SAR and efficiency evaluation of a 900 MHz waveguide chamber for cell exposure

In this work we present the results of numerical and experimental dosimetry carried out for an in vitro exposure device to irradiate sample groups at 900 MHz. The cells are kept in 8 and 15 ml cell cultures, contained, respectively in T25 and T75 rectangular flasks. The dosimetric assessment of the distribution of the specific absorption rate (SAR) is performed for both the bottom of the flask and the whole volume of the sample to provide results for experiments on either the cell layer or the cell suspension. The irradiating chamber is a rectangular waveguide (WG). Different configurations are considered to assess the optimum orientation and positioning of the cell cultures inside the WG. The system performance is optimal when the electric field is parallel to the sample and the WG is terminated by a matched load. In this condition two 15 or four 8 ml cells cultures can be exposed. The efficiency (ratio between the power absorbed by the sample and the incident power) and the non-uniformity degree (ratio between the standard deviation of SAR values and the average SAR over the sample) are calculated and successfully verified through measurements of the scattering parameters and local temperature increases. In the chosen exposure configuration, the efficiency is 0.40 and the non-uniformity degree is 39% for the 15 ml samples. For the 8 ml samples, the efficiency is 0.19 and a low non-uniformity degree (15%) is found.     

Reevaluation and improved design of the TEM cell in vitro exposure unit for replication studies

The transverse electromagnetic (TEM) cell system developed by Litovitz et al. and utilized by Penafiel et al. for the exposure of cells in T25 flasks at 835 MHz has been reevaluated for the purpose of replicating the studies published by Penafiel. The original setup has been reconstructed as closely as possible, with improvements enabling blinded exposures, forced cooling and better repeatable positioning of the flasks, as well as tight exposure and environmental parameter control. The signal unit can simulate the original signal but also enables various other exposure schemes. The setup has been evaluated for four T25 flasks filled with 5 and 10 ml of cell medium by experimental and numerical means. Comparing E field, SAR and temperature measurements resulted in good agreement: <0.4 dB (4.5%) for E field and 0.48 dB (10.5%) for SAR. The overall average SAR within the medium is 6.0 W/kg at 1 W input power with a standard deviation of less than 52%. The temperature increase was determined to be 0.13 degrees C/(W/kg). This can be reduced to 0.045 degrees C/(W/kg) by applying active air flow cooling. The comparison of SAR values from temperature measurements with the corresponding simulated values resulted in excellent agreement. These results do not correspond to the previous study reporting an average SAR within the medium of 2.5 W/kg at an input power of 0.96 W.     

Effect of the meniscus at the solid/liquid interface on the SAR distribution in Petri dishes and flasks

Several authors have reported that the meniscus as occurring at the walls of Petri dishes and flasks (solid-liquid interface) can significantly affect the SAR distribution in the entire dish and flask, while others have ignored this effect. In this study, this effect has been comprehensively analyzed by numerical means supported by theoretical considerations. The focus is on E polarization, which results in the most homogeneous exposure for monolayer cell cultures and therefore is the most often applied polarization in exposure setups for risk assessment studies. This includes setups based on TEM cells, waveguides, radial transmission lines (RTL), and HF chambers. The conclusion of this study is that the meniscus and its size have a significant effect on the strength and homogeneity of the induced SAR distribution. Hence, the meniscus needs to be accounted for in the determination of the averaged SAR as well as in the uncertainty assessment. It has also been demonstrated that a voxel size of less than 0.2 mm is needed to predict the SAR values for a monolayer of cells accurately.     

"Stationary phase aging" of cell culture: an attempt of evaluation of growth medium "age" effect

Cell proliferation rate and 3H-thymidine labeling index of "young" (i. e. harvested in 3 days after subcultivation) cultured Chinese hamster cells (B11 dii-FAF28 line) have been determined in growth medium conditioned by the same cells for various periods of time during their growth and subsequent "stationary phase aging" (medium of different "age"). Cells were serially cultured in Eagle's medium with 10 % bovine serum. The experiment was conducted as follows. The "young" cells were seeded in Carrel's flasks (4500 cells/cm2) with fresh growth medium and placed at 37 degreesC. At definite time intervals, media from 3 randomly selected flasks were filtrated and stored in small glass flasks at 4 degreesC. The cells from all 3 flasks were collected by trypsin treatment and counted with hemocytometer. During the period of 26 day cultivation we collected a set of media of different "age" corresponding to certain points of the growth and "stationary phase aging" curve of the culture. Then, the "young" cells in fresh medium were seeded into tissue culture plates with cover slips placed into wells of the plates (26,600 cells/cm2) and grown at 37degreesC, 5 % CO2 for 2 h. At this point, the medium was replaced with media of different "age". 22 h later (i. e. on the first day after seeding) cell density was evaluated microscopically in all the wells. On the next day (i. e. in 2 days after seeding) 3H-thymidine was added to every well to final concentration 1.85 x 10(4) Bq/ml. After next 24 h (i. e. in 3 days after seeding) cell density was counted again, and the medium was removed. The cover slips were rinsed with Hank's solution and air-dried. Autoradiography was performed in standard manner by photoemulsion exposing for 5 days and subsequent developing in amidol developer. The relative number of nuclei with 10 and more "grains" was revealed microscopically. Based on the obtained results, two basic parameters were evaluated for every "age" medium: 1) cell proliferation activity index calculated as log2 (N3/N1), where N1 - cell density on the first day after seeding, and N3 - the same parameter on the third day after seeding; 2) cell labeling index calculated as percentage of cells with nuclei labeled by 3H-thymidine during incubation from 2nd to 3rd day of cultivation. These two indexes for cell growth in different "age" media appeared to be highly correlating (R = 0.85). Besides, it was found that the observed "age-related" diminishing of ability of the growth media of different "age" to stimulate proliferation of "young" cells cannot completely explain the "stationary phase aging" phenomenon (in particular, even for the "oldest" medium cell labeling index was 65 %). We conclude that the phenomenon is based on exactly intrinsic changes of cells, most likely on molecular level, though environmental effects cannot be entirely excluded. The authors are grateful to the Russian Basic Research Foundation for support (grants 03-04-49030 and 00-04-48049).     

Basis for optimization of in vitro exposure apparatus for health hazard evaluations of mobile communications.

The main objective of this paper is to carefully study the fields induced in flasks exposed to RF electromagnetic fields. The study focuses on the widely used 60 mm Petri dishes and rectangular T-75 flasks for the two following cases: 1) cells in homogeneous suspension and 2) cell monolayers. The dependence of the coupling and the homogeneity of the SAR distribution on frequency (0.7 GHz to 2.5 GHz), polarization (E, H and k polarizations) and the amount of medium (1.9 mm to 4.7 mm medium height) is studied. In addition, the effects of the environment, meniscus and field impedance as well as the distortion of the incident field are discussed. Based on these results, advantages and disadvantages of different fundamental designs of apparatus used in the past are compared. These are TEM cells, HF chambers, radial transmission lines (RTL), waveguides and wire patch cells. Furthermore, the major optimization parameters are identified for the development of highly optimized exposure systems, enabling the conduct of high quality experiments.     

Near-field dosimetry for in vitro exposure of human cells at 60 GHz

Due to the expected mass deployment of millimeter‐wave wireless technologies, thresholds of potential millimeter‐wave‐induced biological and health effects should be carefully assessed. The main purpose of this study is to propose, optimize, and characterize a near‐field exposure configuration allowing illumination of cells in vitro at 60 GHz with power densities up to several tens of mW/cm². Positioning of a tissue culture plate containing cells has been optimized in the near‐field of a standard horn antenna operating at 60 GHz. The optimal position corresponds to the maximal mean‐to‐peak specific absorption rate (SAR) ratio over the cell monolayer, allowing the achievement of power densities up to 50 mW/cm² at least. Three complementary parameters have been determined and analyzed for the exposed cells, namely the power density, SAR, and temperature dynamics. The incident power density and SAR have been computed using the finite‐difference time‐domain (FDTD) method. The temperature dynamics at different locations inside the culture medium are measured and analyzed for various power densities. Local SAR, determined based on the initial rate of temperature rise, is in a good agreement with the computed SAR (maximal difference of 5%). For the optimized exposure setup configuration, 73% of cells are located within the ±3 dB region with respect to the average SAR. It is shown that under the considered exposure conditions, the maximal power density, local SAR, and temperature increments equal 57 mW/cm², 1.4 kW/kg, and 6 °C, respectively, for the radiated power of 425 mW. Bioelectromagnetics 33:55–64, 2012. © 2011 Wiley Periodicals, Inc.     

Influence of Dissolved Oxygen Levels on Production of l-Asparaginase and Prodigiosin by Serratia marcescens

The effect of dissolved oxygen concentrations on the behavior of Serratia marcescens and on yields of asparaginase and prodigiosin produced in shaken cultures and in a 55-liter stainless-steel fermentor was studied. A range of oxygen transfer rates was obtained in 500-ml Erlenmeyer flasks by using internal, stainless-steel baffles and by varying the volume of medium per flask, and in the fermentor by high speed agitation (375 rev/min) or low rates of aeration (1.5 volumes of air per volume of broth per min), or both. Dissolved oxygen levels in the fermentation medium were measured with a membrane-type electrode. Peak yields of asparaginase were obtained in unbaffled flasks (3.0 to 3.8 IU/ml) and in the fermentor (2.7 IU/ml) when the level of dissolved oxygen in the culture medium reached zero. A low rate of oxygen transfer was accomplished by limited aeration. Production of prodigiosin required a supply of dissolved oxygen that was obtainable in baffled flasks with a high rate of oxygen transfer and in the fermentor with a combination of high-speed agitation and low-rate aeration. The fermentation proceeded at a more rapid rate and changes in pH and cell populations were accelerated by maintaining high levels of dissolved oxygen in the growth medium.     

Potential errors in conventional DOT measurement techniques in shake flasks and verification using a rotating flexitube optical sensor

Background  

Dissolved oxygen tension (DOT) is an important parameter for evaluating a bioprocess. Conventional means to measure DOT in shake flasks using fixed Clark-type electrodes immersed in the bulk liquid are problematic, because they inherently alter the hydrodynamics of the systems. Other approaches to measure DOT that apply fluorescing sensor spots fixed at the inside wall of a shake flask are also suboptimal. At low filling volumes for cultivating microorganisms with a high oxygen demand, the measured DOT signal may be erroneous. Here, the sensor spot is sometimes exposed to gas in the head space of the flask. Merely repositioning the sensor spot elsewhere in the flask does not address this problem, since there is no location in the shake flask that is always covered by the rotating bulk liquid. Thus, the aim of this prospective study is first, to verify the systemic error of Clark-type electrodes for measuring DOT in shake flasks. The second principle aim is to use the newly built "flexitube optical sensor" to verify potential errors in conventional optical DOT measurements based on fixed sensor spots.     

Preservation of hematopoietic stem and progenitor cells from umbilical cord blood stored in a surface derivatized with polymer nanosegments

Tissue culture flasks were prepared with immobilized amphiphilic nanosegments of Pluronic F68 and F127, polyethylene oxide (PEO)-polypropylene oxide (PPO)-PEO triblock copolymers, on their surfaces. These so-called "Pluronic-immobilized flasks" were used for the preservation of hematopoietic stem and progenitor cells from umbilical cord blood. The expression ratio of surface markers (CD34) on hematopoietic stem and progenitor cells stored in Pluronic-immobilized flasks was significantly higher than that in polystyrene tissue culture flasks or commercially available bioinert flasks (i.e., low cell-binding cultureware). This was due to the presence of flexible brushlike segments of Pluronic on the Pluronic-immobilized flask. A good correlation was found between the number of CD34+ cells and the ratio of viable CD34+ cells from cord blood in several flasks after five days of storage. Therefore, the high number of CD34+ cells was thought to have originated from the high viability of these cells stored in Pluronic-immobilized flasks. It was found that there was an optimal surface concentration of Pluronic on the Pluronic-immobilized flask surfaces for the preservation (high number and survival) of these stem and progenitor cells. The foregoing results were attributable to the high density of Pluronic nanosegments on the flask surface, limiting the movement of these flexible segments.     

Power consumption in shaking flasks on rotary shaking machines: II. Nondimensional description of specific power consumption and flow regimes in unbaffled flasks at elevated liquid viscosity

This article is the second part of a series presenting and modeling the hydrodynamics and specific power consumption in shaking flasks on rotary (orbital) shaking machines. In part I, a new method was introduced that enables the accurate determination of the specific power consumption in shaking flasks. The method was first applied to investigate unbaffled flasks with a nominal volume of < or =1 L at low viscosity. In part II, the results for the specific power consumption of unbaffled shaking flasks at elevated viscosities are investigated after varying shaking frequency, flask size, filling volume, and shaking diameter. The theory introduced in part I is extended to liquids of elevated viscosities using nondimensional equations. With these results, the specific power consumption in unbaffled shaking flasks can now be fully described. For the first time, the phenomenon of the liquid being "out of phase" is observed and described. This occurs at certain operating conditions and is characterized by an increasing amount of liquid not following the movement of the shaking table, thus reducing the specific power consumption. This, of course, has much relevance for practical work with microbial cultures. The phenomenon of being "out-of-phase" is described in the form of a newly defined nondimensional phase number (Ph) in analogy to a partially filled, rotating horizontal drum. The Ph can be used to determine reasonable operating conditions for shaking flask experiments when using viscous media, avoiding unfavorable "out-of-phase" operation.     

Power consumption in shaking flasks on rotary shaking machines: I. Power consumption measurement in unbaffled flasks at low liquid viscosity

In this first article of a series a new method is introduced that enables the accurate determination of the power consumption in a shaking flask. The method is based on torque measurements in the drive and appropriate compensation of the friction losses. The results for unbaffled shaking flasks at low viscosities are presented after varying shaking frequency, flask size, filling volume, shaking diameter, and surface quality (hydrophilic and hydrophobic) of the inner flask walls. The order of magnitude of the values of power consumption in shaking flasks is equal to, or even higher than, the values typical for agitated tank bioreactors. A physically based model equation for shaking flasks is derived that introduces a modified power number and a resulting constant as the only fitting parameter. With this equation, the measured results are correlated with sufficient accuracy. For the first time, comprehensive data for the power consumption in unbaffled shaking flasks at low viscosity is available, giving a detailed picture of the influences of the different variables.     

Optimization of protein-production by the baculovirus expression vector system in shake flasks

Summary Shake flasks were successfully employed for the cultivation of Spodoptera frugiperda (Sf-9) insect cells and for the production of \-galactosidase, a recombinant model protein, utilizing the baculovirus expression vector system. The culture doubling time and maximal cell density were 20 h and 5 × 10⁶ cells/ml respectively. The optimal liquid volumes for flasks rotating at 100 rpm were 25–40% of the flask total volume. Enzyme production (about 600 mg/l) was best at a multiplicity of infection of between 1 and 20 and at a cell density at time of infection of 0.7 × 10⁶ cells/ml. At a rotation speed of 100 rpm, Pluronic F-68 had no effect on growth and enzyme production. Offprint requests to: Y. Shoham     

Suspension culture of drosophila cells employing a gyratory shaker

Summary To develop a method for culturing a large number of small-scale suspension cultures ofDrosophila melanogaster cells simultaneously, basic conditions were studied using a cell line GM₂ and a gyratory shaker. Under gyration at more than 180 rpm, a majority (>80%) of the cells still remained as suspension and grew normally. Lower speed of gyration caused adhesion of the cells to a substratum. Furthermore, size of the culture vessels was found to affect the pattern of cell growth. Five- or 10-ml Erlenmeyer flasks gave satisfactory results, but the growth curves in 30-ml flasks differed from flask to flask and the saturation level was lower. Besides, the growth curves in the latter case were quite different depending on the volume of the medium. A preliminary experiment showed that the type of flask might affect the pattern of a growth curve. Initial cell densities has to be more than 6×10⁴ cells per ml. Lower densities resulted in the longer doubling time or no increase in the cell number. Therefore the following conditions are recommended as a standard for gyration culture ofD. melanogaster cell, GM₂: speed of gyration, 180 rpm; culture vessel, 5- or 10-ml Erlenmeyer flask of a certain type; initial cell density, 1 to 5×10⁵ per ml. Both D20 and modified Schneider’s medium could be utilized as the medium.     

转瓶内部结构对无血清悬浮培养昆虫细胞的影响

以昆虫细胞为宿主进行基因工程产品的开发是动物细胞培养领域十分有吸引力的研究方向[1] 。由于昆虫细胞对营养要求极高 ,且对培养环境非常敏感 ,所以一般是在含有兼具营养及保护功能的胎牛血清的培养基中进行培养。血清一方面因其高额成本而限制了昆虫细胞大规模培养技术的发展 ,另一方面又因其成分复杂、富含蛋白而给外源基因表达产物的后处理带来困难。因此 ,昆虫细胞无血清培养技术的开发一直是细胞培养工程领域的研究热点 ,采用无血清培养技术取代传统的有血清培养技术已成为昆虫细胞 杆状病毒表达系统的发展趋势[2 ] 。然而 ,昆虫细…     

Different effects of microwave energy and conventional heat on the activity of a thermophilic beta-galactosidase from Bacillus acidocaldarius

The effect of microwave (f = 10.4 GHz) irradiation on a thermostable enzyme was experimentally tested, showing that irreversible inactivation is obtained. Enzymatic solutions (500 microliters, with concentrations between 10-100 micrograms/ml) were exposed at 70 degrees C, at SAR levels of 1.1 and 1.7 W/g for 15, 30, 45, or 60 min, and their activity was compared to that of a sample heated in a water bath at the same temperature. The residual activity of the exposed samples depends on enzyme concentration, microwave power level, and exposure time; activity was reduced to 10% in 10 micrograms/ml solutions treated at 1.7 W/g for 60 min. Microwave effects disappeared at concentrations above 50 micrograms/ml. These results were not found following water bath heating at the same temperature and durations.     

Comparison of Cell Growth and Alkaloid Production of Catharanthus roseus Cells Cultured in Shake Flask and in Bioreactor

The cell growth and alkaloid production of Catharanthus roseus (L.) G. Don cells cultured in the shake flasks with different volumes and in the stirred tank bioreactor (10 L) were compared. Cell dry weight and alkaloid production showed no significant difference in the small volume scale-up shake flasks. When more broths were added to a certain volume in the shake flask, both cell weight and alkaloid production were decreased. The maximum cell dry weight was similar between the cell cultures in the shake flask and the bioreactor, but the alkaloid production of cells was much less in the bioreactor. Gas regime and shear stress were recognized to be the main factors contributing the important effect on alkaloid production during the scale-up processes.     

Noninvasive oxygen measurements and mass transfer considerations in tissue culture flasks

Murine hybridomas were cultivated in tissue culture flasks. Dissolved oxygen tensions in the gas and liquid phases during cell growth were monitored. Oxygen levels were measured noninvasively by interrogating an oxygen-sensitive patch mounted on the interior surface of the tissue culture flask with an optrode from outside the tissue culture flask. Readings were made in tissue culture flasks with caps both cracked open and completely closed. Although the oxygen in the gas phase remained near atmospheric oxygen levels in both flasks, over time the liquid-phase oxygen tension at the bottom of the flasks reached zero during cell growth in both the open and closed tissue culture flasks. These results suggest that the widespread practice of cracking open tissue culture flask caps during cell growth with a view to supplying adequate oxygen to cells is ineffective and probably unnecessary.The mass transfer characteristics of the tissue culture flask were also studied. The dominant resistance to oxygen mass transfer to the sensor and the cells was through the liquid media. The mass transfer rates through the liquid layer under standard laboratory conditions were found to be greater than those predicted by diffusion alone. This suggests that mixing at a microscale occurs. Volumetric and specific oxygen consumption rates were also calculated from the sensor data. These consumption rates were comparable with values published elsewhere. (c) 1996 John Wiley & Sons, Inc.     

Technological problems in cultivation of plant cells at high density. Reprinted from Biotechnology and Bioengineering, Vol. XXII, No. 6, Pages 1203-1218 (1981)

Using Cudrania tricuspidata cells as model plant cells which have high sensitivity to hydrodynamic stress, technological problems in the cultivation of the plant cells at high density were investigated. Using "shake" flasks on a reciprocal shaker and Erlenmeyer flasks on a rotary shaker and with a high supply of oxygen in order to obtain high cell densities in shaken cultures, particle breakdown and damage to the largest cell aggregate group (above 1981 microm in diameter) occurred and normal cell growth became impeded. The mass-transfer coefficient (K) for a model solid-liquid system (beta-naphthol particles and water) in place of a system of plant cells and a liquid medium was proposed as an intensity index of hydrodynamic stress effects on plant cells in suspension cultures under various conditions in the bioreactor systems. Normal cell growth was obtained under culture conditions for K values less than about 4.4 x 10(-3) cm/sec. The characteristics of various bioreactors used until now were investigated by considering the three main technological factors (capacity of oxygen supply, intensity of hydrodynamic stress effects on plant cells, and intensity of culture broth mixing and air-bubble dispersion). The most suitable bioreactor for culturing plant cells at high density was a jar fermentor with a modified paddle-type impeller (J-M). The yield of cell mass in the 10-liter J-M (working volume 5 liter) was about 30 g dry weight per liter of medium.     

Observations consistent with autocrine stimulation of hybridoma cell growth and implications for large-scale antibody production

Existence of autocrine growth factors (aGFs) may influence the serum requirement for growth of hybridoma cells and thus significantly influence process economics. For the murine hybridoma cell line S3H5/2bA2, critical inoculum density (cID) and serum requirement for growth were inversely related for cultivation in both T flasks and spinner flasks. In spinner flasks, an inoculum density of 10⁶ cells/ml was necessary for the cells to grow in RPMI 1640 medium without serum supplement, and an inoculum density of 10³ cell/ml was necessary in RPMI 1640 medium with 10% serum. In T flasks, where the local cell density is higher than in spinner flasks, an inoculum density of 10⁶ cells/ml was necessary for the cells to grow in RPMI 1640 medium without serum supplement, and an inoculum density of 1 cell/ml was also necessary in RPMI 1640 medium with 10% serum. Further, immobilized cells at high local cell density could grow under conditions where cells in T flasks at corresponding overall cell density could not grow. The cells at high inoculum density were less sensitive to shear induced by mechanical agitation than the cells at low inoculum density. Taken together these observations support the existence of secreted aGF(s) by the hybridoma cell line used. Since the specific MAb production rate was independent of cultivation method and inoculum density, the existence of autocrine growth factors would suggest that the use of immobilized cells should improve the economics of MAb production.