RESEARCH ARTICLE | FEBRUARY 10 2023

Effect of pulsed magnetic field in murine T lymphoma EL4

cells
Special Collection: 67th Annual Conference on Magnetism and Magnetic Materials

Hyunsook Lee  ; Boram Lee  ; Sojin Kim; Juyeon Jung

AIP Advances 13, 025344 (2023)

https://doi.org/10.1063/9.0000489

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 AIP Advances ARTICLE scitation.org/journal/adv

Effect of pulsed magnetic field in murine

T lymphoma EL4 cells
Cite as: AIP Advances 13, 025344 (2023); doi: 10.1063/9.0000489
Submitted: 3 October 2022 • Accepted: 20 January 2023 •
Published Online: 10 February 2023

Hyunsook Lee,a) Boram Lee, Sojin Kim, and Juyeon Jung

AFFILIATIONS
Department of Oriental Biomedical Engineering, College of Health Science, Sangji University, Wonju 26339, South Korea

Note: This paper was presented at the 67th Annual Conference on Magnetism and Magnetic Materials.
a)
Author to whom correspondence should be addressed: [email protected]

ABSTRACT
Maintenance of homoeostasis in human body is a very important indicator in all cell activities. When exposed to a disease, various immune
cells are activated due to the inflammatory response, and particularly T cells play a role in inducing apoptosis of mutated cells such as
tumor cells. When the activity of T cells is very low, infection by external invasion is easy, and on the contrary, excessive activation leads
to chronic inflammation caused by autoimmune diseases. Many clinical studies related to pulsed magnetic field (PMF) demonstrated its
efficacy in reducing pain, improving blood circulation, as well as blood’s acid-base balance. Therefore, our study has tried to investigate the

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influence of PMF on the regulation of acid-base homeostasis in EL4 T lymphoma cell. In addition, we have tried to explain the role of PMF
on immune cell activity by measuring the level of pro-inflammatory cytokine, TNF-α in culture supernatants. EL4 cells were cultured in a
DMEM medium supplemented with 10% FBS and 1% penicillin in an incubator at 37 ○ C and 5% CO2 condition. Our PMF stimulator has
the maximum strength of 4700 G at a transition time of 222 μs with pulse intervals of 1 Hz. The homoeostasis in pH was improved as PMF
strength increases. Cell viability decreased by 32% after PMF stimulation of 4700 G. It was observed that the concentration of TNF-α, a
cytokine related to inflammation, also decreased as the strength of PMF increased. These results suggest that PMF stimulation improves the
anti-inflammatory effect, therefore, it is thought to affect the immune system by balancing the activation and suppression of immune cells.
For clinical use, our study might suggest non-invasive PMF can be developed as a medical devices modulating immune system, although it is
necessary to optimize the PMF conditions such as pulse shape, duration, or repetition rate.
© 2023 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY) license
(http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1063/9.0000489

I. INTRODUCTION immune response. They are activated as TCR (T cell antigen recep-
tor) binds to MHC class II molecules of antigen-bearing cells and
Maintenance of homoeostasis in human body is a very impor- transmit signals to the cells.4,5 When the activity of T cells is very low,
tant indicator in all cell activities and its function depends on the infection by external invasion is easy, and on the contrary, excessive
exchange of signals between cells or major organs through chemical activation lead to chronic inflammation caused by autoimmune dis-
substances. In the case of an imbalance in the homeostasis due to eases. Therefore, controlling the balance between suppression and
bacteria and virus, it induces activation of various immune cells to activation of the immune response is very important, and particu-
regulate homeostasis.1 If there is an abnormality in the immune sys- larly T cells play a role in inducing apoptosis of mutated cells such
tem, an autoimmune disease causing the body to mistakenly attack as tumor cells.6
normal cells happens and it can lead to chronic inflammation. This However, in T lymphoma cells, malignant tumor that occurs
might cause tumors by damaging the DNA structure in normal cells in lymphocytes, there are abnormal proliferation and metastasis of
by secreting inflammatory cytokines such as interleukin (IL)-1, IL-2, cancer cell that permit survival beyond its normal life span.7 In
IL-4, tumor necrosis factor α (TNF-α), and interferon γ (IFN-γ).2,3 accordance, immune T cells do not function properly. When cancer
T-cells are one of the essential leukocytes of immune system has spread to the body, various symptoms such as pain, seizures, and
and perform a variety of functions such as helper T cells, memory breathlessness are present. While cancer therapy, involving cyto-
T cells, and killer T cells, and play a central role in the adaptive toxic drugs, kills cells that have a high basal level of proliferation

AIP Advances 13, 025344 (2023); doi: 10.1063/9.0000489 13, 025344-1

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TABLE I. The primer sequences of the genes used in reverse transcription polymerase chain reaction analysis.

Gene Strand Primer sequences (5′ to 3′ direction)

Forward 5′ -GGCAGGTCTACTTTGGAGTCATTGC-3′
TNF-alpha
Reverse 5′ -ACATTCGAGGCTCCAGTGAATTCGG-3′
Forward 5′ -GGAAAGCTGTGGCGTGATG-3′
GAPDH
Reverse 5′ -CTGTTGCTGTAGCCGTATTC-3′

and regeneration, it affects non-tumor cells proliferating rapidly in turns with elliptical shape, and has the maximum strength of 4700 G
the skin, hair, and epithelium.8 Thus, new therapeutic targets and at a transition time of 222 μs with pulse intervals of 1 Hz. PMF stim-
approaches are needed to more specifically treat cancer cells without ulation was applied to the sample at a vertical distance of 1.6 cm
damaging normal host cells. Therefore, in this study, we have tried from the center of the coil, for 3 min in all experiments.14 PMF stim-
to investigate the effect of magnetic field, one of the non-contact ulation with various strength of 1000–4700 G was applied to the EL4
treatment methods, on the balance of pH homeostasis in the cell sample group, in order to confirm the elapsed time when restoring
membrane and the growth inhibition of T-lymphoma cells. pH homeostasis appears most clearly after PMF stimulation, and to
Meanwhile, it is known that magnetic stimulation not only acti- check the conditions for optimization of magnetic field strength to
vates intracellular ions, but also enables non-invasive treatment due maximize the effect of restoring pH homeostasis. Immediately after
to cellular stimulation of induced currents.9 Many clinical studies PMF stimulation, and to give an adaptation time for PMF to affect
related to pulsed magnetic field (PMF) demonstrated its efficacy in the ion channels in the cell membrane, after 2 h, after 18 h of the
the treatment of muscle and nerve diseases, improving blood circu- cell doubling time, and after 24 h, pH changes were measured in
lation, as well as blood’s acid-base balance.10–12 Numerous studies each supernatants of the cultured EL4 cells using pH meter (pH/Ion
have addressed the interaction between magnetic field and Ca2+ meter S220, METTLER TOLEDO) under constant temperature
fluxes, because calcium is a principal regulator of several cellular of 25 ○ C.
processes. It is known that modulations in intracellular Ca2+ con- In order to affirm the apoptosis of T-lymphoma cells with PMF
centrations is closely related to the cohesiveness and deformability stimulation, cell viability was confirmed by counting the number

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of red blood cell (RBC).13 PMF also reduces the Ca2+ level of inner of living cells after dyeing with trypan blue. The number of cells
RBC, and blocks inhibiting Ca2+ pump ATPase activity.14 In addi- was repeatedly measured 10 or more times using a hemocytome-
tion, it was reported that PMF might play a role of the H+ pump in ter for quantitative analysis, and the concentration of living cells
the cell membrane, thereby it is thought that changing the H+ ion was calculated. Also reverse transcription polymerase chain reaction
concentration affects the extracellular pH.15 (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) analy-
Our hypothesis is that PMF might improve unstable pH home- sis were performed to confirm whether PMF stimulation suppressed
ostasis in lymphoma cells and induce apoptosis of cancer cells. For inflammatory cytokine, TNF-α, secreted from T lymphoma cells.
this purpose, we used EL4 T lymphoma cells growing as a solid Total RNA was extracted from all EL4 sample groups using
tumor in C57BL/6J mice not subjected to a variable stress model, an Easy Blue reagent (iNtRON Biotechnology, Korea) according to
and it has been widely used to study T cell activity and function,
due to easy culture and rapid growth. Therefore, the present study
focused to investigate quantitatively the influence of PMF on the
regulation of acid-base homeostasis and apoptosis of murine T lym-
phoma EL4 cells. Moreover, we have tried to elucidate the role of
PMF on anti-inflammatory activity by measuring concentration of
pro-inflammatory cytokine TNF-α in culture supernatants, as well
as TNF-α mRNA expression level in EL4 cells.

II. EXPERIMENTAL METHODS

EL4 cells which are T cells formed through lymphoma gener-
ated by inducing 9,10-dimethyl-1,2-benzanthracene to C57BL mice
were purchased from the Korean Cell Line Bank. They were cultured
in Dulbeccos modified Eagle’s medium (DMEM) supplemented
with 10% fetal bovine serum (FBS) and 1% penicillin G (100 IU/ml),
in an incubator at 37 ○ C and 5% CO2 condition and split every 2–3
days. In order to ensure reproducibility, a concentration of 5.5 × 104
cells/mL was maintained in a cell culture flask. FIG. 1. The change of pH level according to the elapsed time after PMF stimula-
Our PMF device consisted of magnetic field generator fabri- tion in control and PMF groups stimulated by 1000, 2700, 4000 G field strength,
respectively.
cated using silicon-controlled rectifier and single layered coil of 10

AIP Advances 13, 025344 (2023); doi: 10.1063/9.0000489 13, 025344-2

© Author(s) 2023
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normalization of TNF-α concentration in electrophoresis, GAPDH

was used as a loading control.
Secretion of inflammatory cytokine TNF-α in supernatants
of the cultured EL4 cell was quantified with ELISA kit (Mouse
TNF-alpha Quantikine ELISA Kit; R & D Systems) according to the
manufacturer’s instructions and TNF-α cytokine concentration was
calculated according to standard curve.

III. RESULTS AND DISCUSSION

Figure 1 shows the pH change according to PMF strength
and time elapsed after PMF stimulation. For all sample groups,
the homoeostasis in pH was improved from 7.52–7.85 to 7.41–7.78
as PMF strength increases. The pH was close to normal range of
7.35–7.45 when elapsed time is 18–24 h after PMF stimulation. Since
the cell doubling time is the period when the exchange between ions
in the cell membrane becomes active, in order to change the envi-
ronment of the extracellular media sufficiently due to the release
of inflammatory mediators, at least more than doubling time is
required. Therefore, our result for pH measurement according to the
FIG. 2. EL4 cell culture in control and PMF groups were dyed with a trypan blue at elapsed time is consistent with the doubling time of 18–20 h of EL4
a ratio of 1:1. (a) Cell morphology stained with trypan blue in the control group. (b) cells.
Cell morphology in stimulation group. (c) The number of living cells in control group In addition, it can be seen that the pH homeostasis is slowly
and PMF group stimulated with 4000 G was counted using a hemocytometer, restored from 7.85 to 7.52 over elapsed time even in the control
respectively. The number of living cells decreased in the magnetic field stimulated group that is not stimulated with PMF. But it was found that home-
group, compared to control group.
ostasis recovery was faster under PMF stimulation, and the greater

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PMF strength, the clearer the effect was. This means that acid-base
homeostasis can be controlled more effectively by PMF influencing
the pH of the extracellular media. Although there are several ways
the manufacturer’s protocol. RT-PCR was performed using Maxime of intercellular communication used to maintain homeostasis, our
RT-PCR premix kit (iNtRON, Korea) and primers, under the fol- results could be interpreted as ion transport back and forth across
lowing conditions: 30 min at 45 ○ C and 5 min at 94 ○ C (Synthesizing the membrane creating a membrane potential, and controlling H+
cDNA), followed by 35 cycles for 45 sec at 94 ○ C (denaturation), concentration.
40 sec at 57 ○ C (annealing), 1 min at 70 ○ C (extension), and final Figures 2(a) and 2(b) show cell morphology stained with trypan
extension for 5 min at 75 ○ C. The primer sequence is listed in Table I. blue in the control and stimulation groups, respectively. Figure 2(c)
PCR products were separated by 1.2% agarose gel electrophore- shows cell viability in control and PMF group. As the number of
sis. Gels were stained with ethidium bromide and analyzed under living cells decreased by 32% in the PMF group than in the control
ultraviolet light. The mRNA levels were estimated by densitomet- group, it appears that the PMF improved the immune response by
ric analysis of the observed bands using the ImageJ software. For inducing apoptosis of lymphoma EL4 cells. Therefore, it can be seen

FIG. 3. Effect of PMF stimulation (a) on the activation of

TNF-α mRNA in EL4 cell with RT-PCR, (b) on the amount
of TNF-α in the culture supernatants with ELISA analysis.
∗
p < 0.05 vs the Control group (R2 = 0.9716 in standard
curve, y = 0.0027x + 0.0316).

AIP Advances 13, 025344 (2023); doi: 10.1063/9.0000489 13, 025344-3

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that the PMF contributes not only to the regulation of homeostasis ACKNOWLEDGMENTS
but also to the maintenance of the balance of the immune response. This research was supported by National Research Foun-
In Fig. 3(a), RT-PCR analysis revealed lower expression of dation of Korea (NRF) funded by the Ministry of Education
inflammation-related cytokine TNF-α in PMF group of 4700 G, (NRF-2021R1F1A1060167).
compared to control group. Since EL4 cells are T-lymphoma cells,
as predicted, higher TNF-α concentrations, was observed in con-
AUTHOR DECLARATIONS
trol group. Serum levels of TNF-α in the culture supernatants were
shown in Fig. 3(b). Conflict of Interest
Our finding of a significant association from PCR and ELISA
The authors have no conflicts to disclose.
analysis is in agreement with the result showing that TNF-α con-
centration was significantly lower in PMF group of 4700 G than Author Contributions
in control and the other PMF groups. Although the amount of
cytokines measured in serum and the amount of cytokines secreted Hyunsook Lee: Conceptualization (equal); Writing – original draft
from EL4 cells are not exactly proportional, it is clear that the (equal); Writing – review & editing (equal). Boram Lee: Data cura-
secretion of TNF-α decreases as the strength of the magnetic field tion (equal); Validation (equal). Sojin Kim: Methodology (equal).
increases. These results suggest that PMF stimulation improves Juyeon Jung: Methodology (equal).
the anti-inflammatory effect, therefore, it is thought to affect the
immune system of the human body by balancing the activation and DATA AVAILABILITY
suppression of immune cells.
The data that support the findings of this study are available
within the article.
IV. CONCLUSION
REFERENCES
In the present study, we investigated the effect of PMF on pH 1
B. Styr and I. Slutsky, Nature Neuroscience 21(4), 463 (2018).
homeostasis in EL4 T lymphoma cells. There was evidence that PMF 2
N. Xu, X. Li, and Y. Zhong, Mediators of Inflammation 2015(3), 531518.
stimulation results in accelerating the restoration of pH homeosta- 3
J. A. Gracie et al., Journal of Clinical Investigation 104(10), 1393 (1999).
sis. And the apoptosis of lymphoma EL4 cells was more effective in 4
M. G. Rudolph, R. L. Stanfield, and I. A. Wilson, Annual Review of Immunology

04 July 2024 16:03:32

PMF group than in control group. Therefore, it was confirmed that 24(1), 419 (2006).
under the optimal PMF stimulation, not only the regulation of acid- 5
E. M. Behrens, Autoimmunity Reviews 7(4), 305 (2008).
base homeostasis but also cell viability were affected. Also our study 6
P. A. van der Merwe and O. Dushek, Nature Reviews Immunology 11(1), 47
has demonstrated that exposure to PMF may evoke the changes in (2011).
7
the level of cytokine, TNF-α. As PMF strength increased, PMF stim- J. Bhardwaj et al., Immunopharmacology and Immunotoxicology 36(3), 202
ulation decreased the secretion of TNF-α, showing that it had an (2014).
8
anti-inflammatory effect. M. A. Feitelson et al., Seminars in Cancer Biology 35, S25 (2015).
9
Accordingly, this study will contribute toward research on the N. M. Shupak, F. S. Prato, and A. W. Thomas, Neuroscience Letters 363(2), 157
(2004).
development of a non-invasive magnetic field therapy device rather 10
T. Mert et al., Bioelectromagnetics 38(4), 255 (2017).
than a drug therapy with side effects, for alleviating the disease and 11
J. C. McKay, F. S. Prato, and A. W. Thomas, Bioelectromagnetics 28(2), 81
modulating the immune response. For clinical use, it is necessary (2007).
to optimize the PMF conditions such as pulse shape, stimulation 12
S. Bang and H. Lee, AIP Advances 11(1), 015242 (2021).
time, repetition rate, pulse transition time and strength. Future stud- 13
M. Mattsson et al., Cell Biology International 25(9), 901 (2001).
ies will conduct in vivo experiments through animal experiments to 14
M. H. Seo and S. H. Lee, J. Magn. Magn. Mater. 505, 166726 (2020).
confirm the effect of improving the pH homeostasis and immune 15
Y. Choi, S. Bang, and H. Lee, IEEE Transactions on Magnetics 58(2), 5400105
response of PMF. (2021).

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