Inhomogeneous background magnetic field in biological incubators is a potential confounder for experimental variability and reproducibility

This report shows that the background magnetic field in biological incubators can vary by orders of magnitude within and between incubators. These variations can be observed within the same incubator in locations that are centimeters apart from each other as well as between incubators that are identical and located in the same laboratory. Additionally, the values measured were frequently outside the range of magnitudes found naturally on the Earth's surface or ordinary habitation spaces. Exposure to such altered magnetic field environments has been experimentally shown to be sufficient to cause numerous effects in cell cultures. Examples of the effects reported span from differential generation of free radicals and heat shock proteins to differences in cellular proliferation, differentiation, and death. Although the effects are not well established and the molecular mechanism of action is currently under debate, these observations alone support the notion that the inhomogeneity of the background magnetic field in incubators is a potential confounding source of the variability and reproducibility for studies performed on cell cultures. In this regard, it is recommended that special measures be adopted to control the background magnetic fields in incubators when investigating the biological effects of exposure to magnetic fields of comparable characteristics as the ones measured in this study, or when studying small biological effects in general. Bioelectromagnetics 34:337–348, 2013. © 2012 Wiley Periodicals, Inc.     

Chromosomal aberrations in human amniotic cells after intermittent exposure to fifty hertz magnetic fields

Our recent studies have shown a significant increase in the frequency of chromosomal aberrations in human amniotic cells after exposure to a sinusoidal 50 Hz, 30 μT (rms) magnetic field. To evaluate further interactions between chromosomes and electromagnetic fields, we have analyzed the effects of intermittent exposure. Amniotic cells were exposed for 72 h to a 50 Hz, 30 μT (rms) magnetic field in a 15 s on and 15 s off fashion. Eight experiments with cells from different fetuses were performed. The results show a 4% mean frequency of aberrations among exposed cells compared to 2% in sham-exposed cells. The difference is statistically significant, with P < 0.05 both excluding and including gaps. In another series of eight experiments, the cells were exposed in the same way but with the field on for 2 s and off for 20 s. Also in these experiments a similar increase in the frequency of chromosomal aberrations was seen, but only when the analysis included gaps. Continuous exposure for 72 h to 300 μT, 50 Hz, did not increase the frequency of chromosomal aberrations. The background electromagnetic fields at different locations within the two incubators used was carefully checked and was nowhere found to exceed 120 nT. Likewise, the background level of chromosomal aberrations in cells cultured at different locations in the incubators showed no significant interculture differences. © 1994 Wiley-Liss, Inc.     

Piezoelectric and Electrokinetic Effects in Bone Tissue–Review

Power frequency magnetic fields can reach tens of μT in incubators used for in vitro fertilization. This article suggests that this can be one factor connected to the observed increase of congenitial defects in children born after such procedure.     

Effects of static magnetic fields at the cellular level

There have been few studies on the effects of static magnetic fields at the cellular level, compared to those of extremely low frequency magnetic fields. Past studies have shown that a static magnetic field alone does not have a lethal effect on the basic properties of cell growth and survival under normal culture conditions, regardless of the magnetic density. Most but not all studies have also suggested that a static magnetic field has no effect on changes in cell growth rate. It has also been shown that cell cycle distribution is not influenced by extremely strong static magnetic fields (up to a maximum of 10 T). A further area of interest is whether static magnetic fields cause DNA damage, which can be evaluated by determination of the frequency of micronucleus formation. The presence or absence of such micronuclei can confirm whether a particular treatment damages cellular DNA. This method has been used to confirm that a static magnetic field alone has no such effect. However, the frequency of micronucleus formation increases significantly when certain treatments (e.g., X-irradiation) are given prior to exposure to a 10 T static magnetic field. It has also been reported that treatment with trace amounts of ferrous ions in the cell culture medium and exposure to a static magnetic field increases DNA damage, which is detected using the comet assay. In addition, many studies have found a strong magnetic field that can induce orientation phenomena in cell culture.     

Effect of sinusoidal 50 Hz magnetic field on the testosterone production of mouse primary Leydig cell culture

This study evaluated the effect of sinusoidal 50 Hz magnetic field on the basal and human chorionic gonadotropin (hCG)-stimulated testosterone (T) production of 48-h mouse Leydig cell culture. The luteinizing hormone (LH) analog hCG was used to check the T response of the controls and to evaluate the possible effect of the applied magnetic field on the steroidogenic capacity of the exposed cells. Leydig cells were obtained from the testes of 35- to 45-g CFLP mice and isolated by mechanical dissociation without enzyme treatment. The cell cultures were exposed to sinusoidal 50 Hz 100 μT (root mean square) AC magnetic field during the entire time of a 48-h incubation. Testosterone content of the culture media was measured by radioimmunoassay. In cultures exposed to the magnetic field, a marked increase of basal T production was found (P < .05), compared with the unexposed controls, whereas no significant difference was seen between the exposed or unexposed cultures in the presence of maximally stimulating concentration of hCG. These findings demonstrate that sinusoidal 50 Hz 100 μT magnetic fields are able to stimulate the basal T production of primary mouse Leydig cell culture, leaving the steroidogenic responsiveness to hCG unaltered. Bioelectromagnetics 19:429–431, 1998. © 1998 Wiley-Liss, Inc.     

Recurrent Aspergillus contamination in a biomedical research facility: a case study

Fungal contamination of biomedical processes and facilities can result in major revenue loss and product delay. A biomedical research facility (BRF) culturing human cell lines experienced recurring fungal contamination of clean room incubators over a 3-year period. In 2010, as part of the plan to mitigate contamination, 20 fungal specimens were isolated by air and swab samples at various locations within the BRF. Aspergillus niger and Aspergillus fumigatus were isolated from several clean-room incubators. A. niger and A. fumigatus were identified using sequence comparison of the 18S rRNA gene. To determine whether the contaminant strains isolated in 2010 were the same as or different from strains isolated between 2007 and 2009, a novel forensic approach to random amplified polymorphic DNA (RAPD) PCR was used. The phylogenetic relationship among isolates showed two main genotypic clusters, and indicated the continual presence of the same A. fumigatus strain in the clean room since 2007. Biofilms can serve as chronic sources of contamination; visual inspection of plugs within the incubators revealed fungal biofilms. Moreover, confocal microscopy imaging of flow cell-grown biofilms demonstrated that the strains isolated from the incubators formed dense biofilms relative to other environmental isolates from the BRF. Lastly, the efficacies of various disinfectants employed at the BRF were examined for their ability to prevent spore germination. Overall, the investigation found that the use of rubber plugs around thermometers in the tissue culture incubators provided a microenvironment where A. fumigatus could survive regular surface disinfection. A general lesson from this case study is that the presence of microenvironments harboring contaminants can undermine decontamination procedures and serve as a source of recurrent contamination.     

Cell-cycle kinetics of friend erythroleukemia cells in a magnetically shielded room and in a low-frequency/low-intensity magnetic field

This work was undertaken to compare the behavior of Friend erythroleukemia cells in a solenoid, where the magnetic field was 70 μT at 50 Hz (plus 45 μT DC of Earth) with that of the same cells in a magnetically shielded room, where the magnetic field was attenuated to 20 nT DC and 2.5 pT AC. The control laboratory magnetic field corresponded to 45 μT DC and a stray 50 Hz field below 0.2 μT. The culture growth cycle of cells maintained inside the solenoid was slightly accelerated compared with that of cells maintained outside the solenoid (P < .05). This stimulation probably depended on sensitivity of cell cycle to a magnetic field, because, inside the solenoid, the percentage of G₁ cells slightly increased during the culture growth cycle, whereas that of S cells slightly decreased. Acceleration of growth was detected soon after exposure of the cultures to the solenoid field, and growth did not change further if the action of this field continued for a long time, accounting for adaptation. The solenoid field also caused a small increase of cell survival without influencing cell volume. By contrast, the culture growth cycle of cells maintained inside the magnetically shielded room was slightly decelerated compared with that of cells maintained outside the room (P < .05). The essential absence of any field inside the magnetically shielded room also caused a small increase of cell volume, whereas, during the culture growth cycle, the percentage of G₁ cells decreased, and that of S cells increased. The majority of these events did not change in cells induced to differentiate hemoglobin through dimethylsulfoxide. Bioelectromagnetics 18:58–66, 1997. © 1997 Wiley-Liss, Inc.     

磁场对红酵母的生长及类胡萝卜素合成的影响

考察直流电磁场对类胡萝卜素产生前红酵母RG-98的生长及代谢的影响。结果表明,不同的磁场强度、处理时间及处理方式会产生不同的生物效应。将液体种子和发酵培养基一起置于0.10T电磁场处理2h,能使色素的发酵产量提高25％。比较经磁场处理与未经处理的发酵过程,发现磁场处理提高了红酵母对糖的代谢速率,并且在发酵后期促进生物量和色素含量持续地增加。     

ELF magnetic field exposure in a neonatal intensive care unit

In this paper, the magnetic flux density (MFD) distribution in a neonatal intensive care unit is described and MFD values inside a few open infant warming systems and incubators are reported. Typical measured values of the magnetic flux density at power frequency (50 Hz) in the "general environment" (the rooms of the unit) were lower than 0.2 microT, while higher MFD values were detected close to medical equipment and inside the open infant warming systems. In both cases, the magnetic flux density quickly decreases with increasing distance, so that measured values are reduced to "background" (i.e., general environment) levels 20-30 cm away from the sources. The total harmonic content over the 100-800 Hz frequency range was also evaluated. In the general environment, measured values in this band were negligible, while this was not the case close to medical equipment. Field levels inside the open and closed incubators depend on the position of the electronic control system, of the heating power generator and its winding conductor, and of the 220 V main plug. The magnetic flux density was also monitored for a prolonged period of time in a few types of open infant warming systems and incubators under standard intensive care unit operation with premature newborn present.     

Magnetic field direction differentially impacts the growth of different cell types

Magnetic resonance imaging (MRI) machines have horizontal or upright static magnetic field (SMF) of 0.1–3 T (Tesla) at sites of patients and operators, but the biological effects of these SMFs still remain elusive. We examined 12 different cell lines, including 5 human solid tumor cell lines, 2 human leukemia cell lines and 4 human non-cancer cell lines, as well as the Chinese hamster ovary cell line. Permanent magnets were used to provide 0.2–1 T SMFs with different magnetic field directions. We found that an upward magnetic field of 0.2–1 T could effectively reduce the cell numbers of all human solid tumor cell lines we tested, but a downward magnetic field mostly had no statistically significant effect. However, the leukemia cells in suspension, which do not have shape-induced anisotropy, were inhibited by both upward and downward magnetic fields. In contrast, the cell numbers of most non-cancer cells were not affected by magnetic fields of all directions. Moreover, the upward magnetic field inhibited GIST-T1 tumor growth in nude mice by 19.3% (p < 0.05) while the downward magnetic field did not produce significant effect. In conclusion, although still lack of mechanistical insights, our results show that different magnetic field directions produce divergent effects on cancer cell numbers as well as tumor growth in mice. This not only verified the safety of SMF exposure related to current MRI machines but also revealed the possible antitumor potential of magnetic field with an upward direction.     

Design and validation of a pulsatile perfusion bioreactor for 3D high cell density cultures

This study presents the design and validation of a pulsatile flow perfusion bioreactor able to provide a suitable environment for 3D high cell density cultures for tissue engineering applications. Our bioreactor system is mobile, does not require the use of traditional cell culture incubators and is easy to sterilize. It provides real‐time monitoring and stable control of pH, dissolved oxygen concentration, temperature, pressure, pulsation frequency, and flow rate. In this bioreactor system, cells are cultured in a gel within a chamber perfused by a culture medium fed by hollow fibers. Human umbilical vein endothelial cells (HUVEC) suspended in fibrin were found to be living, making connections and proliferating up to five to six times their initial seeding number after a 48‐h culture period. Cells were uniformly dispersed within the 14.40 mm × 17.46 mm × 6.35 mm chamber. Cells suspended in 6.35‐mm thick gels and cultured in a traditional CO₂ incubator were found to be round and dead. In control experiments carried out in a traditional cell culture incubator, the scarcely found living cells were mostly on top of the gels, while cells cultured under perfusion bioreactor conditions were found to be alive and uniformly distributed across the gel. Biotechnol. Bioeng. 2009; 104: 1215–1223. © 2009 Wiley Periodicals, Inc.     

Spatial gradient effects of 120 mT static magnetic field on endothelial tubular formation in vitro

This study investigated the spatial magnetic gradient effects of static magnetic fields (SMF) on endothelial tubular formation by applying the maximum spatial gradient to a target site of culture wells for cell growth. The respective maximum values of magnetic flux density (B(max)), magnetic flux gradient (G(max)) and the magnetic force product of the magnetic flux density and its gradient (a parameter of magnetic force) were 120 mT, 28 mT/mm and 1428 mT(2)/mm. The effects of gradient SMF on tubular formation were compared with those of uniform SMF that has no spatial gradients on the entire bottom area of culture wells. Five experimental groups of 25 samples each were examined: (1) sham exposure (control); (2) peak gradient exposure in the peripheral part; (3) peak gradient exposure in the central part; (4) uniform exposure to 20 mT; (5) uniform exposure to 120 mT. The SMF or sham exposure was carried out for 10 days. Photomicrographs of tubular cells, immunostained with an anti-platelet-endothelial cell adhesion molecule-1 (PECAM-1 [CD31]) antibody as a pan-endothelial marker, were analyzed after the 10-day culture. Gradient SMF in the peripheral or central part was found to significantly promote tubular formation in terms of the area density and length of tubules in each peak gradient/force part of the wells, compared with the sham exposure. In contrast, uniform SMF did not induce any significant change in the tubular formation. These findings suggest that tubule formation can be promoted by applying the peak gradient/force to a target site of culture wells.     

Inexpensive low-oxygen incubators

Although the evidence is overwhelming that ambient oxygen is at least somewhat damaging to most normal cells in culture, the expense and effort involved has resulted in few laboratories growing their cells under physiological oxygen conditions. We here describe how to produce, from commercially available plastic wide-mouth jars, very simple gas-tight containers that can be flushed with prepared gas mixtures to produce low-oxygen environments for standard cell culture. This permits any laboratory to easily try the effects of physiological oxygen on their system without the need for dedicated incubators and substantial monetary investments.     

0.2 T magnetic field inhibits angiogenesis in chick embryo chorioallantoic membrane

Inhibition of angiogenesis is a major target in the fight against cancer and other diseases. Although the effects of static magnetic fields on cancer development and cell growth have been investigated, effects on angiogenesis have received no attention so far. In this study we report the effects on angiogenesis of exposure to 0.2 T static magnetic field. Angiogenesis was analyzed using the chick embryo chorioallantoic membrane assay. Exposure to 0.2 T static magnetic field was achieved by placing the eggs for 3 hr in the isocentre of the magnet of a sectorial magnetic resonance tomograph used in clinical practice. In sham exposed specimens treated with phosphate buffered saline (negative control), no significant vascular reaction was detectable; 3 hr exposure to 0.2 T static magnetic field did not affect the basal pattern of vascularization or chick embryo viability. Prostaglandin E1 and fetal calf serum elicited a strong angiogenic response in sham exposed eggs. This angiogenic response was significantly inhibited by 3 hr exposure to 0.2 T static magnetic field. These findings point to possible use of static magnetic field in inhibiting angiogenesis; this effect could be exploited for treatment of cancer and other diseases where excessive angiogenesis is involved.     

Extremely low frequency (ELF) stray magnetic fields of laboratory equipment: a possible co-exposure conducting experiments on cell cultures

Abstract

Measurements of extremely low frequency (ELF) magnetic fields were conducted in the environment of commercial laboratory equipment in order to evaluate the possible co-exposure during the experimental processes on cell cultures. Three types of device were evaluated: a cell culture CO₂ incubator, a thermostatic water bath and a laboratory shaker table. These devices usually have electric motors, heating wires and electronic control systems, therefore may expose the cell cultures to undesirable ELF stray magnetic fields. Spatial distributions of magnetic field time domain signal waveform and frequency spectral analysis (FFT) were processed. Long- and short-term variation of stray magnetic field was also evaluated under normal use of investigated laboratory devices. The results show that the equipment under test may add a considerable ELF magnetic field to the ambient environmental magnetic field or to the intentional exposure to ELF, RF or other physical/chemical agents. The maximum stray magnetic fields were higher than 3?µT, 20?µT and 75?µT in the CO₂ incubator, in water bath and on the laboratory shaker table, respectively, with high variation of spatial distribution and time domain. Our investigation emphasizes possible confounding factors conducting cell culture studies related to low-level ELF-EMF exposure due to the existing stray magnetic fields in the ambient environment of laboratory equipment.     

Validation of a new live cell strain system: characterization of plasma membrane stress failure.

Motivated by our interest in lung deformation injury, we report on the validation of a new live cell strain system. We showed that the system maintains a cell culture environment equivalent to that provided by conventional incubators and that its strain ouput was uniform and reproducible. With this system, we defined cell deformation dose (i.e., membrane strain amplitude)-cell injury response relationships in alveolar epithelial cultures and studied the effects of temperature on them. Deformation injury occurred in the form of reversible, nonlethal plasma membrane stress failure events and was quantified as the fraction of cells with uptake and retention of fluorescein-labeled dextran (FITC-Dx). The undeformed control population showed virtually no FITC-Dx uptake at any temperature, which was also true for cells strained by 3%. However, when the membrane strain was increased to 18%, ~5% of cells experienced deformation injury at a temperature of 37 degrees C. Moreover, at that strain, a reduction in temperature to 4 degrees C resulted in a threefold increase in the number of cells with plasma membrane breaks (from 4.8 to 15.9%; P < 0.05). Cooling of cells to 4 degrees C also lowered the strain threshold at which deformation injury was first seen. That is, at a 9% substratum strain, cooling to 4 degrees C resulted in a 10-fold increase in the number of cells with FITC-Dx staining (0.7 vs. 7.5%, P < 0.05). At that temperature, A549 cells offered a 50% higher resistance to shape change (magnetic twisting cytometry measurements) than at 37 degrees C. We conclude that the strain-injury threshold of A549 cells is reduced at low temperatures, and we consider temperature effects on plasma-membrane fluidity, cytoskeletal stiffness, and lipid trafficking as responsible mechanisms.     

Cortex reorganization of <Emphasis Type="Italic">Xenopus laevis</Emphasis>eggs in strong static magnetic fields

Observations of magnetic field effects on biological systems have often been contradictory. For amphibian eggs, a review of the available literature suggests that part of the discrepancies might be resolved by considering a previously neglected parameter for morphological alterations induced by magnetic fields – the jelly layers that normally surround the egg and are often removed in laboratory studies for easier cell handling. To experimentally test this hypothesis, we observed the morphology of fertilizable Xenopus laevis eggs with and without jelly coat that were subjected to static magnetic fields of up to 9.4 T for different periods of time. A complex reorganization of cortical pigmentation was found in dejellied eggs as a function of the magnetic field and the field exposure time. Initial pigment rearrangements could be observed at about 0.5 T, and less than 3 T are required for the effects to fully develop within two hours. No effect was observed when the jelly layers of the eggs were left intact. These results suggest that the action of magnetic fields might involve cortical pigments or associated cytoskeletal structures normally held in place by the jelly layers and that the presence of the jelly layer should indeed be included in further studies of magnetic field effects in this system.     

Cleavage and survival of Xenopus embryos exposed to 8 T static magnetic fields in a rotating clinostat

In this study, we examined cleavage and survival of fertilized Xenopus embryos exposed to 8 T static magnetic fields (SMFs). We investigated fertilized Xenopus embryos exposed to magnetic field either in static chamber or in a rotating culture system. Our results showed that the exposure to the strong magnetic field of 8 T changed the third cleavage furrow from the usual horizontal one to a perpendicular one; however, when the direction of gravity was randomized by exposing embryos to magnetic field in a rotating culture system, the third cleavage furrow were formed horizontally, a finding which suggests that the observed distortion of the third cleavage furrow in magnetism-exposed embryos was accomplished by altering gravity effects which were elicited by diamagnetic force due to high gradient magnetic field. Our results also showed that the exposure to the strong magnetic field did not damage survival. These results demonstrate that SMF and altering gravity cause distortion of the third cleavage furrow and show that effects of exposing cleavage embryos to magnetic field were transient and did not affect the post-cleavage development. We also showed that strong magnetic field is not hazardous to the cleavage and blastula-gastrula transition of developing embryonic cells.     

Culture of human T cells in stirred bioreactors for cellular immunotherapy applications: shear, proliferation, and the IL-2 receptor

Ex vivo expansion of T cells is a key step of many cellular immunotherapy protocols, which require large numbers of immune cells to eradicate malignant or virally infected cells. The use of stirred culture systems for T cell expansion offers many potential advantages over the static culture systems commonly used today, including homogeneity of culture conditions, ease of sampling, and implementation of control systems. Primary human T cells as well as the transformed TALL103/2 T cell line were cultured in 100-mL spinner flasks as well as 2-L bioreactors to investigate the effects of shear forces produced by agitation and sparging-based aeration on the expansion of T cells. Primary T cells could be successfully grown at agitation rates of up to 120 rpm in the spinner flasks and to 180 rpm in the bioreactors with no immediate detrimental effects on proliferation. Exposure to agitation and sparging did, however, cause a significantly increased rate of downregulation of the interleukin-2 receptor (IL-2R), resulting in lower overall expansion potential from a single stimulation as compared to static controls, with faster IL-2R downregulation occurring at higher agitation rates. For the primary T cells, no significant effects of agitation were found on expression levels of other key surface receptors (CD3, CD28, or CD62L) examined. No significant effects of agitation were observed on primary T cell metabolism or levels of cellular apoptosis in the cultures. The TALL103/2 T cell line was found to be extremely sensitive to agitation, showing severely reduced growth at speeds above 30 rpm in 100-mL spinner flasks. This unexpected increased fragility in the transformed T cell line as compared to primary T cells points out the importance of carefully selecting a model cell line which will accurately represent the characteristics of the cell system of interest.