined. The mobility and heterogeneity of cultured human embryonic kidney cells with those of fixed rat erythrocytes as model test particle was compared. Electrophoretically separated cell subpopulations with respect to size, viability, and culture characteristics were examined.

  2. Shallow Algal Mass Culture Systems for the Production of Oils: Final Report on Work Carried Out 8/16/84 - 6/15/85

    SciTech Connect

    Laws, E. A.

    1985-01-01

    The objective of this project was to improve the technology of outdoor mass culture of microa1gae for oil production by investigation of species/strains, optimization of culture conditions and development of strategies that increase efficiency and improve yield.

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

    PubMed

    Li, Bo-Jiang; Li, Ping-Hua; Huang, Rui-Hua; Sun, Wen-Xing; Wang, Han; Li, Qi-Fa; Chen, Jie; Wu, Wang-Jun; Liu, Hong-Lin

    2015-08-01

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

  4. Gravity, chromosomes, and organized development in aseptically cultured plant cells

    NASA Technical Reports Server (NTRS)

    Krikorian, Abraham D.

    1993-01-01

    The objectives of the PCR experiment are: to test the hypothesis that microgravity will in fact affect the pattern and developmental progression of embryogenically competent plant cells from one well-defined, critical stage to another; to determine the effects of microgravity in growth and differentiation of embryogenic carrot cells grown in cell culture; to determine whether microgravity or the space environment fosters an instability of the differentiated state; and to determine whether mitosis and chromosome behavior are adversely affected by microgravity. The methods employed will consist of the following: special embryogenically competent carrot cell cultures will be grown in cell culture chambers provided by NASDA; four cell culture chambers will be used to grow cells in liquid medium; two dishes (plant cell culture dishes) will be used to grow cells on a semi-solid agar support; progression to later embryonic stages will be induced in space via crew intervention and by media manipulation in the case of liquid grown cell cultures; progression to later stages in case of semi-solid cultures will not need crew intervention; embryo stages will be fixed at a specific interval (day 6) in flight only in the case of liquid-grown cultures; and some living cells and somatic embryos will be returned for continued post-flight development and 'grown-out.' These will derive from the semi-solid grown cultures.

  5. Algal sensory photoreceptors.

    PubMed

    Hegemann, Peter

    2008-01-01

    Only five major types of sensory photoreceptors (BLUF-proteins, cryptochromes, phototropins, phytochromes, and rhodopsins) are used in nature to regulate developmental processes, photosynthesis, photoorientation, and control of the circadian clock. Sensory photoreceptors of algae and protists are exceptionally rich in structure and function; light-gated ion channels and photoactivated adenylate cyclases are unique examples. During the past ten years major progress has been made with respect to understanding the function, photochemistry, and structure of key sensory players of the algal kingdom.

  6. Cholera toxin stimulation of human mammary epithelial cells in culture

    SciTech Connect

    Stampfer, M.R.

    1982-06-01

    Addition of cholera toxin to human mammary epithelial cultures derived from reduction mammoplasties and primary carcinomas greatly stimulated cell growth and increased the number of times the cells could be successfully subcultured. Other agents known to increase intracellular cAMP levels were also growth stimulatory. The increased growth potential conferred by cholera toxin enhances the usefulness of this cell culture system.

  7. Three-Dimensional Cell Culture: A Breakthrough in Vivo

    PubMed Central

    Antoni, Delphine; Burckel, Hélène; Josset, Elodie; Noel, Georges

    2015-01-01

    Cell culture is an important tool for biological research. Two-dimensional cell culture has been used for some time now, but growing cells in flat layers on plastic surfaces does not accurately model the in vivo state. As compared to the two-dimensional case, the three-dimensional (3D) cell culture allows biological cells to grow or interact with their surroundings in all three dimensions thanks to an artificial environment. Cells grown in a 3D model have proven to be more physiologically relevant and showed improvements in several studies of biological mechanisms like: cell number monitoring, viability, morphology, proliferation, differentiation, response to stimuli, migration and invasion of tumor cells into surrounding tissues, angiogenesis stimulation and immune system evasion, drug metabolism, gene expression and protein synthesis, general cell function and in vivo relevance. 3D culture models succeed thanks to technological advances, including materials science, cell biology and bioreactor design. PMID:25768338

  8. To grow mouse mammary epithelial cells in culture

    PubMed Central

    1984-01-01

    Normal mouse mammary epithelial cells from Balb/c mice were successfully cultivated on tissue culture plastic with lethally irradiated LA7 feeder cells. The feeder cells also promoted colony formation from single mouse mammary cells, and the fraction of cells that formed colonies was proportional to the density of feeder cells. The mouse mammary cells could be passaged at least 8-12 times as long as new feeder cells were added at each passage. The cells now in culture have doubled in number at least 30 times, but the in vitro lifespan is not yet known. The cultures of mouse cells maintained by this technique never became overgrown with fibroblasts and numerous domes formed in the cultures. PMID:6699079

  9. Cell and tissue culture of Miscanthus Sacchariflorus

    SciTech Connect

    Godovikova, V.A.; Moiseyeva, E.A.; Shumny, V.K.

    1995-11-01

    Since recent time search and introduction of new species of plants have paid attention. More perspective are perennial low maintenance landscape plants from genera Phragmites L. and Miscanthus Anderss. known as high speed growing and great amount of cellulose`s containing. Absence of seeds production and limited distribution area prevent from immediately introduction the plants of this species. The main goal of our investigation is the scientific development of the cell and tissue culture methods to get changing clones, salt and cold tolerant plants and their micropogation. At present there are collection of biovariety represented by subspecies, ecotypes and plant regenerants of two species - Miscanthus purpurascens (Anders.) and Miscanthus sacchariflorus (Maxim.). Successful results have been achieved in screening of culture media, prepared on MS base medium and contained a row of tropic components to protect the explant and callus tissue from oxidation and necrosis. Initially the callus was induced from stem segments, apical and nodular meristem of vegetative shoots of elulalia, growing in hydroponic greenhouse. Morphological and cytologic analysis of plant-regenerants have been done.

  10. Reduction of freeze-thaw-induced hemolysis of red blood cells by an algal ice-binding protein.

    PubMed

    Kang, Jae-Shin; Raymond, James A

    2004-01-01

    Antarctic sea ice diatoms produce ice-binding proteins (IBPs) that are strong inhibitors of the recrystallization of ice. Their function may be to reduce cell damage in the frozen state. We show here that an IBP from the diatom Navicula glaciei Vanheurck also has the ability to reduce freeze-thaw damage to red blood cells and that the effect may be due to its ability to inhibit recrystallization of ice.

  11. National Algal Biofuels Technology Roadmap

    SciTech Connect

    Ferrell, John; Sarisky-Reed, Valerie

    2010-05-01

    The framework for National Algal Biofuels Technology Roadmap was constructed at the Algal Biofuels Technology Roadmap Workshop, held December 9-10, 2008, at the University of Maryland-College Park. The Workshop was organized by the Biomass Program to discuss and identify the critical challenges currently hindering the development of a domestic, commercial-scale algal biofuels industry. This Roadmap presents information from a scientific, economic, and policy perspectives that can support and guide RD&D investment in algal biofuels. While addressing the potential economic and environmental benefits of using algal biomass for the production of liquid transportation fuels, the Roadmap describes the current status of algae RD&D. In doing so, it lays the groundwork for identifying challenges that likely need to be overcome for algal biomass to be used in the production of economically viable biofuels.

  12. Skeletal muscle satellite cells cultured in simulated microgravity

    NASA Technical Reports Server (NTRS)

    Molnar, Greg; Hartzell, Charles R.; Schroedl, Nancy A.; Gonda, Steve R.

    1993-01-01

    Satellite cells are postnatal myoblasts responsible for providing additional nuclei to growing or regenerating muscle cells. Satellite cells retain the capacity to proliferate and differentiate in vitro and therefore provide a useful model to study postnatal muscle development. Most culture systems used to study postnatal muscle development are limited by the two-dimensional (2-D) confines of the culture dish. Limiting proliferation and differentiation of satellite cells in 2-D could potentially limit cell-cell contacts important for developing the level of organization in skeletal muscle obtained in vivo. Culturing satellite cells on microcarrier beads suspended in the High-Aspect-Ratio-Vessel (HARV) designed by NASA provides a low shear, three-dimensional (3-D) environment to study muscle development. Primary cultures established from anterior tibialis muscles of growing rats (approximately 200 gm) were used for all studies and were composed of greater than 75 % satellite cells. Different inoculation densities did not affect the proliferative potential of satellite cells in the HARV. Plating efficiency, proliferation, and glucose utilization were compared between 2-D flat culture and 3-D HARV culture. Plating efficiency (cells attached - cells plated x 100) was similar between the two culture systems. Proliferation was reduced in HARV cultures and this reduction was apparent for both satellite cells and non-satellite cells. Furthermore, reduction in proliferation within the HARV could not be attributed to reduced substrate availability since glucose levels in media from HARV and 2-D cell culture were similar. Morphologically, microcarrier beads within the HARVS were joined together by cells into three-dimensional aggregates composed of greater than 10 beads/aggregate. Aggregation of beads did not occur in the absence of cells. Myotubes were often seen on individual beads or spanning the surface of two beads. In summary, proliferation and differentiation of

  13. Three-dimensional tissue culture based on magnetic cell levitation.

    PubMed

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

    2010-04-01

    Cell culture is an essential tool in drug discovery, tissue engineering and stem cell research. Conventional tissue culture produces two-dimensional cell growth with gene expression, signalling and morphology that can be different from those found in vivo, and this compromises its clinical relevance. Here, we report a three-dimensional tissue culture based on magnetic levitation of cells in the presence of a hydrogel consisting of gold, magnetic iron oxide nanoparticles and filamentous bacteriophage. By spatially controlling the magnetic field, the geometry of the cell mass can be manipulated, and multicellular clustering of different cell types in co-culture can be achieved. Magnetically levitated human glioblastoma cells showed similar protein expression profiles to those observed in human tumour xenografts. Taken together, these results indicate that levitated three-dimensional culture with magnetized phage-based hydrogels more closely recapitulates in vivo protein expression and may be more feasible for long-term multicellular studies. PMID:20228788

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

    PubMed Central

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

    2015-01-01

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

  15. Particle Trajectories in Rotating Wall Cell Culture Devices

    NASA Technical Reports Server (NTRS)

    Ramachandran N.; Downey, J. P.

    1999-01-01

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

  16. Phylogenetic analysis of algal symbionts associated with four North American amphibian egg masses.

    PubMed

    Kim, Eunsoo; Lin, Yuan; Kerney, Ryan; Blumenberg, Lili; Bishop, Cory

    2014-01-01

    Egg masses of the yellow-spotted salamander Ambystoma maculatum form an association with the green alga "Oophila amblystomatis" (Lambert ex Wille), which, in addition to growing within individual egg capsules, has recently been reported to invade embryonic tissues and cells. The binomial O. amblystomatis refers to the algae that occur in A. maculatum egg capsules, but it is unknown whether this population of symbionts constitutes one or several different algal taxa. Moreover, it is unknown whether egg masses across the geographic range of A. maculatum, or other amphibians, associate with one or multiple algal taxa. To address these questions, we conducted a phylogeographic study of algae sampled from egg capsules of A. maculatum, its allopatric congener A. gracile, and two frogs: Lithobates sylvatica and L. aurora. All of these North American amphibians form associations with algae in their egg capsules. We sampled algae from egg capsules of these four amphibians from localities across North America, established representative algal cultures, and amplified and sequenced a region of 18S rDNA for phylogenetic analysis. Our combined analysis shows that symbiotic algae found in egg masses of four North American amphibians are closely related to each other, and form a well-supported clade that also contains three strains of free-living chlamydomonads. We designate this group as the 'Oophila' clade, within which the symbiotic algae are further divided into four distinct subclades. Phylogenies of the host amphibians and their algal symbionts are only partially congruent, suggesting that host-switching and co-speciation both play roles in their associations. We also established conditions for isolating and rearing algal symbionts from amphibian egg capsules, which should facilitate further study of these egg mass specialist algae. PMID:25393119

  17. Phylogenetic analysis of algal symbionts associated with four North American amphibian egg masses.

    PubMed

    Kim, Eunsoo; Lin, Yuan; Kerney, Ryan; Blumenberg, Lili; Bishop, Cory

    2014-01-01

    Egg masses of the yellow-spotted salamander Ambystoma maculatum form an association with the green alga "Oophila amblystomatis" (Lambert ex Wille), which, in addition to growing within individual egg capsules, has recently been reported to invade embryonic tissues and cells. The binomial O. amblystomatis refers to the algae that occur in A. maculatum egg capsules, but it is unknown whether this population of symbionts constitutes one or several different algal taxa. Moreover, it is unknown whether egg masses across the geographic range of A. maculatum, or other amphibians, associate with one or multiple algal taxa. To address these questions, we conducted a phylogeographic study of algae sampled from egg capsules of A. maculatum, its allopatric congener A. gracile, and two frogs: Lithobates sylvatica and L. aurora. All of these North American amphibians form associations with algae in their egg capsules. We sampled algae from egg capsules of these four amphibians from localities across North America, established representative algal cultures, and amplified and sequenced a region of 18S rDNA for phylogenetic analysis. Our combined analysis shows that symbiotic algae found in egg masses of four North American amphibians are closely related to each other, and form a well-supported clade that also contains three strains of free-living chlamydomonads. We designate this group as the 'Oophila' clade, within which the symbiotic algae are further divided into four distinct subclades. Phylogenies of the host amphibians and their algal symbionts are only partially congruent, suggesting that host-switching and co-speciation both play roles in their associations. We also established conditions for isolating and rearing algal symbionts from amphibian egg capsules, which should facilitate further study of these egg mass specialist algae.

  18. Phylogenetic Analysis of Algal Symbionts Associated with Four North American Amphibian Egg Masses

    PubMed Central

    Kim, Eunsoo; Lin, Yuan; Kerney, Ryan; Blumenberg, Lili; Bishop, Cory

    2014-01-01

    Egg masses of the yellow-spotted salamander Ambystoma maculatum form an association with the green alga “Oophila amblystomatis” (Lambert ex Wille), which, in addition to growing within individual egg capsules, has recently been reported to invade embryonic tissues and cells. The binomial O. amblystomatis refers to the algae that occur in A. maculatum egg capsules, but it is unknown whether this population of symbionts constitutes one or several different algal taxa. Moreover, it is unknown whether egg masses across the geographic range of A. maculatum, or other amphibians, associate with one or multiple algal taxa. To address these questions, we conducted a phylogeographic study of algae sampled from egg capsules of A. maculatum, its allopatric congener A. gracile, and two frogs: Lithobates sylvatica and L. aurora. All of these North American amphibians form associations with algae in their egg capsules. We sampled algae from egg capsules of these four amphibians from localities across North America, established representative algal cultures, and amplified and sequenced a region of 18S rDNA for phylogenetic analysis. Our combined analysis shows that symbiotic algae found in egg masses of four North American amphibians are closely related to each other, and form a well-supported clade that also contains three strains of free-living chlamydomonads. We designate this group as the ‘Oophila’ clade, within which the symbiotic algae are further divided into four distinct subclades. Phylogenies of the host amphibians and their algal symbionts are only partially congruent, suggesting that host-switching and co-speciation both play roles in their associations. We also established conditions for isolating and rearing algal symbionts from amphibian egg capsules, which should facilitate further study of these egg mass specialist algae. PMID:25393119

  19. High resolution imaging of the ultrastructure of living algal cells using soft x-ray contact microscopy

    SciTech Connect

    Ford, T.W.; Cotton, R.A.; Page, A.M.; Tomie, T.; Majima, T.; Stead, A.D.

    1995-12-31

    Soft x-ray contact microscopy provides the biologist with a technique for examining the ultrastructure of living cells at a much higher resolution than that possible by various forms of light microscopy. Readout of the developed photoresist using atomic force microscopy (AFM) produces a detailed map of the carbon densities generated in the resist following exposure of the specimen to water-window soft x-rays (2--4nm) produced by impact of a high energy laser onto a suitable target. The established high resolution imaging method of transmission electron microscopy (TEM) has inherent problems in the chemical pre-treatment required for producing the ultrathin sections necessary for this technique. Using the unicellular green alga Chlamydomonas the ultrastructural appearance of the cells following SXCM and TEM has been compared. While SXCM confirms the basic structural organization of the cell as seen by TEM (e.g., the organization of the thylakoid membranes within the chloroplast; flagellar insertion into the cytoplasm), there are important differences. These are in the appearance of the cell covering and the presence of carbon-dense spherical cellular inclusions.

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

    PubMed

    Weathers, P J; Zobel, R W

    1992-01-01

    Characteristics of aeroponics are discussed. Contrast is made, where appropriate, with hydroponics and aero-hydroponics as applies to research and commercial applications of nutrient mist technology. Topics include whole plants, plant tissue cultures, cell and microbial cultures, and animal tissue cultures with regard to operational considerations (moisture, temperature, minerals, gaseous atmosphere) and design of apparati. PMID:14540802

  1. Development of Scalable Culture Systems for Human Embryonic Stem Cells

    PubMed Central

    Azarin, Samira M.; Palecek, Sean P.

    2009-01-01

    The use of human pluripotent stem cells, including embryonic and induced pluripotent stem cells, in therapeutic applications will require the development of robust, scalable culture technologies for undifferentiated cells. Advances made in large-scale cultures of other mammalian cells will facilitate expansion of undifferentiated human embryonic stem cells (hESCs), but challenges specific to hESCs will also have to be addressed, including development of defined, humanized culture media and substrates, monitoring spontaneous differentiation and heterogeneity in the cultures, and maintaining karyotypic integrity in the cells. This review will describe our current understanding of environmental factors that regulate hESC self-renewal and efforts to provide these cues in various scalable bioreactor culture systems. PMID:20161686

  2. Genome Sequence of Mycoplasma hyorhinis Isolated from Cell Cultures

    PubMed Central

    Cibulski, Samuel Paulo; Siqueira, Franciele Maboni; Teixeira, Thais Fumaco; Mayer, Fabiana Quoos; Almeida, Luiz Gonzaga

    2016-01-01

    Mycoplasmas are major contaminants of mammalian cell cultures. Here, the complete genome sequence of Mycoplasma hyorhinis recovered from Madin-Darby bovine kidney (MDBK) cells is reported. PMID:27738034

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

    ERIC Educational Resources Information Center

    Bozzone, Donna M.

    1997-01-01

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

  4. Propagation of infectious salmon anaemia (ISA) virus in cell culture.

    PubMed

    Dannevig, B H; Falk, K; Press, C M

    1995-01-01

    A long-term cell line supporting growth of the infectious salmon anaemia (ISA) virus has been established. The cell line (SHK-1) was developed from a culture of head kidney leucocytes from Atlantic salmon, and exhibited macrophage-like enzyme reactivities. By means of transmission experiments, ISA infectivity of cell culture medium could be demonstrated from day 5 after infection of SHK-1 cells with ISA-infective tissue homogenate. ISA infectivity of cell culture medium increased following repeated passages of virus. ISA-infected cell cultures develop cytopathic effects (CPE), making quantitation of virus possible. The development of CPE in ISA virus infected cells was inhibited by ammonium chloride, chloroquine and bafilomycin A, suggesting that infection of SHK-1 cells with ISA virus requires a low-pH step. PMID:8581019

  5. Synthesis of Antihemophilic Factor Antigen by Cultured Human Endothelial Cells

    PubMed Central

    Jaffe, Eric A.; Hoyer, Leon W.; Nachman, Ralph L.

    1973-01-01

    Antihemophilic factor (AHF, Factor VIII) antigen has been demonstrated in cultured human endothelial cells by immunofluorescence studies using monospecific rabbit antibody to human AHF. Control studies with cultured human smooth muscle cells and human fibroblasts were negative. By radioimmunoassay it was demonstrated that cultured human endothelial cells contain AHF antigen which is released into the culture medium. Cultured smooth muscle cells and fibroblasts did not have this property. Cultured endothelial cells incorporated radioactive amino acids into high molecular weight, AHF antigen-rich protein fractions prepared from the culture media, 7% of the radioactive amino acid counts incorporated into this material were precipitated by globulin prepared from rabbit anti-AHF whereas normal rabbit globulin precipitated only 1.5% of the counts. Although cultured endothelial cells actively synthesize AHF antigen, AHF procoagulant activity was not detected in the culture medium. Studies seeking a basis for the lack of procoagulant activity have not clarified this deficiency, but they have established that exogenous AHF procoagulant activity is not inactivated by the tissue culture system. Images PMID:4583980

  6. Algal morphogenesis: modelling interspecific variation in Micrasterias with reaction--diffusion patterned catalysis of cell surface growth

    PubMed Central

    Holloway, D. M.

    1999-01-01

    Semi-cell morphogenesis in unicellular desmid algae of the genus Micrasterias generates a stellar shape by repeated dichotomous branching of growing tips of the cell surface. The numerous species of the genus display variations of the branching pattern that differ markedly in number of branchings, lobe width and lobe length. We have modelled this morphogenesis, following previous work by D. M. Harrison and M. Kolar (1988), on the assumptions that patterning occurs by chemical reaction-diffusion activity within the plasma membrane, leading to morphological expression by patterned catalysis of the extension of the cell surface. The latter has been simulated in simplified form by two-dimensional computations. Our results indicate that for generation of repeated branchings and for the control of diverse species-specific shapes, the loss of patterning activity and of rapid growth in regions separating the active growing tips is an essential feature. We believe this conclusion to be much more general than the specific details of our model. We discuss the limitations of the model especially in terms of what extra features might be addressed in three-dimensional computation.

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

    PubMed

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

    2008-06-01

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

  8. Biotransformation of hyoscyamine into scopolamine in transgenic tobacco cell cultures.

    PubMed

    Moyano, Elisabeth; Palazón, Javier; Bonfill, Mercedes; Osuna, Lidia; Cusidó, Rosa M; Oksman-Caldentey, Kirsi-Marja; Piñol, M Teresa

    2007-04-01

    Hyoscyamine-6beta-hydroxylase (H6H) catalyses the conversion of hyoscyamine into its epoxide scopolamine, a compound with a higher added value in the pharmaceutical market than hyoscyamine. We report the establishment of tobacco cell cultures carrying the Hyoscyamus muticus h6h gene under the control of the promoter CAMV 35S. The cell cultures were derived from hairy roots obtained via genetically modified Agrobacterium rhizogenes carrying the pRi and pLAL21 plasmids. The cultures were fed with hyoscyamine, and 4 weeks later the amount of scopolamine produced was quantified by HPLC. The transgenic cell suspension cultures showed a considerable capacity for the bioconversion of hyoscyamine into scopolamine, and released it to the culture medium. Although the scale-up from shake-flask to bioreactor culture usually results in reduced productivities, our transgenic cells grown in a 5-L turbine stirred tank reactor in a batch mode significantly increased the scopolamine accumulation.

  9. Rabbit uterine epithelial cells: Co-culture with spermatozoa

    SciTech Connect

    Boice, M.L.

    1988-01-01

    A primary culture of rabbit uterine epithelial cells was established and their effects on sperm function were examined in vitro. Epithelial cells were isolated from uteri of estrous rabbits and cultured on floating collagen gels in phenol red-free medium supplemented with 5% fetal bovine serum. Light microscopy and keratin staining showed that the epithelial cell population established in culture had morphological characteristics similar to that seen in the intact endometrium. Cells were cultured with {sup 3}H-leucine and uptake of label by cells and its incorporation into cellular and secretory proteins determined. When compared to cells cultured for 24-48 h, incorporation of label into cellular protein was lower at 72-96 h, but secretion increased. Estradiol 17-{beta} did not affect label uptake or incorporation, but did enhance proliferation of cells as judged by total DNA content of the cell population. Analysis of proteins in media by sodium dodecyl sulfate polyacrylamide gel electrophoresis and fluorography suggested that epithelial and stromal cells synthesis proteins that may be secretory in nature during 72-96 h culture. Twenty-nine to thirty-one h after initiation of epithelial cultures, 1-2 {times} 10{sup 6} sperm were co-incubated with cells and sperm viability, motility, loss of acrosome and fertilizing ability determined.

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

    PubMed

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

    2016-08-01

    Objective To develop an improved method for culturing Schwann cells(SCs) by using both enzyme digestion and explants-culture approaches and compared with traditional explants-culture method and general hemi-explants-culture method. Methods Bilaterally sciatic nerves and brachial plexus nerves were dissected from 3 to 5-day-old neonatal SD rats and explants-culture method,general hemi-explants-culture method,and improved enzyme digestion combined with explants-culture method were adopted to culture SCs,respectively. SCs were digested and passaged after 7 days in culture and counted under the microscope. The purity of SCs was identified by S-100 immunofluorescence staining. Results The SCs of improved method group grew fastest and the total number of cells obtained was(1.85±0.13)×10(6);the SCs of the hemi-explants-culture method group grew slower than the improved method group and the total number of cells obtained was (1.10±0.10)×10(6);the SCs of the explants-culture method group grew slowest and the total number of cells obtained was (0.77±0.03)×10(6).The total number of cells obtained showed significant difference among the three groups(P<0.01). Immunofluorescence staining showed that the SCs purity was (95.73±1.51)% in the improved method group,(84.66±2.68)% in the hemi-explants-culture method group,and (74.50±4.23)% in the explants-culture method group(P<0.01). Conclusion The improved enzyme digestion combined with explants-culture method can obtain sufficient amount of high-purity SCs in a short time and thus may be applied in further research on peripheral nerve regeneration.

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

    PubMed

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

    2016-08-01

    Objective To develop an improved method for culturing Schwann cells(SCs) by using both enzyme digestion and explants-culture approaches and compared with traditional explants-culture method and general hemi-explants-culture method. Methods Bilaterally sciatic nerves and brachial plexus nerves were dissected from 3 to 5-day-old neonatal SD rats and explants-culture method,general hemi-explants-culture method,and improved enzyme digestion combined with explants-culture method were adopted to culture SCs,respectively. SCs were digested and passaged after 7 days in culture and counted under the microscope. The purity of SCs was identified by S-100 immunofluorescence staining. Results The SCs of improved method group grew fastest and the total number of cells obtained was(1.85±0.13)×10(6);the SCs of the hemi-explants-culture method group grew slower than the improved method group and the total number of cells obtained was (1.10±0.10)×10(6);the SCs of the explants-culture method group grew slowest and the total number of cells obtained was (0.77±0.03)×10(6).The total number of cells obtained showed significant difference among the three groups(P<0.01). Immunofluorescence staining showed that the SCs purity was (95.73±1.51)% in the improved method group,(84.66±2.68)% in the hemi-explants-culture method group,and (74.50±4.23)% in the explants-culture method group(P<0.01). Conclusion The improved enzyme digestion combined with explants-culture method can obtain sufficient amount of high-purity SCs in a short time and thus may be applied in further research on peripheral nerve regeneration. PMID:27594149

  12. Autophagic response to cell culture stress in pluripotent stem cells.

    PubMed

    Gregory, Siân; Swamy, Sushma; Hewitt, Zoe; Wood, Andrew; Weightman, Richard; Moore, Harry

    2016-05-01

    Autophagy is an important conserved cellular process, both constitutively as a recycling pathway for long lived proteins and as an upregulated stress response. Recent findings suggest a fundamental role for autophagic processes in the maintenance of pluripotent stem cell function. In human embryonic stem cells (hESCS), autophagy was investigated by transfection of LC3-GFP to visualize autophagosomes and with an antibody to LC3B protein. The presence of the primary cilium (PC) in hESCs as the site of recruitment of autophagy-related proteins was also assessed. HESCs (mShef11) in vitro displayed basal autophagy which was upregulated in response to deprivation of culture medium replacement. Significantly higher levels of autophagy were exhibited on spontaneous differentiation of hESCs in vitro. The PC was confirmed to be present in hESCs and therefore may serve to coordinate autophagy function. PMID:26385182

  13. Environmental feedbacks and engineered nanoparticles: mitigation of silver nanoparticle toxicity to Chlamydomonas reinhardtii by algal-produced organic compounds.

    PubMed

    Stevenson, Louise M; Dickson, Helen; Klanjscek, Tin; Keller, Arturo A; McCauley, Edward; Nisbet, Roger M

    2013-01-01

    The vast majority of nanotoxicity studies measures the effect of exposure to a toxicant on an organism and ignores the potentially important effects of the organism on the toxicant. We investigated the effect of citrate-coated silver nanoparticles (AgNPs) on populations of the freshwater alga Chlamydomonas reinhardtii at different phases of batch culture growth and show that the AgNPs are most toxic to cultures in the early phases of growth. We offer strong evidence that reduced toxicity occurs because extracellular dissolved organic carbon (DOC) compounds produced by the algal cells themselves mitigate the toxicity of AgNPs. We analyzed this feedback with a dynamic model incorporating algal growth, nanoparticle dissolution, bioaccumulation of silver, DOC production and DOC-mediated inactivation of nanoparticles and ionic silver. Our findings demonstrate how the feedback between aquatic organisms and their environment may impact the toxicity and ecological effects of engineered nanoparticles.

  14. Environmental Feedbacks and Engineered Nanoparticles: Mitigation of Silver Nanoparticle Toxicity to Chlamydomonas reinhardtii by Algal-Produced Organic Compounds

    PubMed Central

    Stevenson, Louise M.; Dickson, Helen; Klanjscek, Tin; Keller, Arturo A.; McCauley, Edward; Nisbet, Roger M.

    2013-01-01

    The vast majority of nanotoxicity studies measures the effect of exposure to a toxicant on an organism and ignores the potentially important effects of the organism on the toxicant. We investigated the effect of citrate-coated silver nanoparticles (AgNPs) on populations of the freshwater alga Chlamydomonas reinhardtii at different phases of batch culture growth and show that the AgNPs are most toxic to cultures in the early phases of growth. We offer strong evidence that reduced toxicity occurs because extracellular dissolved organic carbon (DOC) compounds produced by the algal cells themselves mitigate the toxicity of AgNPs. We analyzed this feedback with a dynamic model incorporating algal growth, nanoparticle dissolution, bioaccumulation of silver, DOC production and DOC-mediated inactivation of nanoparticles and ionic silver. Our findings demonstrate how the feedback between aquatic organisms and their environment may impact the toxicity and ecological effects of engineered nanoparticles. PMID:24086348

  15. Technique for Handling Cover Slips Used in Cell Culture Systems *

    PubMed Central

    Bowne, J. G.; Jochim, Michael M.

    1964-01-01

    A stainless steel, grooved rack was designed to handle large numbers of coverslips used in cell cultures. The rack separated and held the coverslips through the washing process and subsequent manipulations. Slips washed by this method allowed consistently good cell sheets to grow with no signs of cytotoxicity to the cultures. ImagesFIGURE 1.FIGURE 2. PMID:17649500

  16. Enhanced performance of an air-cathode microbial fuel cell with oxygen supply from an externally connected algal bioreactor.

    PubMed

    Kakarla, Ramesh; Kim, Jung Rae; Jeon, Byong-Hun; Min, Booki

    2015-11-01

    An algae bioreactor (ABR) was externally connected to air-cathode microbial fuel cells (MFCs) to increase power generation by supplying a high amount of oxygen to cathode electrode. The MFC with oxygen fed from ABR produced maximum cell voltage and cathode potential at a fixed loading of 459 mV and 10 mV, respectively. During polarization analysis, the MFC displayed a maximum power density of 0.63 W/m(2) (at 2.06 A/m(2)) using 39.2% O2 from ABR, which was approximately 30% higher compared with use of atmospheric air (0.44 W/m(2), 20.8% O2,). The cyclic voltammogram analysis exhibited a higher reduction current of -137 mA with 46.5% O2 compared to atmospheric air (-115 mA). Oxygen supply by algae bioreactor to air-cathode MFC could also maintain better MFC performance in long term operation by minimizing cathode potential drop over time.

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

    NASA Technical Reports Server (NTRS)

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

    1991-01-01

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

  18. A digital microfluidic platform for primary cell culture and analysis.

    PubMed

    Srigunapalan, Suthan; Eydelnant, Irwin A; Simmons, Craig A; Wheeler, Aaron R

    2012-01-21

    Digital microfluidics (DMF) is a technology that facilitates electrostatic manipulation of discrete nano- and micro-litre droplets across an array of electrodes, which provides the advantages of single sample addressability, automation, and parallelization. There has been considerable interest in recent years in using DMF for cell culture and analysis, but previous studies have used immortalized cell lines. We report here the first digital microfluidic method for primary cell culture and analysis. A new mode of "upside-down" cell culture was implemented by patterning the top plate of a device using a fluorocarbon liftoff technique. This method was useful for culturing three different primary cell types for up to one week, as well as implementing a fixation, permeabilization, and staining procedure for F-actin and nuclei. A multistep assay for monocyte adhesion to endothelial cells (ECs) was performed to evaluate functionality in DMF-cultured primary cells and to demonstrate co-culture using a DMF platform. Monocytes were observed to adhere in significantly greater numbers to ECs exposed to tumor necrosis factor (TNF)-α than those that were not, confirming that ECs cultured in this format maintain in vivo-like properties. The ability to manipulate, maintain, and assay primary cells demonstrates a useful application for DMF in studies involving precious samples of cells from small animals or human patients.

  19. Optical Oxygen Sensors for Applications in Microfluidic Cell Culture

    PubMed Central

    Grist, Samantha M.; Chrostowski, Lukas; Cheung, Karen C.

    2010-01-01

    The presence and concentration of oxygen in biological systems has a large impact on the behavior and viability of many types of cells, including the differentiation of stem cells or the growth of tumor cells. As a result, the integration of oxygen sensors within cell culture environments presents a powerful tool for quantifying the effects of oxygen concentrations on cell behavior, cell viability, and drug effectiveness. Because microfluidic cell culture environments are a promising alternative to traditional cell culture platforms, there is recent interest in integrating oxygen-sensing mechanisms with microfluidics for cell culture applications. Optical, luminescence-based oxygen sensors, in particular, show great promise in their ability to be integrated with microfluidics and cell culture systems. These sensors can be highly sensitive and do not consume oxygen or generate toxic byproducts in their sensing process. This paper presents a review of previously proposed optical oxygen sensor types, materials and formats most applicable to microfluidic cell culture, and analyzes their suitability for this and other in vitro applications. PMID:22163408

  20. Effects of modified clay on cysts of Scrippsiella trochoidea for harmful algal bloom control

    NASA Astrophysics Data System (ADS)

    Wang, Zhifu; Yu, Zhiming; Song, Xiuxian; Cao, Xihua; Han, Xiaotian

    2014-11-01

    We present results on the effect of modified clay on cyst formation of Scrippsiella trochoidea in harmful algal bloom (HAB). Modified clay (in concentration of 0, 0.1, 0.5, and 1.0 g/L) were added to cultures, and observations were made on cysts of S. trochoidea under controlled laboratory conditions. Results indicate that the removal rate of algal cells reached 97.7% at the clay concentration of 1.0 g/L. The cyst formation rate increased from 4.6% to 24.6% when the concentration of clay was increased from 0 to 1.0 g/L. Two cyst metamorphs were observed: spinal calcareous cysts and smooth noncalcareous ones. The proportion of the spinal cysts decreased from 76.9% to 24.1% when clay concentration increased from 0 to 1.0 g/L. In addition, modified clay affected cyst germination. The germination rate decreased with the increases in the clay concentrations. Non-calcareous cysts had a lower germination rate with a longer germination time. We conclude that modified clay could depress algal cell multiplication and promote formation of temporal cysts of S. trochoidea, which may help in controlling HAB outbreaks.

  1. Investigation of severe UF membrane fouling induced by three marine algal species.

    PubMed

    Merle, Tony; Dramas, Laure; Gutierrez, Leonardo; Garcia-Molina, Veronica; Croué, Jean-Philippe

    2016-04-15

    Reducing membrane fouling caused by seawater algal bloom is a challenge for regions of the world where most of their freshwater is produced by seawater desalination. This study aims to compare ultrafiltration (UF) fouling potential of three ubiquitous marine algal species cultures (i.e., Skeletonema costatum-SKC, Tetraselmis sp.-TET, and Hymenomonas sp.-HYM) sampled at different phases of growth. Results showed that flux reduction and irreversible fouling were more severe during the decline phase as compared to the exponential phase, for all species. SKC and TET were responsible for substantial irreversible fouling but their impact was significantly lower than HYM. The development of a transparent gel layer surrounding the cell during the HYM growth and accumulating in water is certainly responsible for the more severe observed fouling. Chemical backwash with a standard chlorine solution did not recover any membrane permeability. For TET and HYM, the Hydraulically Irreversible Fouling Index (HIFI) was correlated to their biopolymer content but this correlation is specific for each species. Solution pre-filtration through a 1.2 μm membrane proved that cells and particulate algal organic matter (p-AOM) considerably contribute to fouling, especially for HYM for which the HIFI was reduced by a factor of 82.3.

  2. Investigation of severe UF membrane fouling induced by three marine algal species.

    PubMed

    Merle, Tony; Dramas, Laure; Gutierrez, Leonardo; Garcia-Molina, Veronica; Croué, Jean-Philippe

    2016-04-15

    Reducing membrane fouling caused by seawater algal bloom is a challenge for regions of the world where most of their freshwater is produced by seawater desalination. This study aims to compare ultrafiltration (UF) fouling potential of three ubiquitous marine algal species cultures (i.e., Skeletonema costatum-SKC, Tetraselmis sp.-TET, and Hymenomonas sp.-HYM) sampled at different phases of growth. Results showed that flux reduction and irreversible fouling were more severe during the decline phase as compared to the exponential phase, for all species. SKC and TET were responsible for substantial irreversible fouling but their impact was significantly lower than HYM. The development of a transparent gel layer surrounding the cell during the HYM growth and accumulating in water is certainly responsible for the more severe observed fouling. Chemical backwash with a standard chlorine solution did not recover any membrane permeability. For TET and HYM, the Hydraulically Irreversible Fouling Index (HIFI) was correlated to their biopolymer content but this correlation is specific for each species. Solution pre-filtration through a 1.2 μm membrane proved that cells and particulate algal organic matter (p-AOM) considerably contribute to fouling, especially for HYM for which the HIFI was reduced by a factor of 82.3. PMID:26874470

  3. Explantation of mesangial cell 'hillocks': a method for obtaining human mesangial cells in culture.

    PubMed Central

    Muller, E. W.; Kim, Y.; Michael, A. F.; Vernier, R. L.; van der Hem, G. K.; van der Woude, F. J.

    1992-01-01

    A simple method is presented for selective cell culture of human mesangial cells using explantation of mesangial cell hillocks. Glomeruli which had been incubated with collagenase were explanted on plastic tissue culture flasks. Three to 6 weeks after explantation, a rapidly growing multilayer of elongated mesangial cells was observed to grow over the previously established monolayer of glomerular epithelial cells, ultimately forming multiple nodular foci of mesangial cells or 'mesangial cell hillocks'. By explanting mesangial cell hillocks selectively, pure mesangial cell cultures were easily obtained. When compared with mesangial cells grown in mixed cultures from glomerular explants, the hillock-derived cells were identical in morphology, growth characteristics, cell markers and synthesis of extracellular matrix. This system provides a simple method for the isolation of human mesangial cells in culture. Images p12-a Fig. 1 p14-a p15-a p16-a Fig. 2 PMID:1576080

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

    PubMed

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

    2015-01-01

    Pluripotent stem cells, such as embryonic stem cells and induced pluripotent stem (iPS) cells, are regarded as new sources for cell replacement therapy. These cells can unlimitedly expand under undifferentiated conditions and be differentiated into multiple cell types. Automated culture systems enable the large-scale production of cells. In addition to reducing the time and effort of researchers, an automated culture system improves the reproducibility of cell cultures. In the present study, we newly designed a fully automated cell culture system for human iPS maintenance. Using an automated culture system, hiPS cells maintained their undifferentiated state for 60 days. Automatically prepared hiPS cells had a potency of differentiation into three germ layer cells including dopaminergic neurons and pancreatic cells. PMID:26573336

  5. Cultures of mast cell-like (MCL) cells from human pleural exudate cells.

    PubMed

    Krüger, G; Sterry, W; Czarnetzki, B M

    1983-03-01

    Under special culture conditions, rat peritoneal macrophages have previously been shown to transform into mast cells. This method has been adapted here to the human species. Adherent large mononuclear cells from human pleural exudates were cultured in a medium supplemented with horse serum (30%) and fibroblast supernatants (30%). Metachromatic staining (toluidine blue, pH 3.6) of cytoplasmic granules appeared first in a small percentage of cells by days 5-6 of culture and reached a high intensity in 50% of the cells between days 12-22. Histamine levels within the cells increased by a factor of 7 during this same time period and the cell size by a factor of 3. Cultures could be maintained for about three weeks, since viability and total cell number decreased on extended culture. The data suggest that mononuclear cells in inflammatory exudates can transform into mast cell-like cells under the influence of high levels of specific conditioning factors in their microenvironment. PMID:6824794

  6. A Novel Inducer of Roseobacter Motility Is Also a Disruptor of Algal Symbiosis

    PubMed Central

    Sule, Preeti

    2013-01-01

    Silicibacter sp. strain TM1040, a member of the Roseobacter clade, forms a symbiosis with unicellular phytoplankton, which is inextricably linked to the biphasic “swim or stick” lifestyle of the bacteria. Mutations in flaC bias the population toward the motile phase. Renewed examination of the FlaC− strain (HG1016) uncovered that it is composed of two different cells: a pigmented type, PS01, and a nonpigmented cell, PS02, each of which has an identical mutation in flaC. While monocultures of PS01 and PS02 had few motile cells (0.6 and 6%, respectively), coculturing the two strains resulted in a 10-fold increase in the number of motile cells. Cell-free supernatants from coculture or wild-type cells were fully capable of restoring motility to PS01 and PS02, which was due to increased fliC3 (flagellin) transcription, FliC3 protein levels per cell, and flagella synthesis. The motility-inducing compound has an estimated mass of 226 Da, as determined by mass spectrometry, and is referred to as Roseobacter Motility Inducer (RMI). Mutations affecting genes involved in phenyl acetic acid synthesis significantly reduced RMI, while defects in tropodithietic acid (TDA) synthesis had marginal or no effect on RMI. RMI biosynthesis is induced by p-coumaric acid, a product of algal lignin degradation. When added to algal cultures, RMI caused loss of motility, cell enlargement, and vacuolization in the algal cells. RMI is a new member of the roseobacticide family of troponoid compounds whose activities affect roseobacters, by shifting their population toward motility, as well as their phytoplankton hosts, through an algicidal effect. PMID:23161030

  7. Transferrin receptor expression by stimulated cells in mixed lymphocyte culture.

    PubMed Central

    Salmon, M; Bacon, P A; Symmons, D P; Walton, K W

    1985-01-01

    Transferrin receptor (TRFr) expression by cells in mixed lymphocyte culture increases steadily for the first 5 days, but then reaches a plateau. By the sixth day in culture, about 20% of viable cells express TRFr in two-way mixed lymphocyte reactions. This subpopulation of TRFr-positive cells represents the proliferating population; it is heterogeneous, containing T-cell blasts and smaller cells which are a mixture of T and non-T cells. A small group of non-T cells have phenotypic similarity to natural killer (NK) cells. T cells appear to divide earlier in the course of the response than non-T cells. The biphasic nature of this response and the slower non-T reactivity may be due to a secondary stimulation of non-T cells by factors released from activated T cells (such as interleukin-2). PMID:2982734

  8. 21 CFR 876.5885 - Tissue culture media for human ex vivo tissue and cell culture processing applications.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... cell culture processing applications. 876.5885 Section 876.5885 Food and Drugs FOOD AND DRUG... DEVICES Therapeutic Devices § 876.5885 Tissue culture media for human ex vivo tissue and cell culture processing applications. (a) Identification. Tissue culture media for human ex vivo tissue and cell...

  9. 21 CFR 876.5885 - Tissue culture media for human ex vivo tissue and cell culture processing applications.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... cell culture processing applications. 876.5885 Section 876.5885 Food and Drugs FOOD AND DRUG... DEVICES Therapeutic Devices § 876.5885 Tissue culture media for human ex vivo tissue and cell culture processing applications. (a) Identification. Tissue culture media for human ex vivo tissue and cell...

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

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... cell culture processing applications. 876.5885 Section 876.5885 Food and Drugs FOOD AND DRUG... DEVICES Therapeutic Devices § 876.5885 Tissue culture media for human ex vivo tissue and cell culture processing applications. (a) Identification. Tissue culture media for human ex vivo tissue and cell...

  11. 21 CFR 876.5885 - Tissue culture media for human ex vivo tissue and cell culture processing applications.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... cell culture processing applications. 876.5885 Section 876.5885 Food and Drugs FOOD AND DRUG... DEVICES Therapeutic Devices § 876.5885 Tissue culture media for human ex vivo tissue and cell culture processing applications. (a) Identification. Tissue culture media for human ex vivo tissue and cell...

  12. 21 CFR 876.5885 - Tissue culture media for human ex vivo tissue and cell culture processing applications.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... cell culture processing applications. 876.5885 Section 876.5885 Food and Drugs FOOD AND DRUG... DEVICES Therapeutic Devices § 876.5885 Tissue culture media for human ex vivo tissue and cell culture processing applications. (a) Identification. Tissue culture media for human ex vivo tissue and cell...

  13. Differentiation in human amniotic fluid cell cultures: I: Collagen production.

    PubMed Central

    Priest, R E; Priest, J H; Moinuddin, J F; Keyser, A J

    1977-01-01

    The collagen produced by differentiated cells cultured from human amniotic fluid was characterized in two ways. By chain composition and by 4-hydroxyproline:3-hydroxyproline isomer ratio, the collagen synthesized by F-type (fibroblast) cells was indistinguishable from that made by cultured fetal dermal fibroblasts. The predominant cells in young amniotic fluid cultures, termed AF-type, produced collagen with a lower isomer ratio, resembling that of basement membrane collage. The chain composition, as determined by chromatography on carboxymethyl cellulose, varied for different cultures of the AF-type, but the major pattern was consistent with that of basement membrane collagen. On the basis of these characteristics, F cells are of fibroblast origin, whereas most AF cells are of a different origin either endothelial or epithelial. Other evidence (Megaw et al., 1977) suggests an epithelial origin for AF cells. PMID:881704

  14. Mesenchymal Progenitor Cells: Tissue Origin, Isolation and Culture.

    PubMed

    Bourin, Philippe; Gadelorge, Mélanie; Peyrafitte, Julie-Anne; Fleury-Cappellesso, Sandrine; Gomez, Marilyn; Rage, Christine; Sensebé, Luc

    2008-01-01

    SUMMARY: Since the pioneering work of Alexander Friedenstein on multipotent mesenchymal stromal cells (MSCs), a tremendous amount of work has been done to isolate, characterize and culture such cells. Assay of colony forming unit-fibroblasts (CFU-Fs), the hallmark of MSCs, is used to estimate their frequency in tissue. MSCs are adherent cells, so they are easy to isolate, and they show contact inhibition. Thus, several parameters must be taken into account for culture: cell density, number of passages, culture medium, and growth factors used. The purity of the initial material is not a limiting parameter. Similar but not identical cell populations are found in almost all mammal or human tissues. MSCs seem to be very abundant in adipose tissue but at low frequency in blood from umbilical cord or in adult tissue. The culture conditions are very similar, whatever the source of cells. Because of their favorable properties, MSCs are very promising tools for regenerative medicine.

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

    PubMed

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

    1997-09-01

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

  16. Characterization of goldfish fin cells in culture: some evidence of an epithelial cell profile.

    PubMed

    Mauger, P-E; Labbé, C; Bobe, J; Cauty, C; Leguen, I; Baffet, G; Le Bail, P-Y

    2009-03-01

    Comprehensive characterization of cultured cells in fish was little explored and cell origin is often deduced from morphological analogies with either epithelial of fibroblastic cells. This study aims to characterize cell origin in goldfish fin culture using morphological, immunochemical, and molecular approaches. Time lapse analysis revealed that cultured cell morphology changed within minutes. Therefore, cell morphology cannot predict whether cells are from fibroblastic or epithelial origin. The labeling pattern of heterologous anti-cytokeratin and anti-vimentin antibodies against goldfish epithelial cells and fibroblasts was first tested on skin sections and the corresponding labeling of the cultured cells was analyzed. No cell origin specificity could be obtained with the chosen antibodies. In the molecular approach, detection levels of three cytokeratin (CauK8-IIS, CauK49-IE and CauK50-Ie) and one vimentin transcripts were assessed on skin and fin samples. Specificity for epithelial cells of the most abundant mRNA, CauK49-Ie, was thereafter validated on skin sections by in situ hybridization. The selected markers were used afterwards to characterize fin cultures. CauK49-IE riboprobe labeled every cell in young cultures whereas no labeling was observed in older cultures. Accordingly, CauK49-IE transcript levels decreased after 15 days culture while CauK8-IIS ones increased. The use of homologous marker gave evidence that young cultured cells from goldfish fin are homogeneously of epithelial type and that cell characteristics may change over culture time.

  17. Three dimensional spheroid cell culture for nanoparticle safety testing.

    PubMed

    Sambale, Franziska; Lavrentieva, Antonina; Stahl, Frank; Blume, Cornelia; Stiesch, Meike; Kasper, Cornelia; Bahnemann, Detlef; Scheper, Thomas

    2015-07-10

    Nanoparticles are widely employed for many applications and the number of consumer products, incorporating nanotechnology, is constantly increasing. A novel area of nanotechnology is the application in medical implants. The widespread use of nanoparticles leads to their higher prevalence in our environment. This, in turn, raises concerns regarding potential risks to humans. Previous studies have shown possible hazardous effects of some nanoparticles on mammalian cells grown in two-dimensional (2D) cultures. However, 2D in vitro cell cultures display several disadvantages such as changes in cell shape, cell function, cell responses and lack of cell-cell contacts. For this reason, the development of better models for mimicking in vivo conditions is essential. In the present work, we cultivated A549 cells and NIH-3T3 cells in three-dimensional (3D) spheroids and investigated the effects of zinc oxide (ZnO-NP) and titanium dioxide nanoparticles (TiO2-NP). The results were compared to cultivation in 2D monolayer culture. A549 cells in 3D cell culture formed loose aggregates which were more sensitive to the toxicity of ZnO-NP in comparison to cells grown in 2D monolayers. In contrast, NIH-3T3 cells showed a compact 3D spheroid structure and no differences in the sensitivity of the NIH-3T3 cells to ZnO-NP were observed between 2D and 3D cultures. TiO2-NP were non-toxic in 2D cultures but affected cell-cell interaction during 3D spheroid formation of A549 and NIH-3T3 cells. When TiO2-NP were directly added during spheroid formation in the cultures of the two cell lines tested, several smaller spheroids were formed instead of a single spheroid. This effect was not observed if the nanoparticles were added after spheroid formation. In this case, a slight decrease in cell viability was determined only for A549 3D spheroids. The obtained results demonstrate the importance of 3D cell culture studies for nanoparticle safety testing, since some effects cannot be revealed in 2D

  18. Functional activity of mitochondria in cultured neural precursor cells.

    PubMed

    Plotnikov, E Yu; Marei, M V; Podgornyi, O V; Aleksandrova, M A; Zorov, D B; Sukhikh, G T

    2006-01-01

    We studied mitochondrial transmembrane potential of neural precursor cells forming neurospheres in culture. Uneven energization of mitochondria in neurosphere cells was detected. Heterogeneity of cells by the mitochondrial potential increased with neurosphere enlargement during culturing. Decrease in the mitochondrial potential in the central cells in large spheres, presumably caused by insufficient diffusion of oxygen and nutrients, can provoke their damage and death. Population of cells with high mitochondrial potential responded to addition of the nuclear dye by a decrease in mitochondrial potential, which can indicate functioning of ABCG2 complex in these cells, characteristic of undifferentiated stem cells. These data will help to create optimum conditions for culturing of neural stem cells for the maintenance of their maximum functional and proliferative activity. PMID:16929986

  19. Establishment and characterization of Xenopus oviduct cells in primary culture

    SciTech Connect

    Marsh, J.; Tata, J.R. )

    1987-11-01

    Based on previously established procedure of Xenopus hepatocytes, the authors describe tubular oviduct cells in primary culture which continue to secrete substantial quantities of egg jelly for several days, as can be visualized microscopically. Freshly isolated cells exhibited a culture shock response, from which they recovered by the third day in culture. This recovery was characterized by (a) the diminished synthesis of heat shock proteins hsp 70 and hsp 85, (b) the cessation of the drop in number of estrogen receptor, and (c) the enhanced rate of synthesis of cellular and secreted proteins. The oviduct estrogen receptor had the same characteristics as those in other estrogen target tissues and was present in the same amount as in adult female Xenopus hepatocytes. The successful establishment and characterization of primary cultures of both liver and oviduct cells now fulfill the conditions required for investigating the basis for tissue specificity of regulation by estrogen of Xenopus egg protein gene expression in primary cell culture.

  20. Multizone Paper Platform for 3D Cell Cultures

    PubMed Central

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

    2011-01-01

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

  1. Collection and conversion of algal lipid

    NASA Astrophysics Data System (ADS)

    Lin, Ching-Chieh

    Sustainable economic activities mandate a significant replacement of fossil energy by renewable forms. Algae-derived biofuels are increasingly seen as an alternative source of energy with potential to supplement the world's ever increasing demand. Our primary objective is, once the algae were cultivated, to eliminate or make more efficient energy-intensive processing steps of collection, drying, grinding, and solvent extraction prior to conversion. To overcome the processing barrier, we propose to streamline from cultivated algae to biodiesel via algal biomass collection by sand filtration, cell rupturing with ozone, and immediate transesterification. To collect the algal biomass, the specific Chlorococcum aquaticum suspension was acidified to pH 3.3 to promote agglomeration prior to sand filtration. The algae-loaded filter bed was drained of free water and added with methanol and ozonated for 2 min to rupture cell membrane to accelerate release of the cellular contents. The methanol solution now containing the dissolved lipid product was collected by draining, while the filter bed was regenerated by further ozonation when needed. The results showed 95% collection of the algal biomass from the suspension and a 16% yield of lipid from the algae, as well as restoration of filtration velocity of the sand bed via ozonation. The results further showed increased lipid yield upon cell rupturing and transesterified products composed entirely of fatty acid methyl ester (FAME) compounds, demonstrating that the rupture and transesterification processes could proceed consecutively in the same medium, requiring no separate steps of drying, extraction, and conversion. The FAME products from algae without exposure to ozone were mainly of 16 to 18 carbons containing up to 3 double bonds, while those from algae having been ozonated were smaller, highly saturated hydrocarbons. The new technique streamlines individual steps from cultivated algal lipid to transesterified products and

  2. Feeding Frequency Affects Cultured Rat Pituitary Cells in Low Gravity

    NASA Technical Reports Server (NTRS)

    Hymer, W. C.; Grindeland, R. E.; Salada, T.; Cenci, R.; Krishnan, K.; Mukai, C.; Nagaoka, S.

    1996-01-01

    In this report, we describe the results of a rat pituitary cell culture experiment done on STS-65 in which the effect of cell feeding on the release of the six anterior pituitary hormones was studied. We found complex microgravity related interactions between the frequency of cell feeding and the quantity and quality (i.e. biological activity) of some of the six hormones released in flight. Analyses of growth hormone (GH) released from cells into culture media on different mission days using gel filtration and ion exchange chromatography yielded qualitatively similar results between ground and flight samples. Lack of cell feeding resulted in extensive cell clumping in flight (but not ground) cultures. Vigorous fibroblast growth occurred in both ground and flight cultures fed 4 times. These results are interpreted within the context of autocrine and or paracrine feedback interactions. Finally the payload specialist successfully prepared a fresh trypsin solution in microgravity, detached the cells from their surface and reinserted them back into the culture chamber. These cells reattached and continued to release hormone in microgravity. In summary, this experiment shows that pituitary cells are microgravity sensitive and that coupled operations routinely associated with laboratory cel1 culture can also be accomplished in low gravity.

  3. Three Dimensional Culture of Human Renal Cell Carcinoma Organoids

    PubMed Central

    Batchelder, Cynthia A.; Martinez, Michele L.; Duru, Nadire; Meyers, Frederick J.; Tarantal, Alice F.

    2015-01-01

    Renal cell carcinomas arise from the nephron but are heterogeneous in disease biology, clinical behavior, prognosis, and response to systemic therapy. Development of patient-specific in vitro models that efficiently and faithfully reproduce the in vivo phenotype may provide a means to develop personalized therapies for this diverse carcinoma. Studies to maintain and model tumor phenotypes in vitro were conducted with emerging three-dimensional culture techniques and natural scaffolding materials. Human renal cell carcinomas were individually characterized by histology, immunohistochemistry, and quantitative PCR to establish the characteristics of each tumor. Isolated cells were cultured on renal extracellular matrix and compared to a novel polysaccharide scaffold to assess cell-scaffold interactions, development of organoids, and maintenance of gene expression signatures over time in culture. Renal cell carcinomas cultured on renal extracellular matrix repopulated tubules or vessel lumens in renal pyramids and medullary rays, but cells were not observed in glomeruli or outer cortical regions of the scaffold. In the polysaccharide scaffold, renal cell carcinomas formed aggregates that were loosely attached to the scaffold or free-floating within the matrix. Molecular analysis of cell-scaffold constructs including immunohistochemistry and quantitative PCR demonstrated that individual tumor phenotypes could be sustained for up to 21 days in culture on both scaffolds, and in comparison to outcomes in two-dimensional monolayer cultures. The use of three-dimensional scaffolds to engineer a personalized in vitro renal cell carcinoma model provides opportunities to advance understanding of this disease. PMID:26317980

  4. Purification of monolayer cell cultures of the endocrine pancreas.

    PubMed

    Braaten, J T; Järlfors, U; Smith, D S; Mintz, D H

    1975-01-01

    Experimental use of primary cultures of endocrine pancreas is constrained by early, vigorous proliferation of fibroblastoid cells. The addition of heavy metals, sodium ethylmercurithiosalicylate, phenyl mercuric acetate, phenyl mercuric nitrate and sodium aurothiomalate to the culture media selectively destroys these fibroblastoid cells yielding highly enriched, morphologically intact, functionally competent endocrine cells that are capable of cell replication. This action of heavy metals appears to be due to reversible inhibition of sulfhydryl enzymes since glutathione and thioglycolate were demonstrated to completely inhibit the cytotoxic effects of the mercury and gold containing agents, respectively. Certain variables in the application of the mercurial agents to pancreatic endocrine cell cultures were defined, most notably the enhanced sensitivity of fetal vs. neonatal tissue, and in inverse relationship of cell density to effective toxicity. After removal of the heavy metal agent from the culture media, many pancreatic islets send out cytoplasmic projections, containing large numbers of oriented microtubules which serve as bridging units to adjacent endocrine cells. The sustained availability of virtually pure pancreatic endocrine cell cultures, which results from the application of mercury to the culture media will undoubtedly permit many aspects of the cell biology of the endocrine pancreas to be directly and sequentially assailed. PMID:1239830

  5. Algal functional annotation tool

    SciTech Connect

    Lopez, D.; Casero, D.; Cokus, S. J.; Merchant, S. S.; Pellegrini, M.

    2012-07-01

    The Algal Functional Annotation Tool is a web-based comprehensive analysis suite integrating annotation data from several pathway, ontology, and protein family databases. The current version provides annotation for the model alga Chlamydomonas reinhardtii, and in the future will include additional genomes. The site allows users to interpret large gene lists by identifying associated functional terms, and their enrichment. Additionally, expression data for several experimental conditions were compiled and analyzed to provide an expression-based enrichment search. A tool to search for functionally-related genes based on gene expression across these conditions is also provided. Other features include dynamic visualization of genes on KEGG pathway maps and batch gene identifier conversion.

  6. Mammosphere culture of cancer stem cells in a microfluidic device

    NASA Astrophysics Data System (ADS)

    Saadin, Katayoon; White, Ian M.

    2012-03-01

    It is known that tumor-initiating cells with stem-like properties will form spherical colonies - termed mammospheres - when cultured in serum-free media on low-attachment substrates. Currently this assay is performed in commercially available 96-well trays with low-attachment surfaces. Here we report a novel microsystem that features on-chip mammosphere culture on low attachment surfaces. We have cultured mammospheres in this microsystem from well-studied human breast cancer cell lines. To enable the long-term culture of these unattached cells, we have integrated diffusion-based delivery columns that provide zero-convection delivery of reagents, such as fresh media, staining agents, or drugs. The multi-layer system consists of parallel cell-culture chambers on top of a low-attachment surface, connected vertically with a microfluidic reagent delivery layer. This design incorporates a reagent reservoir, which is necessary to reduce evaporation from the cell culture micro-chambers. The development of this microsystem will lead to the integration of mammosphere culture with other microfluidic functions, including circulating tumor cell recovery and high throughput drug screening. This will enable the cancer research community to achieve a much greater understanding of these tumor initiating cancer stem cells.

  7. Experience in primary culture of human peritoneal mesothelial cell.

    PubMed

    Chen, Kuo-Su; Chen, Wen-Shiang

    2012-08-31

    To compare the growth condition between different sources and different culture environments, mesothelial cells were isolated from omentum and peritoneal dialysate effluent (PDE), seeded at different densities (5 × 10⁵, 1 × 10⁵, 5 × 10⁴, 1 × 10⁴, 5 × 10³, 1 × 10³ and 5 × 10² cells/cm², respectively), supported with different fetal calf serum (FCS) concentrations (3%, 6%, 10% and 15%) and grown in dishes with and without gelatin pre-coating. Growth condition was evaluated by simple morphological observation. Cells phenotype was examined by immunofluorescent staining. The results showed that omentum-derived mesothelial cells generally showed a uniform growth pattern with good quality. Alternatively, there was a wide patient-to-patient variation in PDE-derived culture. Heterogeneous colonies composed of a mixture of large, small or abortive mesothelial colonies as well as fibroblastoid colonies were frequently observed. A minimum seeding density of 5 × 10³ cells/cm² is required for the omentum-derived mesothelial cells to grow to confluent monolayer (1-5 × 10⁴ cells/cm² for initial culture from fresh PDE). Appropriate seeding density is always associated with successful culture in omentumbased culture, but not in PDE-based culture. Mesothelial cells could grow to confluency regardless of FCS concentration and gelatin pre-coating. However, growth rate was slower in lower FCS concentrations and on dishes without gelatin coating. Most cells in culture expressed cytokeratin and vimentin, but not VWF. Alpha-smooth muscle actin frequently appeared in cytokeratin+ mesothelial cells, especially in higher FCS concentrations and in PDE-derived culture. Our data demonstrate that PDE, in contrast to omentum, provides a source of mesothelial cells with poor and unstable quality for primary culture. Healthy cell quality and sufficient seeding density seem to be the most important factors for successful culture of mesothelial cells. The frequent occurrence

  8. Optimization of Buffalo (Bubalus bubalis) Embryonic Stem Cell Culture System

    PubMed Central

    Zandi, Mohammad; Muzaffar, Musharifa; Shah, Syed Mohmad; Kumar Singh, Manoj; Palta, Prabhat; Kumar Singla, Suresh; Manik, Radheysham; Chauhan, Manmohan Singh

    2015-01-01

    Objective In order to retain an undifferentiated pluripotent state, embryonic stem (ES) cells have to be cultured on feeder cell layers. However, use of feeder layers limits stem cell research, since experimental data may result from a combined ES cell and feeder cell response to various stimuli. Materials and Methods In this experimental study, a buffalo ES cell line was established from in vitro derived blastocysts and characterized by the Alkaline phosphatase (AP) and immunoflourescence staining of various pluripotency markers. We examined the effect of various factors like fibroblast growth factor 2 (FGF-2), leukemia inhibitory factor (LIF) and Y-27632 to support the growth and maintenance of bubaline ES cells on gelatin coated dishes, in order to establish feeder free culture systems. We also analyzed the effect of feeder-conditioned media on stem cell growth in gelatin based cultures both in the presence as well as in the absence of the growth factors. Results The results showed that Y-27632, in the presence of FGF-2 and LIF, resulted in higher colony growth and increased expression of Nanog gene. Feeder-Conditioned Medium resulted in a significant increase in growth of buffalo ES cells on gelatin coated plates, however, feeder layer based cultures produced better results than gelatin based cultures. Feeder layers from buffalo fetal fibroblast cells can support buffalo ES cells for more than two years. Conclusion We developed a feeder free culture system that can maintain buffalo ES cells in the short term, as well as feeder layer based culture that can support the long term maintenance of buffalo ES cells. PMID:26199905

  9. Growth of melanocytes in human epidermal cell cultures

    SciTech Connect

    Staiano-Coico, L.; Hefton, J.M.; Amadeo, C.; Pagan-Charry, I.; Madden, M.R.; Cardon-Cardo, C. )

    1990-08-01

    Epidermal cell cultures were grown in keratinocyte-conditioned medium for use as burn wound grafts; the melanocyte composition of the grafts was studied under a variety of conditions. Melanocytes were identified by immunohistochemistry based on a monoclonal antibody (MEL-5) that has previously been shown to react specifically with melanocytes. During the first 7 days of growth in primary culture, the total number of melanocytes in the epidermal cultures decreased to 10% of the number present in normal skin. Beginning on day 2 of culture, bipolar melanocytes were present at a mean cell density of 116 +/- 2/mm2; the keratinocyte to melanocyte ratio was preserved during further primary culture and through three subpassages. Moreover, exposure of cultures to mild UVB irradiation stimulated the melanocytes to proliferate, suggesting that the melanocytes growing in culture maintained their responsiveness to external stimuli. When the sheets of cultured cells were enzymatically detached from the plastic culture flasks before grafting, melanocytes remained in the basal layer of cells as part of the graft applied to the patient.

  10. REMOVAL OF HARMFUL ALGAL CELLS (KARENIA BREVIS) AND TOXINS FROM SEAWATER CULTURE BY CLAY FLOCCULATION. (R827090)

    EPA Science Inventory

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  11. A practical guide to hydrogels for cell culture.

    PubMed

    Caliari, Steven R; Burdick, Jason A

    2016-04-28

    There is growing appreciation of the role that the extracellular environment plays in regulating cell behavior. Mechanical, structural, and compositional cues, either alone or in concert, can drastically alter cell function. Biomaterials, and particularly hydrogels, have been developed and implemented to present defined subsets of these cues for investigating countless cellular processes as a means of understanding morphogenesis, aging, and disease. Although most scientists concede that standard cell culture materials (tissue culture plastic and glass) do a poor job of recapitulating native cellular milieus, there is currently a knowledge barrier for many researchers in regard to the application of hydrogels for cell culture. Here, we introduce hydrogels to those who may be unfamiliar with procedures to culture and study cells with these systems, with a particular focus on commercially available hydrogels.

  12. Polydimethylsiloxane SlipChip for mammalian cell culture applications.

    PubMed

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

    2015-11-01

    This paper reports a polydimethylsiloxane (PDMS) SlipChip for in vitro cell culture applications, multiple-treatment assays, cell co-cultures, and cytokine detection assays. The PDMS SlipChip is composed of two PDMS layers with microfluidic channels on each surface that are separated by a thin silicone fluid (Si-fluid) layer. The integration of Si-fluid enables the two PDMS layers to be slid to different positions; therefore, the channel patterns can be re-arranged for various applications. The SlipChip design significantly reduces the complexity of sample handling, transportation, and treatment processes. To apply the developed SlipChip for cell culture applications, human lung adenocarcinoma epithelial cells (A549) and lung fibroblasts (MRC-5) were cultured to examine the biocompatibility of the developed PDMS SlipChip. Moreover, embryonic pluripotent stem cells (ES-D3) were also cultured in the device to evaluate the retention of their stemness in the device. The experimental results show that cell morphology, viability and proliferation are not affected when the cells are cultured in the SlipChip, indicating that the device is highly compatible with mammalian cell culture. In addition, the stemness of the ES-D3 cells was highly retained after they were cultured in the device, suggesting the feasibility of using the SlipChip for stem cell research. Various cell experiments, such as simultaneous triple staining of cells and co-culture of MRC-5 with A549 cells, were also performed to demonstrate the functionalities of the PDMS SlipChip. Furthermore, we used a cytokine detection assay to evaluate the effect of endotoxin (lipopolysaccharides, LPS) treatment on the cytokine secretion of A549 cells using the SlipChip. The developed PDMS SlipChip provides a straightforward and effective platform for various on-chip in vitro cell cultures and consequent analysis, which is promising for a number of cell biology studies and biomedical applications. PMID:26381390

  13. Synchronization of mammalian cell cultures by serum deprivation.

    PubMed

    Langan, Thomas J; Chou, Richard C

    2011-01-01

    Mammalian cells are amenable to study the regulation of cell cycle progression in vitro by shifting them into the same phase of the cycle. Procedures to arrest cultured cells in specific phases of the cell cycle may be termed in vitro synchronization. The procedure described here was developed for the study of primary astrocytes and a glioma cell line, but is applicable to other mammalian cells. Its application allows astrocytes to reenter the cell cycle from a state of quiescence (G(0)), and then, under carefully defined experimental conditions, to move together into subsequent phases such as the G(1) and S phases. A number of methods have been established to synchronize mammalian cell cultures, which include physical separation by centrifugal elutriation and mitotic shake off or chemically induced cell cycle arrest. Yet, there are intrinsic limitations associated with these methods. In the present protocol, we describe a simple, reliable, and reversible procedure to synchronize astrocyte and glioma cultures from newborn rat brain by serum deprivation. The procedure is similar, and generally applicable, to other mammalian cells. This protocol consists essentially of two parts: (1) proliferation of astrocytes under optimal conditions in vitro until reaching desired confluence; and (2) synchronization of cultures by serum downshift and arrested in the G(0) phase of the cell cycle. This procedure has been extended to the examination of cell cycle control in astroglioma cells and astrocytes from injured adult brain. It has also been employed in precursor cloning studies in developmental biology, suggesting wide applicability.

  14. Urokinase production by electrophoretically separated cultured human embryonic kidney cells

    NASA Technical Reports Server (NTRS)

    Kunze, M. E.; Plank, L. D.; Giranda, V.; Sedor, K.; Todd, P. W.

    1985-01-01

    Urokinase is a plasminogen activator found in urine. Relatively pure preparations have been tested in Europe, Japan and the United States for the treatment of deep vein thrombosis and other dangerous blood clots. Human embryonic kidney cell cultures have been found to produce urokinase at much higher concentrations, but less than 5% of the cells in typical cultures are producers. Since human diploid cells become senescent in culture the selection of clones derived from single cells will not provide enough material to be useful, so a bulk purification method is needed for the isolation of urokinase producing cell populations. Preparative cell electrophoresis was chosen as the method, since evidence exists that human embryonic cell cultures are richly heterogeneous with respect to electrophoretic mobility, and preliminary electrophoretic separations on the Apollo-Soyuz space flight produced cell populations that were rich in urokinase production. Similarly, erythropoietin is useful in the treatment of certain anemias and is a kidney cell duct, and electrophoretically enriched cell populations producing this product have been reported. Thus, there is a clear need for diploid human cells that produce these products, and there is evidence that such cells should be separable by free-flow cell electrophoresis.

  15. Surface modified alginate microcapsules for 3D cell culture

    NASA Astrophysics Data System (ADS)

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

    2016-06-01

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

  16. Fundamentals of microfluidic cell culture in controlled microenvironments†

    PubMed Central

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

    2010-01-01

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

  17. Microfluidic perfusion culture of human induced pluripotent stem cells under fully defined culture conditions.

    PubMed

    Yoshimitsu, Ryosuke; Hattori, Koji; Sugiura, Shinji; Kondo, Yuki; Yamada, Rotaro; Tachikawa, Saoko; Satoh, Taku; Kurisaki, Akira; Ohnuma, Kiyoshi; Asashima, Makoto; Kanamori, Toshiyuki

    2014-05-01

    Human induced pluripotent stem cells (hiPSCs) are a promising cell source for drug screening. For this application, self-renewal or differentiation of the cells is required, and undefined factors in the culture conditions are not desirable. Microfluidic perfusion culture allows the production of small volume cultures with precisely controlled microenvironments, and is applicable to high-throughput cellular environment screening. Here, we developed a microfluidic perfusion culture system for hiPSCs that uses a microchamber array chip under defined extracellular matrix (ECM) and culture medium conditions. By screening various ECMs we determined that fibronectin and laminin are appropriate for microfluidic devices made out of the most popular material, polydimethylsiloxane (PDMS). We found that the growth rate of hiPSCs under pressure-driven perfusion culture conditions was higher than under static culture conditions in the microchamber array. We applied our new system to self-renewal and differentiation cultures of hiPSCs, and immunocytochemical analysis showed that the state of the hiPSCs was successfully controlled. The effects of three antitumor drugs on hiPSCs were comparable between microchamber array and 96-well plates. We believe that our system will be a platform technology for future large-scale screening of fully defined conditions for differentiation cultures on integrated microfluidic devices.

  18. Algal Biofuels; Algal Biofuels R&D at NREL (Brochure)

    SciTech Connect

    Not Available

    2010-09-01

    An overview of NREL's algal biofuels projects, including U.S. Department of Energy-funded work, projects with U.S. and international partners, and Laboratory Directed Research and Development projects.

  19. The death mechanism of the harmful algal bloom species Alexandrium tamarense induced by algicidal bacterium Deinococcus sp. Y35

    PubMed Central

    Li, Yi; Zhu, Hong; Lei, Xueqian; Zhang, Huajun; Cai, Guanjing; Chen, Zhangran; Fu, Lijun; Xu, Hong; Zheng, Tianling

    2015-01-01

    Harmful algal blooms (HABs) cause a variety of deleterious effects on aquatic ecosystems, especially the toxic dinoflagellate Alexandrium tamarense, which poses a serious threat to marine economic and human health based on releasing paralytic shellfish poison into the environment. The algicidal bacterium Deinococcus sp. Y35 which can induce growth inhibition on A. tamarense was used to investigate the functional mechanism. The growth status, reactive oxygen species (ROS) content, photosynthetic system and the nuclear system of algal cells were determined under algicidal activity. A culture of strain Y35 not only induced overproduction of ROS in algal cells within only 0.5 h of treatment, also decrease the total protein content as well as the response of the antioxidant enzyme. Meanwhile, lipid peroxidation was induced and cell membrane integrity was lost. Photosynthetic pigments including chlorophyll a and carotenoid decreased along with the photosynthetic efficiency being significantly inhibited. At the same time, photosynthesis-related gene expression showed down-regulation. More than, the destruction of cell nuclear structure and inhibition of proliferating cell nuclear antigen (PCNA) related gene expression were confirmed. The potential functional mechanism of the algicidal bacterium on A. tamarense was investigated and provided a novel viewpoint which could be used in HABs control. PMID:26441921

  20. The death mechanism of the harmful algal bloom species Alexandrium tamarense induced by algicidal bacterium Deinococcus sp. Y35.

    PubMed

    Li, Yi; Zhu, Hong; Lei, Xueqian; Zhang, Huajun; Cai, Guanjing; Chen, Zhangran; Fu, Lijun; Xu, Hong; Zheng, Tianling

    2015-01-01

    Harmful algal blooms (HABs) cause a variety of deleterious effects on aquatic ecosystems, especially the toxic dinoflagellate Alexandrium tamarense, which poses a serious threat to marine economic and human health based on releasing paralytic shellfish poison into the environment. The algicidal bacterium Deinococcus sp. Y35 which can induce growth inhibition on A. tamarense was used to investigate the functional mechanism. The growth status, reactive oxygen species (ROS) content, photosynthetic system and the nuclear system of algal cells were determined under algicidal activity. A culture of strain Y35 not only induced overproduction of ROS in algal cells within only 0.5 h of treatment, also decrease the total protein content as well as the response of the antioxidant enzyme. Meanwhile, lipid peroxidation was induced and cell membrane integrity was lost. Photosynthetic pigments including chlorophyll a and carotenoid decreased along with the photosynthetic efficiency being significantly inhibited. At the same time, photosynthesis-related gene expression showed down-regulation. More than, the destruction of cell nuclear structure and inhibition of proliferating cell nuclear antigen (PCNA) related gene expression were confirmed. The potential functional mechanism of the algicidal bacterium on A. tamarense was investigated and provided a novel viewpoint which could be used in HABs control. PMID:26441921

  1. The death mechanism of the harmful algal bloom species Alexandrium tamarense induced by algicidal bacterium Deinococcus sp. Y35.

    PubMed

    Li, Yi; Zhu, Hong; Lei, Xueqian; Zhang, Huajun; Cai, Guanjing; Chen, Zhangran; Fu, Lijun; Xu, Hong; Zheng, Tianling

    2015-01-01

    Harmful algal blooms (HABs) cause a variety of deleterious effects on aquatic ecosystems, especially the toxic dinoflagellate Alexandrium tamarense, which poses a serious threat to marine economic and human health based on releasing paralytic shellfish poison into the environment. The algicidal bacterium Deinococcus sp. Y35 which can induce growth inhibition on A. tamarense was used to investigate the functional mechanism. The growth status, reactive oxygen species (ROS) content, photosynthetic system and the nuclear system of algal cells were determined under algicidal activity. A culture of strain Y35 not only induced overproduction of ROS in algal cells within only 0.5 h of treatment, also decrease the total protein content as well as the response of the antioxidant enzyme. Meanwhile, lipid peroxidation was induced and cell membrane integrity was lost. Photosynthetic pigments including chlorophyll a and carotenoid decreased along with the photosynthetic efficiency being significantly inhibited. At the same time, photosynthesis-related gene expression showed down-regulation. More than, the destruction of cell nuclear structure and inhibition of proliferating cell nuclear antigen (PCNA) related gene expression were confirmed. The potential functional mechanism of the algicidal bacterium on A. tamarense was investigated and provided a novel viewpoint which could be used in HABs control.

  2. Immunodissection and culture of rabbit cortical collecting tubule cells

    SciTech Connect

    Spielman, W.S.; Sonnenburg, W.K.; Allen, M.L.; Arend, L.J.; Gerozissis, K.; Smith, W.L.

    1986-08-01

    A mouse monoclonal antibody designated IgG3 (rct-30) has been prepared that reacts specifically with an antigen on the surface of all cells comprising the cortical and medullary rabbit renal collecting tubule including the arcades. Plastic culture dishes coated with IgG3 (rct-30) were used to isolate collecting tubule cells from collagenase dispersions of rabbit renal cortical cells by immunoadsorption. Typically, 10W rabbit cortical collecting tubule (RCCT) cells were obtained from 5 g of renal cortex (2 kidneys). Between 20 and 30% of the RCCT cells were reactive with peanut lectin suggesting that RCCT cells are a mixture of principal and intercalated cells. Approximately 10X RCCT cells were obtained after 4 to 5 days in primary culture. Moreover, RCCT cells continued to proliferate after passaging with a doubling time of approx.32 h. RCCT cells passaged once and then cultured 4-5 days were found 1) to synthesize cAMP in response to arginine vasopressin (AVP), prostaglandin E2 (PGE2), isoproterenol, and parathyroid hormone, but not calcitonin, prostaglandin D2, or prostaglandin I, and 2) to release PGE2 in response to bradykinin but not arginine vasopressin or isoproterenol. The results indicate that cultured RCCT cells retain many of the hormonal, histochemical, and morphological properties expected for a mixture of principal and intercalated rabbit cortical collecting tubule epithelia. RCCT cells should prove useful both for studying hormonal interactions in the cortical collecting tubule and as a starting population for isolating intercalated collecting tubule epithelia.

  3. Improved Method for Culturing Guinea-Pig Macrophage Cells

    NASA Technical Reports Server (NTRS)

    Savage, J.

    1982-01-01

    Proper nutrients and periodic changes in culture medium maintain cell viability for a longer period. New method uses a thioglycolate solution, instead of mineral oil, to induce macrophage cells in guinea pigs and also uses an increased percent of fetal-calf bovine serum in cultivation medium. Macrophage cells play significant roles in the body's healing and defense systems.

  4. The Multiparametric Effects of Hydrodynamic Environments on Stem Cell Culture

    PubMed Central

    Kinney, Melissa A.; Sargent, Carolyn Y.

    2011-01-01

    Stem cells possess the unique capacity to differentiate into many clinically relevant somatic cell types, making them a promising cell source for tissue engineering applications and regenerative medicine therapies. However, in order for the therapeutic promise of stem cells to be fully realized, scalable approaches to efficiently direct differentiation must be developed. Traditionally, suspension culture systems are employed for the scale-up manufacturing of biologics via bioprocessing systems that heavily rely upon various types of bioreactors. However, in contrast to conventional bench-scale static cultures, large-scale suspension cultures impart complex hydrodynamic forces on cells and aggregates due to fluid mixing conditions. Stem cells are exquisitely sensitive to environmental perturbations, thus motivating the need for a more systematic understanding of the effects of hydrodynamic environments on stem cell expansion and differentiation. This article discusses the interdependent relationships between stem cell aggregation, metabolism, and phenotype in the context of hydrodynamic culture environments. Ultimately, an improved understanding of the multifactorial response of stem cells to mixed culture conditions will enable the design of bioreactors and bioprocessing systems for scalable directed differentiation approaches. PMID:21491967

  5. The effects of glucocorticoids on cultured human endothelial cells.

    PubMed

    Maca, R D; Fry, G L; Hoak, J C

    1978-04-01

    The effects of hydrocortisone, dexamethasone and prednisone on the morphology, replication, DNA synthesis, cell protein content and protein synthesis of cultured, human endothelial cells were evaluated. After culturing the cells with these glucocorticoids for 24-48 h, the cells covered a greater portion of the culture surface area. The mean surface area of the individual endothelial cell treated with glucocorticoids was 1.53 times greater than that of the untreated control endothelial cell. When compared with controls, the endothelial cover provided by the cells treated with glucocorticoids was more extensive and in many instances covered the entire culture surface. The change in morphology was associated with an increase in protein synthesis and protein content of the cells without an increase in DNA synthesis or cellular replication. Dexamethasone was approximately 10-fold more effective than hydrocortisone, while prednisone was the least effective. Aldosterone, DOCA, testosterone, progesterone, oestradiol and oestriol were ineffective. These studies indicate that glucocorticoids can alter the morphology and biochemistry of cultured endothelial cells and may have implications for the effects of steroids in the treatment of thrombocytopenic states and vascular disorders in man. PMID:646949

  6. Characterization of Amoeboaphelidium protococcarum, an algal parasite new to the cryptomycota isolated from an outdoor algal pond used for the production of biofuel.

    PubMed

    Letcher, Peter M; Lopez, Salvador; Schmieder, Robert; Lee, Philip A; Behnke, Craig; Powell, Martha J; McBride, Robert C

    2013-01-01

    Mass culture of algae for the production of biofuels is a developing technology designed to offset the depletion of fossil fuel reserves. However, large scale culture of algae in open ponds can be challenging because of incidences of infestation with algal parasites. Without knowledge of the identity of the specific parasite and how to control these pests, algal-based biofuel production will be limited. We have characterized a eukaryotic parasite of Scenedesmus dimorphus growing in outdoor ponds used for biofuel production. We demonstrated that as the genomic DNA of parasite FD01 increases, the concentration of S. dimorphus cells decreases; consequently, this is a highly destructive pathogen. Techniques for culture of the parasite and host were developed, and the endoparasite was identified as the Aphelidea, Amoeboaphelidium protococcarum. Phylogenetic analysis of ribosomal sequences revealed that parasite FD01 placed within the recently described Cryptomycota, a poorly known phylum based on two species of Rozella and environmental samples. Transmission electron microscopy demonstrated that aplanospores of the parasite produced filose pseudopodia, which contained fine fibers the diameter of actin microfilaments. Multiple lipid globules clustered and were associated with microbodies, mitochondria and a membrane cisternae, an arrangement characteristic of the microbody-lipid globule complex of chytrid zoospores. After encystment and attachment to the host cells, the parasite injected its protoplast into the host between the host cell wall and plasma membrane. At maturity the unwalled parasite occupied the entire host cell. After cleavage of the protoplast into aplanospores, a vacuole and lipids remained in the host cell. Amoeboaphelidium protococcarum isolate FD01 is characteristic of the original description of this species and is different from strain X-5 recently characterized. Our results help put a face on the Cryptomycota, revealing that the phylum is more

  7. [Cultivation of Neisseria gonorrhoeae in an L-929 cell culture].

    PubMed

    Nurusheva, S M; Lebedeva, M V; Shcherbakova, N I

    1985-12-01

    N. gonorrhoeae strain b has been found to be capable of retaining its viability in medium 199 with 10% of inactivated cattle serum added and in monolayer cell culture L-929 in the above medium. The characteristics obtained in the present investigation permit simulating the mixed association of gonococci and chlamydiae in the culture system used in this work.

  8. 3D Cultures of prostate cancer cells cultured in a novel high-throughput culture platform are more resistant to chemotherapeutics compared to cells cultured in monolayer.

    PubMed

    Chambers, Karen F; Mosaad, Eman M O; Russell, Pamela J; Clements, Judith A; Doran, Michael R

    2014-01-01

    Despite monolayer cultures being widely used for cancer drug development and testing, 2D cultures tend to be hypersensitive to chemotherapy and are relatively poor predictors of whether a drug will provide clinical benefit. Whilst generally more complicated, three dimensional (3D) culture systems often better recapitulate true cancer architecture and provide a more accurate drug response. As a step towards making 3D cancer cultures more accessible, we have developed a microwell platform and surface modification protocol to enable high throughput manufacture of 3D cancer aggregates. Herein we use this novel system to characterize prostate cancer cell microaggregates, including growth kinetics and drug sensitivity. Our results indicate that prostate cancer cells are viable in this system, however some non-cancerous prostate cell lines are not. This system allows us to consistently control for the presence or absence of an apoptotic core in the 3D cancer microaggregates. Similar to tumor tissues, the 3D microaggregates display poor polarity. Critically the response of 3D microaggregates to the chemotherapeutic drug, docetaxel, is more consistent with in vivo results than the equivalent 2D controls. Cumulatively, our results demonstrate that these prostate cancer microaggregates better recapitulate the morphology of prostate tumors compared to 2D and can be used for high-throughput drug testing. PMID:25380249

  9. [Results of mammalian cell culture exposure on artificial earth satellites].

    PubMed

    Sushkov, F V; Portugalov, V V; Rudneva, S V; Bobkova, N N; Iordanishvili, E K

    1976-01-01

    The paper presents the results of an exposure of cells of the Syrian hamster strain VNK-21 to space flight effects. In contrast to the cell culture kept in a thermostat at 29 degrees C, the cell culture that was maintained in thermally uncontrolled conditions developed noticeable structural and physiological changes induced by suboptimal temperatures. It was concluded that a 6-day exposure to weightlessness exerted no adverse effect on mammalian cells in vitro and produced no stable structural or physiological changes. Some changes that were detected in the cell culture--faster ageing, stable tendency to an increase of the number of cells with enlarged nuclei, an increase of the mitotic index at an early stage of cultivation--need further investigation.

  10. Developing defined culture systems for human pluripotent stem cells

    PubMed Central

    Valamehr, Bahram; Tsutsui, Hideaki; Ho, Chih-Ming; Wu, Hong

    2013-01-01

    Human pluripotent stem cells hold promising potential in many therapeutics applications including regenerative medicine and drug discovery. Over the past three decades, embryonic stem cell research has illustrated that embryonic stem cells possess two important and distinct properties: the ability to continuously self-renew and the ability to differentiate into all specialized cell types. In this article, we will discuss the continuing evolution of human pluripotent stem cell culture by examining requirements needed for the maintenance of self-renewal in vitro. We will also elaborate on the future direction of the field toward generating a robust and completely defined culture system, which has brought forth collaborations amongst biologists and engineers. As human pluripotent stem cell research progresses towards identifying solutions for debilitating diseases, it will be critical to establish a defined, reproducible and scalable culture system to meet the requirements of these clinical applications. PMID:21916597

  11. Turgor pressure and water transport properties of suspension-cultured cells of Chenopodium rubrum L.

    PubMed

    Büchner, K H; Zimmermann, U; Bentrup, F W

    1981-01-01

    The turgor pressure and water relation parameters were determined in single photoautotrophically grown suspension cells and in individual cells of intact leaves of Chenopodium rubrum using the miniaturized pressure probe. The stationary turgor pressure in suspension-cultured cells was in the range of betwen 3 and 5 bar. From the turgor pressure relaxation process, induced either hydrostatically (by means of the pressure probe) or osmotically, the halftime of water exchange was estimated to be 20±10 s. No polarity was observed for both ex- and endosmotic water flow. The volumetric elastic modulus, ε, determined from measurements of turgor pressure changes, and the corresponding changes in the fractional cell volume was determined to be in the range of between 20 and 50 bar. ε increases with increasing turgor pressure as observed for other higher plant and algal cells. The hydraulic conductivity, Lp, is calculated to be about 0,5-2·10(-6) cm s(-1) bar(-1). Similar results were obtained for individual leaf cells of Ch. rubrum. Suspension cells immobilized in a cross-linked matrix of alginate (6 to 8% w/w) revealed the same values for the half-time of water exchange and for the hydraulic conductivity, Lp, provided that the turgor pressure relaxation process was generated hydrostatically by means of the pressure probe. Thus, it can be concluded that the unstirred layer from the immobilized matrix has no effect on the calculation of Lp from the turgor pressure relaxation process, using the water transport equation derived for a single cell surrounded by a large external volume. By analogy, this also holds true for Lp-values derived from turgor pressure changes generated by the pressure probe in a single cell within the leaf tissue. The fair similarity between the Lp-values measured in mesophyll cells in situ and mesophyll-like suspension cells suggests that the water transport relations of a cell within a leaf are not fundamentally different from those measured in a

  12. Mechanical algal disruption for efficient biodiesel extraction

    NASA Astrophysics Data System (ADS)

    Krehbiel, Joel David

    Biodiesel from algae provides several benefits over current biodiesel feedstocks, but the energy requirements of processing algae into a useable fuel are currently so high as to be prohibitive. One route to improving this is via disruption of the cells prior to lipid extraction, which can significantly increase energy recovery. Unfortunately, several obvious disruption techniques require more energy than can be gained. This dissertation examines the use of microbubbles to improve mechanical disruption of algal cells using experimental, theoretical, and computational methods. New laboratory experiments show that effective ultrasonic disruption of algae is achieved by adding microbubbles to an algal solution. The configuration studied flows the solution through a tube and insonifies a small section with a high-pressure ultrasound wave. Previous biomedical research has shown effective cell membrane damage on animal cells with similar methods, but the present research is the first to extend such study to algal cells. Results indicate that disruption increases with peak negative pressure between 1.90 and 3.07 MPa and with microbubble concentration up to 12.5 x 107 bubbles/ml. Energy estimates of this process suggest that it requires only one-fourth the currently most-efficient laboratory-scale disruption process. Estimates of the radius near each bubble that causes disruption (i.e. the disruption radius) suggest that it increases with peak negative pressure and is near 9--20 microm for all cases tested. It is anticipated that these procedures can be designed for better efficiency and efficacy, which will be facilitated by identifying the root mechanisms of the bubble-induced disruption. We therefore examine whether bubble expansion alone creates sufficient cell deformation for cell rupture. The spherically-symmetric Marmottant model for bubble dynamics allows estimation of the flow regime under experimental conditions. Bubble expansion is modeled as a point source of

  13. Toxicity of oxygen radicals in cultured pulmonary endothelial cells

    SciTech Connect

    Autor, A.P.; Bonham, A.C.; Thies, R.L.

    1984-01-01

    Superoxide dismutase and catalase, which catalytically remove superoxide radicals and hydrogen perioxide, respectively, each separately protected cultured pulmonary artery endothelial cells from loss of membrane integrity after exposure to oxygen radicals generated either cellularly (polymorphonuclear leukocytes) or chemically (dihydroxyfumarate). Nicotinamide, a precursor of nicotinamide-adenine dinucleotide (NAD) and an inhibitor of ADP-ribose synthetase, also protected cultured endothelial cells from loss of membrane integrity in a concentration-dependent manner after exposure to dihydroxyfumarate.

  14. Preparation of cell cultures and vaccinia virus stocks.

    PubMed

    Earl, P L; Cooper, N; Wyatt, L S; Moss, B; Carroll, M W

    2001-05-01

    This unit describes the maintenance of cell lines used with vaccinia virus, both in monolayer cultures and in suspension. The suspended cell culture is then used in the preparation of vaccinia virus stocks. The preparation of chick embryo fibroblasts (CEF) is also presented for use in the production of the highly attenuated and host range-restricted modified vaccinia virus Ankara (MVA) strain of vaccinia virus. Additionally, support protocols are presented for the titration of standard and MVA vaccinia virus stocks.

  15. Disruption of cultured cells by nitrogen cavitation.

    PubMed

    Simpson, Richard J

    2010-11-01

    Cell disruption by nitrogen decompression from a pressurized vessel is a rapid and effective way to homogenize cells and tissues, to release intact organelles, and to prepare cell membranes. Cells are placed in a pressure vessel and large quantities of oxygen-free nitrogen are dissolved in the cells under high pressure (~5500 kilopascals [kPa], equivalent to 800 pounds per square inch [psi]). When the pressure is released suddenly, the nitrogen bubbles out of solution, rupturing the cell membrane and releasing the cell contents. Nitrogen cavitation is well suited for mammalian and plant cells and fragile bacteria, but is less effective with yeast, fungi, spores, or other cell types with tough cell walls. The chemical and physical stresses imposed by nitrogen cavitation on enzymes and subcellular compartments are minimized compared with ultrasonic and mechanical homogenizing methods. Unlike lysis methods relying on shear stresses and friction, there is no heat damage to proteins and organelles during nitrogen cavitation. Indeed, the method is accompanied by an adiabatic expansion that cools the sample instead. Also, labile cell components are protected from oxidation by the inert nitrogen gas. Furthermore, nitrogen does not alter the pH of the suspending medium. The process is fast and uniform because the same disruptive forces are applied within each cell and throughout the sample, ensuring reproducible cell-free homogenates. Finally, variable sample sizes (e.g., from ~1 mL to 1 L or more) can be accommodated with most commercial systems.

  16. Microfabricated polymeric vessel mimetics for 3-D cancer cell culture

    PubMed Central

    Jaeger, Ashley A.; Das, Chandan K.; Morgan, Nicole Y.; Pursley, Randall H.; McQueen, Philip G.; Hall, Matthew D.; Pohida, Thomas J.; Gottesman, Michael M.

    2013-01-01

    Modeling tumor growth in vitro is essential for cost-effective testing of hypotheses in preclinical cancer research. 3-D cell culture offers an improvement over monolayer culture for studying cellular processes in cancer biology because of the preservation of cell-cell and cell-ECM interactions. Oxygen transport poses a major barrier to mimicking in vivo environments and is not replicated in conventional cell culture systems. We hypothesized that we can better mimic the tumor microenvironment using a bioreactor system for controlling gas exchange in cancer cell cultures with silicone hydrogel synthetic vessels. Soft-lithography techniques were used to fabricate oxygen-permeable silicone hydrogel membranes containing arrays of micropillars. These membranes were inserted into a bioreactor and surrounded by basement membrane extract (BME) within which fluorescent ovarian cancer (OVCAR8) cells were cultured. Cell clusters oxygenated by synthetic vessels showed a ∼100um drop-off to anoxia, consistent with in vivo studies of tumor nodules fed by the microvasculature. We showed oxygen tension gradients inside the clusters oxygenated by synthetic vessels had a ∼100 µm drop-off to anoxia, which is consistent with in vivo studies. Oxygen transport in the bioreactor system was characterized by experimental testing with a dissolved oxygen probe and finite element modeling of convective flow. Our study demonstrates differing growth patterns associated with controlling gas distributions to better mimic in vivo conditions. PMID:23911071

  17. Radiosensitivity of cultured insect cells: II. Diptera

    SciTech Connect

    Koval, T.M.

    1983-10-01

    The radiosensitivity of five dipteran cell lines representing three mosquito genera and one fruit fly genus were examined. These lines are: (1) ATC-10, Aedes aegypti; (2) RU-TAE-14, Toxorhynchites amboinensis; (3) RU-ASE-2A, Anopheles stephensi; (4) WR69-DM-1, Drosophila melanogaster; and (5) WR69-DM-2, Drosophila melanogaster. Population doubling times for these lines range from approximately 16 to 48 hr. Diploid chromosome numbers are six for the mosquito cells and eight for the fruit fly cells D/sub 0/ values are 5.1 and 6.5 Gy for the Drosophila cell lines and 3.6, 6.2, and 10.2 Gy for the mosquito cell lines. The results of this study demonstrate that dipteran insect cells are a few times more resistant to radiation than mammalian cells, but not nearly as radioresistant as lepidopteran cells.

  18. Carbon nanowall scaffold to control culturing of cervical cancer cells

    NASA Astrophysics Data System (ADS)

    Watanabe, Hitoshi; Kondo, Hiroki; Okamoto, Yukihiro; Hiramatsu, Mineo; Sekine, Makoto; Baba, Yoshinobu; Hori, Masaru

    2014-12-01

    The effect of carbon nanowalls (CNWs) on the culturing rate and morphological control of cervical cancer cells (HeLa cells) was investigated. CNWs with different densities were grown using plasma-enhanced chemical vapor deposition and subjected to post-growth plasma treatment for modification of the surface terminations. Although the surface wettability of the CNWs was not significantly dependent on the CNW densities, the cell culturing rates were significantly dependent. Morphological changes of the cells were not significantly dependent on the density of CNWs. These results indicate that plasma-induced surface morphology and chemical terminations enable nanobio applications using carbon nanomaterials.

  19. Progress in the development of shrimp cell cultures in Thailand.

    PubMed

    Kasornchandra, J; Khongpradit, R; Ekpanithanpong, U; Boonyaratpalin, S

    1999-01-01

    Primary shrimp cell cultures were developed from lymphoid organ and ovaries of black tiger shrimp, Penaeus monodon, in double-strength Leibovitz's L-15 medium supplemented with 15% fetal bovine serum, 1% glucose, 5 g/L NaCl, 15% shrimp meat extract. The optimum conditions for primary culture in vitro were obtained in L-15 medium with an osmolality of approximately 730 +/- 10 mmol/kg, a temperature range of 25--28 degrees C and incubation in a normal atmosphere. However, basal medium supplemented with 0.01% cholesterol could enhance good growth and cells performance initiated from lymphoid organ. Both epithelial-like and fibroblastic- like cells were observed from those organs within 2 days incubation. Within 3 days, 80% confluent monolayers were obtained from the lymphoid organ while cultures from other tissues required 5 days. Cultures were maintained for at least 43 days. Only cells from lymphoid organ could be subcultured and confluent monolayers achieved within 10 days post-spilt. Healthy cultures of the lymphoid cells did not persist beyond the third passage. Application of these primary shrimp cell cultures for studying pathogenic viruses of shrimp in vitro will be discussed. PMID:10627677

  20. Progress in the development of shrimp cell cultures in Thailand.

    PubMed

    Kasornchandra, J; Khongpradit, R; Ekpanithanpong, U; Boonyaratpalin, S

    1999-01-01

    Primary shrimp cell cultures were developed from lymphoid organ and ovaries of black tiger shrimp, Penaeus monodon, in double-strength Leibovitz's L-15 medium supplemented with 15% fetal bovine serum, 1% glucose, 5 g/L NaCl, 15% shrimp meat extract. The optimum conditions for primary culture in vitro were obtained in L-15 medium with an osmolality of approximately 730 +/- 10 mmol/kg, a temperature range of 25--28 degrees C and incubation in a normal atmosphere. However, basal medium supplemented with 0.01% cholesterol could enhance good growth and cells performance initiated from lymphoid organ. Both epithelial-like and fibroblastic- like cells were observed from those organs within 2 days incubation. Within 3 days, 80% confluent monolayers were obtained from the lymphoid organ while cultures from other tissues required 5 days. Cultures were maintained for at least 43 days. Only cells from lymphoid organ could be subcultured and confluent monolayers achieved within 10 days post-spilt. Healthy cultures of the lymphoid cells did not persist beyond the third passage. Application of these primary shrimp cell cultures for studying pathogenic viruses of shrimp in vitro will be discussed.

  1. Auxin requirements of sycamore cells in suspension culture.

    PubMed

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

    1983-04-01

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

  2. Contacts between pigmented retina epithelial cells in culture.

    PubMed

    Middleton, C A; Pegrum, S M

    1976-11-01

    The behaviour of primary cultures of dissociated embryonic chick pigmented retina epithelial (PRE) cells has been investigated. Isolated PRE cells have a mean speed of locomotion of 7-16 mum/h. Collisions between the cells normally result in the development of stable contacts between the cells involved. This leads to a gradual reduction in the number of isolated cells and an increase in the number of cells incorporated into islands. Ultrastructural observations of islands of cells after 24 h in culture show that junctional complexes are present between the cells. These complexes consist of 2 components: (a) an apically situated region of focal tight junctions and/or gap junctions, and (b) a more ventrally located zonula adhaerens with associated cytoplasmic filaments forming a band running completely around the periphery of each cell. The intermembrane gap in the region of the zonula is 6-0-12-0 nm. The junctional complexes become more differentiated with time and after 48 h in culture consist of an extensive region of tight junctions and/or gap junctions and a more specialized zonula adhaerens. It is suggested that the development of junctional complexes may be responsible for the stable contacts that the cells display in culture.

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

    PubMed

    Tallman, Gary

    2006-01-01

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

  4. Production of minimally disturbed synchronous cultures of hematopoietic cells

    NASA Technical Reports Server (NTRS)

    Thornton, Maureen; Eward, Kathryn Leigh; Helmstetter, Charles E.; Edward, K. L. (Principal Investigator)

    2002-01-01

    A method is describedforproducing sizable quantities of synchronously dividing, minimally disturbed mammalian cells. Cultures were grown immobilized on surfaces such that cell division within the population resulted in the continuous release of synchronous newborn cells. As judged by the quality and duration of synchronous growth, cell size distributions, and DNA compositions, newborn mouse L1210 cells grew with a very high level of synchrony without overt evidence of growth disturbances. The technology should be applicable to a variety of hematopoietic cells, as evidenced by similar results with human MOLT-4 and U937 cell lines.

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

    PubMed

    Lestard, Nathalia R; Capella, Marcia A M

    2016-01-01

    Although music is part of virtually all cultures in the world, little is known about how it affects us. Since the beginning of this century several studies suggested that the response to music, and to sound in general, is complex and might not be exclusively due to emotion, given that cell types other than auditory hair cells can also directly react to audible sound. The present study was designed to better understand the direct effects of acoustic vibrations, in the form of music, in human cells in culture. Our results suggest that the mechanisms of cell growth arrest and/or cell death induced by acoustic vibrations are similar for auditory and nonauditory cells. PMID:27478480

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

    PubMed Central

    Lestard, Nathalia R.

    2016-01-01

    Although music is part of virtually all cultures in the world, little is known about how it affects us. Since the beginning of this century several studies suggested that the response to music, and to sound in general, is complex and might not be exclusively due to emotion, given that cell types other than auditory hair cells can also directly react to audible sound. The present study was designed to better understand the direct effects of acoustic vibrations, in the form of music, in human cells in culture. Our results suggest that the mechanisms of cell growth arrest and/or cell death induced by acoustic vibrations are similar for auditory and nonauditory cells. PMID:27478480

  7. Cell density determines epithelial migration in culture.

    PubMed Central

    Rosen, P; Misfeldt, D S

    1980-01-01

    The dog kidney epithelial cell line (MDCK) has been shown to exhibit a density-correlated inhibition of growth at approxmately 6.6 X 10(5) cells per cm2. When a confluent monolayer at its maximal density was wounded by removal of a wide swath of cells, migration of the cell sheet into the denuded area occurred. Precise measurements of the rate of migration for 5 day showed that the cells accelerated at a uniform rate of 0.24 micrometer . hr-2 and, by extrapolation, possessed an apparent initial velocity of 2.8 micrometer . hr-1 at the time of wounding. The apparent initial velocity was considered to be the result of a brief (< 10 hr) and rapid acceleration dependent on cell density. To verify this, wounds were made at different densities below the maximum. In these experiments, the cells did not migrate until a "threshold" density of 2.0 X 10(5) cells per cm2 was reached regardless of the density at the time of wounding. At the threshold density, the cell sheet began to accelerate at the previously measured rate (0.24 micrometer . hr-2). Any increase in density by cell division was balanced by cell migration, so that the same threshold density was maintained by the migrating cells. Each migrating cell sustained the movement of the cell sheet at a constant rate of acceleration. It is proposed that an acceleration is, in general, characteristic of the vectorial movement of an epithelial cell sheet. Images PMID:6933523

  8. Hypergravity signal transduction and gene expression in cultured mammalian cells

    NASA Technical Reports Server (NTRS)

    Kumei, Y.; Whitson, P. A.

    1994-01-01

    A number of studies have been conducted during space flight and with clinostats and centrifuges, suggesting that gravity effects the proliferation and differentiation of mammalian cells in vitro. However, little is known about the mechanisms by which mammalian cells respond to changes in gravitational stress. This paper summarizes studies designed to clarify the effects of hypergravity on the cultured human HeLa cells and to investigate the mechanism of hypergravity signal transduction in these cells.

  9. Peroxidases from cell suspension cultures of Brassica napus.

    PubMed

    Agostini, E; de Forchetti, S M; Tigier, H A

    2000-08-01

    Cell suspension cultures of Brassica napus were obtained under different hormonal conditions, using 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin as growth regulators. They were analyzed as a culture system for peroxidase production in vitro to avoid many of the problems that affect the production from field-grown roots. Total peroxidase specific activities reached a maximum at the end of exponential growth phase of the cultures. Cultures obtained with 4 mg/l of 2,4-D an without kinetin or with 1 mg/l of 2,4-D and the same amount of kinetin produced twice the total activity of root extracts and, in addition, they released peroxidases to the culture medium, which would be advantageous for the commercial production of the enzyme. Peroxidase patterns, obtained by isoelectric focusing of cell extracts and of culture medium of cell suspension cultures, differed from those of root crude extracts from field-grown plants with additional bands of higher isoelectric points. These cultures showed interesting properties and could be considered an alternative source of peroxidases for commercial production and/or to be applied as a model for physiological research. PMID:10979611

  10. Sterol phylogenesis and algal evolution

    SciTech Connect

    Nes, W.D.; Norton, R.A.; Crumley, F.G. ); Madigan, S.J.; Katz, E.R. )

    1990-10-01

    The stereochemistry of several sterol precursors and end products synthesized by two fungal-like microorganisms Prototheca wickerhamii (I) and Dictyostelium discoideum (II) have been determined by chromatographic (TLC, GLC, and HPLC) and spectral (UV, MS, and {sup 1}H NMR) methods. From I and II the following sterols were isolated from the cells: cycloartenol, cyclolaudenol, 24(28)-methylenecy-cloartanol, ergosterol, protothecasterol, 4{alpha}-methylergostanol, 4{alpha}-methylclionastanol, clionastanol, 24{beta}-ethylcholesta-8,22-enol, and dictyosterol. In addition, the mechanism of C-24 methylation was investigated in both organisms by feeding to I (2-{sup 3}H)lanosterol, (2-{sup 3}H)cycloartenol, (24{sup 3}H)lanosterol, and (methyl-{sup 2}H{sub 3})methionine and by feeding to II (methyl-{sup 2}H{sub 3})methionine. The results demonstrate that the 24{beta} configuration is formed by different alkylation routes in I and II. The authors conclude that Prototheca is an apoplastic Chlorella (i.e., an alga) and that Dictyostelium as well as the other soil amoebae that synthesize cycloartenol evolved from algal rather than fungal ancestors.

  11. Microflotation performance for algal separation.

    PubMed

    Hanotu, James; Bandulasena, H C Hemaka; Zimmerman, William B

    2012-07-01

    The performance of microflotation, dispersed air flotation with microbubble clouds with bubble size about 50 µm, for algae separation using fluidic oscillation for microbubble generation is investigated. This fluidic oscillator converts continuous air supply into oscillatory flow with a regular frequency to generate bubbles of the scale of the exit pore. Bubble characterization results showed that average bubble size generated under oscillatory air flow state was 86 µm, approximately twice the size of the diffuser pore size of 38 µm. In contrast, continuous air flow at the same rate through the same diffusers yielded an average bubble size of 1,059 µm, 28 times larger than the pore size. Following microbubble generation, the separation of algal cells under fluidic oscillator generated microbubbles was investigated by varying metallic coagulant types, concentration and pH. Best performances were recorded at the highest coagulant dose (150 mg/L) applied under acidic conditions (pH 5). Amongst the three metallic coagulants studied, ferric chloride yielded the overall best result of 99.2% under the optimum conditions followed closely by ferric sulfate (98.1%) and aluminum sulfate with 95.2%. This compares well with conventional dissolved air flotation (DAF) benchmarks, but has a highly turbulent flow, whereas microflotation is laminar with several orders of magnitude lower energy density.

  12. Hydrofocusing Bioreactor for Three-Dimensional Cell Culture

    NASA Technical Reports Server (NTRS)

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

    2003-01-01

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

  13. Recombinant spider silk matrices for neural stem cell cultures.

    PubMed

    Lewicka, Michalina; Hermanson, Ola; Rising, Anna U

    2012-11-01

    Neural stem cells (NSCs) have the capacity to differentiate into neurons, astrocytes, and oligodendrocytes. Accordingly, NSCs hold great promise in drug screening and treatment of several common diseases. However, a major obstacle in applied stem cell research is the limitation of synthetic matrices for culturing stem cells. The objective of this study was to evaluate the suitability of recombinant spider silk (4RepCT) matrices for growth of NSCs. NSCs isolated from the cerebral cortices of mid-gestation rat embryos were cultured on either 4RepCT matrices or conventional poly-L-ornithine and fibronectin (P + F) coated polystyrene plates. From 48 h of culture, no significant differences in cell proliferation or viability were detected in NSC cultures on 4RepCT compared to control matrices (polystyrene plates coated with P + F). The NSCs retained an undifferentiated state, displaying low or no staining for markers of differentiated cells. Upon stimulation NSCs grown on 4RepCT differentiated efficiently into neuronal and astrocytic cells to virtually the same degree as control cultures, but a slightly less efficient oligodendrocyte differentiation was noted. We suggest that recombinant spider silk matrices provide a functional microenvironment and represent a useful tool for the development of new strategies in neural stem cell research. PMID:22863380

  14. The effect of TRAIL molecule on cell viability in in vitro beta cell culture.

    PubMed

    Tekmen, I; Ozyurt, D; Pekçetin, C; Buldan, Z

    2007-06-01

    Insulin-dependent diabetes mellitus (IDDM) is an organ-specific autoimmune disorder triggered by autoreactive T cells directed to pancreas beta-cell antigens. In this disorder, more than 90% of beta cells are destroyed. Cell death may be mediated via soluble or membrane-bound cell death ligands. One of these ligands may be tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the TNF-alpha superfamily. In the present study, we examined whether TRAIL had cytotoxic effects on adult rat pancreas beta cell cultures and INS1-E rat insulinoma cell line cultures or not. In this study, cell destruction models were built with TRAIL concentrations of 10, 100 and 1000 ng. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test was used for evaluating cell viability. It was detected that cell cultures with TRAIL added showed no differences statistically when compared with control cultures containing no toxic additions. These results showed that TRAIL did not have significant cytotoxic effects on pancreas beta cell culture and INS-1E rat insulinoma cell line cultures. Detection of the expression of TRAIL receptors and natural apoptosis inhibitor proteins will be favourable to investigate the resistance mechanisms to TRAIL-induced cell death in this cell culture system.

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

    EPA Science Inventory

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

  16. Mechanical algal disruption for efficient biodiesel extraction

    NASA Astrophysics Data System (ADS)

    Krehbiel, Joel David

    Biodiesel from algae provides several benefits over current biodiesel feedstocks, but the energy requirements of processing algae into a useable fuel are currently so high as to be prohibitive. One route to improving this is via disruption of the cells prior to lipid extraction, which can significantly increase energy recovery. Unfortunately, several obvious disruption techniques require more energy than can be gained. This dissertation examines the use of microbubbles to improve mechanical disruption of algal cells using experimental, theoretical, and computational methods. New laboratory experiments show that effective ultrasonic disruption of algae is achieved by adding microbubbles to an algal solution. The configuration studied flows the solution through a tube and insonifies a small section with a high-pressure ultrasound wave. Previous biomedical research has shown effective cell membrane damage on animal cells with similar methods, but the present research is the first to extend such study to algal cells. Results indicate that disruption increases with peak negative pressure between 1.90 and 3.07 MPa and with microbubble concentration up to 12.5 x 107 bubbles/ml. Energy estimates of this process suggest that it requires only one-fourth the currently most-efficient laboratory-scale disruption process. Estimates of the radius near each bubble that causes disruption (i.e. the disruption radius) suggest that it increases with peak negative pressure and is near 9--20 microm for all cases tested. It is anticipated that these procedures can be designed for better efficiency and efficacy, which will be facilitated by identifying the root mechanisms of the bubble-induced disruption. We therefore examine whether bubble expansion alone creates sufficient cell deformation for cell rupture. The spherically-symmetric Marmottant model for bubble dynamics allows estimation of the flow regime under experimental conditions. Bubble expansion is modeled as a point source of

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

    PubMed

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

    2014-09-01

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

  18. Advances in culture and manipulation of human pluripotent stem cells.

    PubMed

    Qian, X; Villa-Diaz, L G; Krebsbach, P H

    2013-11-01

    Recent advances in the understanding of pluripotent stem cell biology and emerging technologies to reprogram somatic cells to a stem cell-like state are helping bring stem cell therapies for a range of human disorders closer to clinical reality. Human pluripotent stem cells (hPSCs) have become a promising resource for regenerative medicine and research into early development because these cells are able to self-renew indefinitely and are capable of differentiation into specialized cell types of all 3 germ layers and trophoectoderm. Human PSCs include embryonic stem cells (hESCs) derived from the inner cell mass of blastocyst-stage embryos and induced pluripotent stem cells (hiPSCs) generated via the reprogramming of somatic cells by the overexpression of key transcription factors. The application of hiPSCs and the finding that somatic cells can be directly reprogrammed into different cell types will likely have a significant impact on regenerative medicine. However, a major limitation for successful therapeutic application of hPSCs and their derivatives is the potential xenogeneic contamination and instability of current culture conditions. This review summarizes recent advances in hPSC culture and methods to induce controlled lineage differentiation through regulation of cell-signaling pathways and manipulation of gene expression as well as new trends in direct reprogramming of somatic cells.

  19. Mortality in cultures of the dinoflagellate Amphidinium carterae during culture senescence and darkness.

    PubMed Central

    Franklin, Daniel J.; Berges, John A.

    2004-01-01

    The study of cell death in higher plants and animals has revealed the existence of an active ('programmed') process in most types of cell, and similarities in cell death between plants, animals, yeast and bacteria suggest an evolutionarily ancient origin of programmed cell death (PCD). Despite their global importance in primary production, information on algal cell death is limited. Algal cell death could have similarities with metazoan cell death. One morphotype of metazoan PCD, apoptosis, can be induced by light deprivation in the unicellular chlorophyte Dunaliella tertiolecta. The situation in other algal taxa is less clear. We used a model dinoflagellate (Amphidinium carterae) to test whether mortality during darkness and culture senescence showed apoptotic characteristics. Using transmission electron microscopy, fluorescent biomarkers, chlorophyll fluorescence and particulate carbon analysis we analysed the process of cell mortality and found that light deprivation caused mass mortality. By contrast, fewer dead cells (5-20% of the population) were found in late-phase cultures, while a similar degenerate cell morphology (shrunken, chlorotic) was observed. On morphological grounds, our observations suggest that the apoptotic cell death described in D. tertiolecta does not occur in A. carterae. Greater similarity was found with paraptosis, a recently proposed alternative morphotype of PCD. A paraptotic conclusion is supported by inconclusive DNA fragmentation results. We emphasize the care that must be taken in transferring fundamental paradigms between phylogenetically diverse cell types and we argue for a greater consistency in the burden of proof needed to assign causality to cell death processes. PMID:15475328

  20. Automation of 3D cell culture using chemically defined hydrogels.

    PubMed

    Rimann, Markus; Angres, Brigitte; Patocchi-Tenzer, Isabel; Braum, Susanne; Graf-Hausner, Ursula

    2014-04-01

    Drug development relies on high-throughput screening involving cell-based assays. Most of the assays are still based on cells grown in monolayer rather than in three-dimensional (3D) formats, although cells behave more in vivo-like in 3D. To exemplify the adoption of 3D techniques in drug development, this project investigated the automation of a hydrogel-based 3D cell culture system using a liquid-handling robot. The hydrogel technology used offers high flexibility of gel design due to a modular composition of a polymer network and bioactive components. The cell inert degradation of the gel at the end of the culture period guaranteed the harmless isolation of live cells for further downstream processing. Human colon carcinoma cells HCT-116 were encapsulated and grown in these dextran-based hydrogels, thereby forming 3D multicellular spheroids. Viability and DNA content of the cells were shown to be similar in automated and manually produced hydrogels. Furthermore, cell treatment with toxic Taxol concentrations (100 nM) had the same effect on HCT-116 cell viability in manually and automated hydrogel preparations. Finally, a fully automated dose-response curve with the reference compound Taxol showed the potential of this hydrogel-based 3D cell culture system in advanced drug development.

  1. Growth and immobilization of tripterygium wilfordii cultured cells.

    PubMed

    Pépin, M F; Chavarie, C; Archambault, J

    1991-12-20

    The plant Tripterygium wilfordii produces di- and triterpenes of interest for male contraception and treatment of arthritis and skin disorders. Cell line TRP4a obtained form this plant in 1981 was reported to produce these valuable compounds at yields ( approximately 0.04% of the biomass dry weight) higher than found in the plant (0.001%). In order to improve this production, studies were carried out to determine the feasibility of eliminating the troublesome component of coconut milk originally used to culture this cell line. A defined formulation suitable for growth ad maintenance has been developed. This medium consisted of Gamborg's PRL4 or B5 medium supplemented with 2 mg L(-1) 2,4-dichlorophenoxyacetic acid and 20 g L(-1) sucrose. Furthermore, monitoring of carbohydrate uptake revealed that T. wilfordii cells, contrary to many plant cell species, did not hydrolyze sucrose extra-cellularly before uptake. Replacement of this disaccharide by glucose or fructose increased specific growth rate from 0.15 to 0.25 day(-1). As tripdiolide is reported to be present in broth extract in significant amounts, plant cell immobilization technology offers a promising alternative to suspension cultures, especially in view to on line harvesting of the product. Surface immobilized T. wilfordii cell cultures were successfully carried out in 2-L bioreactors. Their biomass production and carbohydrate uptake were comparable to those observed for shake flask grown suspension cultures. Higher nitrate and ammonium uptake were found in immobilized cultures. PMID:18600729

  2. Advanced Cell Culture Techniques for Cancer Drug Discovery

    PubMed Central

    Lovitt, Carrie J.; Shelper, Todd B.; Avery, Vicky M.

    2014-01-01

    Human cancer cell lines are an integral part of drug discovery practices. However, modeling the complexity of cancer utilizing these cell lines on standard plastic substrata, does not accurately represent the tumor microenvironment. Research into developing advanced tumor cell culture models in a three-dimensional (3D) architecture that more prescisely characterizes the disease state have been undertaken by a number of laboratories around the world. These 3D cell culture models are particularly beneficial for investigating mechanistic processes and drug resistance in tumor cells. In addition, a range of molecular mechanisms deconstructed by studying cancer cells in 3D models suggest that tumor cells cultured in two-dimensional monolayer conditions do not respond to cancer therapeutics/compounds in a similar manner. Recent studies have demonstrated the potential of utilizing 3D cell culture models in drug discovery programs; however, it is evident that further research is required for the development of more complex models that incorporate the majority of the cellular and physical properties of a tumor. PMID:24887773

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

    NASA Technical Reports Server (NTRS)

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

    1969-01-01

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

  4. Distribution of heavy metals from flue gas in algal bioreactor

    NASA Astrophysics Data System (ADS)

    Napan, Katerine

    Flue gas from coal-fired power plants is a major source of CO2 to the atmosphere. Microalgae can use this enriched form of CO2 as carbon source and in turn the biomass can be used to produce food, feed, fertilizer and biofuels. However, along with CO2, coal-based flue gas will inevitably introduce heavy metals, which have a high affinity to bind algal cells, could be toxic to the organisms and if transferred to the products could limit their uses. This study seeks to address the distribution and impact of heavy metals present in flue gas on microalgae production systems. To comprehend its effects, algae Scenedesmus obliquus was grown in batch reactors in a multimetal system. Ten heavy metals (Cu, Co, Zn, Pb, As, Se, Cr, Hg, Ni and Cd) were selected and were evaluated at four concentrations (1X, 2X, 5X and 10X). Results show that most heavy metals accumulated mainly in biomass and were found in very low concentrations in media. Hg was shown to be lost from the culture, with low amounts present in the biomass. An upper limit for As uptake was observed, suggesting its likelihood to build-up in the system during medium recycle. The As limited bioaccumulation was overcome by addition of sulfur to the algal medium. Heavy metal at 2X, 5X and 10X inhibited both growth and lipid production, while at the reference concentration both biomass and lipids yields were increased. Heavy metal concentrations in the medium and biomass were time dependent, and at the end of the cultivation most heavy metals in the supernatant solution complied with the recommendations for irrigation water, while biomass was below limits for cattle and poultry feed, fertilizer, plastic and paper. This research shows that bioremediation of CO2 and heavy metals in combination with energy production can be integrated, which is an environmentally friendly form of biotechnology.

  5. Cloning higher plants from aseptically cultured tissues and cells

    NASA Technical Reports Server (NTRS)

    Krikorian, A. D.

    1982-01-01

    A review of aseptic culture methods for higher plants is presented, which focuses on the existing problems that limit or prevent the full realization of cloning plants from free cells. It is shown that substantial progress in clonal multiplication has been made with explanted stem tips or lateral buds which can be stimulated to produce numerous precocious axillary branches. These branches can then be separated or subdivided and induced to root in order to yield populations of genetically and phenotypically uniorm plantlets. Similarly, undifferentiated calluses can sometimes be induced to form shoots and/or roots adventitiously. Although the cell culture techniques required to produce somatic embryos are presently rudimentary, steady advances are being made in learning how to stimulate formation of somatic or adventive embryos from totipotent cells grown in suspension cultures. It is concluded that many problems exist in the producing and growing of totipotent or morphogenetically competent cell suspensions, but the potential benefits are great.

  6. Fabricating gradient hydrogel scaffolds for 3D cell culture.

    PubMed

    Chatterjee, Kaushik; Young, Marian F; Simon, Carl G

    2011-05-01

    Optimizing cell-material interactions is critical for maximizing regeneration in tissue engineering. Combinatorial and high-throughput (CHT) methods can be used to systematically screen tissue scaffolds to identify optimal biomaterial properties. Previous CHT platforms in tissue engineering have involved a two-dimensional (2D) cell culture format where cells were cultured on material surfaces. However, these platforms are inadequate to predict cellular response in a three-dimensional (3D) tissue scaffold. We have developed a simple CHT platform to screen cell-material interactions in 3D culture format that can be applied to screen hydrogel scaffolds. Herein we provide detailed instructions on a method to prepare gradients in elastic modulus of photopolymerizable hydrogels.

  7. Phenotype expression of human bone cells cultured on implant substrates.

    PubMed

    Locci, P; Becchetti, E; Pugliese, M; Rossi, L; Belcastro, S; Calvitti, M; Pietrarelli, G; Staffolani, N

    1997-09-01

    Bone cells derived from the human jaw were cultured on titanium, titanium coated with hydroxyapatite (THA) or with plasma spray (TPS) to study the behaviour of the cells anchored to implant substrates. Bone cells were cultured in MEM with the addition of [3H]-thymidine to evaluate cellular proliferation, and [3H]-glucosamine to evaluate GAG synthesis and accumulation in the extra-cellular matrix (ECM). Moreover, to study the degradation of GAG bone cells were cultured in the presence of NH4Cl, an amine known to inhibit lysosomal activity. Our results show that TPS is the substrate that favours both cellular proliferation and the accumulation of GAG in the ECM. PMID:9377794

  8. Miniature Bioreactor System for Long-Term Cell Culture

    NASA Technical Reports Server (NTRS)

    Gonda, Steve R.; Kleis, Stanley J.; Geffert, Sandara K.

    2010-01-01

    A prototype miniature bioreactor system is designed to serve as a laboratory benchtop cell-culturing system that minimizes the need for relatively expensive equipment and reagents and can be operated under computer control, thereby reducing the time and effort required of human investigators and reducing uncertainty in results. The system includes a bioreactor, a fluid-handling subsystem, a chamber wherein the bioreactor is maintained in a controlled atmosphere at a controlled temperature, and associated control subsystems. The system can be used to culture both anchorage-dependent and suspension cells, which can be either prokaryotic or eukaryotic. Cells can be cultured for extended periods of time in this system, and samples of cells can be extracted and analyzed at specified intervals. By integrating this system with one or more microanalytical instrument(s), one can construct a complete automated analytical system that can be tailored to perform one or more of a large variety of assays.

  9. Microfluidic titer plate for stratified 3D cell culture.

    PubMed

    Trietsch, Sebastiaan J; Israëls, Guido D; Joore, Jos; Hankemeier, Thomas; Vulto, Paul

    2013-09-21

    Human tissues and organs are inherently heterogeneous. Their functionality is determined by the interplay between different cell types, their secondary architecture, vascular system and gradients of signaling molecules and metabolites. Here we propose a stratified 3D cell culture platform, in which adjacent lanes of gels and liquids are patterned by phaseguides to capture this tissue heterogeneity. We demonstrate 3D cell culture of HepG2 hepatocytes under continuous perfusion, a rifampicin toxicity assay and co-culture with fibroblasts. 4T1 breast cancer cells are used to demonstrate invasion and aggregation models. The platform is incorporated in a microtiter plate format that renders it fully compatible with automation and high-content screening equipment. The extended functionality, ease of handling and full compatibility to standard equipment is an important step towards adoption of Organ-on-a-Chip technology for screening in an industrial setting.

  10. Hydrodynamic effects on cells in agitated tissue culture reactors

    NASA Technical Reports Server (NTRS)

    Cherry, R. S.; Papoutsakis, E. T.

    1986-01-01

    The mechanisms by which hydrodynamic forces can affect cells grown on microcarrier beads in agitated cell culture reactors were investigated by analyzing the motion of microcarriers relative to the surrounding fluid, to each other, and to moving or stationary solid surfaces. It was found that harmful effects on cell cultures that have been previously attributed to shear can be better explained as the effects of turbulence (of a size scale comparable to the microcarriers or the spacing between them) or collisions. The primary mechanisms of cell damage involve direct interaction between microcarriers and turbulent eddies, collisions between microcarriers in turbulent flow, and collisions against the impeller or other solid surfaces. The implications of these analytical results for the design of tissue culture reactors are discussed.

  11. Bacterial Cellulose as a Substrate for Microbial Cell Culture

    PubMed Central

    Yin, Na; Santos, Thiago M. A.; Auer, George K.; Crooks, John A.; Oliver, Piercen M.

    2014-01-01

    Bacterial cellulose (BC) has a range of structural and physicochemical properties that make it a particularly useful material for the culture of bacteria. We studied the growth of 14 genera of bacteria on BC substrates produced by Acetobacter xylinum and compared the results to growth on the commercially available biopolymers agar, gellan, and xanthan. We demonstrate that BC produces rates of bacterial cell growth that typically exceed those on the commercial biopolymers and yields cultures with higher titers of cells at stationary phase. The morphology of the cells did not change during growth on BC. The rates of nutrient diffusion in BC being higher than those in other biopolymers is likely a primary factor that leads to higher growth rates. Collectively, our results suggest that the use of BC may open new avenues in microbiology by facilitating bacterial cell culture and isolation. PMID:24441155

  12. Radiosensitivity of cultured insect cells: I. Lepidoptera

    SciTech Connect

    Koval, T.M.

    1983-10-01

    The radiosensitivity of five lepidopteran insect cell lines representing five different genera has been investigated. These lines are: (1) TN-368, Trichoplusia ni; (2) IPLB-SF-1254, Spodoptera frugiperda; (3) IPLB-1075, Heliothis zea; (4) MRRL-CHl, clone GVl, Manduca sexta; and (5) IAL-PID2, Plodia interpunctella. The cell lines grew at different rates and had population doubling times that ranged from 19 to 52 hr. All of the lines are highly heteroploid and have approximate chromosome numbers near or above 100. The chromosomes are very small. All of the lines are extremely radioresistant; cell populations are able to recover from 260 kVp X-ray exposures up to and including 400 Gy, the highest dose examined. Cell survival curves were obtainable for only the TN-368 and IPLB-SF-1254 lines. The TN-368 cells displayed a biphasic survival response with D/sub 0/, d/sub q/, and n values of 65.7 and 130.2 Gy, 9.0 and -36.1 Gy, and 1.2 and 0.8, respectively, for the steep and shallow portions of the curve. The IPLB-SF-1254 cells had a D/sub 0/ of 63.9 Gy. D/sub q/ of 19.0 Gy, and n value of 1.4. These studies provide definitive evidence of the radioresistance of lepidopteran cells, and suggest that this radioresistance is a characteristic of lepidopteran insects.

  13. Scan-Free Absorbance Spectral Imaging A(x, y, λ) of Single Live Algal Cells for Quantifying Absorbance of Cell Suspensions

    PubMed Central

    Isono, Takumi; Yamashita, Kyohei; Momose, Daisuke; Kobayashi, Hiroki; Kitamura, Masashi; Nishiyama, Yusuke; Hosoya, Takahiro; Kanda, Hiroaki; Kudo, Ayane; Okada, Norihide; Yagi, Takafumi; Nakata, Kazuaki; Mineki, Shigeru; Tokunaga, Eiji

    2015-01-01

    Label-free, non-invasive, rapid absorbance spectral imaging A(x,y,λ) microscopy of single live cells at 1.2 μm × 1.2 μm resolution with an NA = 0.85 objective was developed and applied to unicellular green algae Chlamydomonas reinhardtii. By introducing the fiber assembly to rearrange a two-dimensional image to the one-dimensional array to fit the slit of an imaging spectrograph equipped with a CCD detector, scan-free acquisition of three-dimensional information of A(x,y,λ) was realized. The space-resolved absorbance spectra of the eyespot, an orange organelle about 1 μm, were extracted from the green-color background in a chlorophyll-rich single live cell absorbance image. Characteristic absorbance change in the cell suspension after hydrogen photoproduction in C. reinhardtii was investigated to find a single 715-nm absorption peak was locally distributed within single cells. The formula to calculate the absorbance of cell suspensions from that of single cells was presented to obtain a quantitative, parameter-free agreement with the experiment. It is quantitatively shown that the average number of chlorophylls per cell is significantly underestimated when it is evaluated from the absorbance of the cell suspensions due to the package effect. PMID:26061268

  14. Scan-Free Absorbance Spectral Imaging A(x, y, λ) of Single Live Algal Cells for Quantifying Absorbance of Cell Suspensions.

    PubMed

    Isono, Takumi; Yamashita, Kyohei; Momose, Daisuke; Kobayashi, Hiroki; Kitamura, Masashi; Nishiyama, Yusuke; Hosoya, Takahiro; Kanda, Hiroaki; Kudo, Ayane; Okada, Norihide; Yagi, Takafumi; Nakata, Kazuaki; Mineki, Shigeru; Tokunaga, Eiji

    2015-01-01

    Label-free, non-invasive, rapid absorbance spectral imaging A(x,y,λ) microscopy of single live cells at 1.2 μm × 1.2 μm resolution with an NA = 0.85 objective was developed and applied to unicellular green algae Chlamydomonas reinhardtii. By introducing the fiber assembly to rearrange a two-dimensional image to the one-dimensional array to fit the slit of an imaging spectrograph equipped with a CCD detector, scan-free acquisition of three-dimensional information of A(x,y,λ) was realized. The space-resolved absorbance spectra of the eyespot, an orange organelle about 1 μm, were extracted from the green-color background in a chlorophyll-rich single live cell absorbance image. Characteristic absorbance change in the cell suspension after hydrogen photoproduction in C. reinhardtii was investigated to find a single 715-nm absorption peak was locally distributed within single cells. The formula to calculate the absorbance of cell suspensions from that of single cells was presented to obtain a quantitative, parameter-free agreement with the experiment. It is quantitatively shown that the average number of chlorophylls per cell is significantly underestimated when it is evaluated from the absorbance of the cell suspensions due to the package effect. PMID:26061268

  15. Scan-Free Absorbance Spectral Imaging A(x, y, λ) of Single Live Algal Cells for Quantifying Absorbance of Cell Suspensions.

    PubMed

    Isono, Takumi; Yamashita, Kyohei; Momose, Daisuke; Kobayashi, Hiroki; Kitamura, Masashi; Nishiyama, Yusuke; Hosoya, Takahiro; Kanda, Hiroaki; Kudo, Ayane; Okada, Norihide; Yagi, Takafumi; Nakata, Kazuaki; Mineki, Shigeru; Tokunaga, Eiji

    2015-01-01

    Label-free, non-invasive, rapid absorbance spectral imaging A(x,y,λ) microscopy of single live cells at 1.2 μm × 1.2 μm resolution with an NA = 0.85 objective was developed and applied to unicellular green algae Chlamydomonas reinhardtii. By introducing the fiber assembly to rearrange a two-dimensional image to the one-dimensional array to fit the slit of an imaging spectrograph equipped with a CCD detector, scan-free acquisition of three-dimensional information of A(x,y,λ) was realized. The space-resolved absorbance spectra of the eyespot, an orange organelle about 1 μm, were extracted from the green-color background in a chlorophyll-rich single live cell absorbance image. Characteristic absorbance change in the cell suspension after hydrogen photoproduction in C. reinhardtii was investigated to find a single 715-nm absorption peak was locally distributed within single cells. The formula to calculate the absorbance of cell suspensions from that of single cells was presented to obtain a quantitative, parameter-free agreement with the experiment. It is quantitatively shown that the average number of chlorophylls per cell is significantly underestimated when it is evaluated from the absorbance of the cell suspensions due to the package effect.

  16. Effects of carbon monoxide on cardiac muscle cells in culture

    SciTech Connect

    Nag, A.C.; Chen, K.C.; Cheng, Mei General Motors Research Laboratories, Warren, MI )

    1988-09-01

    Embryonic rat cardiac muscle cells grown in the presence of various tensions of CO (5-95%) without the presence of O{sub 2} survived and exhibited reduced cell growth, which was concentration dependent. When cardiac muscle cells were grown in the presence of a mixture of CO (10-20%) and O{sub 2} (10-20%), the growth rate of these cells was comparable to that of the control cells. Cardiac myocytes continued to beat when exposed to varying tensions of CO, except in the case of 95% CO. The cells exposed to different concentrations of CO contained fewer myofibrils of different stages of differentiation compared with the control and the culture exposed to a mixture of 20% O{sub 2} and 20% CO, with cells that contained abundant, highly differentiated myofibrils. There was no significant difference in the structural organization of mitochondria between the control and the surviving experimental cells. It is evident from the present studies that O{sub 2} is required for the optimum in vitro cellular growth of cardiac muscle. Furthermore, CO in combination with O{sub 2} at a concentration of 10 or 20% can produce optimal growth of cardiac muscle cells in culture. To determine maximum labeling index during the labeling period, cells were continuously labeled with ({sup 3}H)thymidine for 24 h before the termination of cultures.

  17. Establishing a stem cell culture laboratory for clinical trials

    PubMed Central

    Sekiya, Elíseo Joji; Forte, Andresa; Kühn, Telma Ingrid Borges de Bellis; Janz, Felipe; Bydlowski, Sérgio Paulo; Alves, Adelson

    2012-01-01

    Adult stem/progenitor cells are found in different human tissues. An in vitro cell culture is needed for their isolation or for their expansion when they are not available in a sufficient quantity to regenerate damaged organs and tissues. The level of complexity of these new technologies requires adequate facilities, qualified personnel with experience in cell culture techniques, assessment of quality and clear protocols for cell production. The rules for the implementation of cell therapy centers involve national and international standards of good manufacturing practices. However, such standards are not uniform, reflecting the diversity of technical and scientific development. Here standards from the United States, the European Union and Brazil are analyzed. Moreover, practical solutions encountered for the implementation of a cell therapy center appropriate for the preparation and supply of cultured cells for clinical studies are described. Development stages involved the planning and preparation of the project, the construction of the facility, standardization of laboratory procedures and development of systems to prevent cross contamination. Combining the theoretical knowledge of research centers involved in the study of cells with the practical experience of blood therapy services that manage structures for cell transplantation is presented as the best potential for synergy to meet the demands to implement cell therapy centers. PMID:23049427

  18. Culturing Cells in 3D Ordered Cellular Solids

    NASA Astrophysics Data System (ADS)

    Lin, Keng-Hui; Lin, Wang-Jung; Lin, Jing-Ying

    2011-03-01

    Constructing a well-defined 3D microenvironment for cell growth is a key step for tissue engineering and mechanobiology. We demonstrate high-throughput fabrication of gelatin-based ordered cellular solids with tunable pore size and solid fraction. This process involves generating monodisperse liquid foam with a cross-flow microfluidic device. The monodisperse liquid foam was further processed into open-cell solid foam, which was used as scaffolds for 3D cell culture. Three distinct cell types were cultured under these conditions and displayed appropriate physiological, morphological, and functional characteristics. Epithelial cells formed cyst-like structures and were polarized inside pores, myoblasts adopted a tubular structure and fused into myotubes, and fibroblasts exhibited wide varieties of morphologies depending on their location inside the scaffolds. These ordered cellular solids therefore make possible the study of pore-size effects on cells.

  19. The replacement of serum by hormones in cell culture media.

    PubMed

    Sato, G; Hayashi, I

    1976-12-01

    The replacement of serum by hormones in cell culture media. (Reemplazo del suero por hormonas en el medio de cultivo de células). Arch. Biol. Med. Exper. 10: 120-121, 1976. The serum used in cell culture media can be replaced by a mixture of hormones and some accesory blood factors. The pituitary cell line GH3 can be grown in a medium in which serum is replaced by triiodothyronine, transferrin, parathormone, tyrotrophin releasing hormone and somatomedins. Hela and BHK cell strains can also be grown in serum free medium supplemented with hormones. Each cell type appears to have different hormonal requirements yet it may found that some hormones are required for most cell types.

  20. Nanopillar based electrochemical biosensor for monitoring microfluidic based cell culture

    NASA Astrophysics Data System (ADS)

    Gangadharan, Rajan

    In-vitro assays using cultured cells have been widely performed for studying many aspects of cell biology and cell physiology. These assays also form the basis of cell based sensing. Presently, analysis procedures on cell cultures are done using techniques that are not integrated with the cell culture system. This approach makes continuous and real-time in-vitro measurements difficult. It is well known that the availability of continuous online measurements for extended periods of time will help provide a better understanding and will give better insight into cell physiological events. With this motivation we developed a highly sensitive, selective and stable microfluidic electrochemical glucose biosensor to make continuous glucose measurements in cell culture media. The performance of the microfluidic biosensor was enhanced by adding 3D nanopillars to the electrode surfaces. The microfluidic glucose biosensor consisted of three electrodes---Enzyme electrode, Working electrode, and Counter electrode. All these electrodes were enhanced with nanopillars and were optimized in their respective own ways to obtain an effective and stable biosensing device in cell culture media. For example, the 'Enzyme electrode' was optimized for enzyme immobilization via either a polypyrrole-based or a self-assembled-monolayer-based immobilization method, and the 'Working electrode' was modified with Prussian Blue or electropolymerized Neutral Red to reduce the working potential and also the interference from other interacting electro-active species. The complete microfluidic biosensor was tested for its ability to monitor glucose concentration changes in cell culture media. The significance of this work is multifold. First, the developed device may find applications in continuous and real-time measurements of glucose concentrations in in-vitro cell cultures. Second, the development of a microfluidic biosensor will bring technical know-how toward constructing continuous glucose

  1. Foetal hepatic progenitor cells assume a cholangiocytic cell phenotype during two-dimensional pre-culture

    PubMed Central

    Anzai, Kazuya; Chikada, Hiromi; Tsuruya, Kota; Ida, Kinuyo; Kagawa, Tatehiro; Inagaki, Yutaka; Mine, Tesuya; Kamiya, Akihide

    2016-01-01

    Liver consists of parenchymal hepatocytes and other cells. Liver progenitor cell (LPC) is the origin of both hepatocytes and cholangiocytic cells. The analyses of mechanism regulating differentiation of LPCs into these functional cells are important for liver regenerative therapy using progenitor cells. LPCs in adult livers were found to form cysts with cholangiocytic characteristics in 3D culture. In contrast, foetal LPCs cannot form these cholangiocytic cysts in the same culture. Thus, the transition of foetal LPCs into cholangiocytic progenitor cells might occur during liver development. Primary CD45−Ter119−Dlk1+ LPCs derived from murine foetal livers formed ALBUMIN (ALB)+CYTOKERATIN (CK)19− non-cholangiocytic cysts within 3D culture. In contrast, when foetal LPCs were pre-cultured on gelatine-coated dishes, they formed ALB−CK19+ cholangiocytic cysts. When hepatocyte growth factor or oncostatin M, which are inducers of hepatocytic differentiation, was added to pre-culture, LPCs did not form cholangiocytic cysts. These results suggest that the pre-culture on gelatine-coated dishes changed the characteristics of foetal LPCs into cholangiocytic cells. Furthermore, neonatal liver progenitor cells were able to form cholangiocytic cysts in 3D culture without pre-culture. It is therefore possible that the pre-culture of mid-foetal LPCs in vitro functioned as a substitute for the late-foetal maturation step in vivo. PMID:27335264

  2. Cytopathogenicity of Naegleria for cultured neuroblastoma cells

    SciTech Connect

    Fulford, D.E.

    1985-01-01

    The cytopathic activity of live Naegleria amoebae and cell-free lysates of Naegleria for B-103 rat neuroblastoma cells was investigated using a /sup 51/Cr release assay. Live amoebae and cell-free lysates of N. fowleri, N. australiensis, N. lovaniensis, and N. gruberi all induced sufficient damage to radiolabeled B-103 cells to cause a significant release of chromium. The cytotoxic activity present in the cell-free lysates of N. fowleri can be recovered in the supernatant fluid following centrifugation at 100,000xg and precipitation of the 100,000xg supernatant fluid with ammonium sulfate. Initial characterization of the cytotoxic factor indicates that it is a heat labile, pH sensitive, soluble protein. The cytotoxic activity is abolished by either extraction, unaffected by repeated freeze-thawing, and is not sensitive to inhibitors of proteolytic enzymes. Phospholipase A activity was detected in the cytotoxic ammonium sulfate precipitable material, suggesting that this enzyme activity may have a role in the cytotoxic activity of the cell-free lysates.

  3. Effects of citric acid on cultured human osteoblastic cells.

    PubMed

    Guimarães, Lizandra Ferrari; Fidalgo, Tatiana Kelly da Silva; Menezes, Gustavo Conde; Primo, Laura Guimarães; Costa e Silva-Filho, Fernando

    2010-11-01

    We investigated the effects of citric acid (CA) on cultured human osteoblastic (HOB) cells by evaluating cell adhesion, proliferation, and cytotoxicity. (3)H-Thymidine-labeled HOB cells were incubated in culture medium supplemented or not with 4%, 6%, 8%, or 10% CA for 1 minute. After incubation, cell morphology was evaluated by Nomarski interferential light microscopy, cell proliferation was accessed by measurements of (3)H-thymidine associated to the cells, and cell lysis was monitored by measuring the amount of (3)H-thymidine released by cells. We observed that most of the CA-treated cells presented numerous atypical vacuoles, and such cells were also highly polymorphic, exhibiting round-shaped cells. Nonetheless, CA at all concentrations assayed did not yield cytotoxicity as measured by (3)H-containing DNA release, although significant decrease in cell proliferation was observed (P > .05). Furthermore, cells which were treated with CA at the lowest concentration assayed (4%) restored normal proliferation rates 3 days after treatment.

  4. Isolation, culture and characterization of primary mouse RPE cells.

    PubMed

    Fernandez-Godino, Rosario; Garland, Donita L; Pierce, Eric A

    2016-07-01

    Mouse models are powerful tools for the study of ocular diseases. Alterations in the morphology and function of the retinal pigment epithelium (RPE) are common features shared by many ocular disorders. We report a detailed protocol to collect, seed, culture and characterize RPE cells from mice. We describe a reproducible method that we previously developed to collect and culture murine RPE cells on Transwells as functional polarized monolayers. The collection of RPE cells takes ∼3 h, and the cultures mimic in vivo RPE cell features within 1 week. This protocol also describes methods to characterize the cells on Transwells within 1-2 weeks by transmission and scanning electron microscopy (TEM and SEM, respectively), immunostaining of vibratome sections and flat mounts, and measurement of transepithelial electrical resistance. The RPE cell cultures are suitable to study the biology of the RPE from wild-type and genetically modified strains of mice between the ages of 10 d and 12 months. The RPE cells can also be manipulated to investigate molecular mechanisms underlying the RPE pathology in the numerous mouse models of ocular disorders. Furthermore, modeling the RPE pathology in vitro represents a new approach to testing drugs that will help accelerate the development of therapies for vision-threatening disorders such as macular degeneration (MD). PMID:27281648

  5. A novel closed cell culture device for fabrication of corneal epithelial cell sheets.

    PubMed

    Nakajima, Ryota; Kobayashi, Toyoshige; Moriya, Noboru; Mizutani, Manabu; Kan, Kazutoshi; Nozaki, Takayuki; Saitoh, Kazuo; Yamato, Masayuki; Okano, Teruo; Takeda, Shizu

    2015-11-01

    Automation technology for cell sheet-based tissue engineering would need to optimize the cell sheet fabrication process, stabilize cell sheet quality and reduce biological contamination risks. Biological contamination must be avoided in clinical settings. A closed culture system provides a solution for this. In the present study, we developed a closed culture device called a cell cartridge, to be used in a closed cell culture system for fabricating corneal epithelial cell sheets. Rabbit limbal epithelial cells were cultured on the surface of a porous membrane with 3T3 feeder cells, which are separate from the epithelial cells in the cell cartridges and in the cell-culture inserts as a control. To fabricate the stratified cell sheets, five different thicknesses of the membranes which were welded to the cell cartridge, were examined. Multilayered corneal epithelial cell sheets were fabricated in cell cartridges that were welded to a 25 µm-thick gas-permeable membrane, which was similar to the results with the cell-culture inserts. However, stratification of corneal epithelial cell sheets did not occur with cell cartridges that were welded to 100-300 µm-thick gas-permeable membranes. The fabricated cell sheets were evaluated by histological analyses to examine the expression of corneal epithelial-specific markers. Immunohistochemical analyses showed that a putative stem cell marker, p63, a corneal epithelial differentiation maker, CK3, and a barrier function marker, Claudin-1, were expressed in the appropriate position in the cell sheets. These results suggest that the cell cartridge is effective for fabricating corneal epithelial cell sheets.

  6. Measuring Cellular-scale Nutrient Distribution in Algal Biofilms with Synchrotron Confocal Infrared Microspectroscopy

    SciTech Connect

    J Murdock; W Dodds; J Reffner; D Wetzel

    2011-12-31

    The microscope and infrared spectrometer are two of the most useful tools for the study of biological materials, and their combined analytical power far exceeds the sum of the two. Performing molecular spectroscopy through a microscope superimposes chemical information onto the physical microstructure obtained from the optical microscope when visible and infrared information are collected under the same conditions. The instrument developments that enable current infrared microspectroscopic studies began with the introduction of the first research-grade infrared microscope, patented in 1989 (1). By 1993, published reports using this method to determine macroalgae (seaweed) cell-wall composition appeared (2-4). Since these initial reports, the use of infrared microspectroscopy (IMS) in microalgal (single cells or groups of cells) research has grown. Primarily, cultured algae have been used to hone IMS methodology and evaluate its capabilities in algal research (5-8). Studies involving natural, mixed species assemblages, which can utilize the spatial resolution potential of this technique fully are rare (9-11). For instance, in a recent review of IMS microalgal ecological research (12), only 3 of the 29 peer-reviewed publications investigated natural algal assemblages. Both thermal and synchrotron infrared sources provide a resolution capable of measuring individual algae in mixed species assemblages, and each has its advantages. For example, thermal source IMS is more accessible, allowing more samples to be analyzed than synchrotron IMS. However, synchrotron IMS with confocal masking provides superior resolution, which can be critical in isolating small or contiguous cells. Algal ecology is the study of the interaction between algae and their environment. Infrared microspectroscopy addresses a major logistical problem in this field, obtaining species-specific cellular biochemical information from natural, mixed-species assemblages (11,12). Benthic (bottom

  7. Novel Micropatterned Cardiac Cell Cultures with Realistic Ventricular Microstructure

    PubMed Central

    Badie, Nima; Bursac, Nenad

    2009-01-01

    Systematic studies of cardiac structure-function relationships to date have been hindered by the intrinsic complexity and variability of in vivo and ex vivo model systems. Thus, we set out to develop a reproducible cell culture system that can accurately replicate the realistic microstructure of native cardiac tissues. Using cell micropatterning techniques, we aligned cultured cardiomyocytes at micro- and macroscopic spatial scales to follow local directions of cardiac fibers in murine ventricular cross sections, as measured by high-resolution diffusion tensor magnetic resonance imaging. To elucidate the roles of ventricular tissue microstructure in macroscopic impulse conduction, we optically mapped membrane potentials in micropatterned cardiac cultures with realistic tissue boundaries and natural cell orientation, cardiac cultures with realistic tissue boundaries but random cell orientation, and standard isotropic monolayers. At 2 Hz pacing, both microscopic changes in cell orientation and ventricular tissue boundaries independently and synergistically increased the spatial dispersion of conduction velocity, but not the action potential duration. The realistic variations in intramural microstructure created unique spatial signatures in micro- and macroscopic impulse propagation within ventricular cross-section cultures. This novel in vitro model system is expected to help bridge the existing gap between experimental structure-function studies in standard cardiac monolayers and intact heart tissues. PMID:19413993

  8. Polyphosphoinositides are present in plant tissue culture cells

    SciTech Connect

    Boss, W.F.; Massel, M.O.

    1985-11-15

    Polyphosphoinositides have been isolated from wild carrot cells grown in suspension culture. This is the first report of polyphosphoinositides in plant cells. The phospholipids were identified by comigration with known standards on thin-layer plates. After overnight labeling of the cells with myo-(2-/sup 3/H) inositol, the phosphoinositides as percent recovered inositol were 93% phosphatidylinositol., 3.7% lysophosphatidylinositol, 1.7% phosphatidylinositol monophosphate, 0.8% phosphatidylinositol bisphosphate.

  9. Oxygen consumption of human heart cells in monolayer culture.

    PubMed

    Sekine, Kaori; Kagawa, Yuki; Maeyama, Erina; Ota, Hiroki; Haraguchi, Yuji; Matsuura, Katsuhisa; Shimizu, Tatsuya

    2014-09-26

    Tissue engineering in cardiovascular regenerative therapy requires the development of an efficient oxygen supply system for cell cultures. However, there are few studies which have examined human cardiomyocytes in terms of oxygen consumption and metabolism in culture. We developed an oxygen measurement system equipped with an oxygen microelectrode sensor and estimated the oxygen consumption rates (OCRs) by using the oxygen concentration profiles in culture medium. The heart is largely made up of cardiomyocytes, cardiac fibroblasts, and cardiac endothelial cells. Therefore, we measured the oxygen consumption of human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs), cardiac fibroblasts, human cardiac microvascular endothelial cell and aortic smooth muscle cells. Then we made correlations with their metabolisms. In hiPSC-CMs, the value of the OCR was 0.71±0.38pmol/h/cell, whereas the glucose consumption rate and lactate production rate were 0.77±0.32pmol/h/cell and 1.61±0.70pmol/h/cell, respectively. These values differed significantly from those of the other cells in human heart. The metabolism of the cells that constitute human heart showed the molar ratio of lactate production to glucose consumption (L/G ratio) that ranged between 1.97 and 2.2. Although the energy metabolism in adult heart in vivo is reported to be aerobic, our data demonstrated a dominance of anaerobic glycolysis in an in vitro environment. With our measuring system, we clearly showed the differences in the metabolism of cells between in vivo and in vitro monolayer culture. Our results regarding cell OCRs and metabolism may be useful for future tissue engineering of human heart.

  10. Rapid measurement of mitotic spindle orientation in cultured mammalian cells

    PubMed Central

    Decarreau, Justin; Driver, Jonathan; Asbury, Charles; Wordeman, Linda

    2014-01-01

    Summary Factors that influence the orientation of the mitotic spindle are important for the maintenance of stem cell populations and in cancer development. However, screening for these factors requires rapid quantification of alterations of the angle of the mitotic spindle in cultured cell lines. Here we describe a method to image mitotic cells and rapidly score the angle of the mitotic spindle using a simple MATLAB application to analyze a stack of Z-images. PMID:24633791

  11. Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research.

    PubMed

    Stover, Alexander E; Herculian, Siranush; Banuelos, Maria G; Navarro, Samantha L; Jenkins, Michael P; Schwartz, Philip H

    2016-01-01

    This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety cabinets (BSCs) and incubators have long been the standard for culturing and expanding cell lines for basic and preclinical research. However, as the focus of many stem cell laboratories shifts from basic research to clinical translation, additional requirements are needed of the cell culturing system. All processes must be well documented and have exceptional requirements for sterility and reproducibility. In traditional incubators, gas concentrations and temperatures widely fluctuate anytime the cells are removed for feeding, passaging, or other manipulations. Such interruptions contribute to an environment that is not the standard for cGMP and GLP guidelines. These interruptions must be minimized especially when cells are utilized for therapeutic purposes. The motivation to move from the standard BSC and incubator system to a closed system is that such interruptions can be made negligible. Closed systems provide a work space to feed and manipulate cell cultures and maintain them in a controlled environment where temperature and gas concentrations are consistent. This way, pluripotent and multipotent stem cells can be maintained at optimum health from the moment of their derivation all the way to their eventual use in therapy. PMID:27341536

  12. Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

    PubMed Central

    Stover, Alexander E.; Herculian, Siranush; Banuelos, Maria G.; Navarro, Samantha L.; Jenkins, Michael P.; Schwartz, Philip H.

    2016-01-01

    This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety cabinets (BSCs) and incubators have long been the standard for culturing and expanding cell lines for basic and preclinical research. However, as the focus of many stem cell laboratories shifts from basic research to clinical translation, additional requirements are needed of the cell culturing system. All processes must be well documented and have exceptional requirements for sterility and reproducibility. In traditional incubators, gas concentrations and temperatures widely fluctuate anytime the cells are removed for feeding, passaging, or other manipulations. Such interruptions contribute to an environment that is not the standard for cGMP and GLP guidelines. These interruptions must be minimized especially when cells are utilized for therapeutic purposes. The motivation to move from the standard BSC and incubator system to a closed system is that such interruptions can be made negligible. Closed systems provide a work space to feed and manipulate cell cultures and maintain them in a controlled environment where temperature and gas concentrations are consistent. This way, pluripotent and multipotent stem cells can be maintained at optimum health from the moment of their derivation all the way to their eventual use in therapy. PMID:27341536

  13. Enhanced power production from microbial fuel cells with high cell density culture.

    PubMed

    Zhai, Dan-Dan; Li, Bing; Sun, Jian-Zhong; Sun, De-Zhen; Si, Rong-Wei; Yong, Yang-Chun

    2016-01-01

    Improvement of power production in a microbial fuel cell (MFC) with a high cell density culture strategy was developed. By using high cell density culture, the voltage output and power density output of the MFC were enhanced about 0.6 and 1.6 times compared to the control, respectively. Further analysis showed that riboflavin concentration in the MFC was dramatically increased from 0.1 mg/L to 1.2 mg/L by high cell density culture. Moreover, the biofilm formation on the anode surface was significantly enhanced by this new strategy. The increased accumulation of electron shuttle (riboflavin) as well as enhanced biofilm formation contributed to the improvement in anodic electrochemical activity and these factors were the underlying mechanism for MFC performance improvement by high cell density culture. This work demonstrated that high cell density culture would be a simple and practical strategy for MFC manipulation.

  14. [Dynamics of the cell cycle in human endothelial cell culture infected with influenza virus].

    PubMed

    Prochukhanova, A R; Lyublinskaya, O G; Azarenok, A A; Nazarova, A V; Zenin, V V; Zhilinskaya, I N

    2015-01-01

    Cell cycle in a culture of endothelial cells EAhy 926 infected with influenza virus was investigated. Cytometric analysis of culture, synchronized using contact inhibition, has shown that the exposure to the influenza virus in cells EAhy 926 lengthened S-phase of the cell cycle. This result has been tested and proven on culture EAhy 926 treated with nocodazole. Compared with lung carcinoma cells A549, in which influenza virus provokes the arrest of G0/G1 phase of the cycle, elongation of S-phase of cycle at a similar infection of endothelial culture EAhy 926 indicates that the influenza virus differently affects the dynamics of the cell cycle according to the origin of the infected culture.

  15. Time evolution of cell size distributions in dense cell cultures

    NASA Astrophysics Data System (ADS)

    Khain, Evgeniy

    2015-03-01

    Living cells in a dense system are all in contact with each other. The common assumption is that such cells stop dividing due to a lack of space. Recent experimental observations have shown, however, that cells continue dividing for a while, but other cells in the system must shrink, to allow the newborn cells to grow to a normal size. Due to these ``pressure'' effects, the average cell size dramatically decreases with time, and the dispersion in cell sizes decreases, too. The collective cell behavior becomes even more complex when the system is expanding: cells near the edges are larger and migrate faster, while cells deep inside the colony are smaller and move slower. This exciting experimental data still needs to be described theoretically, incorporating the distribution of cell sizes in the system. We propose a mathematical model for time evolution of cell size distribution both in a closed and open system. The model incorporates cell proliferation, cell growth after division, cell shrinking due to ``pressure'' from other cells, and possible cell detachment from the interface of a growing colony. This research sheds light on physical and biological mechanisms of cell response to a dense environment and on the role of mechanical stresses in determining the distribution of cell sizes in the system.

  16. Placental-derived stem cells: Culture, differentiation and challenges

    PubMed Central

    Oliveira, Maira S; Barreto-Filho, João B

    2015-01-01

    Stem cell therapy is a promising approach to clinical healing in several diseases. A great variety of tissues (bone marrow, adipose tissue, and placenta) are potentially sources of stem cells. Placenta-derived stem cells (p-SCs) are in between embryonic and mesenchymal stem cells, sharing characteristics with both, such as non-carcinogenic status and property to differentiate in all embryonic germ layers. Moreover, their use is not ethically restricted as fetal membranes are considered medical waste after birth. In this context, the present review will be focused on the biological properties, culture and potential cell therapy uses of placental-derived stem cells. Immunophenotype characterization, mainly for surface marker expression, and basic principles of p-SC isolation and culture (mechanical separation or enzymatic digestion of the tissues, the most used culture media, cell plating conditions) will be presented. In addition, some preclinical studies that were performed in different medical areas will be cited, focusing on neurological, liver, pancreatic, heart, muscle, pulmonary, and bone diseases and also in tissue engineering field. Finally, some challenges for stem cell therapy applications will be highlighted. The understanding of the mechanisms involved in the p-SCs differentiation and the achievement of pure cell populations (after differentiation) are key points that must be clarified before bringing the preclinical studies, performed at the bench, to the medical practice. PMID:26029347

  17. Metabolic measurements in cell culture and tissue constructs

    NASA Astrophysics Data System (ADS)

    Rolfe, P.

    2008-10-01

    This paper concerns the study and use of biological cells in which there is a need for sensors and assemblies for the measurement of a diverse range of physical and chemical variables. In this field cell culture is used for basic research and for applications such as protein and drug synthesis, and in cell, tissue and organ engineering. Metabolic processes are fundamental to cell behaviour and must therefore be monitored reliably. Basic metabolic studies measure the transport of oxygen, glucose, carbon dioxide, lactic acid to, from, or within cells, whilst more advanced research requires examination of energy storage and utilisation. Assemblies are designed to incorporate bioreactor functions for cell culture together with appropriate sensing devices. Oxygen consumption by populations of cells is achieved in a flowthrough assembly that incorporates O2 micro-sensors based on either amperometry or fluorescence. Measurements in single cell are possible with intra-cellular fluorophores acting as biosensors together with optical stimulation and detection. Near infra-red spectroscopy (NIRS) is used for analysis within culture fluid, for example for estimation of glucose levels, as well as within cell populations, for example to study the respiratory enzymes.Â#

  18. Algal Lipids as Quantitative Paleosalinity Proxies

    NASA Astrophysics Data System (ADS)

    Maloney, A.; Shinneman, A.; Hemeon, K.; Sachs, J. P.

    2012-12-01

    The tropics play an important role in driving climate. However it is difficult to uncover past changes in tropical precipitation due to a lack of tree ring records and low accumulation rates of marine sediments. Hydrogen isotope ratios of algal lipids preserved in lacustrine and marine sediments have been used to qualitatively reconstruct tropical paleohydrology. Changes in the hydrologic balance are reflected in salinity and in lake water D/H ratios, which are closely tracked by lipid D/H ratios of algal biomarkers. While useful for determining past periods of "wetter" or "drier" conditions, variability in isotope fractionation in algal lipids during lipid biosynthesis can be exploited to more quantitatively determine how much wetter or drier conditions were in the past. The estuarine diatom, Thalassiosira pseudonnana, was grown in continuous cultures under controlled light, temperature, nutrient, and growth rate conditions to assess the influence of salinity (9-40 PSU) on D/H fractionation between lipids and source water. Three fatty acids, 24-methylcholesta-5,24(28)-dien-3B-ol, and phytol show decreasing fractionation between lipid and source water as salinity increases with 0.8-1.3‰ change in fractionation per salinity unit. These results compliment field-based empirical observations of dinosterol in Chesapeake Bay suspended particles that change 0.99‰ per salinity unit and lipid biomarkers from hyper-saline ponds on Christmas Island that change 0.7-1.1‰ per salinity unit. Biological pathways responsible for the inverse relationship between fractionation and salinity will be discussed.

  19. Specimen Sample Preservation for Cell and Tissue Cultures

    NASA Technical Reports Server (NTRS)

    Meeker, Gabrielle; Ronzana, Karolyn; Schibner, Karen; Evans, Robert

    1996-01-01

    The era of the International Space Station with its longer duration missions will pose unique challenges to microgravity life sciences research. The Space Station Biological Research Project (SSBRP) is responsible for addressing these challenges and defining the science requirements necessary to conduct life science research on-board the International Space Station. Space Station will support a wide range of cell and tissue culture experiments for durations of 1 to 30 days. Space Shuttle flights to bring experimental samples back to Earth for analyses will only occur every 90 days. Therefore, samples may have to be retained for periods up to 60 days. This presents a new challenge in fresh specimen sample storage for cell biology. Fresh specimen samples are defined as samples that are preserved by means other than fixation and cryopreservation. The challenge of long-term storage of fresh specimen samples includes the need to suspend or inhibit proliferation and metabolism pending return to Earth-based laboratories. With this challenge being unique to space research, there have not been any ground based studies performed to address this issue. It was decided hy SSBRP that experiment support studies to address the following issues were needed: Fixative Solution Management; Media Storage Conditions; Fresh Specimen Sample Storage of Mammalian Cell/Tissue Cultures; Fresh Specimen Sample Storage of Plant Cell/Tissue Cultures; Fresh Specimen Sample Storage of Aquatic Cell/Tissue Cultures; and Fresh Specimen Sample Storage of Microbial Cell/Tissue Cultures. The objective of these studies was to derive a set of conditions and recommendations that can be used in a long duration microgravity environment such as Space Station that will permit extended storage of cell and tissue culture specimens in a state consistent with zero or minimal growth, while at the same time maintaining their stability and viability.

  20. Hollow fiber clinostat for simulating microgravity in cell culture

    NASA Technical Reports Server (NTRS)

    Rhodes, Percy H. (Inventor); Miller, Teresa Y. (Inventor); Snyder, Robert S. (Inventor)

    1992-01-01

    A clinostat for simulating microgravity on cell systems carried in a fiber fixedly mounted in a rotatable culture vessel is disclosed. The clinostat is rotated horizontally along its longitudinal axis to simulate microgravity or vertically as a control response. Cells are injected into the fiber and the ends of the fiber are sealed and secured to spaced end pieces of a fiber holder assembly which consists of the end pieces, a hollow fiber, a culture vessel, and a tension spring with three alignment pins. The tension spring is positioned around the culture vessel with its ends abutting the end pieces for alignment of the spring. After the fiber is secured, the spring is decompressed to maintain tension on the fiber while it is being rotated. This assures that the fiber remains aligned along the axis of rotation. The fiber assembly is placed in the culture vessel and culture medium is added. The culture vessel is then inserted into the rotatable portion of the clinostat and subjected to rotate at selected rpms. The internal diameter of the hollow fiber determines the distance the cells are from the axis of rotation.

  1. Isolation, culture, and transplantation of muscle satellite cells.

    PubMed

    Motohashi, Norio; Asakura, Yoko; Asakura, Atsushi

    2014-01-01

    Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors. However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted

  2. A microwell cell culture platform for the aggregation of pancreatic β-cells.

    PubMed

    Bernard, Abigail B; Lin, Chien-Chi; Anseth, Kristi S

    2012-08-01

    Cell-cell contact between pancreatic β-cells is important for maintaining survival and normal insulin secretion. Various techniques have been developed to promote cell-cell contact between β-cells, but a simple yet robust method that affords precise control over three-dimensional (3D) β-cell cluster size has not been demonstrated. To address this need, we developed a poly(ethylene glycol) (PEG) hydrogel microwell platform using photolithography. This microwell cell-culture platform promotes the formation of 3D β-cell aggregates of defined sizes from 25 to 210 μm in diameter. Using this platform, mouse insulinoma 6 (MIN6) β-cells formed aggregates with cell-cell adherin junctions. These naturally formed cell aggregates with controllable sizes can be removed from the microwells for macroencapsulation, implantation, or other biological assays. When removed and subsequently encapsulated in PEG hydrogels, the aggregated cell clusters demonstrated improved cellular viability (>90%) over 7 days in culture, while the β-cells encapsulated as single cells maintained only 20% viability. Aggregated MIN6 cells also exhibited more than fourfold higher insulin secretion in response to a glucose challenge compared with encapsulated single β-cells. Further, the cell aggregates stained positively for E-cadherin, indicative of the formation of cell junctions. Using this hydrogel microwell cell-culture method, viable and functional β-cell aggregates of specific sizes were created, providing a platform from which other biologically relevant questions may be answered. PMID:22320435

  3. Microcystis aeruginosa toxin: cell culture toxicity, hemolysis, and mutagenicity assays.

    PubMed Central

    Grabow, W O; Du Randt, W C; Prozesky, O W; Scott, W E

    1982-01-01

    Crude toxin was prepared by lyophilization and extraction of toxic Microcystis aeruginosa from four natural sources and a unicellular laboratory culture. The responses of cultures of liver (Mahlavu and PCL/PRF/5), lung (MRC-5), cervix (HeLa), ovary (CHO-K1), and kidney (BGM, MA-104, and Vero) cell lines to these preparations did not differ significantly from one another, indicating that toxicity was not specific for liver cells. The results of a trypan blue staining test showed that the toxin disrupted cell membrane permeability within a few minutes. Human, mouse, rat, sheep, and Muscovy duck erythrocytes were also lysed within a few minutes. Hemolysis was temperature dependent, and the reaction seemed to follow first-order kinetics. Escherichia coli, Streptococcus faecalis, and Tetrahymena pyriformis were not significantly affected by the toxin. The toxin yielded negative results in Ames/Salmonella mutagenicity assays. Microtiter cell culture, trypan blue, and hemolysis assays for Microcystis toxin are described. The effect of the toxin on mammalian cell cultures was characterized by extensive disintegration of cells and was distinguishable from the effects of E. coli enterotoxin, toxic chemicals, and pesticides. A possible reason for the acute lethal effect of Microcystis toxin, based on cytolytic activity, is discussed. Images PMID:6808921

  4. Interactions of algal ligands, metal complexation and availability, and cell responses of the diatom Ditylum brightwellii with a gradual increase in copper.

    PubMed

    Rijstenbil, J W; Gerringa, L J A

    2002-01-01

    A continuous culture experiment was conducted to study interactions between copper-binding ligands released by light-limited Ditylum brightwellii, and toxic effects of Cu on this diatom. Over 6 months, the Cu concentration in the medium has been increased in seven steps (3-173 nM). At each Cu addition, Cu speciation, characteristics of Cu sorption to cellular binding sites, and cell characteristics were determined. Physiological effects of Cu were studied, using indicators for metal detoxification (thiols) and lipid peroxidation (malondialdehyde). Minor amounts of Cu (<1.4%) were chelated by a minimum amount of EDTA (57 nM), required to maintain a stable long-term continuous culture. The responses of D. brightwellii to Cu were monitored. (1) From 3 to 47 nM added Cu, decreasing pools of glutathione, increasing malondialdehyde contents, an increased release of lipophilic ligands, and cell lysis indicated the enhancement of lipid peroxidation. (2) From 47 to 94 nM Cu, a 16-fold increase in high-affinity (strong) hydrophilic ligands was measured (conditional stability constants K' approximately 10(12)) that complexed most Cu (maximum 97%); sexual reproduction was stimulated and cell volumes increased. (3) From 126 nM Cu, glutathione pools increased again, whereas cell division rates decreased slightly. (4) At 142 nM Cu, the number of lysed cells reached a maximum, as did the production of lipophilic compounds that complexed approximately 2% Cu. As the binding sites of the strong ligands became Cu-saturated above 142 nM Cu, larger amounts of Cu were bound to low-affinity (weak) dissolved ligands (3-30%) and cellular binding sites (0.2-2.5%). Probably due to saturation of organic complexes at 142 nM Cu, the MINEQL-calculated Cu2+ concentrations increased markedly; pCu values decreased from >11 to approximately 10; division rates were further inhibited; gamma-glutamylcysteine (phytochelatin precursor) was produced. (5) At 157 nM Cu, phytochelatin synthesis started, and

  5. Novel resource utilization of refloated algal sludge to improve the quality of organic fertilizer.

    PubMed

    Huang, Yan; Li, Rong; Liu, Hongjun; Wang, Beibei; Zhang, Chenmin; Shen, Qirong

    2014-08-01

    Without further management, large amounts of refloated algal sludge from Taihu Lake to retrieve nitrogen and phosphorus resources may result in serious secondary environmental pollution. The possibility of utilization of algal sludge to improve the quality of organic fertilizer was investigated in this study. Variations of physicochemical properties, germination index (GI) and microcystin (MC) content were analysed during the composting process. The results showed that the addition of algal sludge improved the contents of nutrients, common free amino acids and total common amino acids in the novel organic fertilizer. Rapid degradation rates of MC-LR and MC-RR, a high GI value and more abundance of culturable protease-producing bacteria were observed during the composting process added with algal sludge. Growth experiments showed that the novel organic fertilizer efficiently promoted plant growth. This study provides a novel resource recovery method to reclaim the Taihu Lake algal sludge and highlights a novel method to produce a high-quality organic fertilizer.

  6. Screening Stress Tolerance Traits in Arabidopsis Cell Cultures.

    PubMed

    Pérez-Salamó, Imma; Boros, Bogáta; Szabados, László

    2016-01-01

    Screening for tolerance traits in plant cell cultures can combine the efficiency of microbial selection and plant genetics. Agrobacterium-mediated transformation can efficiently introduce cDNA library to cell suspension cultures generating population of randomly transformed microcolonies. Transformed cultures can subsequently be screened for tolerance to different stress conditions such as salinity, high osmotic, or oxidative stress conditions. cDNA inserts in tolerant cell lines can be easily identified by PCR amplification and homology search of the determined nucleotide sequences. The described methods have been tested and used to identify regulatory genes controlling salt tolerance in Arabidopsis. As cDNA libraries can be prepared from any plants, natural diversity can be explored by using extremophile plants as gene source. PMID:26867628

  7. Cultivation of vaccinia virus in sheep kidney cell cultures.

    PubMed

    SUBRAMANYAM, P; DIVAKARAN, S; VINODRAJ, P

    1961-01-01

    Attempts to find a suitable tissue for the preparation of cell monolayers for the cultivation of vaccinia virus and for the titration of this virus and its antibodies resulted in the use of sheep kidneys procured from freshly slaughtered healthy young sheep. The cultures are easy and economical to prepare and support the multiplication of the virus well. They can be used for the titration of the virus and its antibody and their sensitivity to virus is comparable to that of the chorio-allantoic membranes of chicken embryos. Preliminary trials indicate that the sheep kidney cell culture virus can be freeze-dried without suffering a significant loss in titre. Studies are in progress to determine the efficacy of a vaccine prepared from sheep kidney cell cultures.

  8. [In vitro cell culture technology in cosmetology research].

    PubMed

    Gojniczek, Katarzyna; Garncarczyk, Agnieszka; Pytel, Agata

    2005-01-01

    For ages the humanity has been looking for all kind of active substances, which could be used in improving the health and the appearance of our skin. People try to find out how to protect the skin from harmful, environmental factors. Every year a lot of new natural and synthetic, chemical substances are discovered. All of them potentially could be used as a cosmetic ingredient. In cosmetology research most of new xenobiotics were tested in vivo on animals. Alternative methods to in vivo tests are in vitro tests with skin cell culture system. The aim of this work was to describe two-dimensional and tree-dimensional skin cell cultures. Additionally, in this work we wanted to prove the usefulness of in vitro skin cell cultures in cosmetology research.

  9. Isolation of Lysosomes from Mammalian Tissues and Cultured Cells.

    PubMed

    Aguado, Carmen; Pérez-Jiménez, Eva; Lahuerta, Marcos; Knecht, Erwin

    2016-01-01

    Lysosomes participate within the cells in the degradation of organelles, macromolecules, and a wide variety of substrates. In any study on specific roles of lysosomes, both under physiological and pathological conditions, it is advisable to include methods that allow their reproducible and reliable isolation. However, purification of lysosomes is a difficult task, particularly in the case of cultured cells. This is mainly because of the heterogeneity of these organelles, along with their low number and high fragility. Also, isolation methods, while disrupting plasma membranes, have to preserve the integrity of lysosomes, as the breakdown of their membranes releases enzymes that could damage all cell organelles, including themselves. The protocols described below have been routinely used in our laboratory for the specific isolation of lysosomes from rat liver, NIH/3T3, and other cultured cells, but can be adapted to other mammalian tissues or cell lines. PMID:27613045

  10. Lingual Epithelial Stem Cells and Organoid Culture of Them.

    PubMed

    Hisha, Hiroko; Tanaka, Toshihiro; Ueno, Hiroo

    2016-01-28

    As tongue cancer is one of the major malignant cancers in the world, understanding the mechanism of maintenance of lingual epithelial tissue, which is known to be the origin of tongue cancer, is unquestionably important. However, the actual stem cells that are responsible for the long-term maintenance of the lingual epithelium have not been identified. Moreover, a simple and convenient culture method for lingual epithelial stem cells has not yet been established. Recently, we have shown that Bmi1-positive cells, residing at the second or third layer of the epithelial cell layer at the base of the interpapillary pit (IPP), were slow-cycling and could supply keratinized epithelial cells for over one year, indicating that Bmi1-positive cells are long-term lingual epithelial stem cells. In addition, we have developed a novel lingual epithelium organoid culture system using a three-dimensional matrix and growth factors. Here, we discuss current progress in the identification of lingual stem cells and future applications of the lingual culture system for studying the regulatory mechanisms of the lingual epithelium and for regenerative medicine.

  11. Biological Effects of Culture Substrates on Human Pluripotent Stem Cells.

    PubMed

    Hayashi, Yohei; Furue, Miho Kusuda

    2016-01-01

    In recent years, as human pluripotent stem cells (hPSCs) have been commonly cultured in feeder-free conditions, a number of cell culture substrates have been applied or developed. However, the functional roles of these substrates in maintaining hPSC self-renewal remain unclear. Here in this review, we summarize the types of these substrates and their effect on maintaining hPSC self-renewal. Endogenous extracellular matrix (ECM) protein expression has been shown to be crucial in maintaining hPSC self-renewal. These ECM molecules interact with integrin cell-surface receptors and transmit their cellular signaling. We discuss the possible effect of integrin-mediated signaling pathways on maintaining hPSC self-renewal. Activation of integrin-linked kinase (ILK), which transmits ECM-integrin signaling to AKT (also known as protein kinase B), has been shown to be critical in maintaining hPSC self-renewal. Also, since naïve pluripotency has been widely recognized as an alternative pluripotent state of hPSCs, we discuss the possible effects of culture substrates and integrin signaling on naïve hPSCs based on the studies of mouse embryonic stem cells. Understanding the role of culture substrates in hPSC self-renewal and differentiation enables us to control hPSC behavior precisely and to establish scalable or microfabricated culture technologies for regenerative medicine and drug development. PMID:27656216

  12. Biological Effects of Culture Substrates on Human Pluripotent Stem Cells

    PubMed Central

    2016-01-01

    In recent years, as human pluripotent stem cells (hPSCs) have been commonly cultured in feeder-free conditions, a number of cell culture substrates have been applied or developed. However, the functional roles of these substrates in maintaining hPSC self-renewal remain unclear. Here in this review, we summarize the types of these substrates and their effect on maintaining hPSC self-renewal. Endogenous extracellular matrix (ECM) protein expression has been shown to be crucial in maintaining hPSC self-renewal. These ECM molecules interact with integrin cell-surface receptors and transmit their cellular signaling. We discuss the possible effect of integrin-mediated signaling pathways on maintaining hPSC self-renewal. Activation of integrin-linked kinase (ILK), which transmits ECM-integrin signaling to AKT (also known as protein kinase B), has been shown to be critical in maintaining hPSC self-renewal. Also, since naïve pluripotency has been widely recognized as an alternative pluripotent state of hPSCs, we discuss the possible effects of culture substrates and integrin signaling on naïve hPSCs based on the studies of mouse embryonic stem cells. Understanding the role of culture substrates in hPSC self-renewal and differentiation enables us to control hPSC behavior precisely and to establish scalable or microfabricated culture technologies for regenerative medicine and drug development. PMID:27656216

  13. Algal Energy Conversion and Capture

    NASA Astrophysics Data System (ADS)

    Hazendonk, P.

    2015-12-01

    We address the potential for energy conversions and capture for: energy generation; reduction in energy use; reduction in greenhouse gas emissions; remediation of water and air pollution; protection and enhancement of soil fertility. These processes have the potential to sequester carbon at scales that may have global impact. Energy conversion and capture strategies evaluate energy use and production from agriculture, urban areas and industries, and apply existing and emerging technologies to reduce and recapture energy embedded in waste products. The basis of biocrude production from Micro-algal feedstocks: 1) The nutrients from the liquid fraction of waste streams are concentrated and fed into photo bioreactors (essentially large vessels in which microalgae are grown) along with CO2 from flue gasses from down stream processes. 2) The algae are processed to remove high value products such as proteins and beta-carotenes. The advantage of algae feedstocks is the high biomass productivity is 30-50 times that of land based crops and the remaining biomass contains minimal components that are difficult to convert to biocrude. 3) The remaining biomass undergoes hydrothermal liquefaction to produces biocrude and biochar. The flue gasses of this process can be used to produce electricity (fuel cell) and subsequently fed back into the photobioreactor. The thermal energy required for this process is small, hence readily obtained from solar-thermal sources, and furthermore no drying or preprocessing is required keeping the energy overhead extremely small. 4) The biocrude can be upgraded and refined as conventional crude oil, creating a range of liquid fuels. In principle this process can be applied on the farm scale to the municipal scale. Overall, our primary food production is too dependent on fossil fuels. Energy conversion and capture can make food production sustainable.

  14. Sterol phylogenesis and algal evolution.

    PubMed Central

    Nes, W D; Norton, R A; Crumley, F G; Madigan, S J; Katz, E R

    1990-01-01

    The stereochemistry of several sterol precursors and end products synthesized by two fungal-like micro-organisms Prototheca wickerhamii (I) and Dictyostelium discoideum (II) have been determined by chromatographic (TLC, GLC, and HPLC) and spectral (UV, MS, and 1H NMR) methods. From I and II the following sterols were isolated from the cells: cycloartenol, cyclolaudenol, 24(28)-methylenecycloartanol, ergosterol, protothecasterol, 4alpha-methylergostanol, 4alpha-methylclionastanol, clionastanol, 24beta-ethylcholesta-8,22-enol, and dictyosterol. In addition, the mechanism of C-24 methylation was investigated in both organisms by feeding to I [2-3H]lanosterol, [2-3H]cycloartenol, [24-3H]lanosterol, and [methyl-2H3]methionine and by feeding to II [methyl-2H3]methionine. The results demonstrate that the 24beta configuration is formed by different alkylation routes in I and II. The Delta25(27) route operates in I while the Delta24(28) route operates in II. Based on what is known in the literature regarding sterol distribution and phylogenesis together with our findings that the stereochemical outcome of squalene oxide cyclization leads to the production of cycloartenol rather than lanosterol (characteristic of the fungal genealogy) and the chirality of the C-24 alkyl group is similar in the two nonphotosynthetic microbes (beta oriented), we conclude that Prototheca is an apoplastic Chlorella (i.e., an alga) and that Dictyostelium as well as the other soil amoebae that synthesize cycloartenol evolved from algal rather than fungal ancestors. PMID:11607106

  15. Identification and quantitation of morphological cell types in electrophoretically separated human embryonic kidney cell cultures

    NASA Technical Reports Server (NTRS)

    Williams, K. B.; Kunze, M. E.; Todd, P. W.

    1985-01-01

    Four major cell types were identified by phase microscopy in early passage human embryonic kidney cell cultures. They are small and large epithelioid, domed, and fenestrated cells. Fibroblasts are also present in some explants. The percent of each cell type changes with passage number as any given culture grows. As a general rule, the fraction of small epithelioid cells increases, while the fraction of fenestrated cells, always small, decreases further. When fibroblasts are present, they always increase in percentage of the total cell population. Electrophoretic separation of early passage cells showed that the domed cells have the highest electrophoretic mobility, fibroblasts have an intermediate high mobility, small epithelioid cells have a low mobility, broadly distributed, and fenestrated cells have the lowest mobility. All cell types were broadly distributed among electrophoretic subfractions, which were never pure but only enriched with respect to a given cell type.

  16. Dynamic cell culture system: a new cell cultivation instrument for biological experiments in space

    NASA Technical Reports Server (NTRS)

    Gmunder, F. K.; Nordau, C. G.; Tschopp, A.; Huber, B.; Cogoli, A.

    1988-01-01

    The prototype of a miniaturized cell cultivation instrument for animal cell culture experiments aboard Spacelab is presented (Dynamic cell culture system: DCCS). The cell chamber is completely filled and has a working volume of 200 microliters. Medium exchange is achieved with a self-powered osmotic pump (flowrate 1 microliter h-1). The reservoir volume of culture medium is 230 microliters. The system is neither mechanically stirred nor equipped with sensors. Hamster kidney (Hak) cells growing on Cytodex 3 microcarriers were used to test the biological performance of the DCCS. Growth characteristics in the DCCS, as judged by maximal cell density, glucose consumption, lactic acid secretion and pH, were similar to those in cell culture tubes.

  17. Flow cytometric cell cycle analysis of somatic cells primary cultures established for bovine cloning.

    PubMed

    Katska, L; Bochenek, M; Kania, G; Ryñska, B; Smorag, Z

    2002-12-01

    An important factor governing developmental rates of somatic cloned embryos is the phase of the cell cycle of donor nuclei. The aim of this experiment was to investigate the distribution of cell cycle phases in bovine cumulus and fibroblast cells cultured using routine treatment, and under cell cycle-arresting treatments. The highest percentages of cumulus cells in the G0 + G1 stage were observed in uncultured, frozen/thawed cells originating from immature oocytes (79.8 +/- 2.2%), fresh and frozen/thawed cells from in vitro matured oocytes (84.1 +/- 6.2 and 77.8 +/- 5.7%, respectively), and in cycling cells (72.7 +/- 16.3 and 78.4 +/- 11.2%, respectively for cumulus cells from immature and in vitro matured oocytes). Serum starvation of cumulus cultures markedly decreased percentages of cells in G0 + G1, and prolonged starvation significantly increased (P < 0.05) percentages of cells in G2 + M phase. Culture of cumulus cells to confluency did not increase percentages of cells in G0 + G1. Contrary to findings in cumulus cells, significantly higher percentages of cells in G0 + G1 were apparent when fibroblast cells were cultured to confluency or serum starved, and significantly increased (P < 0.01) as the starvation period was prolonged. It is concluded that for particular cell types specific strategies should be used to attain improvements in the efficiency of cloning procedures.

  18. Algal taxonomy forum: Algal Taxonomist, Let Serendipity Reign!

    PubMed

    Druehl, Louis

    2013-04-01

    The publication of a mini-review by Olivier De Clerck et al. in this issue of the Journal of Phycology presented an opportunity to open a dialogue on challenges faced by contemporary algal taxonomists. The Editorial Office solicited the following two additional contributions in response to De Clerck et al.'s paper; the responses were edited solely for clarity, space and format.

  19. Three-dimensional cultured glioma cell lines

    NASA Technical Reports Server (NTRS)

    Gonda, Steve R. (Inventor); Marley, Garry M. (Inventor)

    1991-01-01

    Three-dimensional glioma spheroids were produced in vitro with size and histological differentiation previously unattained. The spheroids were grown in liquid media suspension in a Johnson Space Center (JSC) Rotating Wall Bioreactor without using support matrices such as microcarrier beads. Spheroid volumes of greater than 3.5 cu mm and diameters of 2.5 mm were achieved with a viable external layer or rim of proliferating cells, a transitional layer beneath the external layer with histological differentiation, and a degenerative central region with a hypoxic necrotic core. Cell debris was evident in the degenerative central region. The necrotics centers of some of the spheroids had hyaline droplets. Granular bodies were detected predominantly in the necrotic center.

  20. [Culture and control of cells producing bovine leukemia virus].

    PubMed

    Granátová, M

    1987-10-01

    In the field surveys of the occurrence of enzootic bovine leucosis caused by the bovine leucosis virus (BLV), the identification of positive animals is based on the detection of specific antiviral antibodies by serological methods. The reliability of these tests (particularly their sensitivity and specificity) depends on the quality of the virus antigen. The preparation of the antigen is based on the cultivation of BLV virus in cultures of the FLS cell line. A modified procedure of preparing the BLV antigen in the FLS cell culture is described, along with the control of its production by the immunoperoxidase test. PMID:2827363

  1. Synthesis of resveratrol glycosides by cultured plant cells.

    PubMed

    Shimoda, Kei; Hamada, Manabu; Hamada, Hatsuyuki; Takemoto, Mai; Hamada, Hiroki

    2013-07-01

    Incubation of cultured cells of Glycine max with trans-resveratrol gave its 3-O-beta-D- and 4'-O-beta-D-glucosides. Cultured Gossypium hirsutum cells glycosylated trans-resveratrol to its 3-O-beta-D-, 4'-O-beta-D-, and 3,4'-O-beta-D-diglucosides. On the other hand, trans-resveratrol was converted into cis-resveratrol 4'-O-beta-D-glucoside, together with trans-resveratrol 3-O-beta-D-glucoside and trans-resveratrol 4'-O-beta-D-glucoside, by Eucalyptus perriniana.

  2. Human epithelial cell cultures from superficial limbal explants

    PubMed Central

    Basli, E.; Goldschmidt, P.; Pecha, F.; Chaumeil, C.; Laroche, L.; Borderie, V.

    2011-01-01

    Purpose To study the kinetics of growth and the phenotype of cells cultured from human limbal explants in a cholera toxin-free medium with no feeder cell layer. Methods Human organ-cultured corneas were used to prepare limbal explants (full-thickness and superficial limbal explants) and corneal stromal explants. Cell growth kinetics and phenotypes were assessed by cultivating explants in cholera toxin-free Green medium. Epithelial and progenitor cell markers were assessed by immunocytochemistry, flow cytometry, and Reverse Transcription and Polymerase Chain Reaction (RT-PCR). Results The successful epithelial cell growth rates from full thickness limbal explant and superficial limbal explant tissues were 41 and 86%, respectively (p=0.0001). The mean cell area and the percentage of small cells in superficial and full-thickness explant cultures were, respectively, 317 µm2 and 429 µm2, and 8.9% and 1.7% (p<0.001). The percentage of positive cells in superficial and full-thickness limbal explant cultures as assessed by immunocytochemistry were the following: broad spectrum cytokeratins (cytokeratins 4, 5, 6, 8, 10, 13, and 18 [MNF116]), 82%/37% (p=0.01); cytokeratin 3 (CK3), 74%/25% (p=0.009); cytokeratin 19 (CK19), 46%/25% (p=0.19); vimentin, 56%/53% (p=0.48); delta N p63α, 54%/0% (p<0.001); and ABCG2, 5%/0% (p=0.1). Flow cytometry showed a higher percentage of small cells, a higher percentage of MNF116+ cells, and stronger expression of progenitor-associated markers in superficial than in full-thickness explant cultures. For superficial limbal explant cultures, analysis of the expression profiles for various mRNAs at the end of 21 days of culture showed high levels of expression of the mRNAs encoding CK3, vimentin, and CK19. The expression of mRNA of delta N p63α and ABCG2 was weaker. Cultures obtained from full-thickness limbal explants featured no expression of mRNA of CK19, delta N p63α, and ABCG2, whereas mRNAs encoding CK3 and vimentin were detected. Human

  3. Continuous perfusion microfluidic cell culture array for high-throughput cell-based assays.

    PubMed

    Hung, Paul J; Lee, Philip J; Sabounchi, Poorya; Lin, Robert; Lee, Luke P

    2005-01-01

    We present for the first time a microfluidic cell culture array for long-term cellular monitoring. The 10 x 10 array could potentially assay 100 different cell-based experiments in parallel. The device was designed to integrate the processes used in typical cell culture experiments on a single self-contained microfluidic system. Major functions include repeated cell growth/passage cycles, reagent introduction, and real-time optical analysis. The single unit of the array consists of a circular microfluidic chamber, multiple narrow perfusion channels surrounding the main chamber, and four ports for fluidic access. Human carcinoma (HeLa) cells were cultured inside the device with continuous perfusion of medium at 37 degrees C. The observed doubling time was 1.4 +/- 0.1 days with a peak cell density of approximately 2.5*10(5) cells/cm(2). Cell assay was demonstrated by monitoring the fluorescence localization of calcein AM from 1 min to 10 days after reagent introduction. Confluent cell cultures were passaged within the microfluidic chambers using trypsin and successfully regrown, suggesting a stable culture environment suitable for continuous operation. The cell culture array could offer a platform for a wide range of assays with applications in drug screening, bioinformatics, and quantitative cell biology. PMID:15580587

  4. Testicular Sertoli cells influence the proliferation and immunogenicity of co-cultured endothelial cells

    SciTech Connect

    Fan, Ping; He, Lan; Pu, Dan; Lv, Xiaohong; Zhou, Wenxu; Sun, Yining; Hu, Nan

    2011-01-21

    Research highlights: {yields} The proliferation of dramatic increased by co-cultured with Sertoli cells. {yields} VEGF receptor-2 expression of ECs was up-regulated by co-cultured with Sertoli cells. {yields} The MHC expression of ECs induced by INF-{gamma} and IL-6, IL-8 and sICAM induced by TNF-{alpha} decreased respectively after co-cultured with Sertoli cells. {yields} ECs co-cultured with Sertoli cells also didn't increase the stimulation index of spleen lymphocytes. -- Abstract: The major problem of the application of endothelial cells (ECs) in transplantation is the lack of proliferation and their immunogenicity. In this study, we co-cultured ECs with Sertoli cells to monitor whether Sertoli cells can influence the proliferation and immunogenicity of co-cultured ECs. Sertoli cells were isolated from adult testicular tissue. ECs were divided into the control group and the experimental group, which included three sub-groups co-cultured with 1 x 10{sup 3}, 1 x 10{sup 4} or 1 x 10{sup 5} cell/ml of Sertoli cells. The growth and proliferation of ECs were observed microscopically, and the expression of vascular endothelial growth factor (VEGF) receptor-2 (KDR) was examined by Western blotting. In another experiment, ECs were divided into the control group, the single culture group and the co-culture group with the optimal concentration of Sertoli cells. After INF-{gamma} and TNF-{alpha} were added to the culture medium, MHC II antigen expression was detected by immunofluorescence staining and western blotting; interleukin (IL)-6, IL-8 and soluble intercellular adhesion molecule (sICAM) were measured in the culture medium by ELISA. We demonstrated that 1 x 10{sup 4} cell/ml Sertoli cells promoted the proliferation of co-cultured ECs more dramatically than that in other groups (P < 0.05). Western blotting showed that 1 x 10{sup 4} cell/ml of the Sertoli cells was most effective in the up-regulation of KDR expression in the co-cultured ECs (P < 0.05). Sertoli cells

  5. Polyamines in Relation to Growth in Carrot Cell Cultures 1

    PubMed Central

    Fallon, Kevin M.; Phillips, Richard

    1988-01-01

    Changes in polyamine metabolism were investigated in relation to growth of cell suspension cultures of carrot (Daucus carota, cv Chantenay). Changes in levels of the major amines putrescine and spermidine throughout the culture period correlated poorly with changes in fresh weight, but a closer correlation with the minor component spermine was observed. The arginine decarboxylase (ADC) inhibitor difluoromethylarginine (DFMA) strongly and specifically inhibited ADC activity in the supernatant, reduced the major amine (putrescine) by 95% and the total amine content by 80%. It had no effect on cell number and stimulated fresh weight by over 25% through increased cell expansion. Spermine content, in contrast, increased with DFMA concentration in parallel with fresh weight increases. Difluoromethylornithine strongly inhibited ornithine decarboxylase activity in the pellet, but had little effect on either polyamine levels or culture growth. It was concluded that little evidence for a correlation between free polyamines and cell number in carrot cultures could be detected, but that a possible correlation between spermine content and cell expansion was observed. PMID:16666271

  6. Polyamines in relation to growth in carrot cell cultures.

    PubMed

    Fallon, K M; Phillips, R

    1988-09-01

    Changes in polyamine metabolism were investigated in relation to growth of cell suspension cultures of carrot (Daucus carota, cv Chantenay). Changes in levels of the major amines putrescine and spermidine throughout the culture period correlated poorly with changes in fresh weight, but a closer correlation with the minor component spermine was observed. The arginine decarboxylase (ADC) inhibitor difluoromethylarginine (DFMA) strongly and specifically inhibited ADC activity in the supernatant, reduced the major amine (putrescine) by 95% and the total amine content by 80%. It had no effect on cell number and stimulated fresh weight by over 25% through increased cell expansion. Spermine content, in contrast, increased with DFMA concentration in parallel with fresh weight increases. Difluoromethylornithine strongly inhibited ornithine decarboxylase activity in the pellet, but had little effect on either polyamine levels or culture growth. It was concluded that little evidence for a correlation between free polyamines and cell number in carrot cultures could be detected, but that a possible correlation between spermine content and cell expansion was observed.

  7. Ex vivo Co-culture of Lymphoid Tissue Stromal Cells and T Cells

    PubMed Central

    Zeng, Ming; Haase, Ashley T.

    2016-01-01

    Stromal cells within lymphoid tissues produce IL-7, which is critical for the survival and function of T cells. This protocol is to be used to isolate primary human lymphoid tissue stromal cells to study their impact on the survival of T cells in an ex vivo co-culture system.

  8. Arsenic exposure induces the Warburg effect in cultured human cells

    SciTech Connect

    Zhao, Fei; Severson, Paul; Pacheco, Samantha; Futscher, Bernard W.; Klimecki, Walter T.

    2013-08-15

    Understanding how arsenic exacts its diverse, global disease burden is hampered by a limited understanding of the particular biological pathways that are disrupted by arsenic and underlie pathogenesis. A reductionist view would predict that a small number of basic pathways are generally perturbed by arsenic, and manifest as diverse diseases. Following an initial observation that arsenite-exposed cells in culture acidify their media more rapidly than control cells, the report here shows that low level exposure to arsenite (75 ppb) is sufficient to induce aerobic glycolysis (the Warburg effect) as a generalized phenomenon in cultured human primary cells and cell lines. Expanded studies in one such cell line, the non-malignant pulmonary epithelial line, BEAS-2B, established that the arsenite-induced Warburg effect was associated with increased accumulation of intracellular and extracellular lactate, an increased rate of extracellular acidification, and inhibition by the non-metabolized glucose analog, 2-deoxy-D-glucose. Associated with the induction of aerobic glycolysis was a pathway-wide induction of glycolysis gene expression, as well as protein accumulation of an established glycolysis master-regulator, hypoxia-inducible factor 1A. Arsenite-induced alteration of energy production in human cells represents the type of fundamental perturbation that could extend to many tissue targets and diseases. - Highlights: • Chronic arsenite exposure induces aerobic glycolysis, dubbed the “Warburg effect”. • Arsenite-induced Warburg effect is a general phenomenon in cultured human cells. • HIF-1A may mediate arsenite induced Warburg effect.

  9. Contraction-induced cluster formation in cardiac cell culture

    NASA Astrophysics Data System (ADS)

    Harada, Takahiro; Isomura, Akihiro; Yoshikawa, Kenichi

    2008-11-01

    The evolution of the spatial arrangement of cells in a primary culture of cardiac tissue derived from newborn rats was studied experimentally over an extended period. It was found that cells attract each other spontaneously to form a clustered structure over the timescale of several days. These clusters exhibit spontaneous rhythmic contraction and have been confirmed to consist of cardiac muscle cells. The addition of a contraction inhibitor (2,3-butanedione-2-monoxime) to the culture medium resulted in the inhibition of both the spontaneous contractions exhibited by the cells as well as the formation of clusters. Furthermore, the formation of clusters is suppressed when high concentrations of collagen are used for coating the substratum to which the cells adhere. From these experimental observations, it was deduced that the cells are mechanically stressed by the tension associated with repeated contractions and that this results in the cells becoming compact and attracting each other, finally resulting in the formation of clusters. This process can be interpreted as modulation of a cellular network by the activity associated with contraction, which could be employed to control cellular networks by modifying the dynamics associated with the contractions in cardiac tissue culture.

  10. Cytotoxicity of voriconazole on cultured human corneal endothelial cells.

    PubMed

    Han, Sang Beom; Shin, Young Joo; Hyon, Joon Young; Wee, Won Ryang

    2011-10-01

    The purpose of the present study was to evaluate the toxicity of voriconazole on cultured human corneal endothelial cells (HCECs). HCECs were cultured and exposed to various concentrations of voriconazole (5.0 to 1,000 μg/ml). Cell viability was measured using a Cell Counting Kit-8 (CCK-8) and live/dead viability/cytotoxicity assays. Cell damage was assessed using phase-contrast microscopy after 24 h of exposure to voriconazole. To analyze the effect of voriconazole on the intercellular barrier, immunolocalization of zonula occludens 1 (ZO1) was performed. A flow cytometric assay was performed to evaluate the apoptotic and necrotic effects of voriconazole on HCECs. Cytotoxicity tests demonstrated the dose-dependent toxic effect of voriconazole on HCECs. Voriconazole concentrations of ≥100 μg/ml led to a significant reduction in cell viability. The morphological characteristics of HCECs also changed in a dose-dependent manner. Increasing concentrations of voriconazole resulted in fading staining for ZO1. Higher concentrations of voriconazole resulted in an increased number of propidium iodide (PI)-positive cells, indicating activation of the proapoptotic pathway. In conclusion, voriconazole may have a dose-dependent toxic effect on cultured HCECs. The results of this study suggest that although voriconazole concentrations of up to 50 μg/ml do not decrease cell viability, intracameral voriconazole concentrations of ≥100 μg/ml may increase the risk of corneal endothelial damage.

  11. Cell culture media impact on drug product solution stability.

    PubMed

    Purdie, Jennifer L; Kowle, Ronald L; Langland, Amie L; Patel, Chetan N; Ouyang, Anli; Olson, Donald J

    2016-07-01

    To enable subcutaneous administration of monoclonal antibodies, drug product solutions are often needed at high concentrations. A significant risk associated with high drug product concentrations is an increase in aggregate level over the shelf-life dating period. While much work has been done to understand the impact of drug product formulation on aggregation, there is limited understanding of the link between cell culture process conditions and soluble aggregate growth in drug product. During cell culture process development, soluble aggregates are often measured at harvest using cell-free material purified by Protein A chromatography. In the work reported here, cell culture media components were evaluated with respect to their impact on aggregate levels in high concentration solution drug product during accelerated stability studies. Two components, cysteine and ferric ammonium citrate, were found to impact aggregate growth rates in our current media (version 1) leading to the development of new chemically defined media and concentrated feed formulations. The new version of media and associated concentrated feeds (version 2) were evaluated across four cell lines producing recombinant IgG4 monoclonal antibodies and a bispecific antibody. In all four cell lines, the version 2 media reduced aggregate growth over the course of a 12 week accelerated stability study compared with the version 1 media, although the degree to which aggregate growth decreased was cell line dependent. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:998-1008, 2016.

  12. Testing of serum atherogenicity in cell cultures: questionable data published

    PubMed Central

    Jargin, Sergei V.

    2012-01-01

    In a large series of studies was reported that culturing of smooth muscle cells with serum from atherosclerosis patients caused intracellular lipid accumulation, while serum from healthy controls had no such effect. Cultures were used for evaluation of antiatherogenic drugs. Numerous substances were reported to lower serum atherogenicity: statins, trapidil, calcium antagonists, garlic derivatives etc. On the contrary, beta-blockers, phenothiazines and oral hypoglycemics were reported to be pro-atherogenic. Known antiatherogenic agents can influence lipid metabolism and cholesterol synthesis, intestinal absorption or endothelium-related mechanisms. All these targets are absent in cell monocultures. Inflammatory factors, addressed by some antiatherogenic drugs, are also not reproduced. In vivo, relationship between cholesterol uptake by cells and atherogenesis must be inverse rather than direct: in familial hypercholesterolemia, inefficient clearance of LDL-cholesterol by cells predisposes to atherosclerosis. Accordingly, if a pharmacological agent reduces cholesterol uptake by cells in vitro, it should be expected to elevate cholesterol in vivo. Validity of clinical recommendations, based on serum atherogenicity testing in cell monocultures, is therefore questionable. These considerations pertain also to the drugs developed on the basis of the cell culture experiments. PMID:22355278

  13. Algal growth response to particle-bound orthophosphate and zinc

    SciTech Connect

    Kuwabara, J.S.; Davis, J.A.; Chang, C.C.Y.

    1986-05-01

    Dissolved nutrient concentrations in natural waters may at times be controlled by interactions between particulate and solution phases. Effects of Zn (0-1 ..mu..M total Zn(II)) and orthophosphate (8-12 ..mu..M total P) additions on growth indices for the chlorophyte Selenastrum capricornutum Printz were examined in a synthetic growth medium containing 50 mg liter/sup -1/ colloidal titania. Over the Zn(II) concentration range used, detrimental growth and yield effects were observed. Addition of P to a synthetic growth medium (S-3) increased stationary phase cell density, but had minimal effect on growth rate and duration of lag phase. Presence of TiO/sub 2/ particles in culture media significantly reduced Zn and P dissolved fractions. Although adsorbed Zn and P were less available to Selenastrum, desorption of both solutes increased their availability. Rapid desorption of Zn(II) from TiO/sub 2/ particles served in effect to buffer Zn/sup 2 +/ free ion concentration, until Zn became partitioned primarily with the algal fraction as cell concentration approached stationary phase density. Although phosphate desorption from TiO/sub 2/ in nonbiological systems was negligible, Selenastrum was able to scavenge some P initially adsorbed onto TiO/sub 2/. Accurate primary productivity predictions in nature may therefore require an understanding of equilibrium and reaction rates involved in the partitioning of nutrients and toxic substances between dissolved and particulate phases.

  14. Culture of Oral Mucosal Epithelial Cells for the Purpose of Treating Limbal Stem Cell Deficiency.

    PubMed

    Utheim, Tor Paaske; Utheim, Øygunn Aass; Khan, Qalb-E-Saleem; Sehic, Amer

    2016-01-01

    The cornea is critical for normal vision as it allows allowing light transmission to the retina. The corneal epithelium is renewed by limbal epithelial cells (LEC), which are located in the periphery of the cornea, the limbus. Damage or disease involving LEC may lead to various clinical presentations of limbal stem cell deficiency (LSCD). Both severe pain and blindness may result. Transplantation of cultured autologous oral mucosal epithelial cell sheet (CAOMECS) represents the first use of a cultured non-limbal autologous cell type to treat this disease. Among non-limbal cell types, CAOMECS and conjunctival epithelial cells are the only laboratory cultured cell sources that have been explored in humans. Thus far, the expression of p63 is the only predictor of clinical outcome following transplantation to correct LSCD. The optimal culture method and substrate for CAOMECS is not established. The present review focuses on cell culture methods, with particular emphasis on substrates. Most culture protocols for CAOMECS used amniotic membrane as a substrate and included the xenogeneic components fetal bovine serum and murine 3T3 fibroblasts. However, it has been demonstrated that tissue-engineered epithelial cell sheet grafts can be successfully fabricated using temperature-responsive culture surfaces and autologous serum. In the studies using different substrates for culture of CAOMECS, the quantitative expression of p63 was generally poorly reported; thus, more research is warranted with quantification of phenotypic data. Further research is required to develop a culture system for CAOMECS that mimics the natural environment of oral/limbal/corneal epithelial cells without the need for undefined foreign materials such as serum and feeder cells. PMID:26938569

  15. Culture of Oral Mucosal Epithelial Cells for the Purpose of Treating Limbal Stem Cell Deficiency

    PubMed Central

    Paaske Utheim, Tor; Aass Utheim, Øygunn; Khan, Qalb-E-Saleem; Sehic, Amer

    2016-01-01

    The cornea is critical for normal vision as it allows allowing light transmission to the retina. The corneal epithelium is renewed by limbal epithelial cells (LEC), which are located in the periphery of the cornea, the limbus. Damage or disease involving LEC may lead to various clinical presentations of limbal stem cell deficiency (LSCD). Both severe pain and blindness may result. Transplantation of cultured autologous oral mucosal epithelial cell sheet (CAOMECS) represents the first use of a cultured non-limbal autologous cell type to treat this disease. Among non-limbal cell types, CAOMECS and conjunctival epithelial cells are the only laboratory cultured cell sources that have been explored in humans. Thus far, the expression of p63 is the only predictor of clinical outcome following transplantation to correct LSCD. The optimal culture method and substrate for CAOMECS is not established. The present review focuses on cell culture methods, with particular emphasis on substrates. Most culture protocols for CAOMECS used amniotic membrane as a substrate and included the xenogeneic components fetal bovine serum and murine 3T3 fibroblasts. However, it has been demonstrated that tissue-engineered epithelial cell sheet grafts can be successfully fabricated using temperature-responsive culture surfaces and autologous serum. In the studies using different substrates for culture of CAOMECS, the quantitative expression of p63 was generally poorly reported; thus, more research is warranted with quantification of phenotypic data. Further research is required to develop a culture system for CAOMECS that mimics the natural environment of oral/limbal/corneal epithelial cells without the need for undefined foreign materials such as serum and feeder cells. PMID:26938569

  16. A Microwell Cell Culture Platform for the Aggregation of Pancreatic β-Cells

    PubMed Central

    Bernard, Abigail B.; Lin, Chien-Chi

    2012-01-01

    Cell–cell contact between pancreatic β-cells is important for maintaining survival and normal insulin secretion. Various techniques have been developed to promote cell–cell contact between β-cells, but a simple yet robust method that affords precise control over three-dimensional (3D) β-cell cluster size has not been demonstrated. To address this need, we developed a poly(ethylene glycol) (PEG) hydrogel microwell platform using photolithography. This microwell cell-culture platform promotes the formation of 3D β-cell aggregates of defined sizes from 25 to 210 μm in diameter. Using this platform, mouse insulinoma 6 (MIN6) β-cells formed aggregates with cell–cell adherin junctions. These naturally formed cell aggregates with controllable sizes can be removed from the microwells for macroencapsulation, implantation, or other biological assays. When removed and subsequently encapsulated in PEG hydrogels, the aggregated cell clusters demonstrated improved cellular viability (>90%) over 7 days in culture, while the β-cells encapsulated as single cells maintained only 20% viability. Aggregated MIN6 cells also exhibited more than fourfold higher insulin secretion in response to a glucose challenge compared with encapsulated single β-cells. Further, the cell aggregates stained positively for E-cadherin, indicative of the formation of cell junctions. Using this hydrogel microwell cell-culture method, viable and functional β-cell aggregates of specific sizes were created, providing a platform from which other biologically relevant questions may be answered. PMID:22320435

  17. Measurement and analysis of calcium signaling in heterogeneous cell cultures.

    PubMed

    Richards, Gillian R; Jack, Andrew D; Platts, Amy; Simpson, Peter B

    2006-01-01

    High-content imaging platforms capable of studying kinetic responses at a single-cell level have elevated kinetic recording techniques from labor-intensive low-throughput experiments to potential high-throughput screening assays. We have applied this technology to the investigation of heterogeneous cell cultures derived from primary neural tissue. The neuronal cultures mature into a coupled network and display spontaneous oscillations in intracellular calcium, which can be modified by the addition of pharmacological agents. We have developed algorithms to perform Fourier analysis and quantify both the degree of synchronization and the effects of modulators on the oscillations. Functional and phenotypic experiments can be combined using this approach. We have used post-hoc immunolabeling to identify subpopulations of cells in cocultures and to dissect the calcium responses of these cells from the population response. The combination of these techniques represents a powerful tool for drug discovery.

  18. Influence of three laser wavelengths on human fibroblasts cell culture.

    PubMed

    Crisan, Bogdan; Soritau, Olga; Baciut, Mihaela; Campian, Radu; Crisan, Liana; Baciut, Grigore

    2013-02-01

    Although experimental studies in vitro and vivo have been numerous, the effect of laser wavelength irradiation on human fibroblast cell culture is poorly understood. This emphasizes the need of additional cellular and molecular research into laser influence with low energy and power. The aim of this study was to assess the influence of three different laser wavelengths on the human skin fibroblasts cell culture. We wanted to evaluate if near infrared lasers had any influence in healing of wounds by stimulating mitochondrial activity of fibroblasts. The cells were irradiated using 830-, 980- and 2,940-nm laser wavelengths. The irradiated cells were incubated and their mitochondrial activity was assessed by the MTT assay at 24, 48 and 72 h. Simultaneously, an apoptosis assay was assessed on the irradiated fibroblasts. It can be concluded that laser light of the near-infrared region (830 and 980 nm) influences fibroblasts mitochondrial activity compared to the 2,940-nm wavelength which produces apoptosis.

  19. Cultured meat from stem cells: challenges and prospects.

    PubMed

    Post, Mark J

    2012-11-01

    As one of the alternatives for livestock meat production, in vitro culturing of meat is currently studied. The generation of bio-artificial muscles from satellite cells has been ongoing for about 15 years, but has never been used for generation of meat, while it already is a great source of animal protein. In order to serve as a credible alternative to livestock meat, lab or factory grown meat should be efficiently produced and should mimic meat in all of its physical sensations, such as visual appearance, smell, texture and of course, taste. This is a formidable challenge even though all the technologies to create skeletal muscle and fat tissue have been developed and tested. The efficient culture of meat will primarily depend on culture conditions such as the source of medium and its composition. Protein synthesis by cultured skeletal muscle cells should further be maximized by finding the optimal combination of biochemical and physical conditions for the cells. Many of these variables are known, but their interactions are numerous and need to be mapped. This involves a systematic, if not systems, approach. Given the urgency of the problems that the meat industry is facing, this endeavor is worth undertaking. As an additional benefit, culturing meat may provide opportunities for production of novel and healthier products. PMID:22543115

  20. Cultured meat from stem cells: challenges and prospects.

    PubMed

    Post, Mark J

    2012-11-01

    As one of the alternatives for livestock meat production, in vitro culturing of meat is currently studied. The generation of bio-artificial muscles from satellite cells has been ongoing for about 15 years, but has never been used for generation of meat, while it already is a great source of animal protein. In order to serve as a credible alternative to livestock meat, lab or factory grown meat should be efficiently produced and should mimic meat in all of its physical sensations, such as visual appearance, smell, texture and of course, taste. This is a formidable challenge even though all the technologies to create skeletal muscle and fat tissue have been developed and tested. The efficient culture of meat will primarily depend on culture conditions such as the source of medium and its composition. Protein synthesis by cultured skeletal muscle cells should further be maximized by finding the optimal combination of biochemical and physical conditions for the cells. Many of these variables are known, but their interactions are numerous and need to be mapped. This involves a systematic, if not systems, approach. Given the urgency of the problems that the meat industry is facing, this endeavor is worth undertaking. As an additional benefit, culturing meat may provide opportunities for production of novel and healthier products.

  1. Biliary epithelial cells proliferate during oxygenated ex situ liver culture

    PubMed Central

    Bian, Congwen; Du, Yiqi; Ding, Rui; Huang, Jun; Dai, Yan; Bao, Sujin; Zhao, Lijuan; Shen, Hefang; Dong, Jing; Xu, Jianjian; Xiong, Qiru; Xu, Lili

    2016-01-01

    Biliary complications remain a major source of morbidity in liver transplant patients. Among these complications, nonanastomotic biliary strictures (NAS) are especially common and they are frequently therapy resistant in part because biliary epithelial cells are more sensitive to warm ischemic injury than hepatocytes. It has been a challenge to maintain the physiological function of biliary epithelial cells during liver transplantation. In this work, we have examined the effect of oxygen on proliferation of biliary epithelial cells in the rat livers obtained from donation after circulatory death (DCD). Twelve rat livers from DCD were divided into two groups. Livers in the control group were isolated following a standard procedure without oxygen supply. Livers in the experimental group were isolated with a constant supply of oxygen. All livers were then connected to an ex situ liver culture system in the presence of bromodeoxyuridine (BrdU), a thymidine analogue and a marker for cell proliferation. After 6 hours of normothermic ex situ liver culture, morphology and DNA replication in hepatocytes and biliary epithelial cells were assessed and compared between the two groups. We found that about 4.5% of the biliary epithelial cells in the experimental group proliferated compared with only 0.4% of cells in the control based on BrdU staining. No significant change in cell morphology was observed in those cells between the two groups. Thus, our results indicate that oxygen supply is required for maintenance of the physiological function of biliary epithelial cells during liver transplant and suggest that a constant oxygen supply during liver isolation along with ex situ liver organ culture can enhance the repair of biliary epithelial cell injury during liver transplantation. PMID:27725875

  2. Hybridoma cell behaviour in continuous culture under hyperosmotic stress.

    PubMed

    Cherlet, M; Marc, A

    1999-01-01

    In this paper, we propose an alternative strategy to the ones proposed before (Oh et al., 1993; Øyaas et al., 1994a) to get real increases of global final antibody titer and production at hyperosmotic stress, by reducing the detrimental effect of such a stress on cell growth, and conserving the stimulating effect on antibody production. It consists of cultivating the cells in continuous culture and increasing the osmolality stepwise. In this way, the cells could progressively adapt to the higher osmolality at each step and antibody titers could be nearly doubled at 370 and 400 mOsm kg-1, compared to the standard osmolality of 335 mOsm kg-1. Surprisingly, the stimulation of antibody production was not confirmed for higher osmolalities, 425 and 450 mOsm kg- 1, despite the minor negative effect on cell growth. Intracellular IgG analysis by flow cytometry revealed at these osmolalities a significant population of non-producing cells. However, even when taking into account this non-producing population, a stimulating effect on antibody production could not be shown at these highest osmolalities. It seems to us that osmolality has a significant effect on the appearance of these non-producing cells, since they were not observed in continuous cultures at standard osmolality, of comparable duration and at an even higher dilution rate. The appearance of the non-producing cells coincides furthermore with modifications of the synthesised antibody, as shown by electrophoretic techniques. It is however not really clear if these two observations reflect actually the same phenomenon. Hyperosmolality affects the cell behaviour in continuous culture in multiple ways, independently of the growth rate, counting all at least partially for the observed stimulation of antibody production: acceleration of the amino acid, and in particular the glutamine metabolism, increase of the cell volume, increase of the intracellular pH and accumulation of cells in the G1 cell cycle phase. PMID

  3. Cannabinoids induce incomplete maturation of cultured human leukemia cells

    SciTech Connect

    Murison, G.; Chubb, C.B.H.; Maeda, S.; Gemmell, M.A.; Huberman, E.

    1987-08-01

    Monocyte maturation markers were induced in cultured human myeloblastic ML-2 leukemia cells after treatment for 1-6 days with 0.03-30 ..mu..M ..delta../sup 9/-tetrahydrocannabinol (THC), the major psychoactive component of marijuana. After a 2-day or longer treatment, 2- to 5-fold increases were found in the percentages of cells exhibiting reactivity with either the murine OKM1 monoclonal antibody of the Leu-M5 monoclonal antibody, staining positively for nonspecific esterase activity, and displaying a promonocyte morphology. The increases in these differentiation markers after treatment with 0.03-1 ..mu..M THC were dose dependent. At this dose range, THC did not cause an inhibition of cell growth. The THC-induced cell maturation was also characterized by specific changes in the patterns of newly synthesized proteins. The THC-induced differentiation did not, however, result in cells with a highly developed mature monocyte phenotype. However, treatment of these incompletely matured cells with either phorbol 12-myristate 13-acetate of 1..cap alpha..,25-dihydroxycholecalciferol, which are inducers of differentiation in myeloid leukemia cells (including ML-2 cells), produced cells with a mature monocyte morphology. The ML-2 cell system described here may be a useful tool for deciphering critical biochemical events that lead to the cannabinoid-induced incomplete cell differentiation of ML-2 cells and other related cell types. Findings obtained from this system may have important implications for studies of cannabinoid effects on normal human bone-marrow progenitor cells.

  4. Microtable Arrays for Culture and Isolation of Cell Colonies

    PubMed Central

    Pai, Jeng-Hao; Xu, Wei; Sims, Christopher E.; Allbritton, Nancy L.

    2010-01-01

    Cell microarrays with culture sites composed of individually removable microstructures or micropallets have proven benefits for isolation of cells from a mixed population. The laser energy required to selectively remove these micropallets with attached cells from the array depends on the microstructure surface area in contact with the substrate. Laser energies sufficient to release micropallets greater than 100 μm resulted in loss of cell viability. A new 3-dimensional culture site similar in appearance to a table was designed and fabricated using a simple process that relied on a differential sensitivity of two photoresists to UV-mediated photopolymerization. With this design, the larger culture area rests on four small supports to minimize the surface area in contact with the substrate. Microtables up to 250 × 250 μm were consistently released with single 10 μJ pulses to each of the 4 support structures. In contrast, microstructures with a 150 × 150 μm surface area in contact with the substrate could not be reliably released at pulse energies up to 212 μJ. Cassie-Baxter wetting is required to provide a barrier of air to localize and sequester cells to the culture sites. A second asset of the design was an increased retention of this air barrier under conditions of decreased surface tension and after prolonged culture of cells. The improved air retention was due to the hydrophobic cavity created beneath the table and above the substrate which entrapped air when an aqueous solution was added to the array. The microtables proved an efficient method for isolating colonies from the array with 100% of selected colonies competent to expand following release from the array. PMID:20644916

  5. Potential of carbon nanotubes in algal biotechnology.

    PubMed

    Lambreva, Maya Dimova; Lavecchia, Teresa; Tyystjärvi, Esa; Antal, Taras Kornelievich; Orlanducci, Silvia; Margonelli, Andrea; Rea, Giuseppina

    2015-09-01

    A critical mass of knowledge is emerging on the interactions between plant cells and engineered nanomaterials, revealing the potential of plant nanobiotechnology to promote and support novel solutions for the development of a competitive bioeconomy. This knowledge can foster the adoption of new methodological strategies to empower the large-scale production of biomass from commercially important microalgae. The present review focuses on the potential of carbon nanotubes (CNTs) to enhance photosynthetic performance of microalgae by (i) widening the spectral region available for the energy conversion reactions and (ii) increasing the tolerance of microalgae towards unfavourable conditions occurring in mass production. To this end, current understanding on the mechanisms of uptake and localization of CNTs in plant cells is discussed. The available ecotoxicological data were used in an attempt to assess the feasibility of CNT-based applications in algal biotechnology, by critically correlating the experimental conditions with the observed adverse effects. Furthermore, main structural and physicochemical properties of single- and multi-walled CNTs and common approaches for the functionalization and characterization of CNTs in biological environment are presented. Here, we explore the potential that nanotechnology can offer to enhance functions of algae, paving the way for a more efficient use of photosynthetic algal systems in the sustainable production of energy, biomass and high-value compounds.

  6. Digital image processing-an alternate tool for monitoring of pigment levels in cultured cells with special reference to green alga Haematococcus pluvialis.

    PubMed

    Kamath, Sandesh B; Chidambar, Shalini; Brinda, B R; Kumar, M A; Sarada, R; Ravishankar, G A

    2005-11-15

    A method for analyzing carotenoid content in Haematococcus pluvialis, a green alga was developed using digital image processing (DIP) and an artificial neural network (ANN) model. About 90 images of algal cells in various phases of growth were processed with the tools of DIP. A good correlation of R(2)=0.967 was observed between carotenoid content as estimated by analytical method and DIP. Similar correlation was also observed in case of chlorophyll. Since the conventional methods of carotenoid estimation are time consuming and result in loss of pigments during analysis, DIP method was found to be an effective online monitoring method. This method will be useful in measurement of pigments in cultured cells.

  7. Patterns of plasminogen activator production in cultured normal embryonic cells

    PubMed Central

    1977-01-01

    Cultured normal low-passage embryo fibroblasts, from a number of species, and two untransformed clones of a Balb/3T3 line elaborate increasing amounts of plasminogen activator (PA) as they approach confluence; the low-passage cells then lose this PA activity after reaching confluence, while the 3T3 cells retain it indefinitely. Even at their peaks, however, the PA activities of the low-passage cells remain well below those of the corresponding virally or spontaneously transformed cells. The PA increases in normal cells are probably a result of PA production rather than of adsorption of secreted PA to the cell surface, or of changes in cell-associated protease inhibitors. The elaboration of PA by normal cells is dependent upon their metabolic activity, such that the level of serum supplementation and the growth phase of the culture directly influence the level of cell-associated PA observed. In addition, there may be a component of serum which exerts a negative control on PA production and which is not an acid-labile protease inhibitor. PMID:21193

  8. Microfluidic approaches for epithelial cell layer culture and characterisation

    PubMed Central

    Thuenauer, Roland; Rodriguez-Boulan, Enrique; Römer, Winfried

    2014-01-01

    In higher eukaryotes, epithelial cell layers line most body cavities and form selective barriers that regulate the exchange of solutes between compartments. In order to fulfil these functions, the cells assume a polarised architecture and maintain two distinct plasma membrane domains, the apical domain facing the lumen and the basolateral domain facing other cells and the extracellular matrix. Microfluidic biochips offer the unique opportunity to establish novel in vitro models of epithelia in which the in vivo microenvironment of epithelial cells is precisely reconstituted. In addition, analytical tools to monitor biologically relevant parameters can be directly integrated on-chip. In this review we summarise recently developed biochip designs for culturing epithelial cell layers. Since endothelial cell layers, which line blood vessels, have similar barrier functions and polar organisation as epithelial cell layers, we also discuss biochips for culturing endothelial cell layers. Furthermore, we review approaches to integrate tools to analyse and manipulate epithelia and endothelia in microfluidic biochips, including methods to perform electrical impedance spectroscopy, methods to detect substances undergoing trans-epithelial transport via fluorescence, spectrophotometry, and mass spectrometry, techniques to mechanically stimulate cells via stretching and fluid flow-induced shear stress, and methods to carry out high-resolution imaging of vesicular trafficking with light microscopy. Taken together, this versatile microfluidic toolbox enables novel experimental approaches to characterise epithelial monolayers. PMID:24668405

  9. Three-Dimensional Cultures of Mouse Mammary Epithelial Cells

    PubMed Central

    Mroue, Rana; Bissell, Mina J.

    2013-01-01

    The mammary gland is an ideal “model organism” for studying tissue specificity and gene expression in mammals: it is one of the few organs that develop after birth and it undergoes multiple cycles of growth, differentiation and regression during the animal’s lifetime in preparation for the important function of lactation. The basic “functional differentiation” unit in the gland is the mammary acinus made up of a layer of polarized epithelial cells specialized for milk production surrounded by myoepithelial contractile cells, and the two-layered structure is surrounded by basement membrane. Much knowledge about the regulation of mammary gland development has been acquired from studying the physiology of the gland and of lactation in rodents. Culture studies, however, were hampered by the inability to maintain functional differentiation on conventional tissue culture plastic. We now know that the microenvironment, including the extracellular matrix and tissue architecture, plays a crucial role in directing functional differentiation of organs. Thus, in order for culture systems to be effective experimental models, they need to recapitulate the basic unit of differentiated function in the tissue or organ and to maintain its three-dimensional (3D) structure. Mouse mammary culture models evolved from basic monolayers of cells to an array of complex 3D systems that observe the importance of the microenvironment in dictating proper tissue function and structure. In this chapter, we focus on how 3D mouse mammary epithelial cultures have enabled investigators to gain a better understanding of the organization, development and function of the acinus, and to identify key molecular, structural, and mechanical cues important for maintaining mammary function and architecture. The accompanying chapter of Vidi et al. describes 3D models developed for human cells. Here, we describe how mouse primary epithelial cells and cell lines—essentially those we use in our

  10. Sex stratified neuronal cultures to study ischemic cell death pathways.

    PubMed

    Fairbanks, Stacy L; Vest, Rebekah; Verma, Saurabh; Traystman, Richard J; Herson, Paco S

    2013-01-01

    Sex differences in neuronal susceptibility to ischemic injury and neurodegenerative disease have long been observed, but the signaling mechanisms responsible for those differences remain unclear. Primary disassociated embryonic neuronal culture provides a simplified experimental model with which to investigate the neuronal cell signaling involved in cell death as a result of ischemia or disease; however, most neuronal cultures used in research today are mixed sex. Researchers can and do test the effects of sex steroid treatment in mixed sex neuronal cultures in models of neuronal injury and disease, but accumulating evidence suggests that the female brain responds to androgens, estrogens, and progesterone differently than the male brain. Furthermore, neonate male and female rodents respond differently to ischemic injury, with males experiencing greater injury following cerebral ischemia than females. Thus, mixed sex neuronal cultures might obscure and confound the experimental results; important information might be missed. For this reason, the Herson Lab at the University of Colorado School of Medicine routinely prepares sex-stratified primary disassociated embryonic neuronal cultures from both hippocampus and cortex. Embryos are sexed before harvesting of brain tissue and male and female tissue are disassociated separately, plated separately, and maintained separately. Using this method, the Herson Lab has demonstrated a male-specific role for the ion channel TRPM2 in ischemic cell death. In this manuscript, we share and discuss our protocol for sexing embryonic mice and preparing sex-stratified hippocampal primary disassociated neuron cultures. This method can be adapted to prepare sex-stratified cortical cultures and the method for embryo sexing can be used in conjunction with other protocols for any study in which sex is thought to be an important determinant of outcome. PMID:24378980

  11. B35 neuroblastoma cells: an easily transfected, cultured cell model of central nervous system neurons.

    PubMed

    Otey, Carol A; Boukhelifa, Malika; Maness, Patricia

    2003-01-01

    A panel of neuronal cell lines was derived from tumors of the neonatal rat central nervous system (CNS) in 1974, and two of these lines are in wide use today. Both the B35 and B50 lines offer a number of advantages to researchers who study CNS neurons in culture: they are simple to grow, to differentiate, and to transfect. B50 cells have been used extensively in the study of neuronal cell death, toxicology, and differentiation, whereas B35 cells have proven useful in the molecular analysis of endocytosis and of signaling pathways, in particular those that guide axonal outgrowth and cell motility. This chapter provides protocols for growing and transfecting B35 cells, selecting stable transfectants, exploring protein function using an antisense approach, and assaying cell motility in a Transwell chamber. All of these protocols have been written for researchers who have some skill in basic cell culture techniques, but previous experience with cultured neurons is not required.

  12. Salt tolerance in cultured cells of Spartina pectinata.

    PubMed

    Warren, R S; Baird, L M; Thompson, A K

    1985-04-01

    Suspension cultures with cell doubling times of ca. 2 days were developed from the halophytic grass Spartina pectinata. Maximum rates of exponential growth measured by direct cell counts and by total culture packed-cell-volume were not significantly reduced by NaCl up to 200 mM but dropped beyond this point. In contrast, total cell production over a one week culture cycle, by both measures, was reduced in a roughly linear fashion between 0 and 500 mM NaCl. The pattern of growth in relation to NaCl is very similar to that of previously described cell suspensions derived from another halophyte, Distichlis spicata. In the field the latter is much more salt tolerant. The basis for the whole plant differences is not clear. They do not appear to reflect effectiveness of cell based salt tolerance or the presence of salt glands, which are reported here for the first time in S. pectinata and are found on the leaves of both species.

  13. Endocytic activity of Sertoli cells grown in bicameral culture chambers

    SciTech Connect

    Dai, R.X.; Djakiew, D.; Dym, M.

    1987-07-01

    Immature rat Sertoli cells were cultured for 7 to 14 days on Millipore filters impregnated with a reconstituted basement membrane extract in dual-environment (bicameral) culture chambers. Electron microscopy of the cultured cells revealed the presence of rod-shaped mitochondria, Golgi apparatus, rough endoplasmic reticulum, and Sertoli-Sertoli tight junctions, typical of these cells in vivo. The endocytic activity of both the apical and basal surfaces of the Sertoli cells was examined by either adding alpha 2-macroglobulin (alpha 2-M) conjugated to 20 nm gold particles to the apical chamber or by adding /sup 125/I labeled alpha 2-M to the basal chamber. During endocytosis from the apical surface of Sertoli cells, the alpha 2-M-gold particles were bound initially to coated pits and then internalized into coated vesicles within 5 minutes. After 10 minutes, the alpha 2-M-gold was found in multi-vesicular bodies (MVBs) and by 30 minutes it was present in the lysosomes. The proportion of alpha 2-M-gold found within endocytic cell organelles after 1 hour of uptake was used to estimate the approximate time that this ligand spent in each type of organelle. The alpha 2-M-gold was present in coated pits, coated vesicles, multivesicular bodies, and lysosomes for approximately 3, 11, 22, and 24 minutes, respectively. This indicates that the initial stages of endocytosis are rapid, whereas MVBs and lysosomes are relatively long-lived.

  14. Sequential cancer mutations in cultured human intestinal stem cells.

    PubMed

    Drost, Jarno; van Jaarsveld, Richard H; Ponsioen, Bas; Zimberlin, Cheryl; van Boxtel, Ruben; Buijs, Arjan; Sachs, Norman; Overmeer, René M; Offerhaus, G Johan; Begthel, Harry; Korving, Jeroen; van de Wetering, Marc; Schwank, Gerald; Logtenberg, Meike; Cuppen, Edwin; Snippert, Hugo J; Medema, Jan Paul; Kops, Geert J P L; Clevers, Hans

    2015-05-01

    Crypt stem cells represent the cells of origin for intestinal neoplasia. Both mouse and human intestinal stem cells can be cultured in medium containing the stem-cell-niche factors WNT, R-spondin, epidermal growth factor (EGF) and noggin over long time periods as epithelial organoids that remain genetically and phenotypically stable. Here we utilize CRISPR/Cas9 technology for targeted gene modification of four of the most commonly mutated colorectal cancer genes (APC, P53 (also known as TP53), KRAS and SMAD4) in cultured human intestinal stem cells. Mutant organoids can be selected by removing individual growth factors from the culture medium. Quadruple mutants grow independently of all stem-cell-niche factors and tolerate the presence of the P53 stabilizer nutlin-3. Upon xenotransplantation into mice, quadruple mutants grow as tumours with features of invasive carcinoma. Finally, combined loss of APC and P53 is sufficient for the appearance of extensive aneuploidy, a hallmark of tumour progression.

  15. Process control in cell culture technology using dielectric spectroscopy.

    PubMed

    Justice, C; Brix, A; Freimark, D; Kraume, M; Pfromm, P; Eichenmueller, B; Czermak, P

    2011-01-01

    In the biopharmaceutical industry, mammalian and insect cells as well as plant cell cultures are gaining worldwide importance to produce biopharmaceuticals and as products themselves, for example in stem cell therapy. These highly sophisticated cell-based production processes need to be monitored and controlled to guarantee product quality and to satisfy GMP requirements. With the process analytical technology (PAT) initiative, requirements regarding process monitoring and control have changed and real-time in-line monitoring tools are now recommended. Dielectric spectroscopy (DS) can serve as a tool to satisfy some PAT requirements. DS has been used in the medical field for quite some time and it may allow real-time process monitoring of biological cell culture parameters. DS has the potential to enable process optimization, automation, cost reduction, and a more consistent product quality. Dielectric spectroscopy is reviewed here as a tool to monitor biochemical processes. Commercially available dielectric sensing systems are discussed. The potential of this technology is demonstrated through examples of current and potential future applications in research and industry for mammalian and insect cell culture.

  16. Cementum matrix formation in vivo by cultured dental follicle cells.

    PubMed

    Handa, K; Saito, M; Yamauchi, M; Kiyono, T; Sato, S; Teranaka, T; Sampath Narayanan, A

    2002-11-01

    Dental follicle is the fibrous tissue that surrounds the developing tooth germ, and it is believed to contain progenitors for cementoblasts, periodontal ligament cells, and osteoblasts. In this study, we report the presence of cementoblast progenitors in cultures of bovine dental follicle cells and demonstrate their differentiation capacity. Bovine dental follicle cells (BDFC) obtained from tooth germs by collagenase digestion were compared with bovine alveolar bone osteoblasts (BAOB) and bovine periodontal ligament cells (BPDL) in vitro and in vivo. In culture, BDFC exhibited low levels of alkaline phosphatase activity and expressed mRNA for osteopontin (OP) and type I collagen (COLI), as well as low levels of osteocalcin (OC) mRNA. In contrast, cultured BAOB exhibited high alkaline phosphatase activity levels and expressed mRNA for OC, OP, COLI, and bone sialoprotein (BSP). To elucidate the differentiation capacity of BDFC in vivo, cells were transplanted into severe combined immunodeficiency (SCID) mice and analyzed after 4 weeks. Transplanted BDFC formed fibrous tissue and cementum-like matrix, which stained positive for anti-cementum attachment protein (CAP) monoclonal antibody (3G9), and expressed mRNA for OC, OP, COLI, and BSP. On the other hand, transplanted BAOB formed bone-like matrix, but were negative for anti-CAP monoclonal antibody. The BPDL transplants formed fibrous tissue that contained a few cells expressing CAP. These results indicate that cementoblast progenitors are present in BDFC, which can provide a useful model for investigating the molecular mechanisms of cementogenesis. PMID:12477575

  17. Apple Derived Cellulose Scaffolds for 3D Mammalian Cell Culture

    PubMed Central

    Modulevsky, Daniel J.; Lefebvre, Cory; Haase, Kristina; Al-Rekabi, Zeinab; Pelling, Andrew E.

    2014-01-01

    There are numerous approaches for producing natural and synthetic 3D scaffolds that support the proliferation of mammalian cells. 3D scaffolds better represent the natural cellular microenvironment and have many potential applications in vitro and in vivo. Here, we demonstrate that 3D cellulose scaffolds produced by decellularizing apple hypanthium tissue can be employed for in vitro 3D culture of NIH3T3 fibroblasts, mouse C2C12 muscle myoblasts and human HeLa epithelial cells. We show that these cells can adhere, invade and proliferate in the cellulose scaffolds. In addition, biochemical functionalization or chemical cross-linking can be employed to control the surface biochemistry and/or mechanical properties of the scaffold. The cells retain high viability even after 12 continuous weeks of culture and can achieve cell densities comparable with other natural and synthetic scaffold materials. Apple derived cellulose scaffolds are easily produced, inexpensive and originate from a renewable source. Taken together, these results demonstrate that naturally derived cellulose scaffolds offer a complementary approach to existing techniques for the in vitro culture of mammalian cells in a 3D environment. PMID:24842603

  18. Enhanced production of green tide algal biomass through additional carbon supply.

    PubMed

    de Paula Silva, Pedro H; Paul, Nicholas A; de Nys, Rocky; Mata, Leonardo

    2013-01-01

    Intensive algal cultivation usually requires a high flux of dissolved inorganic carbon (Ci) to support productivity, particularly for high density algal cultures. Carbon dioxide (CO2) enrichment can be used to overcome Ci limitation and enhance productivity of algae in intensive culture, however, it is unclear whether algal species with the ability to utilise bicarbonate (HCO3 (-)) as a carbon source for photosynthesis will benefit from CO2 enrichment. This study quantified the HCO3 (-) affinity of three green tide algal species, Cladophora coelothrix, Cladophora patentiramea and Chaetomorpha linum, targeted for biomass and bioenergy production. Subsequently, we quantified productivity and carbon, nitrogen and ash content in response to CO2 enrichment. All three species had similar high pH compensation points (9.7-9.9), and grew at similar rates up to pH 9, demonstrating HCO3 (-) utilization. Algal cultures enriched with CO2 as a carbon source had 30% more total Ci available, supplying twenty five times more CO2 than the control. This higher Ci significantly enhanced the productivity of Cladophora coelothrix (26%), Chaetomorpha linum (24%) and to a lesser extent for Cladophora patentiramea (11%), compared to controls. We demonstrated that supplying carbon as CO2 can enhance the productivity of targeted green tide algal species under intensive culture, despite their clear ability to utilise HCO3 (-).

  19. Enhanced Production of Green Tide Algal Biomass through Additional Carbon Supply

    PubMed Central

    de Paula Silva, Pedro H.; Paul, Nicholas A.; de Nys, Rocky; Mata, Leonardo

    2013-01-01

    Intensive algal cultivation usually requires a high flux of dissolved inorganic carbon (Ci) to support productivity, particularly for high density algal cultures. Carbon dioxide (CO2) enrichment can be used to overcome Ci limitation and enhance productivity of algae in intensive culture, however, it is unclear whether algal species with the ability to utilise bicarbonate (HCO3−) as a carbon source for photosynthesis will benefit from CO2 enrichment. This study quantified the HCO3− affinity of three green tide algal species, Cladophora coelothrix, Cladophora patentiramea and Chaetomorpha linum, targeted for biomass and bioenergy production. Subsequently, we quantified productivity and carbon, nitrogen and ash content in response to CO2 enrichment. All three species had similar high pH compensation points (9.7–9.9), and grew at similar rates up to pH 9, demonstrating HCO3− utilization. Algal cultures enriched with CO2 as a carbon source had 30% more total Ci available, supplying twenty five times more CO2 than the control. This higher Ci significantly enhanced the productivity of Cladophora coelothrix (26%), Chaetomorpha linum (24%) and to a lesser extent for Cladophora patentiramea (11%), compared to controls. We demonstrated that supplying carbon as CO2 can enhance the productivity of targeted green tide algal species under intensive culture, despite their clear ability to utilise HCO3−. PMID:24324672

  20. Disposable Bioreactors for Plant Micropropagation and Mass Plant Cell Culture

    NASA Astrophysics Data System (ADS)

    Ducos, Jean-Paul; Terrier, Bénédicte; Courtois, Didier

    Different types of bioreactors are used at Nestlé R&D Centre - Tours for mass propagation of selected plant varieties by somatic embryogenesis and for large scale culture of plants cells to produce metabolites or recombinant proteins. Recent studies have been directed to cut down the production costs of these two processes by developing disposable cell culture systems. Vegetative propagation of elite plant varieties is achieved through somatic embryogenesis in liquid medium. A pilot scale process has recently been set up for the industrial propagation of Coffea canephora (Robusta coffee). The current production capacity is 3.0 million embryos per year. The pre-germination of the embryos was previously conducted by temporary immersion in liquid medium in 10-L glass bioreactors. An improved process has been developed using a 10-L disposable bioreactor consisting of a bag containing a rigid plastic box ('Box-in-Bag' bioreactor), insuring, amongst other advantages, a higher light transmittance to the biomass due to its horizontal design. For large scale cell culture, two novel flexible plastic-based disposable bioreactors have been developed from 10 to 100 L working volumes, validated with several plant species ('Wave and Undertow' and 'Slug Bubble' bioreactors). The advantages and the limits of these new types of bioreactor are discussed, based mainly on our own experience on coffee somatic embryogenesis and mass cell culture of soya and tobacco.

  1. Using Haworthia Cultured Cells as an Aid in Teaching Botany

    ERIC Educational Resources Information Center

    Majumdar, Shyamal K.; Castellano, John M.

    1977-01-01

    Callus induction from species of Haworthia can be done quickly in the laboratory with minimal equipment to study tissue dedifferentiation and cellular redifferentiation. It is shown that the cultured cell can also be used to study and evaluate the effects of various mutagens, carcinogens, and pesticides in controlled environments. (Author/MA)

  2. Mutagenicity of instant coffee on cultured Chinese hamster lung cells.

    PubMed

    Nakasato, F; Nakayasu, M; Fujita, Y; Nagao, M; Terada, M; Sugimura, T

    1984-10-01

    Coffee showed mutagenic activity in cultured Chinese hamster lung (CHL) cells as assessed by using diphtheria toxin resistance as a selective marker. Most of the mutagenicity was suppressed in the presence of sodium bisulfite. The contribution of methylglyoxal to the total mutagenicity of coffee was less than 3%.

  3. Morphological characteristics of cultured fresh and thawed pericardium cells.

    PubMed

    Maslova, Olga; Fedevych, Oleg; Shuvalova, Nadiia; Deryabina, Olena; Zhovnir, Volodymyr; Novak, Miroslav; Kruzliak, Peter

    2016-06-01

    The need for selection of the optimal material for the manufacturing of cardio-patches can be resolved by the use of cryostored autologous pericardial tissue. This short communication is a concise fragment of a large-scale research and demonstrates only the efficiency of cell culturing before and after pericardial preservation in the low temperature conditions.

  4. DIVERSITY OF ARSENIC METABOLISM IN CULTURED HUMAN CANCER CELL LINES

    EPA Science Inventory

    Diversity of arsenic metabolism in cultured human cancer cell lines.

    Arsenic has been known to cause a variety of malignancies in human. Pentavalent As (As 5+) is reduced to trivalent As (As3+) which is further methylated by arsenic methyltransferase(s) to monomethylarson...

  5. Oxidative Stress Induces Senescence in Cultured RPE Cells

    PubMed Central

    Aryan, Nona; Betts-Obregon, Brandi S.; Perry, George; Tsin, Andrew T.

    2016-01-01

    The aim of this research is to determine whether oxidative stress induces cellular senescence in human retinal pigment epithelial cells. Cultured ARPE19 cells were subjected to different concentrations of hydrogen peroxide to induce oxidative stress. Cells were seeded into 24-well plates with hydrogen peroxide added to cell medium and incubated at 37°C + 5% CO2 for a 90-minute period [at 0, 300, 400 and 800 micromolar (MCM) hydrogen peroxide]. The number of viable ARPE19 cells were recorded using the Trypan Blue Dye Exclusion Method and cell senescence was measured by positive staining for senescence-associated beta-galactosidase (SA-beta-Gal) protein. Without hydrogen peroxide treatment, the number of viable ARPE19 cells increased significantly from 50,000 cells/well to 197,000 within 72 hours. Treatment with hydrogen peroxide reduced this level of cell proliferation significantly (to 52,167 cells at 400 MCM; to 49,263 cells at 800 MCM). Meanwhile, cells with a high level of positive senescence-indicator SA-Beta-Gal-positive staining was induced by hydrogen peroxide treatment (from a baseline level of 12% to 80% at 400 MCM and at 800 MCM). Our data suggests that oxidative stress from hydrogen peroxide treatment inhibited ARPE19 cell proliferation and induced cellular senescence. PMID:27651846

  6. Oxidative Stress Induces Senescence in Cultured RPE Cells.

    PubMed

    Aryan, Nona; Betts-Obregon, Brandi S; Perry, George; Tsin, Andrew T

    2016-01-01

    The aim of this research is to determine whether oxidative stress induces cellular senescence in human retinal pigment epithelial cells. Cultured ARPE19 cells were subjected to different concentrations of hydrogen peroxide to induce oxidative stress. Cells were seeded into 24-well plates with hydrogen peroxide added to cell medium and incubated at 37°C + 5% CO2 for a 90-minute period [at 0, 300, 400 and 800 micromolar (MCM) hydrogen peroxide]. The number of viable ARPE19 cells were recorded using the Trypan Blue Dye Exclusion Method and cell senescence was measured by positive staining for senescence-associated beta-galactosidase (SA-beta-Gal) protein. Without hydrogen peroxide treatment, the number of viable ARPE19 cells increased significantly from 50,000 cells/well to 197,000 within 72 hours. Treatment with hydrogen peroxide reduced this level of cell proliferation significantly (to 52,167 cells at 400 MCM; to 49,263 cells at 800 MCM). Meanwhile, cells with a high level of positive senescence-indicator SA-Beta-Gal-positive staining was induced by hydrogen peroxide treatment (from a baseline level of 12% to 80% at 400 MCM and at 800 MCM). Our data suggests that oxidative stress from hydrogen peroxide treatment inhibited ARPE19 cell proliferation and induced cellular senescence. PMID:27651846

  7. Oxidative Stress Induces Senescence in Cultured RPE Cells

    PubMed Central

    Aryan, Nona; Betts-Obregon, Brandi S.; Perry, George; Tsin, Andrew T.

    2016-01-01

    The aim of this research is to determine whether oxidative stress induces cellular senescence in human retinal pigment epithelial cells. Cultured ARPE19 cells were subjected to different concentrations of hydrogen peroxide to induce oxidative stress. Cells were seeded into 24-well plates with hydrogen peroxide added to cell medium and incubated at 37°C + 5% CO2 for a 90-minute period [at 0, 300, 400 and 800 micromolar (MCM) hydrogen peroxide]. The number of viable ARPE19 cells were recorded using the Trypan Blue Dye Exclusion Method and cell senescence was measured by positive staining for senescence-associated beta-galactosidase (SA-beta-Gal) protein. Without hydrogen peroxide treatment, the number of viable ARPE19 cells increased significantly from 50,000 cells/well to 197,000 within 72 hours. Treatment with hydrogen peroxide reduced this level of cell proliferation significantly (to 52,167 cells at 400 MCM; to 49,263 cells at 800 MCM). Meanwhile, cells with a high level of positive senescence-indicator SA-Beta-Gal-positive staining was induced by hydrogen peroxide treatment (from a baseline level of 12% to 80% at 400 MCM and at 800 MCM). Our data suggests that oxidative stress from hydrogen peroxide treatment inhibited ARPE19 cell proliferation and induced cellular senescence.

  8. A microfluidic device for uniform-sized cell spheroids formation, culture, harvesting and flow cytometry analysis.

    PubMed

    Patra, Bishnubrata; Chen, Ying-Hua; Peng, Chien-Chung; Lin, Shiang-Chi; Lee, Chau-Hwang; Tung, Yi-Chung

    2013-01-01

    Culture of cells as three-dimensional (3D) aggregates, named spheroids, possesses great potential to improve in vitro cell models for basic biomedical research. However, such cell spheroid models are often complicated, cumbersome, and expensive compared to conventional Petri-dish cell cultures. In this work, we developed a simple microfluidic device for cell spheroid formation, culture, and harvesting. Using this device, cells could form uniformly sized spheroids due to strong cell-cell interactions and the spatial confinement of microfluidic culture chambers. We demonstrated cell spheroid formation and culture in the designed devices using embryonic stem cells, carcinoma cells, and fibroblasts. We further scaled up the device capable of simultaneously forming and culturing 5000 spheroids in a single chip. Finally, we demonstrated harvesting of the cultured spheroids from the device with a simple setup. The harvested spheroids possess great integrity, and the cells can be exploited for further flow cytometry assays due to the ample cell numbers. PMID:24396525

  9. A microdroplet cell culture based high frequency somatic embryogenesis system for pigeonpea, Cajanus cajan (L.) Millsp.

    PubMed

    Kumar, Nagan Udhaya; Gnanaraj, Muniraj; Sindhujaa, Vajravel; Viji, Maluventhen; Manoharan, Kumariah

    2015-09-01

    A protocol for high frequency production of somatic embryos was worked out in pigeonpea, Cajanus cajan (L.) Millsp. The protocol involved sequential employment of embryogenic callus cultures, low density cell suspension cultures and a novel microdroplet cell culture system. The microdroplet cell cultures involved culture of a single cell in 10 μI of Murashige and Skoog's medium supplemented with phytohormones, growth factors and phospholipid precursors. By employing the microdroplet cell cultures, single cells in isolation were grown into cell clones which developed somatic embryos. Further, 2,4-dichlorophenoxyacetic acid, kinetin, polyethylene glycol, putrescine, spermine, spermidine, choline chloride, ethanolamine and LiCl were supplemented to the low density cell suspension cultures and microdroplet cell cultures to screen for their cell division and somatic embryogenesis activity. Incubation of callus or the inoculum employed for low density cell suspension cultures and microdroplet cell cultures with polyethylene glycol was found critical for induction of somatic embryogenesis. Somatic embryogenesis at a frequency of 1.19, 3.16 and 6.51 per 10(6) cells was achieved in the callus, low density cell suspension cultures and microdroplet cell cultures, respectively. Advantages of employing microdroplet cell cultures for high frequency production of somatic embryos and its application in genetic transformation protocols are discussed. PMID:26548080

  10. A microdroplet cell culture based high frequency somatic embryogenesis system for pigeonpea, Cajanus cajan (L.) Millsp.

    PubMed

    Kumar, Nagan Udhaya; Gnanaraj, Muniraj; Sindhujaa, Vajravel; Viji, Maluventhen; Manoharan, Kumariah

    2015-09-01

    A protocol for high frequency production of somatic embryos was worked out in pigeonpea, Cajanus cajan (L.) Millsp. The protocol involved sequential employment of embryogenic callus cultures, low density cell suspension cultures and a novel microdroplet cell culture system. The microdroplet cell cultures involved culture of a single cell in 10 μI of Murashige and Skoog's medium supplemented with phytohormones, growth factors and phospholipid precursors. By employing the microdroplet cell cultures, single cells in isolation were grown into cell clones which developed somatic embryos. Further, 2,4-dichlorophenoxyacetic acid, kinetin, polyethylene glycol, putrescine, spermine, spermidine, choline chloride, ethanolamine and LiCl were supplemented to the low density cell suspension cultures and microdroplet cell cultures to screen for their cell division and somatic embryogenesis activity. Incubation of callus or the inoculum employed for low density cell suspension cultures and microdroplet cell cultures with polyethylene glycol was found critical for induction of somatic embryogenesis. Somatic embryogenesis at a frequency of 1.19, 3.16 and 6.51 per 10(6) cells was achieved in the callus, low density cell suspension cultures and microdroplet cell cultures, respectively. Advantages of employing microdroplet cell cultures for high frequency production of somatic embryos and its application in genetic transformation protocols are discussed.

  11. High-affinity binding of fibronectin to cultured Kupffer cells

    SciTech Connect

    Cardarelli, P.M.; Blumenstock, F.A.; McKeown-Longo, P.J.; Saba, T.M.; Mazurkiewicz, J.E.; Dias, J.A. )

    1990-11-01

    Hepatic Kupffer cells are a major component of the reticuloendothelial or macrophage system. They were the first phagocytic cell type whose phagocytosis was shown to be influenced by plasma fibronectin, a dimeric opsonic glycoprotein. In the current study, the binding of soluble radioiodinated fibronectin purified from rat serum to isolated rat hepatic Kupffer cells was investigated using a cultured Kupffer cell monolayer technique. Binding was specific, since unlabeled purified fibronectin competed in a dose-dependent manner with the 125I-fibronectin for binding to the Kupffer cells. Addition of gelatin enhanced the binding of 125I-fibronectin to Kupffer cells. The phagocytosis of gelatinized-coated red cells by Kupffer cells was increased either by preopsonizing the target particles with purified fibronectin or by the addition of purified fibronectin to the culture medium. In contrast, exposure of the Kupffer cells to medium containing purified fibronectin followed by wash-removal of the fibronectin did not increase the uptake of gelatin-coated red blood cells, even though fibronectin was detected on the surface of the Kupffer cells by immunofluorescence. Trypsinized monolayers expressed decreased capacity to bind 125I-fibronectin as well as fibronectin-coated sheep erythrocytes. The binding of 125I-fibronectin-gelatin complexes was inhibited by excess unlabeled fibronectin. We calculated that specific high-affinity (Kd = 7.46 x 10(-9) M) binding sites for fibronectin exist on Kupffer cells. There are approximately 2,800-3,500 binding sites or putative fibronectin receptors per Kupffer cell. These sites appear to mediate the enhanced phagocytosis of gelatin-coated particles opsonized by fibronectin.

  12. Electrolytic Valving Isolation for Cell Co-Culture Microenvironment with Controlled Cell Pairing Ratios

    PubMed Central

    Chen, Yu-Chih; Ingram, Patrick; Yoon, Euisik

    2016-01-01

    Cancer-stromal interaction is a critical process in tumorigenesis. Conventional dish-based co-culture assays simply mix two cell types in the same dish; thus, they are deficient in controlling cell locations and precisely tracking single cell behavior from heterogeneous cell populations. Microfluidic technology can provide a good spatial temporal control of microenvironments, but the control has been typically realized by using external pumps, making long-term cultures cumbersome and bulky. In this work, we present a cell-cell interaction microfluidic platform that can accurately control co-culture microenvironment by using a novel electrolytic cell isolation scheme without using any valves or pneumatic pumps. The proposed microfluidic platform can also precisely control the number of interacting cells and pairing ratios to emulate cancer niches. More than 80% of the chambers captured the desired number of cells. The duration of cell isolation can be adjusted by electrolytic bubble generation and removal. We verified that electrolytic process has a negligible effect on cell viability and proliferation in our platform. To the best of our knowledge, this work is the first attempt to incorporate electrolytic bubble generation as a cell isolation method in microfluidics. For proof of feasibility, we performed cell-cell interaction assays between prostate cancer (PC3) cells and myoblast (C2C12) cells. The preliminary results demonstrated the potential of using electrolysis for micro-environmental control during cell culture. Also, the ratio controlled cell-cell interaction assays was successfully performed showing that the cell pairing ratios of PC3 to C2C12 affected the proliferation rate of myoblast cells due to increased secretion of growth factors from prostate cancer cells. PMID:25118341

  13. Differential Effect of Culture Temperature and Specific Growth Rate on CHO Cell Behavior in Chemostat Culture

    PubMed Central

    Vergara, Mauricio; Becerra, Silvana; Berrios, Julio; Osses, Nelson; Reyes, Juan; Rodríguez-Moyá, María; Gonzalez, Ramon; Altamirano, Claudia

    2014-01-01

    Mild hypothermia condition in mammalian cell culture technology has been one of the main focuses of research for the development of breeding strategies to maximize productivity of these production systems. Despite the large number of studies that show positive effects of mild hypothermia on specific productivity of r-proteins, no experimental approach has addressed the indirect effect of lower temperatures on specific cell growth rate, nor how this condition possibly affects less specific productivity of r-proteins. To separately analyze the effects of mild hypothermia and specific growth rate on CHO cell metabolism and recombinant human tissue plasminogen activator productivity as a model system, high dilution rate (0.017 h−1) and low dilution rate (0.012 h−1) at two cultivation temperatures (37 and 33°C) were evaluated using chemostat culture. The results showed a positive effect on the specific productivity of r-protein with decreasing specific growth rate at 33°C. Differential effect was achieved by mild hypothermia on the specific productivity of r-protein, contrary to the evidence reported in batch culture. Interestingly, reduction of metabolism could not be associated with a decrease in culture temperature, but rather with a decrease in specific growth rate. PMID:24699760

  14. Arraying heterotypic single cells on photoactivatable cell-culturing substrates.

    PubMed

    Kikuchi, Yukiko; Nakanishi, Jun; Shimizu, Takahiro; Nakayama, Hidekazu; Inoue, Satoshi; Yamaguchi, Kazuo; Iwai, Hideo; Yoshida, Yasuhiko; Horiike, Yasuhiro; Takarada, Tohru; Maeda, Mizuo

    2008-11-18

    This article describes a photochemical method for the site-selective assembly of heterotypic cells on a glass substrate modified with a silane coupling agent having a caged functional group. Silane coupling agents having a carboxyl (COOH), amino (NH 2), hydroxyl (OH), or thiol (SH) group protected by a photocleavable 2-nitrobenzyl group were synthesized to modify the surfaces of glass coverslips. The caged substrates were first coated by the adsorption of a blocking agent, bovine serum albumin (BSA), to make the entire surface non-cell-adhesive and then irradiated at 365 nm under a standard fluorescence microscope. The photocleavage reaction on the surface was followed by contact angle measurements and X-ray photoelectron spectroscopy. When COS7, NIH3T3, and HEK293 cells were seeded onto these substrates in a serum-free medium, the cells adhered selectively and efficiently to the irradiated regions on the caged NH 2 substrate, whereas the other caged COOH, SH, and OH substrates were nonphotoactivatable for cell adhesion. Qualitative and quantitative analysis of BSA adsorbed to the uncaged substrates revealed that this highly efficient photoactivation on the caged NH 2 substrate arose because of the following reasons: (i) upon photoactivation, BSA adsorbed in advance on the 2-nitrobenzyl groups was readsorbed onto the uncaged functional groups and (ii) BSA readsorbed onto the NH 2 groups became unable to passivate the surface against cell adhesion whereas BSA on the other groups still had normal passivating activity. It was also demonstrated that heterotypic single COS7, NIH3T3, and HEK293 cells were positioned at any desired arrangement on the caged NH 2 substrate by repeating the UV irradiation at optimized array spot sizes and cell seeding in optimized cell concentrations. The present method will be particularly useful in studying the dynamic processes of cell-cell interactions at a single-cell level.

  15. Modulation of the cancer cell transcriptome by culture media formulations and cell density

    PubMed Central

    KIM, SEUNG WOOK; KIM, SUN-JIN; LANGLEY, ROBERT R.; FIDLER, ISAIAH J.

    2015-01-01

    We investigated how varying the composition of cell culture formulations and growing cancer cells at different densities might affect tumor cell genotype. Specifically, we compared gene expression profiles generated by human MDA-MB-231 breast cancer cells cultured in different media [minimum essential medium (MEM), Dulbecco’s modified Eagle’s medium (DMEM), or Roswell Park Memorial Institute (RPMI)-1640 medium] containing different concentrations of fetal bovine serum (FBS) or different sera (equine or bovine) that were grown at different cell densities. More than 2,000 genes were differentially modulated by at least a 2-fold difference when MDA-MB-231 cancer cells were 90% confluent and compared with cultures that were 50% confluent. Altering the concentration of serum produced an even more pronounced effect on MDA-MB-231 cancer cell gene expression in that 2,981 genes were differentially expressed in a comparison between cells cultured in 0.1% FBS and same cell density cultures that were maintained in 10% FBS. A comparison between MDA-MB-231 cancer cells that were 90% confluent in MEM, DMEM, or RPMI-1640 media, all containing 10% FBS, resulted in 8,925 differentially expressed genes. Moreover, one-quarter (25.6%) of genes from our genome-wide expression analysis were expressed at significantly different levels by cells grown in MEM, DMEM, or RPMI-1640 media. Genes associated with epithelial-mesenchymal transition (EMT) were among the genes that were differentially modulated by cells grown in different cell culture formulations and these genes were verified at the protein level. Collectively, these results underscore the importance of accurate reporting and maintenance of uniform culture conditions to ensure reproducible results. PMID:25776572

  16. Comparison of defined culture systems for feeder cell free propagation of human embryonic stem cells

    PubMed Central

    Akopian, Veronika; Beil, Stephen; Benvenisty, Nissim; Brehm, Jennifer; Christie, Megan; Ford, Angela; Fox, Victoria; Gokhale, Paul J.; Healy, Lyn; Holm, Frida; Hovatta, Outi; Knowles, Barbara B.; Ludwig, Tenneille E.; McKay, Ronald D. G.; Miyazaki, Takamichi; Nakatsuji, Norio; Oh, Steve K. W.; Pera, Martin F.; Rossant, Janet; Stacey, Glyn N.; Suemori, Hirofumi

    2010-01-01

    There are many reports of defined culture systems for the propagation of human embryonic stem cells in the absence of feeder cell support, but no previous study has undertaken a multi-laboratory comparison of these diverse methodologies. In this study, five separate laboratories, each with experience in human embryonic stem cell culture, used a panel of ten embryonic stem cell lines (including WA09 as an index cell line common to all laboratories) to assess eight cell culture methods, with propagation in the presence of Knockout Serum Replacer, FGF-2, and mouse embryonic fibroblast feeder cell layers serving as a positive control. The cultures were assessed for up to ten passages for attachment, death, and differentiated morphology by phase contrast microscopy, for growth by serial cell counts, and for maintenance of stem cell surface marker expression by flow cytometry. Of the eight culture systems, only the control and those based on two commercial media, mTeSR1 and STEMPRO, supported maintenance of most cell lines for ten passages. Cultures grown in the remaining media failed before this point due to lack of attachment, cell death, or overt cell differentiation. Possible explanations for relative success of the commercial formulations in this study, and the lack of success with other formulations from academic groups compared to previously published results, include: the complex combination of growth factors present in the commercial preparations; improved development, manufacture, and quality control in the commercial products; differences in epigenetic adaptation to culture in vitro between different ES cell lines grown in different laboratories. PMID:20186512

  17. Differentiation of cultured epithelial cells: Response to toxic agents

    SciTech Connect

    Rice, R.H.; LaMontagne, A.D.; Petito, C.T.; Rong, Xianhui )

    1989-03-01

    Cell culture systems are instrumental in elucidating regulation of normal function and mechanisms of its perturbation by toxic substances. To this end, three applications of epithelial cells cultured with 3T3 feeder layer support are described. First, treatment of the premalignant human epidermal keratinocyte line SCC-12F2 with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate suppressed cell growth and differentiation. This agent produced a biphasic growth response greatly inhibiting cell growth at 1 to 10 nM, but much less above 100 nM. Expression of the differentiated functions involucrin and transglutaminase was found to be inhibited markedly at concentrations above 10 nM. Second, 3-methylcholanthrene toxicity was surveyed in a variety of rat epithelial cell types. The two most sensitive to growth inhibition were epidermal and mammary epithelial cells, while those from bladder, prostate, thyroid, and endometrium were insensitive to growth inhibition. Finally, expression of estrogen receptors in rat endometrial cells was shown to be stimulated by the cAmP-elevating agent forskolin. Maximal stimulation of 3- to 6-fold occurred in 6 hr, compatible with a requirement for protein synthesis. Pursuit of such results will aid in understanding differences in response among cell types and species, in elucidating mechanisms of action of known toxic substances and, ultimately, in predicting toxicity of less well understood agents.

  18. DNA Analysis of Algal Endosymbionts of Ciliates Reveals the State of Algal Integration and the Surprising Specificity of the Symbiosis.

    PubMed

    Hoshina, Ryo; Kusuoka, Yasushi

    2016-04-01

    Many freshwater protists harbor unicellular green algae within their cells, but little is known of their degree of integration and specificity. Using algae-targeted PCR of whole ciliate cells collected at irregular intervals over 15 months from Lake Biwa, Japan, we explored the SSU-ITS rDNA of the endosymbiotic algae and its changes over time, obtaining sequences of algal rDNA fragments from four ciliate species. A high proportion of clonal algae was evident within the ciliate cells. The differences observed in those sequences from the SSU through to the ITS region were less than 1%. The name 'Chlorb' is proposed for these algae, with the implication that they represent a single 'species.' The sequences of the algal DNA fragments were identical for any given host species throughout the collection period, thus we conclude that these four ciliates stably retain their algae over long term. In contrast, algal DNA fragments obtained from Didinium sp. were variable within each sample, which indicates that this ciliate only temporarily holds its algal cells. The ITS1 sequences of Chlorb populations are close (at intraspecific level) to those of algae isolated from ciliates in Austria, which raises the possibility that Chlorb algae are universally shared as symbionts among various ciliates.

  19. Effects of vibration on differentiation o