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ELECTRO- AND MAGNETOBIOLOGY, 20(3), 263280 (2001)

MAGNETIC FIELD STIMULATION OF HYBRIDOMA CELLS IN VITRO


Debatosh Datta,* Prabuddha K. Kundu, and Atul Deshpande Cellular Engineering Laboratory, School of Biomedical Engineering, Indian Institute of Technology-Bombay, Powai, Bombay 400 076, India

ABSTRACT
Non-ionizing physical eld interactions with cells, both in situ and in vitro, is of current interest globally. This is from various directionsstarting from their abilities to induce permanent modications in cell behavior in situ, through carcinogenesis and mutagenesis, to utilizing eld effects for possibly enhancing the viable cell population in vitro. This results in parallel increase in some high-value, low-volume biochemical production. In the present study, screening experiments were carried out with a unique cell linehybridoma (OKT3) (secreting monoclonal antibodies [MAbs] against T3 surface antigens of human peripheral CD4 cells)for a possible enhancement in the yield of extremely high value product (MAb). Overall, in the absence of any such data globally, there is apparently an urgent need for screening of such eld effects on various other cell types in vitro for various reasons; e.g., low cost of manipulation, nonpolluting nature of interactions, distinct possibility of enhancement of produced biochemical titers, etc. In the present study, we observed various responses of the cell population both to magnetic elds alone and in combination with other known chemical stimulants of viable biomass (mono- and poly-lysine). Fifty hertz, 0.8 mT magnetic eld and below, in conjunction with bulkier poly-lysine molecules, needs to be investigated further for a possible resonance-induced anti-interaction between these known mitogens and their cell surface receptors, which possibly could be extrapolated
* Corresponding author. E-mail: ddatta@cc.iitb.ernet.in Current address: Bio-Rad Laboratories (I) Ltd., New Delhi, India

263 Copyright 2001 by Marcel Dekker, Inc. www.dekker.com

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to other growth factor-receptor interactions in magnetic eld environments, in situ. Key Words: Antibody titer; Hybridoma cells; Magnetic eld

INTRODUCTION Scarcity of research done on the biological effects of magnetic elds (MFs) stands in sharp contrast to the abundance of bioelectric effects (1). There are several reasons. First, living systems are operating in an aqueous environment in which ions and polyelectrolytes play major functional roles. With the exception of bird orientation and navigation and bacterial geotactic responses, biomagnetic effects have not been known to play any major physiological roles. Second, interactions of the Earths MF (about 0.5 G or 50 T) with biomolecules or tissues approach the energy level of the inherent thermal energy. Third, there are inherent difculties in investigating biomagnetic effects. Many of the earlier experiments on MF effects in living tissue or organisms were not reproducible. However, with the recent increase in electrical gadgets and appliances, and their ubiquitous presence around us, the induced surrounding MFs in our environment have brought about attention to possible health hazards, especially the link between exposure to such radiation and the incidence of cancer (2). Moreover, recent studies have shown that the environmental effects originate primarily from the magnetic component of the electromagnetic eld (EMF) (1). Therefore, there has been a urry of research activity in this area, though the results have been far from conclusive. This is not to belie the fact that there is some concrete evidence as to the effects, but the clinching hypothesis is yet to be determined. The effects include healing of fractured bones (3), skin regeneration of experimental animals (4), increase in the permeability of cell membranes (5), effects on cellular metabolism (6), increase in Na K ATPase activity (7), and enhancement of transcription and translation (8,9). Two decades have elapsed since the rst chronically unhealed fracture (nonunion) patient was treated successfully at Columbia with time-varying magnetic elds (3). In the interval since then, more than a quarter of a million such patients have been treated with this method throughout the world. Success rates and time to bony union are comparable to those produced by bone grafting. Unlike surgical repair, EMFs carry no known risks (tangible), cause no discomfort, and are considerably more cost effective. The cellular effects of appropriately congured EMFs address specic aspects of the pathology underlying a fracture nonunion. These include modications of cellular calcium metabolism and mineralization (10,11), synthesis of the extracellular matrix (1214), and new blood vessel formation (angiogenesis) (15). Interest in the cellular effects of EMFs originated from research in nucleated red blood cells (16). Available data point toward the presence of two general

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features, among others. First, a relationship exists between the electrical and magnetic perturbation and the redistribution of rst messengers (e.g., Ca2 ions) on the cell surface and/or their inux into a cell. Second, a consequent global process of chromatin decondensation and nuclear volume increase occurs, as an early reactivation step, before the onset of protein, RNA, and DNA syntheses (17). Forgacs et al. (18), Antonopoulos et al. (19), and Santoro et al. (20) studied the cytological effects of low frequency (LF) 50-Hz EMFs on animal and human cells in vitro. On the other end of the spectrum, Bras et al. (21) studied the orientational behavior of microtubules assembled in the presence of strong MFs, and Norimura et al. (22) undertook experiments in order to verify whether a strong MF would have any biological effects on the cell growth, viability, and radiation response of mammalian cells. Results suggest that exposure to a static MF of 4 T or stronger may lead to physiological and growth abnormalities at the cellular level. Similarly, Zhang et al. (23) reported that exposure to static MFs of 6.34 T coupled with ionizing radiation decreased cell survival, whereas elds of 0.3 and 0.7 T had no effect on cell survival. They concluded that (i) MFs decreased the colony-forming abilities of cultured mammalian cells; (ii) MFs could affect the cell cycle; (iii) a stronger MF strength does not always have stronger biological effects; and (iv) the gradient of an MF may be an important factor when combined with ionizing radiation. Raylman et al. (24) also reported that strong static MFs inhibit the growth of cancer cells in vitro. This has immense application to the understanding and treatment of cancers. The presence of a magneto-sensor system, e.g., proteins, must be considered when looking into the plethora of molecular-level phenomena taking place. Even though the sensor remains elusive, the pathway for the events may be correlated to the increase in Ca2 ion concentration as a second messenger, leading to elevation of transcription and translation (1). Kim et al. (25) suggested that the mechanism of biological effects of extremely low frequency (ELF) electric and MFs may involve induced changes of Ca2 transport through plasma membrane ion channels. Results demonstrate that low-intensity electric elds can alter calcium distribution in cells, most probably due to the effect on receptor-operated Ca2 and/or ion channels. Once EMFs have acted upon key eventsmost of which occur within 6 hr from binding of the mitogen to membraneall processes related to the cell proliferation, such as RNA synthesis, gene expression, and subsequent DNA synthesis (i.e., late events), are affected. Data and physical modeling strongly support the hypothesis that biological and physical windows must exist for effects to occur. Reproducibility of results depends on the extent to which the variables are considered. Exposure conditions, as well as the type and physiological status of the cell to be exposed to EMFs, are important. Despite strong and reproducible effects on cell proliferation, low-intensity EMFs seem not to have any genotoxic effects. Later research, however, has proposed that exposure of cells to 60-Hz elds does not initiate tumor development, but rather may promote the process

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once initiated. This view is supported by studies that show that exposure to EMFs accelerates tumorigenesis in experimental animals exposed to carcinogens (26,27). The effects of EMFs on translational activity are even more dramatic. The remarkable feature is the observation that the molecular weight distribution of polypeptides synthesized during EMF exposure and sudden elevated heat shock proteins (e.g., hsp 70) are virtually identical. The fact that some heat shock proteins are affected by exposure to EMFs may provide another model for determining the mechanism at the cellular level (9). This also indicates that exposure to EMFs increases the transcription rate of certain proteins relative to the unexposed population, and that their amino acid composition is altered due to this. Our present work deals with low-frequency magnetic eld (LFMF) exposure to hybridoma cells. There is little research reported on the effects of magnetic eld exposure to hybridoma cells, and its subsequent effect on cellular growth and secretion. Hybridoma cells secrete monoclonal antibodies (MAbs) which constitute the heart of all in vitro diagnostic kits. In addition, the molecules are widely used for in vivo diagnostic imaging and drug delivery maneuvers. One of the major drawbacks of these molecules widespread usage is the prohibitive cost. In the current work, physical (EMF) stimulation of hybridoma cells as a means of MAb production enhancement was studied. It was earlier observed by Datta et al. (28) that LFMF has a denite role in hybridoma growth and secretion. Thus, in the present work, we probed whether exposure to LFMF in the presence or absence of chemical growth stimulants, e.g., l-lysine (29) and homopolymers of l-lysine of varying molecular weights (10007500), could minimize the growth phase and maximize secretion by the cells in culture, thereby enhancing efciency of the production cycle. MATERIALS AND METHODS Cell Line, Media, and Additives The cell line used for screening was OKT3, a mousemouse hybridoma, secreting MAb (IgG) against CD3 peripheral molecule of a T cell (NCCS, Pune, India). The OKT3 cell line was maintained in Iscoves modication of Dulbeccos media (IMDM) (Sigma Chemical Co., St. Louis, MO) supplemented with 0.044 M NaHCO3, 0.025 M glucose, 40 units/ml penicillin G, 400 g/ml streptomycin sulfate, 0.002 ml/ml gentamycin sulfate (v/v), and 400 g/ml mycostatin. A 10% fetal calf serum (FCS, Gibco BRL, Grand Island, NY) supplement was added to the culture. The cells were then cultured in 5% CO2 (v/v) atmosphere in an incubator (Jouan, France) at 37C and 100% relative humidity. The experimental population of cells was cultured inside a magnetic coil, excited by a current. After the predetermined time of exposure to an MF, the culture was harvested and assays were conducted. The main equipment comprised a coil, current amplier with dual DC voltage supply, and function generators (ElectroLab, India). The current was delivered by applying a voltage as deter-

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mined by the required MF, for a mean time of 6.5 hr daily. An oscilloscope (Hewlett Packard, Palo Alto, CA) was used to determine the voltage (Vp-p and Vrms) and frequency of the signal across the coil. The axial magnetic eld was measured by a gaussmeter (Bell Technologies Inc., Orlando, FL). Coil for Generation of an MF An air core coil (solenoid) was fabricated on a polyvinyl chloride (PVC) pipe of length 0.30 m and 0.136 m internal diameter. Four layers of wire were wound on the PVC tubing and all were connected in parallel to obtain an adequate magnetic eld. The four layers (separated from each other by presban paper) were enamel-coated copper wires. Of the four wires, two were 33 SWG with a diameter of 0.254 mm (current capacity 94 mA) and with 35.1 turns/cm. The remaining two were 36 SWG (0.193 mm diameter, 54 mA current capacity) and had 45.70 turns/cm. Resistance of each layer of the coil was 135, 144, 300, and 326 ohm. All four layers were connected in parallel so that the total resistance was 50.2 , and the net current carrying capacity of the coil became 300 mA. The eld strength on the axis of the coil (the length of which was 2.5 times its diameter) was observed to be fairly constant in a segment of 0.13 m (sufcient for exposing a 24-well plate to uniform MF). After the circuit was set up and the coil was activated with the proper signal, the MF inside the coil was measured by a gaussmeter tted with the Hall probe. After standardizing the probe, it was inserted into the coil for measurement of the MF. The eld was found to be stable over 0.13 m with an input voltage of 32.5 V (Fig. 1). The coil was swabbed with hot water/dettol mixture and absolute alcohol each time before work was started.

Figure 1. MF strength along x axis which shows stable generation of MF along the middle region of the coil with an input voltage of 32.5 V.

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The MF Parameters and Protocol for Screening The cells were cultured in 24-well culture plates (Flow Labs, U.S.A.) in 5% CO2 atmosphere and 37C in a water-jacketed incubator (Jouan, France). The experimental plate was exposed to the predetermined MF for 67 hr and the other plate was kept as control. In this set of experiments, physical stimulation was studied in isolation as well as in various combinations with l-lysine, mono- and poly-mediated chemical stimulations. The objective of such physical and physicochemical stimulations was to get higher viable cell counts and product concentrations.

Analytical Methods Viable cell counts were made using a cell counting chamber (Rohem, India) and the trypan blue dye (0.4%) exclusion technique, and the stimulation index (SI) was calculated. The SI is dened as the ratio of population of the experimental cells to the population of control cells at a given time. Because it is a ratio of the same physical parameters, it does not have any units. It gives an insight into the effect (stimulatory or repressive) that a molecule has over the cells under experimentation versus the control population. SI Experimental(T) Control(T)

The culture media were isolated, centrifuged at 1000 rpm, and the supernatants stored at 20C for further analyses. Culture supernatants were analyzed qualitatively and quantitatively for identication of changes in the metabolite patterns and secretion prole of the cell line by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), enzyme-linked immunosorbent assay (ELISA), and fast protein liquid chromatography (FPLC). The percentage of gel used was 8%, the volume of sample loaded in each lane was in the range 1015 l. The current applied during the passage of samples through stacking gel was 15 mA and that during the running gel was 20 mA. The gels were stained with Coomassie brilliant blue. FPLC (Pharmacia, Sweden) parameters were as follows: 1 MPa pressure at a ow rate of 0.4 ml/min. Absorbance of the eluted volume was screened (280 nm) in a UV spectrophotometer. The column used was Superose-12 (Pharmacia, Sweden) and the volume loaded was 100 l. Antibody titer was measured by direct ELISA technique for the detection and quantitation of monoclonal antibody: 50 l samples from OKT3 supernatants were incubated in separate wells of the microtiter plate at 4C overnight. Washings were done with phosphate buffer saline (PBS)Tween-20, and then 0.2% bovine serum albumin (w/v) was added to the wells and incubated at 37C for 2 hr. Washing was again done with PBSTween-20 to remove unbound components.

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Anti-mouserabbit antibody conjugated with enzyme alkaline phosphatase was added to the wells and incubated at 37C for 1.5 hr. Any nonspecic binding was removed by washing with PBSTween-20 for ve to six times, and substrate solution (p-nitrophenol phosphate) was added to each well, followed by incubation at 37C for 10 min. Control samples of known IgG content were assayed simultaneously. Absorbance was taken at 405 nm. RESULTS AND DISCUSSION Growth curves derived from viable cell counts of the exposed cells from all studied parameters showed signicant differences in their growth patterns versus the controls. Study of secretory patterns also showed an elevated production of MAb by the exposed population. However, the synergistic effect of both chemical and physical stimulation was not very evident in the experiments, but we did get some interesting results. All of the exposed cell populations were exposed to mean times of 6 hr, 34 min7 hr. With exposure to 76 Hz and 0.8 mT MF, the exposed population of cells has a denite growth advantage over the control population, with the MF llysine (7 g/ml) population peaking over the MF-exposed population after 24 hrs (Fig. 2). The SI for MF lysine was 2.23 and for MF alone was 1.84. PAGE, ELISA, and FPLC plots conrm the elevated secretory status of MF l-lysine over both MF and control (Fig. 3, Table 1, Fig. 4a). With exposure to 90 Hz and 0.6 mT MF, though the exposed cell populations had a higher cell density than the controls, there was not much of a difference between the two exposed populations, i.e., the one containing l-lysine (7 g/ml) and the other. Though the MF-exposed population had a higher viable count at 72 hr, they more or less converged at 96 hr (Fig. 5). The SI values for the

Figure 2. Growth prole of OKT3 cells cultured in complete IMDM/10% FCS supplemented with monomeric l-lysine (7 g/ml) in presence of 76 Hz, 0.8 mT magnetic eld.

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Figure 3. Eight percent SDS-PAGE of the spent media (supernatants) of OKT3 cells grown in MF (76 Hz, 0.8 mT and 90 Hz, 0.6 mT) in complete IMDM for 96 hr: lane (2) MF (76 Hz, 0.8 mT); lane (3) MF (76 Hz, 0.8 mT) l-lysine (7 g/ml); lane (4) MF (90 Hz, 0.6 mT); lane (5) MF (90 Hz, 0.6 mT) l-lysine (7 g/ml).

Table 1.
Cell Line for Exposure
OKT3 OKT3 OKT3 OKT3 OKT3

Experimentation Protocol for Studying OKT3 Cells Under MF and Added Lysine
Magnetic Field Strength (mT)
0.8 0.6 0.8 0.8 0.8

Frequency of Exposure (Hz)


76 90 50 35 15

Voltage Required (Vrms)


12.90 10.20 12.20 11.51 11.16

Mean Time of Exposure (hr, min)


6, 6, 6, 7, 6, 34 30 38 20 49

In Presence and Absence of lLysine (7 g/ml)

OKT3 cells were incubated in the presence and absence of LFMF and l-lysine HCl in 24-well culture plate in complete IMDM/10% FCS/4% antibiotics. Cell counts were taken daily, and PAGE, ELISA, and FPLC analyses of the supernatants were carried out for product characterization and quantication. Seeding densities varied from 1 104 to 4 104 cells/ml in different sets of experiments.

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Figure 4. FPLC analyses of spent media of OKT3 cells cultured in the presence of MF. A 100 l sample was loaded on Superose 12 prepacked 10/30 gel-ltration column and eluted with 0.1 M phosphate buffer, pH 7.4, at a ow rate of 0.4 ml/min and UV sensitivity of 00.1. (Each division corresponds to 4 ml.)

MF lysine were 1.96 and for MF 1.72. This was denitely less than in the previous experiment. ELISA and FPLC patterns showed that the MF-exposed cell population had a marginally higher secretory status than the MF l-lysine population (Fig. 3, Table 1, Fig. 4b) (lysine-induced cellular growth could be one of the reasons for this lowered secretory response, because the MAb secretion process is

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Figure 5. Growth prole of OKT3 cells cultured in complete IMDM/10% FCS supplemented with monomeric l-lysine (7 g/ml) in presence of 90 Hz, 0.6 mT magnetic eld.

a non-growth-associated phenomenon). This probably indicates also that higher frequencies do not support promotion of cellular growth (Fig. 5). It can possibly be concluded, with respect to the above coupled experiments, that MF coupled with l-lysine had a positive effect on cellular growth, though it lowered functional secretory status. With exposure to 50 Hz and 0.8 mT MF, the exposed cell populations denitely had a higher population, but the order was totally reversed. Here, it was observed that MF-exposed cells were of a higher density than MF l-lysine cells (Fig. 6). This is in contrast to the earlier observations, where the physicochemically stimulated population had attained a higher population than the MFexposed population. At 48 hr, SI was 1.43 for the MF-exposed population and

Figure 6. Repeat growth prole of OKT3 cells cultured in complete IMDM/10% FCS supplemented with mono-l-lysine (7 g/ml) in the presence of 50 Hz, 0.8 mT MF and nonexposed cells. OKT3 cells were also cultured in l-lysine to observe native chemical stimulation.

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Figure 7. Eight percent SDS-PAGE of the spent media (supernatants) of OKT3 cells grown in MF (50 Hz, 0.8 mT) in complete IMDM: lane (1) control (OKT3 cells IMDM) 72 hr; lane (2) OKT3 cells IMDM l-lysine (7 g/ml) 72 hr; lane (3) MF (50 Hz, 0.8 mT) 72 hr; lane (4) MF (50 Hz, 0.8 mT) l-lysine (7 g/ml) 72 hr; lane (5) control (OKT3 cells IMDM) 96 hr; lane (6) OKT3 cells IMDM l-lysine (7 g/ml) 96 hr; lane (7) MF (50 Hz, 0.8 mT) 96 hr; lane (8) MF (50 Hz, 0.8 mT) l-lysine (7 g/ml) 96 hr.

1.33 for MF l-lysine population, whereas at 96 hr, we had 1.72 for MF exposed cells vs. 1.56 for MF l-lysine population. This itself was quite remarkable, in that at 96 hr, in 2 ml of conned media, the cells were still being activated and stimulated compared to the control population, which was already on the decline. This is consistent with the reported data that 5060 Hz is optimal for stimulation of cells (though no data are yet available for hybridoma cultures). The MF cell population was signicantly more elevated than the MF l-lysine population ( 0.05). This either indicates that the combination of MF and llysine is not costimulatory at 50 Hz, 0.8 mT or mutually repressive, with the MFalone population being far more enhanced than the coupled population, whereas we observed that at 76 Hz, 0.8 mT, the effect was costimulatory, both in terms of cell population and product concentration. The obvious questions became, why was the physical and chemical stimulation not synergistic for 50 Hz, 0.8 mT MF, as was observed for the other elds, and second, as compared to lysine in absence of magnetic eld, why did MF and l-lysine MF give a very low cell growth pattern? (Fig. 6). Testing at lower frequencies than 50 Hz at 0.8 mT was performed to screen if the phenomenon was unique or if 50 Hz acted as a cut-off point. SDS-PAGE (Fig. 7), ELISA, and FPLC data (Fig. 4c) were analyzed, and interesting observations were obtained which showed that the secretory pattern was affected. ELISA results showed that MF, though giving a higher cell count, did not have greater MAb productivity compared to MF l-lysine (Table 1). FPLC data corroborated this observation.

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With exposure to MF of 35 Hz, 0.8 mT, cell growth patterns followed the previous trend, where the MF l-lysine had a higher cell count compared to only l-lysine and only MF, though at 24 hr, l-lysine and MF l-lysine had exactly the same counts (Fig. 8). But at 48 hr, MF l-lysine had a higher cell count compared to MF and only l-lysine (Figs. 8 and 9). The SI values at 48 hr also verify this fact: for MF l-lysine, SI was 1.786; for l-lysine, SI was 1.571; and for MF, SI was 1.429. Note that at 72 hr, the MF cell population overtook the other two and peaked at 96 hr over the rest. This could be an artifact of experimentation or a real trend. SDS-PAGE analysis did not reveal signicant changes. ELISA values showed that l-lysine had the highest values, followed by MF l-lysine and MF. Here it was seen that the MF and l-lysine acted in synergy where production of MAb was concerned, though cell counts may have suggested otherwise (Table 1). For 15 Hz, 0.8 mT MF, we observed that MF l-lysine had a higher cell count than l-lysine itself at 48 hr, though this was not statistically signicant (Figs. 10 and 11). On the other hand, the MF cell population did not show any stimulation (rather, after 24 hr, the SI values were less than 1, which is indicative of lesser growth than the control population). Though again, this was found to be statistically insignicant. The SI values for l-lysine and MF l-lysine at 24 hr were the same, and at 72 hr nearly the same. At 96 hr, the SI value of l-lysine (1.4) was higher than that of MF l-lysine (1.1). SDS-PAGE did not show any signicant changes in pattern, whereas in ELISA (Table 1), the quantitative antibody values are comparatively on the lower side because the cells were not being stimulated. This was corroborated by FPLC (Fig. 4). Thus, we might deduce that

Figure 8. Growth prole of OKT3 cells cultured in complete IMDM/10% FCS supplemented with monomeric l-lysine (7 g/ml) in presence of 35 Hz, 0.8 mT magnetic eld. l-lysine was run to observe for chemical stimulation.

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Figure 9. Eight percent SDS-PAGE of the spent media (supernatants) of OKT3 cells grown in MF (35 Hz, 0.8 mT) in complete IMDM: lane (1) control (OKT3 cells IMDM) 72 hr; lane (2) OKT3 cells IMDM l-lysine (7 g/ml) 72 hr; lane (3) MF (35 Hz, 0.8 mT) 72 hr; lane (4) MF (35 Hz, 0.8 mT) l-lysine (7 g/ml) 72 hrs; lane (5) control (OKT3 cells IMDM) 96 hr; lane (6) OKT3 cells IMDM l-lysine (7 g/ml) 96 hr; lane (7) MF (35 Hz, 0.8 mT) 96 hr; lane (8) MF (35 Hz, 0.8 mT) l-lysine (7 g/ml) 96 hr.

at 15 Hz, the MF effect ceased to act synergistically in OKT3 hybridoma cells. Whether the same can be said about other cells remains to be seen. As the scanning of the frequencies was carried out, the most interesting results were obtained at 50 Hz, 0.8 mT MF. The question now became, what would be the effect of increasing chain length of poly-lysine molecules on cell popula-

Figure 10. Growth prole of OKT3 cells cultured in complete IMDM/supplemented with monomeric l-lysine (7 g/ml) in presence of 15 Hz, 0.8 mT MF.

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Figure 11. Eight percent SDS-PAGE of the spent media (supernatants) of OKT3 cells grown in MF (15 Hz, 0.8 mT) in complete IMDM: lane (1) control (OKT3 cells IMDM); lane (2) OKT3 cells IMDM l-lysine (7 g/ml) 72 hr; lane (3) MF (15 Hz, 0.8 mT) 72 hr; lane (4) MF (15 Hz, 0.8 mT) l-lysine (7 g/ml) 72 hr; lane (5) control (OKT3 cells IMDM) 96 hr; lane (6) OKT3 cells IMDM l-lysine (7 g/ml) 96 hr; lane (7) MF (15 Hz, 0.8 mT) 96 hr; lane (8) MF (15 Hz, 0.8 mT) l-lysine (7 g/ml) 96 hr.

tions at 50 Hz and 0.8 mT MF (Fig. 12) (Table 2)? As observed previously at 50 Hz, 0.8 mT MF, the cellular growth was not stimulated in the presence of MF l-lysine, whereas MF-only exposed cells were stimulated (with MAb titer being higher for MF l-lysine). We observed that in the non-MF-exposed cell plate, the cell populations followed the following trend. Poly-l-lysine (1000) gave the highest cell count at all points, followed by monomeric l-lysine, poly-d-lysine, poly-l-lysine (4000), and then poly-l-lysine (7500) (29) (though there was not much of difference between 4000 and 7500 poly-l-lysine). But in presence of magnetic eld, as the chain length increased, the cell counts decreased, though there were no signicant differences between molecular weights of 1000 and above (Fig. 13). The results had all become independent of chain length in the presence of an MF of 50 Hz, 0.8 mT. This is in stark contrast to the earlier experiments, where we observed that in the absence of MF, chain length had a signicant contribution up to molecular weight 1000. Thus, we could possibly conclude that at 50 Hz, 0.8 mT, coupling of poly-l-lysine molecules with serum-derived growth factors were inhibited or at least not facilitated, and MF inhibits the mitotic promotion property of poly-lysine molecules. No signicant changes were evident in SDS-PAGE patterns. Thus, we concluded that possibly there exists a window of frequencies, or rather a very specic band width where MF has some modulating effect, in and around 50 Hz. Similar observations were seen with media-supplemented oligomeric peptides of lysine, poly-lysines (mol. wt. 10007500) in the presence of 50 Hz, 0.8 mT MF. MF in the absence of any supplementation showed

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Table 2. Experimental Protocol for Studying the Effects of MF Coupled with Various Chain Lengths of Oligolysines Cell Line for Exposure OKT-3 OKT-3 OKT-3 OKT-3 OKT-3 MF Strength (mT) 0.8 0.8 0.8 0.8 0.8 Frequency of Exposure (Hz) 50 50 50 50 50 Voltage Required (Vrms) 11.72 11.68 11.69 11.73 11.70 Mean Time of Exposure (hr, min) 7, 54 7, 50 7, 45 7, 34 7, 40 Additive (7 g/ml) l-Lysine HCl l-Lysine HCl (1000) d-Lysine HCl (1000) l-Lysine HCl (4000) l-Lysine HCl (7500)

To determine the effect of chain length of polymeric lysine molecules (oligopeptides) and their effect on the cells in conjunction with MF of xed parameters, OKT3 cells were incubated in the presence and absence of LFMF and l-lysine HCl and its various polymers in a 24-well culture plate in complete IMDM/10% FCS/4% antibiotics. Cell counts were taken daily while PAGE, ELISA, and FPLC analyses were carried out with spent media. Seeding density was 2.1 104 cells/ml.

Figure 12. Eight percent SDS-PAGE of the spent media (supernatants) of OKT3 cells grown in MF (50 Hz, 0.8 mT) in complete IMDM for 96 hr: lane (1) OKT3 cells IMDM MF ; lane (2) OKT3 cells MF l-lysine (7 g/ml); lane (3) OKT3 cells poly-l-lysine (1000) (7 g/ ml); lane (4) OKT3 cells magnetic eld poly-d-lysine (1000) (7 g/ml); lane (6) OKT3 cells MF l-lysine (7500) (7 g/ml).

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Figure 13. Growth prole of OKT3 cells cultured in complete IMDM/10% FCS supplemented with l-lysine, poly-l-lysine (1000), poly-d-lysine (1000), poly-l-lysine (4500), and poly-l-lysine (7500) in presence of 50 Hz, 0.8 mT magnetic eld. Note that the growth of cells in magnetic eld is highest over l-lysine, whereas poly-l-lysine (1000) growth-promoting ability is inhibited.

the highest cell count, with decreasing cell counts as the molecular weight increased, (even poly-l-lysine 1000, as in earlier experiments, could not stimulate OKT3 cells in presence of 50 Hz, 0.8 mT MF). This could possibly be because of a resonance-induced anti-interaction between the cell membrane components (phospholipids and membrane-inserted bulkier protein molecules) and the longer versions of the bridge molecule, and further experimental probing is needed (Fig. 12). The implication of this cannot be ignored given the omnipresent MF generated by the electrical and induced magnetic environment in our immediate surroundings. If there is any deleterious or enhancing effect, it must be probed thoroughly to assess the situation and its potency or lack thereof.

ACKNOWLEDGMENT The authors are indebted to the support provided by the BRNS grant no. BR97013.

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