Magnetic field exposure system and its use

Abstract

The present invention relates to a magnetic field exposure system for use in exposing at least a portion of organic cells, tissue, or a human subject to a low-frequency magnetic field. The system includes a magnetic field generator and an amplifier supplying one or more electromagnetic coils configured to generate a magnetic field that varies in accordance with an amplitude-modulated signal having a carrier frequency between 360 Hz and 450 Hz, the modulation frequency being between 0.5 Hz and 100 Hz, and the magnetic field strength being between 0.5 μT and 250 μT. The present invention also relates to a method for improving well-being, reducing stress, promoting concentration, reducing anxiety, reducing skin aging, promoting tissue regeneration, promoting wound healing, promoting immune function, and / or preventing aging-related diseases in a subject by exposing at least a portion of the subject's tissue to the low-frequency magnetic field, as well as a method for promoting the in vitro proliferation of stem cells, progenitor cells, and other primary cells by exposing the cells to the low-frequency magnetic field.

Description

[Technical Field]

[0001] The present invention relates to a magnetic field exposure system for use in exposing at least a portion of an organic cell, tissue, or subject to a modulated low frequency magnetic field (LF-MF).

[0002] The present invention also relates to methods for improving well-being, reducing stress, promoting focus, reducing anxiety, reducing skin aging, promoting tissue regeneration, promoting wound healing, treating cancer, and / or promoting immune function in a subject, particularly by exposing at least a portion of the subject's tissue to the LF-MF using the magnetic field (MF) exposure system of the present invention.

[0003] The present invention further relates to a method for promoting the in vitro proliferation of cells, particularly human epidermal stem cells, human epidermal progenitor cells, and other primary cells, by exposure to the above-mentioned LF-MF. [Background technology]

[0004] Electrical signals are known to drive biochemical signals during the body's healing process (Zhao et al., Nature 442:457-460, 2006). It has been shown that nerve sprouting, nerve growth direction, and epithelial wound healing rate are coordinately regulated by wound-induced electric fields (Song et al., Journal of Cell Science 117, 4681-4690, 2004; doi:10.1242 / jcs.01341). Furthermore, application of pulsed electric fields has been shown to promote skin rejuvenation by inducing significant epidermal keratinocyte proliferation, capillary vasculature formation, and collagen secretion (Golberg et al., Nature Scientific Reports, 5:10187, 2014).

[0005] A drawback of electrotherapy is that electric fields cannot penetrate deep into the skin and the body when applied non-invasively. According to Faraday's law and Maxwell's equations, electric fields are coupled with magnetic fields, which allow the low range of the electromagnetic spectrum to penetrate unhindered into tissue. A permanent magnet generates a static magnetic field, while a time-varying magnetic field is generated by passing an alternating current (AC) through a coil or wire. Unlike a static magnetic field, a time-varying electromagnetic field induces a voltage difference in tissue, and the voltage difference induces ion flow. Therefore, time-varying electromagnetic fields can be used to alleviate the drawbacks of pure electric and magnetic fields, and the use of time-varying electromagnetic fields (EMFs) has been suggested for various medical applications.

[0006] There are a variety of frequency definitions used in this field. The International Commission on Non-Ionizing Radiation Protection (ICNIRP) defines low-frequency magnetic fields (EMF) as time-varying electromagnetic fields (EMF) and magnetic fields (MF) with frequencies in the range of 1 Hz to 100 kHz, and high-frequency magnetic fields (EMF / MF) as time-varying electromagnetic fields (EMF / MF) with frequencies in the range of 100 kHz to 300 GHz. The International Telecommunication Union (ITU) defines time-varying electromagnetic fields (EMF) and magnetic fields (MF) with frequencies in the range of 3 Hz to 30 Hz as extremely low frequency, time-varying electromagnetic fields (EMF) and magnetic fields (MF) with frequencies in the range of 30 Hz to 300 Hz as extremely low frequency, time-varying electromagnetic fields (EMF) and magnetic fields (MF) with frequencies in the range of 300 Hz to 3 kHz as extremely low frequency, time-varying electromagnetic fields (EMF) and magnetic fields (MF) with frequencies in the range of 3 kHz to 30 kHz as very low frequency, and time-varying electromagnetic fields (EMF) and magnetic fields (MF) with frequencies in the range of 30 kHz to 300 kHz as low frequency EMF / MF.

[0007] As used herein, the term "extremely low frequency" (ELF) refers to an (electro)magnetic field having a frequency in the range of 1 Hz to 300 Hz.

[0008] As used herein, the term "low frequency" (LF) refers to magnetic fields having frequencies in the range of 300 Hz to 100 kHz.

[0009] Specific low-frequency sounds are considered potentially beneficial for well-being due to their effects on the heart and brain. For example, music tuned to a frequency of 432 Hz reduces human heart rate more than music tuned to 440 Hz (Calamassi et al., Explore, vol. 15, issue 4, pp. 283–290, July–August 2019; doi:10.1016 / j.explore.2019.04.001). Music tuned to 432 Hz has also been shown to have a significant sedative effect, reflected by increased alpha-band brain activity, without significantly affecting sleep latency during daytime naps (Dubey et al., “Effect of music of specific frequencies upon the sleep architecture and electroencephalographic pattern of individuals with delayed sleep latency: A daytime nap study,” Journal of Family Medicine and Primary Care, 2019;8:3915–399).

[0010] ELF-EMF also promotes human bone marrow stem / progenitor cell differentiation (Maziarz et al. Stem Cell Research & Therapy, 7:54, 2016, doi:10.1186 / s13287-016-0312-5). Furthermore, specific EMF parameters, frequency, intensity, and exposure time, have been suggested to influence human bone marrow stem / progenitor cell differentiation in vitro (Ross et al. Stem Cell Res., July, 15(1):96-108, 2015, doi:10.1016 / j.scr.2015.04.009). ELF-EMF has also been suggested to promote skin wound healing. EMF is thought to have an anti-inflammatory effect through modulation of cytokine profiles that drive a transition from a chronic pro-inflammatory state to an anti-inflammatory state during the healing process (Madduri et al., Defense Life Science Journal, Vol. 3, No. 3, pp. 293-300, July 2018, doi:10.14429 / dlsj.3.12032; and Rosado et al., Front. Public Health, 26 March 2018; doi https: / / doi.org / 10.3389 / fpubh.2018.00085. Vianale et al. (Br. J. Dermatol. 158(6), 1189-1196, 2008, doi:10.1111 / j.1365-2133.2008.08540.x) showed that ELF-EMF exposure (1 mT, 50 Hz, 48 h) increased the proliferative activity of the transformed human keratinocyte cell line HaCaT, without detectable differences in cell viability.

[0011] Makarov et al. ("Design and analysis of a whole body non-contact electromagnetic stimulation device with field modulation", doi:https: / / doi.org / 10.1101 / 416065, September 2018) describe a whole body non-contact electromagnetic stimulation device based on the concept of a conventional MRI radiofrequency (RF) resonant coil with a resonant (carrier) frequency of 100 kHz to 150 kHz, magnetic field modulation (amplitude modulation) options of 0.5 to 100 Hz, and input power up to 3 kW. A potential clinical application of this device is suggested as the treatment of chronic pain.

[0012] The 7.83 Hz frequency corresponds to a naturally occurring magnetic field known as the Schumann resonance. This frequency is thought to have beneficial effects on human health (Guerriero, F. et al., Extremely low frequency electromagnetic fields stimulation modulates autoimmunity and immune responses: a possible immunomodulatory therapeutic effect in neurodegenerative diseases; Neural Regen Res 11, 1888-1895 (2016)). NASA-sponsored research (NASA Grant NSG-259-62) demonstrated that this frequency, both naturally occurring and anthropogenic, regulates the human circadian clock (Wever, R., Influence of weak electromagnetic fields on the circadian periodicity of humans; Die Naturwissenschaften 55, 29-32 (1968)). Several other studies have demonstrated that brain activity in individuals exposed to this frequency is synchronized to adapt to a similar frequency range, the alpha band frequency of 8-13 Hz, thereby producing significant physiological effects (Salansky, N. et al., Responses of the nervous system to low frequency stimulation and EEG rhythms: clinical implications. Neurosci Biobehav Rev 22, 395-409 (1998); Wang, C. E. et al., Transduction of the Geomagnetic Field as Evidenced from Alpha-Band Activity in the Human Brain; eNeuro6 (2019)).

[0013] Alpha-band brain waves have been shown to reduce psychological stress, support a relaxed mood state, and even increase attention and memory (Cherry, NJ, Human intelligence: the brain, an electromagnetic system synchronized by the Schumann Resonance signal. Med Hypotheses 60, 843-844 (2003); and Wang, CX et al., supra). These brain waves also increase serotonin production, reduce cortisol levels, and enhance the immune system (Guerriero, supra). Reduced psychological stress has been shown to enhance regenerative processes such as wound healing, in large part through beneficial effects on the immune system resulting from reduced stress levels (Britteon, P. et al., Association between psychological health and wound complications after surgery. Br. J Surg. 104, 769-776 (2017)).

[0014] Stimulating serotonin production and reducing stress through cortisol suppression improve well-being (Kraus, C., Castren, E., Kasper, S., Lanzenberger, R., "Serotonin and neuroplasticity—Links between molecular, functional, and structural pathophysiology in depression," 10.1016 / j.neubiorev.2017.03.007, March 2017). In rats, 15 days of daily 10-Hz treatment at 690–720 μT intensity induced an increase in serotonin in the raphe nucleus (Shahbazi-Gahrouei, D., Shiri, L., Alaei, H., Naghdi, N., "The effect of continuous ELF-MFs on the level of 5-HIAA in the raphe nucleus of the rat," 10.1093 / jrr / rrv093, January 2016).

[0015] U.S. Patent Application Publication No. 2003 / 231126A1 describes a device for generating a natural alternating electromagnetic field in the range of several hundred Hz up to 20 kHz in the vicinity of a user's body to compensate for electrical stress acting on the user. The device comprises an alternating magnetic field generating means and an alternating magnetic field transmitting means. These two means may be housed in a common housing, which may be worn directly on the user's body over clothing, or integrated into furniture such as a chair. It has also been suggested to mix the natural alternating electromagnetic field with an artificial alternating electromagnetic field, e.g., a very low-frequency signal such as a broadband pulse in the frequency range of 0.01 Hz to 40 Hz.

[0016] WO 2008 / 127011A2 relates to a low-frequency magnetic field physical therapy device comprising a magnetic field induction coil, a pulse signal controller, and a magnetic field induction coil driver. This device can be manufactured in the form of a mattress. The device further includes a frequency modulation program that modulates input frequencies (six brain waves: Schumann resonance waves designated as 12.5 Hz, 16.3 Hz, 23.4 Hz, 39.6 Hz, 87 Hz, 250 Hz, and 8.7 Hz) through the magnetic field induction coil to generate several pulse magnetic signals less than 100 μT.

[0017] WO 2013 / 139915 A1 relates to a method and system for exposing organic cells in a region of a subject to an extremely low frequency magnetic field. A generator generates a sinusoidal, non-harmonic current signal having a predetermined frequency of 7.5 Hz to 7.9 Hz and electromagnetic radiation of substantially 0.7 mT to 3 mT. A resonant medium is operably connected to the generator, energized by the signal, and positioned adjacent to the organic cells in the region for a predetermined period of time, thereby subjecting the organic cells in the region to a constant magnetic field of less than 1 mT and a frequency of substantially 7.5 Hz to 7.9 Hz for the predetermined period of time. At least one resonant medium may be attached to a support medium selected from the group including a belt-like member, a mask-like member, a bandage, a mattress, a pillow, and a helmet. Oral keratinocyte stem cells were stimulated using the device of the invention for 7 days, i.e., subjected to a non-modulating, constant ELF magnetic field of 7.692 Hz and 0.75 mT, and cell development was assessed at 3 and 7 days. The system is suggested for use in the regeneration of tissues composed of keratin-containing cells and in the proliferation of undifferentiated progenitor or stem cells in tissues.

[0018] German Patent No. 26 55 723 discloses a device for generating a pulsed magnetic field having a fundamental frequency of 30 Hz to 100 Hz and a superimposed oscillation of 300 Hz to 600 Hz, clocked in a frequency package ranging from 1 Hz to 30 Hz. The device is said to provide health-promoting effects to subjects exposed to the magnetic field.

[0019] US Patent Application Publication No. 2012 / 0203131 discloses an EEG device for treating tinnitus that generates a low frequency electromagnetic field having a frequency between 1 Hz and 100 Hz and a field strength of less than 20 mT.

[0020] US Pat. No. 6,461,289 discloses a device for generating a magnetic field having a static component and a pulsed magnetic field having an alternating component. Summary of the Invention [Problem to be solved by the invention]

[0021] There remains a need for a system that uses magnetic field therapy and / or magnetic fields to promote beneficial effects in organic cells, particularly stem and progenitor cells (e.g., in tissue homeostasis, or regeneration, or ex-vivo cell culture), to promote well-being (e.g., reduce psychological stress) in subjects exposed to the magnetic field.

[0022] An object of the present invention is to provide an improved magnetic field (MF) exposure system for use in exposing at least a portion of an organic cell (in vitro), cellular tissue (in vitro or in vivo), or subject (e.g., a human) to amplitude-modulated low-frequency magnetic fields (LF-MF). Exposing cells (e.g., stem or progenitor cells) to MFs generated by the MF exposure system disclosed herein significantly improves cell viability and / or increases cell proliferation and / or reduces cell stress and / or reduces cell senescence after long-term exposure (e.g., 1 to 4 weeks), such as 1 to 10 weeks.

[0023] Another object of the present invention is to provide a modulated low frequency magnetic field (LF-MF) exposure system for use in reducing stress, increasing focus, reducing anxiety, and promoting well-being in subjects exposed to LF-MF, for example, by inducing increased alpha brain wave activity and strengthening the immune system. [Means for solving the problem]

[0024] In a first aspect of the present invention, there is provided an MF exposure system for use in exposing at least a portion of a subject (e.g., a human) to organic cells, cellular tissue, or LF-MF, the system comprising a magnetic field generator having one or more coils configured to generate a magnetic field that varies in accordance with an amplitude modulated signal having a carrier frequency between 360 Hz and 450 Hz, the modulation frequency being between 0.5 Hz and 100 Hz, and the MF having a field strength between 0.5 μT and 250 μT.

[0025] Preferably, the MF varies according to an amplitude modulated signal having a carrier frequency in the range of 400 Hz to 450 Hz, preferably around 432 Hz.

[0026] In certain embodiments, the modulation frequency is less than 50 Hz, for example, less than 30 Hz. Preferably, the modulation frequency is between 0.5 Hz and 50 Hz, for example, between 0.5 Hz and 30 Hz, between 0.5 Hz and less than 30 Hz, between 3 Hz and 30 Hz, between 3 Hz and less than 30 Hz, or between 3 Hz and 28 Hz. Preferably, the modulation frequency corresponds to the Schumann resonance mode, specifically about 7.83 Hz. Preferably, the MF field strength is between 5 μT and 200 μT, for example, between 10 μT and 200 μT. Preferably, the MF field strength is less than or equal to about 200 μT, for example, about 100 μT, about 30 μT, or about 11 μT. Preferably, the LF-MF generated by the present system is a continuous field (i.e., the LF-MF is non-clocked or non-pulsed).

[0027] The term "modulation frequency," as used herein, may also be referred to as "amplitude-varying frequency." The amplitude-varying waveform is preferably a sinusoidal amplitude-varying waveform. In use, one or more coils are supplied with a time-varying current that varies according to the amplitude-modulated signal, whereby the MF intensity at a given point in space varies proportionally to the instantaneous current value. The MF exposure system of the present invention may be used to expose at least some organic cells, cellular tissue of a subject (e.g., a human) to the LF-MF and promote cell regeneration in the cellular tissue for any of the uses and methods described herein. Specifically, the MF exposure system may be used to expose human skin tissue to the LF-MF to promote cell regeneration and / or tissue homeostasis. The application may be therapeutic (including treatment, symptom suppression, or prevention) or non-therapeutic (e.g., cosmetic), as further described below.

[0028] Also disclosed is a MF exposure system for use in exposing at least a portion of an organic cell, tissue, or subject (e.g., a human) to the LF-MF, the system comprising a MF generator having one or more coils configured to generate a MF that varies in accordance with an amplitude-modulated signal having a carrier frequency in the range of 400 Hz to 450 Hz, preferably 432 Hz, and the MF has an MF strength in the range of 10 μT to 200 μT, preferably 200 μT. More specifically, the MF varies in accordance with an amplitude-modulated signal having a carrier frequency in the range of 400 Hz to 450 Hz, preferably 432 Hz, a modulation frequency in the range of 3 Hz to 30 Hz, preferably 7.83 Hz, such as a modulation frequency corresponding to a Schumann resonance mode, and an MF strength in the range of 10 μT to 200 μT, preferably 200 μT.

[0029] The Schumann resonance describes an electromagnetic phenomenon in the Earth's atmosphere that is associated with Earth's lightning activity. The naturally occurring Schumann resonance is not measurable in the presence of "electromagnetic smog" from electronic devices and is attenuated by concrete and steel buildings. In 1952, Winfried Otto Schumann discovered that the resonance is 7.49[n(n+1)] 1 / 2Hz, where n is an integer. However, as measured by several observations across the globe (e.g., in Tomsk), the actual Schumann frequency modes vary slightly from this calculation, with a fundamental Schumann resonance at approximately 7.83 Hz, with harmonics occurring at approximately 14.1 Hz, 20.3 Hz, 26.4 Hz, 32.4 Hz, etc. However, there are geographic, diurnal, and seasonal variations in the Schumann resonances of the different modes.

[0030] Surprisingly, exposure of stem and progenitor cells (e.g., human stem and progenitor cells, particularly human epidermal keratinocytes) to LF-MFs modulated according to an amplitude-modulated signal with a carrier frequency of 432 Hz, a sinusoidal modulation frequency of 7.83 Hz, and an MF intensity of 200 μT has been found to significantly improve cell survival, increase cell proliferation, reduce cellular senescence, and / or reduce cellular stress compared to exposure to 7.83 Hz MFs alone at 200 μT or unmodulated LF-MFs at 432 Hz at 200 μT. Enhancing the body's ability to efficiently mobilize its own stem cells, instead of multiplying cells that have already replicated numerous times while accumulating DNA mutations and other damage, represents a powerful means of regenerative tissue homeostasis, eliminating chronic dysfunction and disease, including cancer.

[0031] As shown in the Examples, the modulated LF-MF herein is particularly effective in promoting the in vitro proliferation of stem and progenitor cells (e.g., keratinocyte stem or progenitor cells, particularly human keratinocyte stem or progenitor cells). Thus, in use, the system may be configured to provide a cell culture system that provides improved cell culture / population growth by exposing cells to the modulated LF-MF, thereby increasing cell proliferation and / or increasing cell viability and / or reducing cell stress.

[0032] Accordingly, in one embodiment, there is provided a MF exposed cell culture system comprising: a cell culture vessel defining a cell culture cavity within the vessel; and a magnetic field generator having one or more coils configured to generate a MF within the cell culture cavity that varies according to an amplitude modulated signal having a carrier frequency between 360 Hz and 450 Hz, wherein the modulation frequency is between 0.5 Hz and 100 Hz, and the MF has a field strength between 0.5 μT and 250 μT.

[0033] In a further embodiment, the MF-exposed cell culture system further comprises one or more organic cells (preferably a population of stem cells, progenitor cells, or other primary cells) in the cell culture cavity. Suitably, the cell culture cavity further comprises cell culture medium. In use, the MF-exposed cell culture system exposes cells in the cell culture vessel to the modulated LF-MF described herein, thereby increasing cell proliferation, reducing cell stress, improving cell viability, and / or preventing cell senescence.

[0034] In a second aspect, there is provided a method of improving well-being, reducing stress (e.g., psychological stress), increasing concentration, and / or reducing anxiety in a subject (e.g., a healthy human) by exposing at least the brain of the subject to an LF-MF that varies according to an amplitude modulation frequency having a carrier frequency between 360 Hz and 450 Hz, wherein the modulation frequency is between 0.5 Hz and 100 Hz, and the MF has a magnetic field strength between 0.5 μT and 250 μT.

[0035] Preferably, the MF varies according to an amplitude-modulated signal having a carrier frequency in the range of 400 Hz to 450 Hz, preferably about 432 Hz. The modulation frequency may be less than 50 Hz, preferably less than 30 Hz. Preferably, the modulation frequency is 0.5 Hz to 50 Hz, for example, 0.5 Hz to 30 Hz, 0.5 Hz to less than 30 Hz, 3 Hz to 30 Hz, or 3 Hz to 28 Hz. Preferably, the modulation frequency corresponds to the Schumann resonance mode, more preferably about 7.83 Hz. Preferably, the MF field strength is 0.5 μT to 200 μT, for example, 5 μT to 200 μT, for example, 10 μT to 200 μT, 10 μT to 100 μT, 10 μT to 50 μT, 10 μT to 50 μT, 0.5 μT to 40 μT, 0.5 μT to 15 μT, or 0.5 μT to 12 μT. Therefore, the MF field strength can be about 200 μT, about 100 μT, about 30 μT, or about 11 μT.

[0036] Also disclosed are methods for improving well-being, reducing stress (e.g., psychological stress), increasing concentration, and / or reducing anxiety in a subject, particularly a healthy human, by exposing at least a portion of the subject to an LF-MF that varies in accordance with an amplitude-modulated signal having a carrier frequency in the range of 400 Hz to 450 Hz, preferably 432 Hz, where the MF has an MF strength in the range of 10 μT to 200 μT, preferably 200 μT. More specifically, the MF varies in accordance with an amplitude-modulated signal having a carrier frequency in the range of 400 Hz to 450 Hz (preferably 432 Hz), a modulation frequency in the range of 3 Hz to 30 Hz, such as a modulation frequency corresponding to the Schumann resonance mode (preferably 7.83 Hz), and an MF strength in the range of 10 μT to 200 μT (e.g., up to about 11 μT, up to about 30 μT, or up to about 200 μT). In some embodiments of the second aspect, the method is a non-therapeutic method. In some embodiments, the method of the second aspect is a therapeutic method.

[0037] In a third aspect, there is provided the use of an LF-MF varied according to an amplitude-modulated signal having a carrier frequency of 360 Hz to 450 Hz, the modulation frequency being 0.5 Hz to 100 Hz, and the MF having a magnetic field strength of 0.5 μT to 250 μT. By exposing at least the brain of the subject to the LF-MF, the subject (e.g., a healthy human) can improve their well-being, reduce stress (e.g., psychological stress), increase their concentration, and / or reduce their anxiety. Preferably, the MF is varied according to an amplitude-modulated signal having a carrier frequency in the range of 400 Hz to 450 Hz, preferably 432 Hz. The modulation frequency can be less than 50 Hz, preferably less than 30 Hz. Preferably, the modulation frequency can be between 0.5 Hz and 50 Hz, for example, between 0.5 Hz and 30 Hz, between 0.5 Hz and less than 30 Hz, between 3 Hz and 30 Hz, or between 3 Hz and 28 Hz. Preferably, the modulation frequency corresponds to the Schumann resonance mode, more preferably about 7.83 Hz. Preferably, the MF field strength is between 5 μT and 200 μT, for example between 10 μT and 200 μT, between 10 μT and 100 μT, between 10 μT and 50 μT, between 10 μT and 50 μT. Thus, the MF field strength may be about 200 μT, about 100 μT, about 30 μT, or about 11 μT.

[0038] Also disclosed herein is a non-therapeutic use of LF-MFs varied according to an amplitude-modulated signal having a carrier frequency in the range of 400 Hz to 450 Hz (preferably 432 Hz) and an MF intensity in the range of 10 μT to 200 μT (preferably 200 μT), whereby exposing at least a portion of the subject to the LF-MFs improves the well-being of the subject, particularly healthy humans, by reducing stress (e.g., psychological stress), increasing concentration, and / or reducing anxiety. More specifically, the MFs are varied according to an amplitude-modulated signal having a carrier frequency in the range of 400 Hz to 450 Hz (preferably 432 Hz), a modulation frequency in the range of 3 Hz to 30 Hz, such as a modulation frequency corresponding to the Schumann resonance mode (preferably 7.83 Hz), and an MF intensity in the range of 0.5 μT to 250 μT (e.g., 0.5 μT to 200 μT or 10 μT to 200 μT (preferably 200 μT)).

[0039] In some embodiments of the second and third aspects, the method or use is a non-therapeutic method. In some embodiments of the second and third aspects, the method or use is a therapeutic use or a therapeutic method of treatment.

[0040] In the second and third aspects, a subject may be exposed to modulated LF-MF for, for example, up to 8 hours per day (e.g., up to 8 hours, up to 5 hours, up to 4 hours, up to 3 hours, up to 2 hours, or up to 1 hour). Thus, a subject may be exposed to LF-MF for 10 minutes to 1 hour per day for a time period of 1 to 8 weeks, such as 4 weeks. Specifically, the MF is varied according to an amplitude-modulated signal having a carrier frequency of 360 Hz to 450 Hz (e.g., in the range of 400 Hz to 450 Hz, preferably about 432 Hz), a modulation frequency in the range of 3 Hz to 30 Hz (e.g., 0.5 Hz to less than 30 Hz), such as a modulation frequency corresponding to the Schumann resonance mode (preferably about 7.83 Hz), and an MF strength of 0.5 μT to 250 μT (e.g., 0.5 μT to 200 μT or 10 μT to 200 μT, e.g., in the range of 10 μT to 200 μT).

[0041] In a fourth aspect, there is provided a cosmetic method for treating a disease associated with skin stem cells or skin progenitor cells in a subject (e.g., a healthy human), the method comprising exposing at least a portion of the subject's cellular tissue to an LF-MF that varies according to an amplitude-modulated signal having a carrier frequency of 360 Hz to 450 Hz, the modulation frequency being 0.5 Hz to 100 Hz, and the MF having a magnetic field strength of 0.5 μT to 250 μT.

[0042] In a fifth aspect, there is provided a non-therapeutic use of LF-MF varied according to an amplitude-modulated signal having a carrier frequency of 360 Hz to 450 Hz, the modulation frequency being 0.5 Hz to 100 Hz, and the MF having a magnetic field strength of 0.5 μT to 250 μT, to treat a disease associated with skin stem cells or skin progenitor cells in a subject (e.g., a healthy human) by exposing at least a portion of the subject's cellular tissue to the LF-MF.

[0043] The diseases associated with skin stem cells or skin progenitor cells in the fourth and fifth aspects may be associated with skin stem cells selected from, for example, epithelial stem cells, melanocyte stem cells, hair follicle stem cells, keratinocyte stem cells, and keratinocyte precursor cells or fibroblasts. Preferably, the diseases are associated with keratinocyte stem cells or keratocyte precursor cells.

[0044] In the fourth and fifth aspects, the non-therapeutic (cosmetic) use may be selected from, for example, maintaining skin homeostasis, reducing skin aging, promoting skin rejuvenation, preventing or reducing skin wrinkles, increasing skin elasticity, preventing or reducing skin stretch marks, preventing or reducing cellulitis, increasing skin hydration, reducing rough skin, reducing skin pore size, inducing hair growth, preventing or reducing hair loss, inhibiting hair graying, inducing hair darkening, maintaining skin pigmentation, and inducing nail growth. Thus, the use may be inducing hair growth. The use may be preventing or reducing hair loss. The use may be reducing skin aging.

[0045] Preferably, in the fourth and fifth aspects, the MF varies according to an amplitude-modulated signal having a carrier frequency in the range of 400 Hz to 450 Hz, preferably about 432 Hz. The modulation frequency may be less than 50 Hz, preferably less than 30 Hz. Preferably, the modulation frequency is 3 Hz to 28 Hz. Preferably, the modulation frequency corresponds to the Schumann resonance mode, preferably about 7.83 Hz. Preferably, the MF field strength is 5 μT to 200 μT, for example 10 μT to 200 μT. Preferably, the MF field strength is about 200 μT.

[0046] Also disclosed is a cosmetic method for reducing skin aging in a subject, particularly a healthy human (e.g., by promoting skin rejuvenation, reducing wrinkles, increasing skin elasticity, reducing stretch marks, and / or reducing cellulitis), by exposing at least a portion of the subject's cellular tissue to an LF-MF that varies in accordance with an amplitude-modulated signal having a carrier frequency in the range of 400 Hz to 450 Hz (preferably 432 Hz) and an MF strength in the range of 0.5 μT to 250 μT (e.g., 0.5 μT to 200 μT or 10 μT to 200 μT, preferably 200 μT).

[0047] It is believed that the reduction of skin aging is achieved through stem cell activation, such as promoting the proliferation of human keratinocyte stem cells and keratinocyte precursor cells. More specifically, the MF is varied according to an amplitude-modulated signal having a carrier frequency in the range of 400 Hz to 450 Hz, a modulation frequency in the range of 3 Hz to 30 Hz, such as a modulation frequency corresponding to the Schumann resonance mode (preferably 7.83 Hz), and an MF strength in the range of 0.5 μT to 250 μT (e.g., 0.5 μT to 200 μT or 10 μT to 200 μT).

[0048] Also disclosed herein is a non-therapeutic cosmetic use of LF-MF, which varies according to an amplitude-modulated signal having a carrier frequency in the range of 400 Hz to 450 Hz (preferably 432 Hz) and an MF intensity in the range of 10 μT to 200 μT (preferably 200 μT), to reduce skin aging in a subject, particularly a healthy human, by exposing at least a portion of the subject's tissue (e.g., tissue in the subject's face) to the LF-MF (e.g., by promoting skin rejuvenation, reducing wrinkles, increasing skin elasticity, reducing stretch marks, and / or reducing cellulitis). For example, at least a portion of the subject's tissue can be exposed to the LF-MF for 10 minutes to 1 hour per day for a period of 1 to 8 weeks, such as 4 weeks. More specifically, the MF varies according to an amplitude modulation signal having a carrier frequency of 400 Hz to 450 Hz, a modulation frequency in the range of 3 Hz to 30 Hz, such as a modulation frequency corresponding to the Schumann resonance mode (preferably approximately 7.83 Hz), and an MF strength of 0.5 μT to 250 μT (e.g., 0.5 μT to 200 μT or 10 μT to 200 μT).

[0049] In a sixth aspect, there is provided a method of treating a medical disorder associated with stem or progenitor cells in a subject (e.g., a human) by exposing at least a portion of the subject's cellular tissue to an LF-MF that varies according to an amplitude-modulated signal having a carrier frequency between 360 Hz and 450 Hz, wherein the modulation frequency is between 0.5 Hz and 100 Hz and the MF has a magnetic field strength between 0.5 μT and 250 μT.

[0050] In a seventh aspect, there is provided a therapeutic use of an LF-MF varied according to an amplitude modulated signal having a carrier frequency of 360 Hz to 450 Hz, wherein the modulation frequency is 0.5 Hz to 100 Hz, and the MF has a magnetic field strength of 0.5 μT to 250 μT, for treating a medical disorder associated with stem or progenitor cells in a subject (e.g., a human) by exposing at least a portion of the subject to the LF-MF.

[0051] In the sixth and seventh aspects, the medical disease may be associated with adult stem cells (also called somatic stem cells). For example, the medical disease may be associated with muscle stem cells, hematopoietic stem cells, epithelial stem cells, neural stem cells, mesenchymal stem cells, mammary stem cells, intestinal stem cells, mesodermal stem cells, endothelial stem cells, skin stem cells, olfactory stem cells, neural crest stem cells, dental pulp stem cells, or fibroblasts. In particular, the medical disease may be associated with skin stem cells, e.g., keratinocyte stem cells, such as human epidermal keratinocyte stem cells.

[0052] In the sixth and seventh aspects, the medical condition can be tissue regeneration, wound healing, bone regeneration, inflammatory diseases, cardiovascular diseases, neurodegenerative diseases, cognitive disorders, autoimmune diseases, osteoarthritis, tissue fibrosis, periodontal disease, or skin diseases. In certain embodiments, the medical condition is wound healing (including healing of chronic injuries), such as healing of surgical wounds, traumatic wounds, abrasions, burns (including radiation burns, e.g., burns resulting from sunburn or radiation therapy), blisters, ulcers (including diabetic leg ulcers and venous leg ulcers), or preventing or minimizing scarring (e.g., by preventing or inhibiting skin fibrosis). In further embodiments, LF-MF promotes tissue regeneration in a subject, e.g., regeneration of a tissue selected from muscle tissue (e.g., for maintaining or increasing muscle mass or muscle repair), connective tissue, joint tissue, epithelial tissue, endothelial tissue, nervous tissue, brain tissue, adipose tissue, skin tissue, lung tissue, liver tissue, bladder tissue, kidney tissue, heart tissue, stomach tissue, intestinal tissue, spinal tissue, eye tissue, fibrous tissue, dentin, bone, and bone marrow.

[0053] In an eighth aspect, there is provided a therapeutic use of LF-MF, which is varied according to an amplitude-modulated signal having a carrier frequency in the range of 360 Hz to 450 Hz (e.g., 400 Hz to 450 Hz, preferably about 432 Hz) and an MF intensity in the range of 0.5 μT to 250 μT (e.g., 10 μT to 200 μT, preferably 200 μT), to promote tissue regeneration (e.g., post-operative tissue healing), promote wound healing, treat cancer, and / or enhance immune function in a subject in need thereof by exposing at least a portion of the subject's tissue to the LF-MF. For example, at least a portion of the subject's tissue may be exposed to the LF-MF for 10 minutes to 24 hours per day for a period of 1 to 8 weeks, such as 4 weeks. More specifically, the MF varies according to an amplitude modulation signal having a carrier frequency of 400 Hz to 450 Hz, a modulation frequency within the range of 0.5 Hz to 100 Hz (e.g., 3 Hz to 30 Hz or less than 3 Hz to 30 Hz, such as a modulation frequency corresponding to the Schumann resonance mode (preferably 7.83 Hz)), and an MF strength within the range of 0.5 μT to 250 μT (e.g., 0.5 μT to 200 μT or 10 μT to 200 μT).

[0054] In a ninth aspect, a method is provided for promoting tissue regeneration in a subject in need thereof by exposing at least a portion of the subject's cellular tissue to an LF-MF that varies in accordance with an amplitude-modulated signal having a carrier frequency in the range of 360 Hz to 450 Hz (e.g., 400 Hz to 450 Hz, preferably 432 Hz) and an MF intensity in the range of 0.5 μT to 250 μT (e.g., 10 μT to 200 μT, preferably 200 μT). The promotion of tissue regeneration is believed to be achieved through stem cell activation. More specifically, the MF varies in accordance with an amplitude-modulated signal having a carrier frequency in the range of 400 Hz to 450 Hz, a modulation frequency in the range of 0.5 Hz to 100 Hz (e.g., 3 Hz to 30 Hz or less than 3 Hz), such as a modulation frequency corresponding to the Schumann resonance mode (preferably 7.83 Hz), and an MF intensity in the range of 0.5 μT to 250 μT (e.g., 0.5 μT to 200 μT or 10 μT to 200 μT).

[0055] In a tenth aspect, there is provided a method of promoting wound healing in a subject in need thereof by exposing at least a portion of the subject's tissue to an LF-MF that varies in accordance with an amplitude-modulated signal having a carrier frequency in the range of 360 Hz to 450 Hz (e.g., 400 Hz to 450 Hz, preferably 432 Hz) and an MF strength in the range of 0.5 μT to 250 μT (e.g., 10 μT to 200 μT, preferably 200 μT). Without wishing to be limited by theory, it is believed that the promotion of wound healing is achieved through stem cell activation. More specifically, the MF varies according to an amplitude modulation signal having a carrier frequency in the range of 400 Hz to 450 Hz, a modulation frequency in the range of 0.5 Hz to 100 Hz (e.g., 3 Hz to 30 Hz or less than 3 Hz to 30 Hz, such as a modulation frequency corresponding to the Schumann resonance mode (preferably 7.83 Hz)), and an MF intensity in the range of 0.5 μT to 250 μT (e.g., 0.5 μT to 200 μT or 10 μT to 200 μT).

[0056] In an eleventh aspect, there is provided a method for treating, including preventing and / or reducing, cancer in a subject in need thereof by exposing at least a portion of the subject's cellular tissue to an LF-MF that varies in accordance with an amplitude-modulated signal having a carrier frequency in the range of 360 Hz to 450 Hz (e.g., 400 Hz to 450 Hz, preferably 432 Hz) and an MF strength in the range of 0.5 μT to 250 μT (e.g., 10 μT to 200 μT, preferably 200 μT). It is believed that the prevention and reduction of cancer is achieved through stem cell activation. More specifically, the MF varies according to an amplitude-modulated signal having a carrier frequency in the range of 400 Hz to 450 Hz, a modulation frequency in the range of 0.5 Hz to 100 Hz (e.g., 3 Hz to 30 Hz or less than 3 Hz to 30 Hz, such as a modulation frequency corresponding to the Schumann resonance mode (preferably 7.83 Hz)), and an MF intensity in the range of 0.5 μT to 250 μT (e.g., 0.5 μT to 200 μT or 10 μT to 200 μT). In other embodiments, the systems and methods described herein are not used to prevent and / or reduce cancer in a subject.

[0057] In a twelfth aspect, a method is provided for enhancing immune function in a subject in need thereof by exposing at least a portion of the subject's cellular tissue to an LF-MF that varies in accordance with an amplitude-modulated signal having a carrier frequency in the range of 360 Hz to 450 Hz (e.g., 400 Hz to 450 Hz, preferably 432 Hz) and an MF intensity in the range of 0.5 μT to 250 μT (e.g., 10 μT to 200 μT, preferably 200 μT). Enhancement of immune function is believed to be achieved through epidermal and mesenchymal stem cell activation. More specifically, the MF varies in accordance with an amplitude-modulated signal having a carrier frequency in the range of 400 Hz to 450 Hz, a modulation frequency in the range of 0.5 Hz to 100 Hz (e.g., 3 Hz to 30 Hz or less, such as a modulation frequency corresponding to the Schumann resonance mode (preferably 7.83 Hz)), and an MF intensity in the range of 10 μT to 200 μT.

[0058] In a thirteenth aspect, there is provided a method of promoting proliferation of organic cells, particularly stem cells, progenitor cells, or other primary cells, by exposing the cells (e.g., stem or progenitor cells) to an LF-MF that is varied in accordance with an amplitude-modulated signal having a carrier frequency between 360 Hz and 450 Hz, wherein the modulation frequency is between 0.5 Hz and 100 Hz, and the MF has a magnetic field strength between 0.5 μT and 250 μT.

[0059] When the cells are stem or progenitor cells, they may be any of the stem or progenitor cells disclosed herein, and in particular, any of the human stem or progenitor cells. The stem cells are preferably adult stem cells, although other stem cell types, such as iPS stem cells, amniotic stem cells, or embryonic stem cells, may also be used in the methods. Examples of adult stem cells include muscle stem cells, hematopoietic stem cells, epithelial stem cells, neural stem cells, mesenchymal stem cells, mammary stem cells, intestinal stem cells, mesodermal stem cells, endothelial stem cells, skin stem cells, melanocyte stem cells, hair follicle stem cells, olfactory stem cells, neural crest stem cells, and dental pulp stem cells. In particular, the stem or progenitor cells are skin stem cells or skin progenitor cells, such as keratinocyte stem cells or keratinocyte progenitor cells. In another embodiment, the stem cells are mesenchymal stem cells.

[0060] The method may be an in vivo method in which a subject is exposed to LF-MF, thereby increasing the proliferation of stem or progenitor cells in the subject. Increased stem cell proliferation is expected, for example, to promote tissue regeneration in the subject. Preferably, the LF-MF is directed to a specific area of the subject's body containing stem cells, such as a body joint such as a knee or hip joint, an injured site on or within the subject's body, or an area of the subject's skin or hair. In certain embodiments of the method, a wound in the subject is exposed to LF-MF to promote wound healing. For example, the method may increase the rate of wound closure and / or prevent or reduce the formation of scar tissue.

[0061] The method can be an in vitro method in which one or more organic cells (e.g., stem cells, progenitor cells, or other primary cells) are exposed to LF-MF, thereby increasing proliferation of the cells (e.g., stem cells or progenitor cells) in vitro (e.g., in a cell culture vessel) and expanding the population of cells. The in vitro method can further include differentiating the stem or progenitor cells into specific tissue cells, such as osteoblasts, fibroblasts (e.g., gingival fibroblasts), skin fibroblasts, keratinocytes, or chondrocytes. The method can also be used to generate specific tissues from stem or progenitor cells, for example, by differentiating the stem or progenitor cells into skin, cartilage, ligament, tendon, or bone. Formation of specific tissues in vitro can be carried out by culturing the stem or progenitor cells in the presence of a scaffold suitable for promoting tissue formation / structure. The scaffold can be, for example, a collagen or polymer scaffold or matrix. Cells can be exposed to LF-MF during both the cell proliferation and differentiation stages of the method. Alternatively, LF-MF may be applied only during stem or progenitor cell proliferation or only during cell differentiation.

[0062] Expanded stem or progenitor cells prepared according to the present in vitro method can be used for regenerative tissue therapy, for example, by administering the cells locally to a subject's site in need of cell or tissue regeneration. For example, stem or progenitor cells prepared according to the present method can be administered to muscle tissue, connective tissue, joint tissue, epithelial tissue, endothelial tissue, nervous tissue, adipose tissue, skin tissue, lung tissue, liver tissue, bladder tissue, kidney tissue, cardiac tissue, pancreatic tissue, stomach tissue, intestinal tissue, spinal tissue, brain tissue, eye tissue, fibrous tissue, dentin, bone, or bone marrow. For example, chondrocyte precursor cells prepared according to the present method can be injected into the hip joint of a subject's knee to promote cartilage regeneration or growth in the subject. Preferably, the cells administered to the subject are autologous cells. However, administration of allogeneic cells prepared according to the present method is also contemplated.

[0063] In a further embodiment, the method may be used to prepare an in vitro cultured autograft, the method comprising culturing skin stem or progenitor cells (preferably keratinocyte stem or progenitor cells and / or fibroblasts), wherein the stem or progenitor cells are exposed to LF-MF to promote cell proliferation and cell confluence to form an epithelial layer.

[0064] Culturing keratinocytes to form epithelial sheets can be carried out using known conditions, for example, as described by Hynds et al. (EMBO Mol Med. 2018 Feb;10(2):139-150) and Green et al. (Formation of epidermis by serially cultivated human epidermal cells transplanted as an epithelium to athymic mice; 1980, Transplantation 29:308-313). Preferably, keratinocyte stem cells or keratinocyte progenitor cells are cultured on a suitable substrate, such as fibrin or a polymer matrix (Pellegrini et al., (1999), Transplantation 68:868-879 and Zhu et al., (2005), Eur J Plast. Surg. 28:319-330).

[0065] The epithelial autografts produced may be used to treat wounds, burns, ulcers (including diabetic ulcers, particularly diabetic leg ulcers), and the like.

[0066] A further embodiment is a method of regenerating tissue in a subject, comprising: (i) culturing in vitro one or more stem or progenitor cells from the subject, wherein the cells are exposed to a LF-MF that varies in accordance with an amplitude-modulated signal having a carrier frequency of 360 Hz to 450 Hz, the modulation frequency being between 0.5 Hz and 100 Hz, and the MF having a magnetic field strength between 0.5 μT and 250 μT during at least a portion of the culturing, thereby expanding a population of stem or progenitor cells; (ii) harvesting the cells; (iii) administering the cells to a subject; The present invention provides a method comprising:

[0067] In a further embodiment of this process after step (i), the expanded population of stem or progenitor cells from step (i) are differentiated into target cells. The target cells are then harvested and administered to a subject according to steps (ii) and (iii) of the process. Differentiation of the stem or progenitor cells may be achieved by culturing the cells to confluence. Optionally, LF-MF is applied to the cells during differentiation.

[0068] In step (i) of the process, stem or progenitor cells are optionally exposed to LF-MF throughout the culture step. The stem or progenitor cells used in the process may be any of the stem or progenitor cells disclosed herein.

[0069] The in vitro methods for promoting cell proliferation described herein are suitable for use with a wide range of organic cells, including stem cells, progenitor cells, and other primary cells. Therefore, the methods may be suitable for promoting the proliferation of primary cells in vitro, such as epithelial cells, endothelial cells, fibroblasts, melanocytes, keratinocytes, neurons, astrocytes, hepatocytes, skeletal muscle cells, smooth muscle cells, osteoblasts, myocytes, chondrocytes, adipocytes, synoviocytes, hair cells, or blood cells. Primary cells are cells obtained from organ tissue and cultured in vitro in the presence of a modulated LF-MF as described herein to promote cell proliferation and expand the cell population. Primary cells obtained according to the methods can be used in a variety of applications, such as providing cells for use in cell-based assays for drug screening, toxicity assays, or cell biology studies.

[0070] Therefore, there is a great deal of interest in culturing primary cells (or primary tissue cultures) because primary cell cultures are generally considered to be more representative of in vivo cells than immortalized cell lines. However, primary cells can be difficult to culture in vitro because they have limited dividing capacity and often enter senescence after only a few cycles. This is particularly true for primary cells that terminally differentiate into specific tissue types. As shown in the Examples herein, it has been found that the modulated LF-MF described herein can suppress cellular senescence and can also provide other benefits to cell culture, such as reduced cellular stress and / or enhanced cell viability, and therefore may be beneficial for the culture of organic cells in general, and stem cells, progenitor cells, and other primary cells in particular. Therefore, there is also provided an in-vitro method of culturing organic cells (e.g., stem cells, progenitor cells, or other primary cells) by exposing the cells to a LF-MF that is varied in accordance with an amplitude-modulated signal having a carrier frequency of 360 Hz to 450 Hz, wherein the modulation frequency is 0.5 Hz to 100 Hz and the MF has a magnetic field strength of 0.5 μT to 250 μT.

[0071] The in vitro method of culturing cells using the modulated LF-MF described herein can also be useful for culturing cell lines, i.e., immortalized cells. Cells can be immortalized naturally or as a result of genetic modification. Cell lines are well known and include, by way of example and not limitation, mammalian cell lines (e.g., HEK cells, HeLa cells, Namalwa cells, Chinese hamster ovary (CHO) cells and their derivatives (e.g., CHO-K1, CHO DG44, or CHO DXB11), baby hamster kidney (BHK-21) cells, 293 transformed kidney cells, WI-38 (normal human diploid embryonic lung cells), MRC-5 (normal human diploid embryonic lung cells), HepG2 (hepatocellular carcinoma transformed cells), myeloma cells (e.g., NS / O), or Vero cells. Thus, the in vitro method of culturing cells using the modulated LF-MF described herein can also be used to promote the culture and growth of cell lines used in the production of recombinant proteins, viruses, and vaccines.

[0072] In a further aspect of the present invention, a method for promoting in vitro proliferation of human epidermal stem cells or human epidermal progenitor cells is provided by exposing the cells to an LF-MF that varies in accordance with an amplitude-modulated signal having a carrier frequency in the range of 360 Hz to 450 Hz (e.g., 400 Hz to 450 Hz, preferably 432 Hz) and an MF intensity in the range of 0.5 μT to 250 μT (e.g., 10 μT to 200 μT, preferably 200 μT). More specifically, the MF varies in accordance with an amplitude-modulated signal having a carrier frequency in the range of 400 Hz to 450 Hz, a modulation frequency in the range of 0.5 Hz to 100 Hz (e.g., 3 Hz to 30 Hz or 3 Hz to less than 30 Hz, such as the modulation frequency corresponding to the Schumann resonance mode (preferably 7.83 Hz)), and an MF intensity in the range of 0.5 μT to 250 μT (e.g., 0.5 μT to 200 μT or 10 μT to 200 μT).

[0073] Disclosed herein is the use of LF-MF, which is varied according to an amplitude-modulated signal having a carrier frequency in the range of 360 Hz to 450 Hz (e.g., 400 Hz to 450 Hz, preferably 432 Hz) and an MF intensity in the range of 0.5 μT to 250 μT (e.g., 10 μT to 200 μT, preferably 200 μT), to promote in vitro proliferation of human epithelial stem cells and human epithelial progenitor cells by exposing the cells to the LF-MF. For example, cells can be continuously exposed to the LF-MF for a time period of 1 to 8 weeks, such as 4 weeks. More specifically, the MF is varied according to an amplitude-modulated signal having a carrier frequency in the range of 400 Hz to 450 Hz, a modulation frequency in the range of 0.5 Hz to 100 Hz (e.g., 3 Hz to 30 Hz or less), such as a modulation frequency corresponding to the Schumann resonance mode (preferably 7.83 Hz), and an MF intensity in the range of 0.5 μT to 200 μT, e.g., 10 μT to 200 μT. The Examples herein demonstrate that exposing stem and progenitor cells to LF-MF promotes cell proliferation while simultaneously reducing cell stress and / or minimizing cell senescence. These characteristics of LF-MF are expected to be particularly beneficial to the in vitro cell culture methods described herein, for example, by maximizing the proliferation potential of cells, improving cell survival, and / or improving cell viability during the cell culture process.

[0074] In the Examples herein, it has been shown that stem and progenitor cells exposed to the modulated LF-MF according to the present invention exhibit reduced telomere length shortening following multiple cell divisions compared to cells not exposed to the modulated MF. Telomere shortening is associated with cellular senescence, leading to cellular senescence. Cell senescence and the accumulation of senescent cells in the body are associated with several medical diseases, particularly age-related diseases (Zhao et al., Telomere length maintenance, shortening, and lengthening. J Cell Physiol. 229, 1323-1329 (2014)).

[0075] Accordingly, a further aspect provides a method for inhibiting cellular senescence, comprising exposing cells to an LF-MF that varies according to an amplitude-modulated signal having a carrier frequency between 360 Hz and 450 Hz, wherein the modulation frequency is between 0.5 Hz and 100 Hz, and the MF has a magnetic field strength between 0.5 μT and 250 μT.

[0076] The cells can be somatic cells. More specifically, the cells are stem or progenitor cells, such as any of the stem, progenitor, or other primary cells disclosed herein, preferably human stem or progenitor cells. Even more specifically, the cells are skin stem or progenitor cells, such as keratinocyte stem or progenitor cells, preferably human keratinocyte stem or progenitor cells.

[0077] In some embodiments, the method of inhibiting cellular senescence is an in vitro method.

[0078] In some embodiments, the method of inhibiting cellular senescence is an in vivo method performed in a subject. When performed in vivo, at least a portion of the subject's tissue is exposed to LF-MF. For example, the LF-MF can be directed to a specific region of the body or targeted to a specific organ or tissue type, e.g., the LF-MF can be directed to the head, torso, arms, legs, brain, eyes, liver, kidneys, muscle, bone, skin, hair, brain, heart, stomach, intestines, or large intestine.

[0079] In a further embodiment, a method of treating an aging-related disease in a subject is provided, comprising exposing at least a portion of cellular tissue of the subject to an LF-MF that varies according to an amplitude-modulated signal having a carrier frequency between 360 Hz and 450 Hz, wherein the modulation frequency is between 0.5 Hz and 100 Hz, and the MF has a magnetic field strength between 0.5 μT and 250 μT.

[0080] The aging-related disease can be an age-related disease or illness. For example, the aging-related disease can be selected from cardiovascular disease (e.g., associated with arteriosclerosis (e.g., atherosclerosis)), pulmonary disease (e.g., idiopathic pulmonary fibrosis, asthma, chronic obstructive pulmonary disease), osteoarthritis, aging-related skin disease or disorder, neurodegenerative disease (e.g., multiple sclerosis, Alzheimer's disease, Parkinson's disease, Huntington's disease, or motor neuron disease), eye disease (e.g., age-related macular degeneration, glaucoma, diabetic retinopathy, or cataract), diabetes (e.g., pancreatic diabetes), liver disease (e.g., nonalcoholic steatohepatitis (NASH), primary biliary cholangitis (PBC), or primary sclerosing cholangitis (PSC)), sarcopenia, and benign prostatic hyperplasia.

[0081] Guerrio et al., supra, investigated the effects of low-frequency electromagnetic fields on various medical disorders, including autoimmune and immunomodulatory disorders and neurodegenerative disorders. The study also showed that low-frequency electromagnetic fields may be beneficial for the treatment of cognitive disorders, psychiatric disorders, and anti-inflammatory effects in diseases such as rheumatoid arthritis. LF-MFs have been described to have an effect on macrophage activity, which may lead to beneficial immunomodulation in subjects exposed to LF-MFs.

[0082] Thus, there is also provided a system according to the first aspect for use in treating a disease selected from an autoimmune disease, a neurodegenerative disease, an inflammatory disease, a cognitive disorder, and a psychiatric disease in a subject.

[0083] Also provided is a method of treating a disorder selected from an autoimmune disorder, a neurodegenerative disorder, an inflammatory disorder, a cognitive disorder, and a psychiatric disorder in a subject, the method comprising exposing at least a portion of the subject to an LF-MF that varies according to an amplitude-modulated signal having a carrier frequency between 360 Hz and 450 Hz, wherein the modulation frequency is between 0.5 Hz and 100 Hz, and the MF has a magnetic field strength between 0.5 μT and 250 μT.

[0084] There is further provided a use of an LF-MF varied according to an amplitude modulated signal having a carrier frequency of 360 Hz to 450 Hz, for treating a disorder selected from an autoimmune disorder, a neurodegenerative disorder, an inflammatory disorder, a cognitive disorder, and a psychiatric disorder in a subject, wherein the modulation frequency is 0.5 Hz to 100 Hz and the MF has a magnetic field strength of 0.5 μT to 250 μT, the method comprising exposing at least a portion of the subject to the LF-MF.

[0085] Characteristics of modulated low-frequency magnetic fields In any of the systems, devices, uses, and methods described herein, the carrier frequency may be between 360 Hz and 450 Hz, for example, between 365 Hz and 450 Hz, between 370 Hz and 445 Hz, between 390 Hz and 440 Hz, or between 427 Hz and 438 Hz. Preferably, the carrier frequency is 432 Hz.

[0086] In any of the systems, devices, uses, and methods described herein, the modulation frequency may be between 0.5 Hz and 100 Hz. For example, the modulation frequency may be less than 50 Hz or less than 30 Hz. Preferably, the modulation frequency is between 0.5 Hz and 30 Hz, between 0.5 Hz and less than 30 Hz, between 3 Hz and 30 Hz, between 3 Hz and less than 30 Hz, between 3 Hz and 28 Hz, between 6.5 Hz and 11.5 Hz, or between 7 Hz and 10.5 Hz. Preferably, the modulation frequency corresponds to a Schumann resonance mode. Therefore, the modulation frequency may be within the range of 7.50 Hz and 8.00 Hz, such as 7.83 Hz; or within the range of 14.0 Hz and 14.5 Hz, such as 14.1 Hz; or within the range of 20.0 Hz and 21.0 Hz, such as 20.3 Hz; or within the range of 26.0 Hz and 27.5 Hz, such as 26.4 Hz. Therefore, the modulation frequency may be selected from about 7.83 Hz, about 14.1 Hz, about 20.3 Hz, and about 26.4 Hz. Most preferably, the modulation frequency is 7.83 Hz.

[0087] In any of the systems, devices, uses, and methods described herein, the MF has a magnetic field strength between 0.5 μT and 250 μT. Thus, the MF can be less than 230 μT, such as 200 μT or less, 100 μT or less, 50 μT or less, 30 μT or less, 20 μT or less, 15 μT or less, or 11 μT or less. Preferably, the MF is between 1 μT and 250 μT, 5 μT and 250 μT, 5 μT and 220 μT, 5 μT and 200 μT, 10 μT and 200 μT, 15 μT and 200 μT, 25 μT and 200 μT, 25 μT and 200 μT, 20 μT and 120 μT, 20 μT and 100 μT, 20 μT and 50 μT, 20 μT and 40 μT, 0.5 μT and 50 μT, 0.5 μT and 40 μT, 0.5 μT and 30 μT, 0.5 μT and 20 μT, 0.5 μT and 15 μT, 0.5 μT and 11 μT, or 0.5 μT and 12 μT. For example, the MF field strength can be about 200 μT, about 100 μT, about 30 μT, or about 11 μT.

[0088] In any of the systems, devices, uses, and methods described herein, the carrier frequency is preferably 432 Hz. The maximum MF intensity is preferably 200 μT. For example, the MF is preferably converted according to an amplitude modulation signal having a carrier frequency in the range of 400 Hz to 450 Hz and a modulation frequency in the range of 3 Hz to 30 Hz, such as a modulation frequency corresponding to a Schumann resonance mode, for example, in the range of 7.50 Hz to 8.00 Hz, such as 7.83 Hz; or in the range of 14.0 Hz to 14.5 Hz, such as 14.1 Hz; or in the range of 20.0 Hz to 21.0 Hz, such as 20.3 Hz; or in the range of 26.0 Hz to 27.5 Hz, such as 26.4 Hz. The modulation frequency is preferably in the range of 3 Hz to 30 Hz, such as less than 3 Hz, for example, in the range of 7.0 Hz to 28.0 Hz, more preferably 7.83 Hz. The amplitude modulation can be of any waveform type, such as sinusoidal, square, sawtooth, or triangular. However, the modulation frequency is preferably a sinusoidal modulation frequency, more preferably a sinusoidal modulation frequency in the range of 3 Hz to 30 Hz, such as 7.83 Hz.

[0089] In certain embodiments of any of the systems, devices, uses, and methods described herein, the carrier frequency is 427 Hz to 438 Hz, the modulation frequency is 7.50 Hz to 8.00 Hz (preferably, the modulation frequency has a sinusoidal waveform), and the magnetic field strength is 0.5 μT to 250 μT (e.g., the maximum magnetic field is about 200 μT, about 100 μT, about 30 μT, or about 11 μT). Thus, the magnetic field can be 1 μT to about 200 μT, 1 μT to about 100 μT, about 1 μT to 30 μT, or 1 μT to about 11 μT. In certain embodiments of the systems, devices, uses, and methods described herein, the carrier frequency is about 432 Hz, the modulation frequency is about 7.83 Hz (preferably the modulation frequency has a sinusoidal waveform), and the magnetic field strength is between 0.5 μT and 250 μT (e.g., a maximum magnetic field of about 200 μT, about 100 μT, about 30 μT, or about 11 μT). Thus, the magnetic field can be between 1 μT and about 200 μT, between 1 μT and about 100 μT, between about 1 μT and 30 μT, or between 1 μT and about 11 μT.

[0090] In preferred embodiments of any of the systems, devices, uses, and methods described herein, the carrier frequency is 432 Hz and the modulation frequency is about 7.83 Hz. Suitably, in these embodiments, the maximum MF field strength is 200 μT, e.g., 0.5 μT to 200 μT, 0.5 μT to 100 μT, 0.5 μT to 50 μT, 0.5 μT to 30 μT, 0.5 μT to 12 μT, or 0.5 μT to 11 μT, e.g., about 100 μT, about 30 μT, or about 11 μT.

[0091] In any of the embodiments of the systems, devices, uses, and methods described herein, the modulated LF-MF may be applied in a continuous or pulsed manner. When the modulated LF-MF is applied in a pulsed manner, there are distinct periods when the carrier signal and / or modulation signal are not present, and packets or pulses of the modulated LF-MF are applied. For example, the modulated LF-MF may be applied as a pulse followed by a period when the carrier signal and modulation signal are not applied. When the modulated LF-MF is applied continuously, both the carrier signal and modulation signal are applied continuously (i.e., there are no distinct "off" periods of the carrier signal and / or modulation signal). As an example, when the carrier frequency is modulated using sinusoidal modulation, the carrier frequency is sinusoidally modulated to the modulation frequency throughout the entire exposure without any distinct breaks in the applied modulated LF-MF. Figures 6A and 6B illustrate such a continuously modulated LF-MF. In a preferred embodiment, the modulated LF-MF is a continuously modulated LF-MF.

[0092] In any of the systems, devices, uses, and methods described herein, a subject, organic cell, or cellular tissue can be exposed to modulated LF-MF for a time suitable to provide the desired effect. The specific duration and frequency of exposure will depend on the particular situation and the desired effect of the modulated LF-MF. For example, when the system is used in cosmetic or non-therapeutic applications, the subject or sample can be exposed to LF-MF for 5 minutes to 4 hours, e.g., 5 minutes to 2 hours or 10 minutes to 1 hour. When the system is used in therapeutic applications, the subject or a portion of the subject can be exposed to LF-MF substantially continuously (e.g., during wound healing or to promote tissue regeneration). Thus, in therapeutic applications, a subject or area of a subject can be exposed to LF-MF for 1 hour or more, 2 hours or more, 4 hours or more, 8 hours or more, 12 hours or more, 24 hours or more, 1 week or more, 1 month or more, 3 months or more. For example, 1 hour to 3 months or more, 1 day to 3 months, 1 week to 3 months, 1 week to 2 months, or 1 week to 1 month. When the system is used to promote ex vivo expansion of stem or progenitor cells, for example, in cell culture vessels, the cells are preferably exposed to LF-MF substantially continuously throughout the entire process to maximize ex vivo cell expansion. However, shorter exposure times are also contemplated, in which the cells are exposed to LF-MF for only a portion of the ex vivo process, for example, from 10 minutes to 3 months, 12 hours to 1 month, 24 hours to 1 week, 1 day to 3 months, 1 week to 12 weeks, or 1 week to 10 weeks.

[0093] Embodiments of the present invention will now be further described with reference to the accompanying drawings. [Brief explanation of the drawings]

[0094] [Figures 1A-1C] 1A and 1B illustrate schematic diagrams of an embodiment of the MF exposure system disclosed herein in the form of a chair. [Figure 2] 10 shows a schematic diagram of another embodiment of the MF exposure system disclosed herein in the form of a room. [Figure 3] FIG. 1 is a schematic diagram illustrating the circuitry used to generate MF in one embodiment of the MF exposure system disclosed herein. [Figure 4] 10A and 10B schematically illustrate a further embodiment of the MF exposure system disclosed herein in the form of an oval chair. [Figure 5] FIG. 1 presents the three main maturation phases involved in epithelial homeostasis in vivo and in vitro. [Figure 6] 6A and 6B show amplitude modulation carrier frequencies (drive frequencies) such as 432 / 7.83 Hz as described herein, with Figure 6A showing 100% amplitude modulation and Figure 6B showing approximately 80% amplitude modulation. [Figures 7A-7B] Figure 7A shows extrapolated cell numbers on a 106 scale for primary human keratinocyte stem and progenitor cells from juvenile (Figure 7A) and adult (Figure 7B) donors grown in an electromagnetic coil with no MF generation (coil off) or with continuous 432 / 7.83 Hz MF at 200 μT. Cells were continuously exposed during proliferation phase 1 of epithelial homeostasis (see Figure 5) during which they expanded to 80% confluence over 6 passages; subgraphs show expansion scales for each passage from 0 to 1, 1 to 2, 2 to 3, and 4 to 5. [Figure 8A-8B] Relative cell numbers of primary human keratinocyte stem and progenitor cells from juvenile (Fig. 8A) and adult donors (Fig. 8B) exposed to no MF (coil-off) or continuously exposed to 7.83 Hz or 432 / 7.83 Hz MF at 200 μT during expansion to 80% confluence over one passage (i.e., phase 1 in Fig. 5). [Figure 9] Figure 5 shows cumulative cell counts of human keratinocyte primary stem and progenitor cells from a young donor continuously exposed to 432 Hz or 432 / 7.83 Hz MF, with no MF exposure (coil-off), during expansion of 36,000 cells per passage to 80% confluence over six passages (phase 1 in Figure 5). Traces marked with ** are cells exposed to 432 Hz. Traces marked with *** are cells exposed to 432 / 7.83 Hz MF. [Figures 10A-10F]The effects of various (10A, 10B) modulation frequencies, (10C, 10D) driver frequencies, and (10E, 10F) magnetic field strengths on the proliferation (phase 1 in Figure 5) of continuously exposed juvenile and adult primary stem and progenitor cells are compared, respectively. Figures 10A and 10B show cells exposed to no MF (coil off), 432 / 7.83 Hz, 432 / 30 Hz, 432 / 50 Hz, and 432 / 100 Hz, respectively. Figure 10 Figures 10C and 10D show the effects of various driver frequencies on cells not exposed to MF (coil off), 368 / 7.83 Hz, 400 / 7.83 Hz, 432 / 7.83 Hz, and 464 / 7.83 Hz, and Figures 10E and 10F show the effect of magnetic field strength on cells not exposed to MF (coil off) or exposed to 368 / 7.83 Hz MF at 10 μT, 30 μT, 200 μT, or 250 μT. [Figures 11A-11B] Relative cell death counts are shown for primary human keratinocyte stem and progenitor cells from juvenile (FIG. 11A) and adult (FIG. 11B) donors exposed to no MF (coil-off) or continuous 7.83 Hz or 432 / 7.83 Hz MF during growth to 80% confluence at passage 1 (phase 1 in FIG. 5). [Figures 12A-12B] Figure 12A shows contact inhibition and turnover of primary stem and progenitor human keratinocytes from juvenile (Figure 12A) and adult (Figure 12B) donors continuously exposed to no MF (coil-off) or 7.83 Hz or 432 / 7.83 Hz MF during all three metabolic phases (see Figure 5) from passage 1 to 4. [Figure 13] Figure 1 shows the relative shortening of telomere length (readout for senescence) (mean ratio of ddCt values) over six passages in primary human keratinocyte stem and progenitor cells from young donors exposed to no MF (coil-off) or 432 / 7.83 Hz MF. [Figures 14A-14B]Relative protein levels of heat shock protein 47 (Hsp47) (Figure 14A) and Hsp90 (Figure 14B), normalized to tubulin, are shown for human keratinocyte primary stem and progenitor cells from young donors exposed to 432 / 7.83 Hz MF or no MF (coil-off) in fully supplemented medium or starvation medium composed of 25% median / 75% PBS. [Figure 15] 1 shows one embodiment of the MF exposure system disclosed herein in the form of a chair used in the EEG pilot study described in the Examples. [Figures 16A-16B] Figure 16 shows results from a blind crossover pilot study of EEG measured in four individuals over 18 sessions showing differences primarily in the alpha band at the parent's occipital scalp in the eyes-open (EC) and eyes-open (EO) conditions under the true (432 / 7.83 Hz at 30 μT) condition compared to the sham (no MF) condition. (Figure 16A) is a graphical representation of the mean power in each frequency band under the sham condition overlaid with the true (solid curve) condition. (Figure 16B) is a t-map of the changes in the low and high alpha bands and beta 1 bands across the scalp (prefrontal cortex toward the top of the page; occipital cortex toward the bottom of the page). Levels above 1 and below -1 were calculated using the MATLAB add-on "Randomization Graphical User Interface" (Ragu). (Habermann, M., Weusmann, D., Stein, M. & Koenig, TA Student's Guide to Randomization Statistics for Multichannel Event-Related Potentials Using Ragu. Front Neurosci 12,355,doi:10.3389 / fnins.2018.00355(2018)). DETAILED DESCRIPTION OF THE INVENTION

[0095] As used herein, the term "subject" refers to a human or an animal (e.g., a non-human primate; a domestic animal or livestock animal such as a dog, horse, cat, cow, sheep, mouse, rat, or rabbit). Preferably, the subject is a human.

[0096] As used herein, the term "healthy subject" means a subject (e.g., a human) who is free of any clinical signs of physical disease or infection.

[0097] The term "stem cell" is used herein to refer to a cell that has both the capacity for self-renewal and the ability to generate differentiated progeny (see Morrison et al. (1997) Cell 88:287-298). Generally, stem cells also have one or more of the following properties: asynchronous or asymmetric replication, i.e., the ability of two daughter cells after division to have different phenotypes; broad self-renewal capacity; the ability to exist in a mitotically quiescent form; and the ability of hematopoietic stem cells to reconstitute all hematopoietic lineages in all tissues in which they reside. "Progenitor cells" differ from stem cells in that they typically do not have broad self-renewal capacity and are often capable of regenerating only a subset of the lineages of the tissue from which they originate. In certain embodiments, the stem cells are multipotent stem cells, which include adult stem cells that can differentiate into specific cell types, including, for example, hematopoietic stem cells, mesenchymal stem cells, and neural stem cells. In certain embodiments, the stem cells are pluripotent stem cells, i.e., cells that can differentiate into any type of cell, including embryonic stem cells, perinatal stem cells (umbilical cord stem cells), and iPS cells (iPSCs). In certain embodiments, the stem cells are multipotent stem cells or iPS cells. In certain embodiments, the stem cells are not embryonic stem cells.

[0098] The stem or progenitor cells may be mammalian stem or progenitor cells, where the term "mammal" refers to any animal classified as a mammal, including humans, non-human primates, domestic animals, and livestock (e.g., dogs, cats, horses, cows, sheep, mice, rats, or rabbits). Preferably, the stem cells are human stem cells.

[0099] Stem cells include adult stem cells. Adult stem cells are also called somatic stem cells or tissue stem cells. Adult stem cells reside in differentiated tissues but retain the ability to self-renew and give rise to multiple cell types, usually cell types typical of the tissue in which they are found. Many examples of somatic stem cells are known to those skilled in the art, including muscle stem cells, hematopoietic stem cells, epithelial stem cells, neural stem cells, mesenchymal stem cells, mammary stem cells, intestinal stem cells, mesodermal stem cells, endothelial stem cells, olfactory stem cells, dental pulp stem cells, or neural crest stem cells.

[0100] In some embodiments, the stem cells are hematopoietic stem cells (HSCs). HSCs are mesodermally derived cells that can be isolated from bone marrow, blood, umbilical cord blood, fetal liver, and yolk sac. HSCs can repopulate erythroid, neutrophil-macrophage, megakaryocytic, and lymphoid vascular cell lineages in vivo. In vitro, HSCs can be induced to undergo at least some self-renewal cell differentiation and can be induced to differentiate into the same lineages found in vivo. Thus, HSCs can be induced to divide into one or more of erythroid cells, megakaryocytes, neutrophils, macrophages, and lymphoid cells.

[0101] In some other embodiments, the stem cells are neural stem cells (NSCs). Neural stem cells (NSCs) are capable of differentiating into neurons and glial cells (including oligodendrocytes and astrocytes). Neural stem cells are multipotent stem cells capable of multiple divisions and, under certain conditions, can generate daughter cells that are neural stem cells or neural progenitor cells, which can be neuroblasts or glioblasts, e.g., cells committed to becoming one or more types of neurons and glioblasts, respectively.

[0102] In other embodiments, the stem cells are mesenchymal stem cells (MSCs). Originally derived from embryonic mesoderm and isolated from adult bone marrow, MSCs can differentiate to form muscle, bone, cartilage, fat, marrow matrix, and tendon.

[0103] In a preferred embodiment, the stem cells are keratinocyte stem cells.

[0104] In certain embodiments, the stem cells are aged adult stem cells. For example, aged adult stem cells are adult stem cells obtained from or present in a human individual, for example, over 30 years old, over 40 years old, over 50 years old, over 60 years old, or over 70 years old. For example, aged adult stem cells obtained from or present in a human individual between the ages of 30 and 70 years old.

[0105] In certain embodiments, the stem cells are young adult stem cells, e.g., adult stem cells obtained from or present in a human individual aged less than 10 years or less than 6 years, e.g., 0 months to 10 years or 1 month to 6 years.

[0106] Reference to "primary cells" refers to cells obtained from a subject. Examples of primary cells include stem or progenitor cells of epithelial cells, endothelial cells, fibroblasts, melanocytes, keratinocytes, neurons, astrocytes, hepatocytes, skeletal muscle cells, smooth muscle cells, osteoblasts, myocytes, chondrocytes, adipocytes, synovial cells, hair cells, and blood cells. Primary cells may be obtained using well-known methods, such as through tissue biopsy followed by separation / isolation of the primary cells of interest. Cell separation and isolation methods are well-known and include immunomagnetic cell separation, fluorescence-activated cell sorting, centrifugation methods (e.g., density gradient centrifugation, immunodensity cell separation, cell sedimentation, cell adhesion methods, or microfluidic cell separation).

[0107] As used herein, the terms "treatment," "treating," and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, meaning that a disease or its symptoms are completely or partially prevented, and / or may be therapeutic, meaning that the disease and / or its associated adverse effects are partially or completely cured. "Treatment," as used herein, encompasses any treatment of a disease in a mammal, e.g., a human, including (a) preventing the onset of the disease in a subject who may be predisposed to the disease but has not yet been diagnosed with the disease, (b) suppressing the disease, i.e., preventing the onset of the disease, and (c) remission of the disease, i.e., causing regression of the disease.

[0108] Reference to a condition being "associated with" stem cells or progenitor cells means that the condition is caused directly or indirectly by stem cells or progenitor cells, e.g., the disease or condition results from or is alleviated by decreased stem cell proliferation, stem cell or progenitor cell senescence, stem cell or progenitor cell stress, stem cell or progenitor cell aging, and / or decreased stem cell or progenitor cell differentiation.

[0109] Unless otherwise specified, references herein to "magnetic field strength," "MF strength," "magnetic field intensity," or "field strength" refer to the root mean square (rms) of the magnetic flux intensity (B) generated by the systems described herein, which is a vector quantity that determines the force on one or more moving charges (currents). Magnetic flux density is expressed in Tesla (T).

[0110] The term "about" as used herein refers to + / -10% of the cited number.

[0111] Throughout the description and claims of this specification, the words "comprise" and "include" and variations thereof mean "including, but not limited to" and are not intended to (and do not) exclude other elements, integers, or steps.

[0112] Throughout the description and claims of this specification, the singular includes the plural unless the context requires otherwise. In particular, where the indefinite article is used, the designation is to be understood as including the plural and the singular unless the context requires otherwise.

[0113] It is to be understood that any feature, integer, characteristic, or group described in connection with a particular aspect, embodiment, or example of the invention is applicable to any other aspect, embodiment, or example described herein, except where incompatible. All of the features disclosed in this specification (including any accompanying claims, abstract, and drawings) and / or all of the steps of any method or process so disclosed may be combined in any combination, except combinations in which at least some of such features and / or steps are mutually exclusive. The invention is not limited to the details of any of the above embodiments. The invention extends to any novel one or any novel combination of features disclosed in this specification (including any accompanying claims, abstract, and drawings), or to any novel one or any novel combination of steps of any method or process so disclosed.

[0114] The reader's attention is directed to all articles and documents in connection with this application that have been filed contemporaneously with or prior to this application and that are open to public inspection along with this application, the entire contents of which are incorporated herein by reference.

[0115] Magnetic Field Exposure System Disclosed herein is an LF-MF exposure system for use in exposing at least a portion of an organic cell, tissue, or subject to LF-MF. For example, the system can be a ring or cylinder, or a chair, room, or chamber configured to surround, for example, the arm or leg of the subject to be exposed.

[0116] The system may include a magnetic field generator having one or more coils configured to generate a magnetic field that varies according to an amplitude-modulated signal having a carrier frequency in the range of 360 Hz to 450 Hz (e.g., 400 Hz to 450 Hz), the magnetic field having a maximum magnetic field strength in the range of 0.5 μT to 250 μT (e.g., 10 μT to 200 μT). The magnetic field generator may include a generator and amplifier supplying the one or more coils configured to generate a magnetic field that varies according to an amplitude-modulated signal having a carrier frequency in the range of 360 Hz to 450 Hz (e.g., 400 Hz to 450 Hz) and a magnetic field strength in the range of 0.5 μT to 250 μT (e.g., 0.5 μT to 200 μT or 10 μT to 200 μT). The amplitude-modulated magnetic field has a modulation frequency of 0.5 Hz to 100 Hz, e.g., 3 Hz to less than 30 Hz, or 3 Hz to 28 Hz.

[0117] In a preferred embodiment, the system comprises a magnetic field generator comprising a generator and amplifier supplying one or more coils configured to generate a magnetic field that varies according to an amplitude-modulated signal having a carrier frequency in the range of 400 Hz to 450 Hz, a modulation frequency corresponding to a Schumann resonance mode (preferably 7.83 Hz), and a magnetic field strength in the range of 0.5 μT to 250 μT (e.g., 0.5 μT to 200 μT or 10 μT to 200 μT).

[0118] One or more coils of the MF exposure system may be configured to at least partially surround a subject exposed to the LF-MF.

[0119] The coil or coils of the MF exposure system may be one or more copper coils.

[0120] The size, shape, and type of coil system can be determined according to the biological system to be exposed. Circular coil systems, such as Helmholtz coils, can be used to generate MF over a small volume, while rectangular coil systems, such as Merritt and Ruben coils, can generate MF over a larger volume, for example, for whole-body exposure or exposure in a room or chamber. For whole-body exposure, it is preferable that the generated MF be substantially uniform.

[0121] Many coil configurations and geometries can be used to generate modulated LF-MF. The specific coil geometry used depends on the desired application and the magnitude of the LF-MF required. Design and optimization of the coil geometry and orientation can be performed using known coil configurations and methods (e.g., as described in Kirschvink JL, Uniform magnetic fields and double-wrapped coil systems: improved techniques for the design of bioelectromagnetic experiments. Bioelectromagnetics 13:401-411). In certain embodiments, the coils are Helmholtz coils, in which two or more coils of substantially the same diameter are spaced coaxially. In other embodiments, Merritt coils can be used, in which rectangular coils are arranged to provide a uniform magnetic field within the coil (Merritt et al., (1983) Uniform magnetic field produced by three, four, and five square coils. Rev Sci Instrum 54:879-882). For example, Merritt coils can be arranged orthogonally, with, for example, two to five (or six or more) coils spaced axially from one another. This arrangement provides an interior volume defined by the coil in which the modulated LF magnetic field produced by current passing through the coil is substantially uniform.

[0122] In certain embodiments, one or more coils are Helmholtz coils. A Helmholtz coil comprises two substantially identical circular coils spaced apart on the same axis, with the coil spacing being approximately the same as the radius of the coils. The coils are supplied with the same input current, which results in the generation of a substantially uniform magnetic field in the space between the two coils. The use of Helmholtz coils in the system allows a subject (or a portion of a subject) or a sample (e.g., cells) to be positioned in the space between the coils such that the subject or sample is exposed to a substantially uniform magnetic field.

[0123] The maximum MF intensity generated by the MF generator of the MF exposure system can be set within acceptable limits according to nationally recommended exposure levels, for example, as described in the 1998 and 2010 guidelines of the International Commission on Non-Ionizing Radiation Protection (ICNIRP). Preferably, the MF intensity of the LF-MF when the system is used to expose human subjects to modulated LF-MF is in the range of 0.5 μT to 200 μT, e.g., 1 μT to 200 μT, 5 μT to 200 μT, 10 μT to 200 μT, 25 μT to 200 μT, 50 μT to 200 μT, 100 μT to 200 μT, 150 μT to 200 μT, 1 μT to 40 μT, 1 μT to 20 μT, 1 μT to 15 μM, or 1 μT to 11 μT. In certain embodiments, the maximum magnetic field strength of the LF-MF is 200 μT. In other embodiments, the maximum magnetic field strength is lower, e.g., about 100 μT, about 30 μT, about 30 μT, about 20 μT, about 15 μT, about 12 μT, or about 11 μT. When the system is used, for example, to expose cells ex-vivo to modulated LF-MF (e.g., to promote cell growth in vitro), higher maximum magnetic field strengths can be used, e.g., any of the magnetic field strengths disclosed herein up to 250 μT. Maximum magnetic field strength refers to the root mean square (rms) magnetic field strength. Magnetic field strength can be determined using known methods, e.g., using a suitable Gaussmeter.

[0124] References herein to modulated LF-MF refer to the modulated magnetic field generated by a coil when a time-varying current is passed through the coil. Therefore, references herein to magnetic field strength refer to the magnetic field generated by the current passing through the coil of the systems described herein. As one skilled in the art will appreciate, other electric and magnetic fields may also be present, for example, as a result of the Earth's magnetic field and background fields from electrical equipment. In certain embodiments, the effects of external electric and / or magnetic background may be reduced or eliminated, for example, by placing the system within a Faraday cage and / or using a mu-metal layer surrounding the system to block unwanted external electric and / or magnetic background.

[0125] The MF exposure systems disclosed herein may be configured to expose at least 50%, e.g., 50-75%, of a subject's body surface to the MF. For example, the MF exposure system may be configured to expose the subject's upper body, including the head and torso, to the MF. In some embodiments, the MF exposure system may be configured for full-body exposure of the subject. In such embodiments, the MF exposure system provides cranial and peripheral MF exposure of the subject.

[0126] Alternatively, the MF exposure systems disclosed herein may be configured to provide MF exposure of only the subject's skull, in such embodiments, only brain regions of the subject are exposed to the MFs.

[0127] Further alternatively, the MF exposure systems disclosed herein may be configured to provide only peripheral (transcutaneous) MF exposure in a subject. In such embodiments, the subject's brain is not exposed to the MF.

[0128] 1A illustrates an MF exposure system according to one embodiment of the present invention. In the illustrated embodiment, the MF exposure system comprises a chair 100A having a backrest 102 with a coil 104 integrated into the backrest 102. The coil 104 is electrically connected to a driver circuit 300, which includes a generator and an amplifier, as shown in FIG. 3, and is configured to apply a time-varying current to the coil 104.

[0129] As will be appreciated by those skilled in the art, when a current passes through the coil 104, an MF is generated in and around the coil with a strength proportional to the magnitude of the current. Thus, a time-varying current passing through the coil generates a time-varying MF.

[0130] In particular, coil 104 is positioned so that current passing through coil 104 generates an MF in and around region 106A where at least a portion of the subject's head is likely to be located when the subject is seated in chair 100.

[0131] Figure 1B shows a chair 100B in an embodiment similar to that shown in Figure 1A. However, in Figure 1B, three separate coils 104 are provided within the backrest 102. In this way, the region 106B in which MFs are generated is expanded to include, for example, the region where a subject's head and torso are likely to be located when the subject is seated in the chair 100B.

[0132] 1C shows a chair 100C in a further embodiment of the present invention, in which coils 104 are positioned in the seat 108 and top 110 of the chair 100C. In this way, a substantially uniform MF can be generated in region 106C, which is likely to include the entire head and torso of a subject seated in the chair 100C.

[0133] In particular, at least a portion (e.g., head and / or torso) of a subject seated in any of chairs 100A, 100B, or 100C is exposed to MFs that vary in accordance with an amplitude-modulated signal having a carrier frequency within a range defined herein, e.g., 400 Hz to 450 Hz (e.g., 432 Hz), a modulation frequency corresponding to a Schumann resonance mode (e.g., 7.83 Hz), and an MF intensity within a range of 10 μT to 200 μT. More specifically, at least a portion (e.g., head and / or torso) of a subject seated in any of chairs 100A, 100B, or 100C is exposed to MFs that vary in accordance with an amplitude-modulated signal having a carrier frequency within a range of 400 Hz to 450 Hz (e.g., 432 Hz), a modulation frequency corresponding to a Schumann resonance mode (e.g., 7.83 Hz), and an MF intensity within a range of 0.5 μT to 250 μT (e.g., 10 μT to 200 μT).

[0134] While the chair 100C shown in FIG. 1C is shown with a separate upper portion 110, it will be understood that in some embodiments, side walls may be provided to allow at least a portion of a subject to be positioned within a cavity defined by the backrest 102, seat portion 108, upper portion 110, and side walls (not shown). Coils may be positioned on the side walls and / or backrest in addition to or as an alternative to the coils positioned in the seat portion 108 and upper portion 110. The system in FIG. 15 shows a seat portion 1801 positioned within a cavity formed between two side walls 1802. Circular coils 1803 of the same diameter are positioned on each side wall 1802, with the coils 1803 arranged coaxially, thereby forming Helmholtz coils. The coils 1803 are electrically connected to a drive circuit 300, as shown in FIG. 3, which is configured to pass a time-varying current through the coils 1803. When a current passes through the coils 1803, a substantially uniform magnetic field is generated in the space between the two coils 1803. In use, when a subject sits in chair 1801, the subject is exposed to the modulated LF-MF generated between coils 1803. The size of the coils can be configured to expose only parts of the body, such as the head or torso. Alternatively, larger coils may be used to expose substantially all of the subject to the modulated LF-MF.

[0135] Furthermore, in some embodiments, the cavity in which at least a portion of the subject may be placed may be defined by a single curved wall with one or more coils disposed therein, and a chair with such a cavity may be referred to as an Eggpod chair.

[0136] In one embodiment, the system is a chair with a hollow oval body forming a seat and a backrest, and one or more coils are integrated into the hollow oval body of the chair to generate the LF-MF.

[0137] FIG. 4 shows a chair 400 according to a further embodiment of the present invention. The chair 400 comprises a hollow oval-shaped body forming a seat 408 and a backrest 410, within which are integrated a plurality of coils 404. In this manner, a substantially uniform MF is generated in an area 406 that should include the entire head and torso of a subject sitting in the chair 400. The chair in FIG. 4 may comprise a plurality of copper coils 404. The plurality of coils 404 are electrically connected to a driver circuit 300 as shown in FIG. 3, which is configured to pass a time-varying current through the coils 404.

[0138] In some embodiments, the portion of a chair (e.g., the Eggpod chair shown in FIG. 4, the chair 100C shown in FIG. 1C, or the chair shown in FIG. 15) that defines a cavity in which a user can sit may be provided with an outer lining that shields the user from ambient EMFs, thereby ensuring that the user experiences only well-defined stimuli generated by the chair 100C or 400 while using the chair. Such a lining may be formed, for example, of mu-metal, a nickel-iron soft ferromagnetic alloy used to shield sensitive electronic devices from static or low-frequency MFs. An exemplary composition includes 77% nickel, 16% iron, 5% copper, and 2% chromium or molybdenum.

[0139] Thus, in a further embodiment, the system is a chair comprising a hollow oval body forming a seat and a backrest, wherein one or more coils are integrated into the hollow oval body of the chair to generate the LF-MF, and wherein the hollow oval body of the chair is electromagnetically shielded.

[0140] In a further embodiment, the system is a chair comprising a hollow oval body forming a seat and a backrest, wherein one or more coils are integrated into the hollow oval body of the chair to generate the LF-MF, and the hollow oval body has a lining formed of mu-metal.

[0141] In another embodiment, the system is a chair with a hollow oval body forming a seat and a backrest, and at least two orthogonal copper coils are integrated into the hollow oval body of the chair to generate LF-MF. The hollow oval body of the chair may be electromagnetically shielded, and in particular the oval body may have a lining made of mu metal.

[0142] In a further embodiment, the system is a chair comprising a hollow oval body forming a seat and a backrest, wherein a first plurality of copper coils (e.g., 2-5 copper coils) and a second plurality of copper coils (e.g., 2-5 copper coils) are integrated into the hollow oval body of the chair, the first plurality of copper coils extending orthogonally to the second plurality of copper coils. The hollow oval body of the chair may be electromagnetically shielded, in particular the oval body may have a lining made of mu-metal.

[0143] 2 shows a room (or chamber) 200 in a building in a further embodiment of the present invention. Side walls 202, 204 of room 200 each include a plurality of coils 206 such that when a time-varying current passes through the coils 206, a corresponding time-varying MF is generated within room 200. When a time-varying current passes through all coils 206 simultaneously, the corresponding time-varying MF can substantially fill room 200.

[0144] The coils may be arranged so that the direction of MF through all coils at a given time is the same (i.e., vertically up or down), thereby reducing spatial variation of MF. A subject in room or chamber 200 is exposed to MFs that vary according to an amplitude-modulated signal having a carrier frequency within a range described herein (e.g., 400 Hz to 450 Hz (e.g., 432 Hz)) and an MF intensity within a range of 0.5 μT to 250 μT (e.g., 10 μT to 200 μT). In particular, a subject in room or chamber 200 is exposed to MFs that vary according to an amplitude-modulated signal having a carrier frequency within a range of 400 Hz to 450 Hz (e.g., 432 Hz), a modulation frequency corresponding to a Schumann resonance mode (e.g., 7.83 Hz), and an MF intensity within a range of 10 μT to 200 μT. More specifically, a subject in room or chamber 200 may be exposed to a homogeneous MF that varies according to an amplitude-modulated signal having a carrier frequency in the range of 400 Hz to 450 Hz (e.g., 432 Hz), a modulation frequency corresponding to the Schumann resonance mode (e.g., 7.83 Hz), and an MF strength in the range of 0.5 μT to 250 μT (e.g., 10 μT to 200 μT).

[0145] The walls, floor, and ceiling of room 200 may all be provided with a shielding outer layer to block interference from ambient EMF originating outside the room. Again, the shielding layer may be formed from mu-metal.

[0146] 1 and 2 show one or more coils distributed around a target area where a subject is to be placed for MF exposure, it will be understood that other configurations are possible. For example, in some embodiments, a single large coil may surround the target area such that all or part of the subject may be placed within the coil. This may be particularly useful when the MF exposure system includes a room in which the subject may be exposed to MF or a chair (e.g., an egg pod chair) that at least partially surrounds the subject.

[0147] In a further embodiment, the system comprises a horizontal platform (e.g., a bed) and one or more coils surrounding the platform such that when a time-varying current is passed through the coils, a corresponding time-varying MF is generated around the horizontal platform. In use, when a subject lies on the bed, at least a portion of the subject is exposed to the modulated LF-MF. The system may be configured such that the multiple coils surround the horizontal platform. For example, the multiple coils (e.g., circular coils) surrounding the horizontal platform may be coaxially arranged along the horizontal axis of the platform, thereby exposing at least a portion, or preferably the entire body, of a subject lying on the platform to the LF-MF. In a further embodiment, the system comprises a horizontal platform (e.g., a bed) and one or more coils arranged above and / or below the platform.

[0148] To expose smaller areas of the body, the system may include a housing with one or more coils disposed within the housing such that a time-varying current is passed through the coil to generate a corresponding time-varying MF. The housing can then be positioned to expose specific body parts or tissues of the subject to the LF-MF. The housing may be handheld so that the LF-MF can be manually positioned to direct the LF-MF to a target area of the subject. Alternatively, the housing may be or include a frame that can be moved to direct the LF-MF to specific parts of the subject.

[0149] In a further embodiment, the system is in the form of a cell culture system, said system comprising: a cell culture vessel defining a cell culture cavity within the vessel; and a MF generator having one or more coils configured to generate a MF within the cell culture cavity that varies in accordance with an amplitude modulated signal having a carrier frequency between 360 Hz and 450 Hz, wherein the modulation frequency is between 0.5 Hz and 100 Hz, and the MF has a magnetic field strength between 0.5 μT and 250 μT.

[0150] A cell culture cavity is a volume within a cell culture vessel suitable for containing cells to be grown (cultured), such that, in use, cells contained within the cell culture cavity are exposed to the modulated LF-MF described herein.

[0151] The cell culture vessel can be any vessel suitable for culturing cells, such as stem and progenitor cells. For example, the vessel can be a Petri dish, flask, multiwell plate (e.g., 96-well plate), bottle, conical tube, stacked culture vessel, 2D tissue culture vessel, or 3D suspension culture vessel, or a larger-scale component reaction vessel. One or more coils are positioned such that, when an electric current is applied to the coil, an LF-MF is generated in the cell culture cavity. In use, cells are contained within the cell culture cavity and exposed to the LF-MF. In certain embodiments, a pair of coils can be coaxially spaced apart (e.g., as Helmholtz coils), and the reaction vessel is positioned between the coils such that, in use, when an electric current is passed through the coil, the cell culture cavity within the cell culture vessel is exposed to a substantially uniform LF-MF. In further embodiments, the one or more coils are attached to the wall of the cell culture vessel and configured to generate an LF-MF in the cell culture cavity. The one or more coils can be integrated into the wall of the cell culture vessel. In a further embodiment, the cell culture system further comprises one or more organic cells (e.g., one or more stem cells, progenitor cells, or primary cells) within the cell culture cavity, and optionally further comprises cell culture medium in the cell culture cavity.

[0152] 3 shows a schematic representation of an MF generating circuit 300 arranged to generate a time-varying MF. The illustrated circuit comprises multiple coils 302 arranged in series and connected to a generator 306, and an amplifier 304 that controls the electrical power and frequency to the coils. A microcontroller 308 may be included to regulate use. It will be appreciated that the coils may be equally arranged in parallel or in any other suitable circuit arrangement.

[0153] The generator 306 is fed via a power supply 310, the output frequency of which is controlled by an amplifier 304. The power supply 310 can be a DC power source, such as a battery, or an AC power source, such as a mains connection.

[0154] Generator 306, under the control of amplifier 304, is operable to output a predefined, constant, time-varying voltage or current signal. In particular, generator 306 and amplifier 304 may be configured to output an amplitude-modulated AC signal having a carrier frequency (e.g., 400 Hz-450 Hz) and a sinusoidal modulation frequency as described herein, as shown in FIG. 6. As described in more detail below with respect to Example 1, particularly beneficial effects have been observed in vitro when cells are exposed to a time-varying MF corresponding to an 80% to 100% amplitude-modulated signal, as shown in FIGS. 6A and 6B. In particular, such beneficial effects have been observed when the amplitude is modulated at 100%, the modulating signal is a sine wave having a modulation frequency corresponding to a Schumann resonance mode, such as 7.83 Hz, and the carrier signal has a frequency of 400 Hz-450 Hz, e.g., 432 Hz.

[0155] The first Schumann resonance mode occurs at approximately 7.83 Hz, although any frequency within the range of approximately 7.0-10.0 Hz, particularly 7.5-8.0 Hz, may be considered to correspond to the first Schumann resonance within the scope of the present disclosure. Additional Schumann resonance frequencies occur within at least the following frequency ranges: 14.0 Hz to 14.5 Hz, particularly 14.1 Hz; 20.0 Hz to 21.0 Hz, particularly 20.3 Hz; and 26.0 Hz to 27.5 Hz, particularly 26.4 Hz.

[0156] In some embodiments, the MF may be substantially continuously variable, and therefore the voltage or current signal is also generally non-pulsed and continuously variable.

[0157] In addition to determining the current or voltage waveform, the amplifier 304 can also be configured to determine the length of time the system exposes the subject to the time-varying MF. For example, the MF may be generated only during a user-defined time period each day, such as a 15-minute to 1-hour time period per day. Alternatively, the system may be configured to generate the MF substantially continuously over a predetermined time period.

[0158] The respective amplitudes of the carrier signal and the amplitude modulation (also called amplitude variation) signal may be selected to generate a magnetic field having a maximum MF strength or flux density of 200 μT in accordance with ICNIRP Recommendation 2010 in the target region where the subject is expected to be located (e.g., regions 106A-C shown in FIGS. 1A-1C). In some embodiments, as described herein, lower maximum magnetic field strengths may be used, e.g., maximum magnetic fields of 100 μT, 50 μT, 30 μT, or 11 μT. The magnitude of the current or voltage signal required to generate such a MF is empirically determined for a particular configuration of coils and frequency and final MF as confirmed by a gaussmeter. Furthermore, the position of the coil may be empirically determined and confirmed by control means to generate a substantially homogeneous MF strength in the target region.

[0159] In an embodiment, the amplitudes of the amplitude modulation signal and the carrier signal may be selected such that the peak amplitude of the amplitude modulation signal is between 0% and 100% of the maximum amplitude, as shown in FIG. 6. Preferably, however, the peak amplitude is at least four times the minimum amplitude. Preferably, the modulation index (i.e., modulation signal amplitude / carrier signal amplitude) is between 1 (i.e., 100% modulation) and 0.6 (60% modulation), and preferably, the modulation index is between 80% and 100%, more preferably between 90% and 100%, and most preferably about 100%. As will be appreciated by those skilled in the art, an amplitude modulation signal is generated by modulating a carrier signal (generally a sinusoidal waveform) with the amplitude modulation signal, thereby generating a waveform similar to that shown in FIG. 6.

[0160] An MF exposure system disclosed herein, such as the chair shown in any one of Figures 1A-1C, 4, or 15, the room or chamber shown in Figure 2, or a bed, can be used to expose at least a portion of a subject to low-frequency MFs as disclosed herein that vary according to an amplitude-modulated signal having a carrier frequency in the range of 400 Hz to 450 Hz and an MF intensity in the range of 0.5 μT to 200 μT (e.g., 10 μT to 200 μT). More specifically, the room or chamber shown in Figure 2 can be used to expose at least a portion of a subject to low-frequency MFs as disclosed herein that vary according to an amplitude-modulated signal having a carrier frequency in the range of 400 Hz to 450 Hz, a modulation frequency in the range of 3 Hz to 30 Hz, such as a modulation frequency corresponding to a Schumann resonance mode, and an MF intensity in the range of 0.5 μT to 200 μT (e.g., 10 μT to 200 μT).

[0161] The Examples herein demonstrate that the low-frequency modulated MFs described herein promote the proliferation and survival of stem and progenitor cells (e.g., human epithelial stem and progenitor cells). Thus, the MF exposure systems disclosed herein are believed to be useful for reducing skin aging, promoting tissue regeneration, promoting wound healing, treating cancer, and / or enhancing immune function in a subject by exposing at least a portion of the subject to said low-frequency MFs.

[0162] A low frequency modulated magnetic field (LF-MF) exposure system may be used in any of the methods or uses described herein. The system may be for use in promoting in vitro proliferation of stem and progenitor cells or other primary cells. The system may be for use in promoting in vivo proliferation of stem and progenitor cells. The system may be for use in cosmetic treatment of a condition associated with skin stem cells or skin progenitor cells in a subject. The system may be for use in reducing skin aging. The system may be for use in treating a medical disorder associated with stem or progenitor cells in a subject. The system may be for use in promoting tissue regeneration. The system may be for use in promoting wound healing. The system may be for use in promoting tissue regeneration. The system may be for use in preventing or treating cancer. The system may be for use in a method of inhibiting cellular senescence. The system may be for use in a method of treating an aging-related disorder in a subject. The system may be for use in treating a disorder selected from an autoimmune disorder, a neurodegenerative disorder, an inflammatory disorder, a cognitive disorder, and a psychiatric disorder.

[0163] The systems, methods, and uses described herein provide non-invasive methods and uses for the treatment or prevention of therapeutic and non-therapeutic diseases, including those described herein, by exposing at least a portion of a subject to modulated LF-MF.

[0164] Cell proliferation The MF exposure systems, methods, and uses disclosed herein are useful for promoting the in vitro proliferation of organic cells, particularly stem, progenitor, or other primary cells, more particularly human epithelial stem and progenitor cells, by exposing the cells to the above-described LF-MF.

[0165] The proliferation and differentiation of stem and progenitor cells are crucial for tissue homeostasis and tissue regeneration, for example, during injury or wound healing. Stem / progenitor cell activation, proliferation, maintenance, and differentiation can be dysregulated in certain diseases and conditions, particularly age-related conditions. The modulated LF-MFs described herein provide beneficial effects to stem and progenitor cells, for example, by promoting proliferation, reducing stem / progenitor cell senescence, and / or reducing stem / progenitor cell stress. Thus, exposing stem and progenitor cells to the modulated LF-MFs described herein is expected to be beneficial in many therapeutic and non-therapeutic / cosmetic applications, including those described above in the overview of the present invention. In particular, the modulated LF-MFs are expected to be beneficial in treating or preventing disorders associated with keratinocyte stem cells and keratinocyte progenitor cells.

[0166] wound healing The systems, methods and uses of modulated LF-MF described herein are expected to be beneficial in wound healing.

[0167] Wound healing is a complex and dynamic process involving, among other parameters, the interaction of dermal and epithelial cells. Shortly after wounding, typically 2 to 10 days after wounding, keratinocyte proliferation increases, and the formation of a continuous keratinocyte monolayer is rate-limiting for successful wound healing (Harding, BMJ. 2002 Jan 19;324(7330):160-163). Therefore, increased proliferation of keratinocyte stem and progenitor cells exposed to the modulated LF-MF described herein is expected to be beneficial in this process. For example, modulated LF-MF may increase the rate of wound closure, thereby preventing or reducing wound contracture, preventing or reducing scarring, and / or promoting tissue remodeling, and ultimately shortening the time required for wound healing. Modulated LF-MF can also stimulate the rare event of hair follicle neogenesis (Ito, M., Nature. 2007 May 17;447(7142):316-320).

[0168] The modulated LF-MF may be applied to nearly any subject to promote wound healing, however, preferably the modulated LF-MF is applied locally to the wound area, thereby exposing cells and tissue at and near the wound site to the modulated LF-MF described herein.

[0169] In certain embodiments, wound healing may be treated by locally applying ex vivo cultured wound stem or progenitor cells according to the methods described herein, in which stem and progenitor cells are exposed to a modulated LF-MF ex vivo to promote cell proliferation and, optionally, differentiation. The cells may be applied directly to the wound site, for example, by topical administration or injection. The cells may be applied to the wound, for example, using a suitable wound dressing or patch coated or impregnated with the cells. The cells may also be introduced into the wound together with a scaffold or matrix (e.g., a biodegradable polymer matrix), thereby further promoting tissue regeneration in the wound. The matrix of the scaffold may be impregnated or coated with stem / progenitor cells. It is also possible to culture stem / progenitor cells in the presence of the scaffold or matrix and the modulated LF-MF, thereby directly promoting cell growth on or within the scaffold or matrix.

[0170] The systems, methods, and uses utilizing the modulated LF-MFs described herein are expected to be beneficial for promoting wound healing in a wide range of tissues, including but not limited to epidermal wounds. For example, the systems, methods, and uses utilizing the modulated LF-MFs described herein may be useful for promoting wound healing (e.g., surgical wounds, injuries, or other trauma) in tissues selected from muscle tissue, connective tissue, joint tissue, epithelial tissue, endothelial tissue, nervous tissue, adipose tissue, skin tissue, lung tissue, liver tissue, bladder tissue, kidney tissue, cardiac tissue, pancreatic tissue, stomach tissue, intestinal tissue, spinal tissue, brain tissue, eye tissue, fibrous tissue, dentin, bone, or bone marrow. Thus, in certain embodiments, the systems, methods, and uses described herein may be used to promote wound healing in a subject undergoing neurosurgery, in which case the subject is exposed to the modulated LF-MFs described herein to promote healing of brain or nervous tissue. Preferably, in this embodiment, the subject's head is exposed to the modulated LF-MFs described herein when the subject undergoes neurosurgery. Localized application of modulated LF-MFs is also contemplated, where specific portions of the brain or neural tissue are exposed to the modulated LF-MFs.

[0171] in vitro cell culture As discussed in the overview of the invention, cells, particularly stem cells, progenitor cells, or other primary cells, can be cultured in vivo, and the cells (e.g., stem or progenitor cells) are exposed to modulated LF-MF during the culture and expansion of the cell population.

[0172] In vitro cell culture methods and suitable culture media are well known to those skilled in the art and are illustrated in the Examples herein. Cell culture media suitable for the proliferation and / or differentiation of stem or progenitor cells are well known (Dakhore, S. et al., Human Pluripotent Stem Cell Culture: Current Status, Challenges, and Advancement. Stem Cells International, 2018, 7396905). For example, the proliferation medium can be Dulbecco's Modified Eagle Medium (DMEM) or Minimum Essential Medium Eagle Alpha Modification (α-MEM), which are modified basal culture media based on Minimum Essential Medium (MEM). The stem cell proliferation medium is a suitable serum-free stem cell proliferation medium. Stem cell proliferation medium is commercially available. For the differentiation of stem cells or progenitor cells, a differentiation medium can be used; alternatively, the proliferation medium can be supplemented with growth factors to promote the differentiation of stem cells or progenitor cells.

[0173] Cells are suitably cultured and grown in a suitable culture vessel. A cell culture cavity is a volume within the cell culture vessel that contains the cells to be propagated, e.g., cell culture medium containing the cells. In use, the cells contained within the cell culture cavity are exposed to the modulated LF-MF described herein.

[0174] The culture vessel can be any vessel suitable for culturing stem or progenitor cells, such as a 2D tissue culture vessel or a 3D suspension culture vessel. In some embodiments, the culture vessel is selected from a Petri dish, a multi-well plate, a stacked cell culture vessel, a conical tube, or a 3D suspension culture vessel.

[0175] In 2D culture vessels, the vessel is typically coated with a suitable protein such as cadherin, laminin, or vitronectin. Stem cells attach to the coated surface and grow in colonies on the surface. The cells are passaged before the colonies become too dense, for example, when they reach more than 50%, more than 60%, more than 70%, more than 80%, or more than 90% confluence and / or when the cells show increased differentiation. Once the desired confluence is reached, the cells are dissociated from the vessel surface using a suitable method (e.g., shaking, mechanical scraping, enzymatic dissociation (e.g., trypsin-EDTA)). The collected cells are then replated in the vessel with culture medium at a lower surface density to continue growing. Passage can be performed multiple times to provide the required population of cells.

[0176] In the 3D culture vessel, cells can be suspended in a suitable culture medium and form aggregates, typically spherical, as a suspension in the culture medium. 3D culture can also be performed using a suitable gel or scaffold in which the cells are suspended. In the 3D culture vessel, cells are passaged when the average size of the cell aggregates reaches 100 μm to 500 μm, and / or when the cells reach 50%, 60%, 70%, 80%, or 90% confluence, and / or when the cells show increased differentiation. The cell aggregates are collected and dissociated (e.g., using a suitable enzyme) and diluted to the required density / concentration for continued growth.

[0177] In certain embodiments, cells are passaged multiple times while exposed to a modulated LF-MF as described herein. Thus, cells (e.g., stem or progenitor cells) can be passaged at least two times, at least three times, at least four times, at least five times, at least six times, at least seven times, or at least eight times. For example, cells can be passaged three to eight times or three to six times, with the cells being exposed to a modulated LF-MF during each passage.

[0178] Cell growth according to the methods described herein may be monitored using conventional methods, for example, using a light microscope, to determine the degree of confluence.

[0179] Stem and progenitor cells and differentiated cells generated according to the methods described herein can also be used to create in-vitro models of tissues for in vitro modeling of disease and for testing and screening drug candidates for efficacy and / or toxicity. Such cell models, or "diseases in a dish," have been used to study cardiomyocytes in models of cardiac disease and to model neurological disorders.

[0180] Treatment of autoimmune diseases, neurodegenerative diseases, inflammatory diseases, cardiovascular diseases, cognitive disorders, and psychiatric disorders As described in the overview of the present invention, the work by Guerriero et al. suggests that low frequency magnetic fields may be beneficial in the treatment of several conditions, including autoimmune diseases, neurodegenerative diseases, inflammatory diseases, cognitive disorders, psychiatric disorders, spinal injuries, and eye diseases.

[0181] The modulated LF-MFs described herein can be for the treatment of a neurodegenerative disease selected from, for example, Alzheimer's disease, amyotrophic lateral sclerosis, motor neuron disease, movement disorders, Parkinson's disease, Huntington's disease, or multiple sclerosis.

[0182] The modulated LF-MF described herein can be for the treatment of cognitive dysfunction, for example, cognitive dysfunction associated with any of the above neurodegenerative diseases.

[0183] The modulated LF-MF described herein may be for the treatment of a disorder selected from a mood disorder, post-traumatic stress syndrome, attention deficit disorder, attention deficit hyperactivity disorder, depression, or a depressive disorder.

[0184] Modulated LF-MF may be suitable for the treatment of blistering skin diseases such as autoimmune diseases of the pemphigus group or genetic disorders such as epidermal excoriation simplex.

[0185] Modulated LF-MF may be suitable for the treatment of spinal cord injury, for example, by promoting the regeneration of new nerve cells and the growth of new nerve fibers and / or improving nerve function.

[0186] Modulated LF-MF may be suitable for treating ocular diseases, such as damaged cornea, regeneration of the retinal optic nerve, age-related macular degeneration (AMD), glaucoma, or retinitis pigmentosa.

[0187] Modulated LF-MF may be suitable for the treatment of autoimmune diseases, for example for the treatment of type 1 diabetes by replenishing pancreatic beta cells or for the treatment of multiple sclerosis (for example for the regeneration of neurons together with myelin sheaths).

[0188] Modulated LF-MF may be suitable for the treatment of cardiovascular diseases, for example for myocardial regeneration or cardiovascular repair resulting from, for example, cardiac ischemia.

[0189] Brain wave modulation Electroencephalogram (EEG) studies show that brain waves can be divided into five distinct bandwidths. Alpha-band waves, with frequencies between 8.5 Hz and 12.5 Hz, have been shown to reduce psychological stress, induce a relaxed mood state, improve attention, and improve memory (Klimesch, W. Alpha-band oscillations, attention, and controlled access to stored information. Trends Cogn. Sci. 16, 606-617, doi:10.1016 / j.tics.2012.10.007 (2012); Hammerschlag, Biofield Physiology: A Framework for an Emerging Discipline., Glob Adv Health Med. 4, 35-41, doi:10.7453 / gahmj.2015.015.suppl (2015)).Alpha-band waves also increase serotonin production, decrease cortisol levels, and boost the immune system (Yu, Activation of the anterior prefrontal cortex and serotonergic system is associated with improvements in mood and EEG changes induced by Zen meditation practice in novices. Int J Psychophysiol 80, 103-111, doi:10.1016 / j.ijpsycho.2011.02.004 (2011); Puig, M.V. & Gulledge, A.T. Serotonin and prefrontal cortex function: neurons, networks, and circuits. Mol Neurobiol 44, 449-464, doi:10.1007 / s12035-011-8214-0 (2011); and Lozano-Soldevilla, D. On the Physiological Modulation and Potential Mechanisms Underlying Parieto-Occipital Alpha Oscillations.Front Comput Neurosci 12,23,doi:10.3389 / fncom.2018.00023(2018)).

[0190] Beta-band waves have a frequency between 12.5 Hz and 30 Hz and are associated with attention, focus, and cognition. The examples herein demonstrate that when subjects are exposed to the modulated LF-MF described herein, both alpha-band and beta-band waves increase. Tested subjects described an increased state of relaxation, consistent with increased alpha-band activity. Normally, alpha-band waves are not detected when the eyes are open. Surprisingly, subjects exposed to modulated LF-MF exhibited increased and sustained alpha-band activity, particularly in the parieto-occipital cortex, even with their eyes open.

[0191] The observation of increased both alpha and beta band activity resulting from exposure to modulated LF-MF suggests that modulated LF-MF may be useful in reducing anxiety and stress, increasing attention, increasing focus, and / or enhancing cognition. In certain embodiments, LF-MF is used to reduce psychological stress.

[0192] Thus, the modulated LF-MFs described herein may be useful for treating or preventing attention deficit disorders (e.g., attention deficit hyperactivity disorder (ADHD)), post-traumatic stress syndrome, cognitive dysfunction, and / or anxiety disorders.

[0193] In some embodiments, the modulated LF-MF is for use in treating or preventing anxiety disorders. Anxiety is a persistent feeling of apprehension or fear caused by an individual's perception of persistent and unrelenting stress. Anxiety is typically accompanied by a variety of physical symptoms, including twitching, trembling, muscle tension, headache, sweating (e.g., night sweats), dry mouth, or difficulty swallowing. Examples of anxiety disorders include separation anxiety disorder, selective mutism, specific phobia, social anxiety disorder (social phobia), panic disorder, panic attacks, agoraphobia, post-traumatic stress disorder (PTSD), generalized anxiety disorder, substance / medication-induced anxiety disorder, anxiety disorder caused by another medical disorder, or obsessive-compulsive disorder. There is.

[0194] Furthermore, the examples herein demonstrate that exposure of cells to modulated LF-MF reduced markers of cellular stress and cellular senescence, suggesting that LF-MF provides beneficial effects to subjects exposed to modulated LF-MF.

[0195] Furthermore, the MF exposure systems disclosed herein are believed to be useful for improving well-being, reducing stress, increasing concentration, and / or reducing anxiety in a subject by exposing at least a portion of the subject to the LF-MF. The systems are believed to be useful for non-therapeutic improvement of well-being, reducing stress, including psychological stress, inducing relaxation, increasing concentration, increasing focus, increasing attention, enhancing cognition, and / or reducing anxiety.

[0196] Accordingly, there is also provided a non-therapeutic method for improving well-being, reducing stress, including psychological stress, inducing relaxation, improving concentration, increasing focus, increasing attention, enhancing cognition, and / or reducing anxiety in a subject, said method comprising exposing at least a portion of the subject to LF-MF.

[0197] In embodiments herein relating to modulation of electroencephalograms, it is preferred that at least the brain of the subject is exposed to LF-MF.

[0198] Further embodiments The present invention is further illustrated by the following numbered clauses: 1. A magnetic field exposure system for exposing at least a portion of an organic cell, cellular tissue, or subject to a low frequency magnetic field, the system comprising: a magnetic field generator having one or more coils configured to generate a magnetic field that varies according to an amplitude modulated signal having a carrier frequency in the range of 400 Hz to 450 Hz, the magnetic field having a maximum field strength in the range of 10 μT to 200 μT. 2. A magnetic field exposure system according to clause 1, wherein the magnetic field is varied according to an amplitude modulated signal having a carrier frequency in the range of 400Hz to 450Hz and a modulation frequency in the range of 3Hz to 30Hz. 3. A magnetic field exposure system according to clause 1 or 2, the carrier frequency of which is 432 Hz. 4. A magnetic field exposure system according to any one of paragraphs 1-3, with a maximum magnetic field strength of 200 μT. 5. A magnetic field exposure system according to any one of clauses 1 to 4, wherein the magnetic field is varied according to an amplitude modulated signal having a carrier frequency in the range of 400 Hz to 450 Hz and a modulation frequency corresponding to a Schumann resonance mode. 6. A magnetic field exposure system according to any one of clauses 2 to 5, wherein the modulation frequency is within the range of 7.50 Hz to 8.00 Hz, such as 7.83 Hz. 7. A magnetic field exposure system according to any one of clauses 2 to 5, wherein the modulation frequency is within the range of 14.0 Hz to 14.5 Hz, such as 14.1 Hz, or within the range of 20.0 Hz to 21.0 Hz, such as 20.3 Hz, or within the range of 26.0 Hz to 27.5 Hz, such as 26.4 Hz. 8. A magnetic field exposure system according to any one of clauses 2 to 7, wherein the modulation frequency is a sine wave. 9. The magnetic field exposure system according to any one of clauses 1-8, wherein the one or more coils are configured to at least partially surround a subject to be exposed to the low frequency magnetic field. 10. The magnetic field exposure system according to any one of clauses 1-9, wherein the system is a chair having a hollow oval body forming a seat and a backrest, and wherein the one or more coils are integrated into the hollow oval body of the chair. 11. A magnetic field exposure system according to clause 10, wherein the hollow oval body of the chair is electromagnetically shielded, preferably the hollow oval body of the chair is provided with a lining formed of mu-metal. 12. A magnetic field exposure system according to clause 10 or 11, wherein at least two orthogonal copper coils are integrated into the hollow oval body of the chair. 13. A method for improving well-being, reducing stress, increasing concentration, and / or reducing anxiety in a subject by exposing at least a portion of the subject to a low frequency magnetic field that is varied in accordance with an amplitude modulated signal having a carrier frequency within 400Hz to 450Hz and a magnetic field strength in the range of 10μT to 200μT, preferably wherein the magnetic field is varied in accordance with an amplitude modulated signal having a carrier frequency within 400Hz to 450Hz and a modulation frequency in the range of 3Hz to 30Hz. 14. A method for inhibiting skin aging in a subject by exposing at least a portion of the subject's cellular tissue to a low-frequency magnetic field that varies according to an amplitude-modulated signal having a carrier frequency within 400 Hz to 450 Hz and a magnetic field strength within a range of 10 μT to 200 μT, preferably wherein the magnetic field varies according to an amplitude-modulated signal having a carrier frequency within 400 Hz to 450 Hz and a modulation frequency within a range of 3 Hz to 30 Hz. 15. A method for promoting in vitro proliferation of human epithelial stem and progenitor cells by exposing the cells to a low-frequency magnetic field that is varied according to an amplitude-modulated signal having a carrier frequency within 400 Hz to 450 Hz and a magnetic field strength within the range of 10 μT to 200 μT, preferably wherein the magnetic field is varied according to an amplitude-modulated signal having a carrier frequency within 400 Hz to 450 Hz and a modulation frequency within the range of 3 Hz to 30 Hz. [Example]

[0199] Unless otherwise specified, the following applies: Pooled juvenile (under 8 years old; 3 donors) or adult human primary keratinocyte stem and progenitor cells (the major cell type in the epidermis) were cultured in 2 ml of CnT-07 (CnT-BM.1 (CnT-07.S) supplemented with low calcium (0.07 mM)) basal cell culture medium at an initial cell density of 4,000 cells / cm. 2 were seeded onto 6-well plates. Pooled young human primary fibroblasts (the major cell type found in the dermis) were cultured at an initial cell density of 2,000 cells / cm in 2 ml of CnT-PrimeF. 2 were seeded onto 6-well plates. All cells, media, and supplements were purchased from CELLnTEC Advanced Cell Systems AG (Switzerland). The culture medium did not contain antibiotics or antimycotics and was changed every 2 to 3 days. Cells were cultured at 37°C ± 0.1°C, 90% ± 5% humidity, and 5% ± 0.1% CO2. All groups contain at least three sample values (at least triplicates) from at least three independent experiments. Once any group reached 80% confluence (phase 1 as described in Figure 5), all cells from all groups were harvested, except for metabolic activity studies (Figure 12). ·Cells were detached using CnT-Accutase, and cells were counted and passaged. Live and dead cells (proliferating and viable, respectively) were counted using trypan blue and a Thermo Fisher "Countess II" instrument. Cells were exposed to a magnetic field as described in each example using a classical Helmholtz coil (radius: 15 cm) placed in the incubator and powered by a generator and amplifier. All treatment conditions were initiated on the same sowing date. All references to "coil off" or "off" indicate that cells were cultured in the coil without electrical connection. Results for "coil off" and no coil were indistinguishable (data not shown). The MF exposure system was configured to generate 7.83 Hz (320 mV), 432 Hz (570 mV), or 432 Hz drive frequencies with 7.83 Hz amplitude modulation (1050 mV) at an average intensity of 200 μT (in accordance with ICNIRP 2010 guidelines). Figure 6A shows the 432 Hz driver / carrier waveform with 7.83 Hz 100% amplitude modulation frequency used in the study (also referred to herein as "432 / 7.83 Hz" and "combined MF"). The MF exposure system was configured and continuously tested to generate a pure and stable magnetic field (with ±2% variation) for the entire duration of the experiment, with the incubator at 37°C and 90% humidity (with ±2% variation). Cell turnover in phases 1–3 (described in Figure 12) was measured using a colorimetric assay based on the conversion of MTS tetrazolium compounds to colored monoazo dyes by NAD(P)H-dependent dehydrogenases in metabolically active cells (MTS assay) according to the protocol provided by the supplier (Abcam). After MF exposure, telomere length was determined using a quantitative PCR-based assay as described in Cawthon, RM, “Telomere measurement by quantitative PCR”, 10.1093 / nar / 30.10.e47, May 2002). All results are presented as mean ± SEM. The coil-off group was set to 1 to normalize the results. Statistical analysis: When comparing data between two groups, a two-tailed unpaired Student's t-test was used. When comparing data between three or more groups, an unpaired one-way ANOVA followed by Tukey's multiple comparison test was used. Analysis was performed using GraphPad Prism 8 software. Asterisks indicate statistical significance ( * P < 0.05; ** P < 0.01; and *** P<0.001). Cell morphology based on cell size, cell shape, and granularity was assessed blindly by two experienced researchers through visual inspection and picturing via light microscopy.

[0200] Figure 5 shows the three main maturation phases involved in epithelial homeostasis in vivo and in vitro: in phase 1, keratinocyte stem and progenitor cells proliferate, in phase 2, keratinocytes exit the cell cycle (GO) upon confluence and commit to differentiation in phase 3 (adapted from Kolly, C. et al., J Invest Dermatol 124, 1014-1025 (2005)).

[0201] During skin homeostasis, keratinocytes in the basal cell layer undergo continuous proliferation, exit the cell cycle, and differentiation while migrating to the suprabasal layers.

[0202] A key event in cell cycle withdrawal involves the repression of c-Myc, which sets the starting point for the initiation of differentiation. Accordingly, during terminal differentiation, the expression of differentiation markers such as keratin 10, Dsg1, involucrin, and loricrin increases in cultured cells and epidermis (Y. Poumay et al., J Invest Dermatol 104:271-276 (1995); Kolly, C. et al., J Invest Dermatol 124, 1014-1025 (2005)). An increase in extracellular calcium (switch) accelerates the process.

[0203] Under standard conditions, keratinocytes cease proliferation at 100% confluence following contact inhibition and cell cycle withdrawal. In contrast, uncontrolled growth that ignores contact inhibition is a hallmark of cancer and other hyperproliferative diseases.

[0204] Example 1: Effect of 432 / 7.83 Hz MF on proliferation of stem and progenitor cells Primary keratinocytes from young and adult donors were seeded and then exposed in coils without generating MF (coil off) or without generating 432 / 7.83 Hz MF.

[0205] This experiment was continued for a total of six passages to compare the short-term and long-term effects of LF-MF on cell proliferation. The extrapolated cell number (Figure 7) was calculated as follows: C = n * 2 PD where "C" is the calculated cell number, "n" is the number of cells seeded, and "PD" is the cumulative population doubling at each passage. "PD" is 3.33 * The significant increase in cell count in primary keratinocytes from juvenile (Figure 7A) and adult (Figure 7B) donors exposed to 432 / 7.83 Hz MF remained constant with each passage over the entire time course of the experiment (Figure 7).

[0206] Example 2: Effect of 432 / 7.83 Hz modulated MF compared to 7.83 Hz alone Primary keratinocytes from young and adult donors were seeded and then exposed to coil-off, 7.83 Hz, or 432 / 7.83 Hz MF.

[0207] After one passage of continuous MF exposure, a significant increase in the number of young cells ( Fig. 8A ) was observed in both the 7.83 Hz and 432 / 7.83 Hz groups compared with the coil-off group.

[0208] The effect of continuous MF exposure was examined in parallel on primary adult donor keratinocytes after one passage (Fig. 8B).

[0209] As observed with young primary cells (Figure 8A), the number of adult cells also increased significantly in both the 7.83 Hz and 432 / 7.83 Hz groups compared to the OFF MF group (Figure 8B). Interestingly, adult keratinocytes exposed to 432 / 7.83 Hz proliferated significantly better than cells exposed to 7.83 Hz.

[0210] Furthermore, similar to young donor primary cells, cells exposed to 432 / 7.83 Hz had the additional beneficial effect of increasing cell viability by an average of 45% (see Figure 11).

[0211] In terms of morphology, adult donor keratinocytes also showed improvement when exposed to 7.83 Hz and 432 / 7.83 Hz, with the healthiest morphology seen in the 432 / 7.83 Hz exposed group.

[0212] Results in adult donor primary cells coupled with observations in young donor primary cells show that 432 / 7.83Hz MF can increase cell proliferation when compared to the 7.83Hz and off groups.

[0213] 8A-8B demonstrate improved growth of human keratinocyte primary stem and progenitor cells in phase 1 when the cells were exposed to 432 / 7.83 Hz MF for one passage.

[0214] Example 3: 432 / 7.83 Hz modulated MF compared to 432 Hz only unmodulated driving frequency on stem and progenitor cell proliferation Young donor primary keratinocytes were seeded and then exposed to either coil-off, 432 Hz, or 432 / 7.83 Hz MF.

[0215] After one passage, keratinocytes exposed to 432 Hz showed significantly higher cell counts than the coil-off control group, however, the 432 / 7.83 Hz MF group showed higher proliferation (Figure 9).

[0216] Over the first two passages, there was a small increase in cell count in the 432 Hz driving frequency exposed group, but after the third passage, this turned negative and the number of viable cells began to decline in this group. After six passages of exposure, cells in the 432 Hz driving frequency group showed a significant decrease in cell count compared to the coil-off control group. In contrast, cells in the 432 / 7.83 Hz MF group showed a significant increase in proliferation compared to the coil-off control (Figure 9).

[0217] These results indicate that modulated 432 / 7.83 Hz MF led to higher proliferation of young donor primary keratinocytes compared to cells exposed to the 432 Hz driving frequency alone.

[0218] Example 4: Effects of modulation frequency, driving frequency, and magnetic field strength on stem and progenitor cell proliferation Primary keratinocytes from young and adult donors were seeded and then exposed to the following MF conditions to assess the effect of modulation frequency, driving frequency, and magnetic field strength on proliferation.

[0219] Example 4.1: Effect of modulation frequency on proliferation During cell culture, the cells were exposed to the following conditions: Coil off (control) 432Hz amplitude modulated at 3Hz 432Hz amplitude modulated at 7.83Hz 432Hz amplitude modulated at 10Hz 432Hz amplitude modulated at 30Hz

[0220] For young donor primary keratinocytes, modulation frequencies of 432 / 30 Hz, 432 / 50 Hz, and 432 / 100 Hz significantly increased proliferation compared to the control (coil off) (Figure 10A). A highly significant proliferation rate was observed in young donor cells exposed to 432 / 7.83 Hz. For adult keratinocytes, a significant increase in proliferation was observed in cells exposed to 432 / 7.83 Hz. A non-significant increase in proliferation was observed at 432 / 30 Hz. A decrease in proliferation occurred in the groups exposed to 432 / 50 Hz and 432 / 100 Hz (Figure 10B).

[0221] Example 4.2: Effect of driving frequency on proliferation During cell culture, the cells were exposed to the following conditions: Coil off (control) 368Hz amplitude modulated at 7.83Hz 400Hz amplitude modulated at 7.83Hz 432Hz amplitude modulated at 7.83Hz 464Hz amplitude modulated at 7.83Hz

[0222] A significant increase in proliferation was observed in young donor primary keratinocytes exposed to 368 / 7.83 Hz and 400 / 7.83 Hz. A highly significant increase in proliferation was observed in young donor primary cells exposed to 432 / 7.83 Hz. A non-significant effect was observed in the 464 / 7.83 Hz group (Figure 10C).

[0223] In adult donor primary keratinocytes, a significant increase in acceleration was observed in the 400 / 7.83 Hz and 432 / 7.83 Hz groups (FIG. 10D).

[0224] Example 4.3: Effect of magnetic field strength on proliferation During cell culture, the cells were exposed to the following conditions: Coil off (control) 10µT, 432Hz amplitude modulated at 7.83Hz, 55mVpp 30µT, 432Hz amplitude modulated at 7.83Hz, 550mVpp 200µT, 432Hz amplitude modulated at 7.83Hz, 1050mVpp 250µT, 432Hz amplitude modulated at 7.83Hz, 1250mVpp

[0225] Compared to control cells, a significant increase in proliferation was observed in all groups of young donor primary keratinocytes (Figure 10E). For adult donor primary keratinocytes, a significant increase in proliferation was observed in cells exposed to 30 μT and 200 μT (Figure 10F).

[0226] Example 5: Effect of MF treatment on cell survival The same juvenile and adult donor primary keratinocytes described in Example 8 below were seeded and then exposed to coil-off, 7.83 Hz, or 432 / 7.83 Hz MF.

[0227] After one passage of continuous MF exposure to 432 / 7.83 Hz, the viability of young cells increased significantly ( Figure 11 A).

[0228] Keratinocytes also showed better morphology in both the 7.83Hz and 432 / 7.83Hz groups, with the healthiest looking cells observed in the 432 / 7.83Hz group.

[0229] Similar to the young donor cells, 432 / 7.83 Hz exposure also significantly increased the viability of adult cells (Figure 11B).

[0230] Adult donor primary keratinocytes also showed improved cell morphology when exposed to 7.83 Hz and 432 / 7.83 Hz frequencies, with the healthiest morphology seen in the 432 / 7.83 Hz group.

[0231] Results in adult donor cells, aligned with observations in young donor primary cells, show that 432 / 7.83 Hz MF significantly improves cell viability when compared to the 7.83 Hz and coil-off groups.

[0232] Figures 11A and 11B demonstrate that the viability of human primary keratinocyte stem and progenitor cells in phase 1 was improved when the cells were exposed to 432 / 7.83 Hz MF for one passage.

[0233] Example 6: Effect of MF treatment on contact inhibition A high-throughput assay was set up to measure cell number based on metabolic activity (MTS) through phase 1 (80% confluence), phase 2 (100% confluence), and phase 3 (5 days after reaching 100% confluence and raising the calcium concentration to 1.2 mM) (Figure 5). When contact inhibition occurs in phase 2, cells are expected to stop proliferating, during which metabolic activity will decline and be similar across all treatment groups.

[0234] Growth correlated with metabolic activity and was assessed by the MTS assay.

[0235] Juvenile and adult donor primary keratinocytes were seeded and then exposed to coil-off, 7.83 Hz, or 432 / 7.83 Hz MF.

[0236] Figure 12 shows juvenile primary keratinocytes in phase 1 after one passage (Figure 12A) and adult primary keratinocytes across all maturation states (phases 1, 2, and 3; Figure 5) (Figure 12B). The graph represents substrate conversion relative to coil-off MF in phase 1, which is set to 1.

[0237] After one passage, a significant increase in metabolic activity in phase 1 was observed in young donor primary keratinocytes exposed to 432 / 7.83 Hz or 7.83 Hz and in adult keratinocytes exposed to 432 / 7.83 Hz MF (Figures 12A and 12B).

[0238] When the time course was followed for phases 2 and 3 by allowing cells to become confluent (phase 2) and differentiate (phase 3), a decrease in metabolic turnover in phase 2 and no difference between treatment groups was observed (Figure 12B shown for adult donor keratinocytes). In phase 3, metabolic activity increased slightly again as cells began to differentiate.

[0239] Taken together, the results shown in Figures 12A and 12B demonstrate that human primary keratinocyte stem and progenitor cells treated with 432 / 7.83 Hz MF exhibited normal contact inhibition and turnover, thus suggesting that no neoplastic overgrowth occurred.

[0240] Example 7: Benefits of MF treatment on aging Telomere shortening is a sign of aging cells (Zhao et al., Telomere length maintenance, shortening, and lengthening. J. Cell Physiol. 229, 1323-1329 (2014)). Cellular senescence (the cessation of cell differentiation) is thought to be involved in a number of diseases and medical conditions, such as carcinogenesis, aging, and tissue repair.

[0241] Quantitative PCR was used to measure telomere repeat number in human keratinocyte stem and progenitor cells using a method similar to that described by Cawthon, RM Telomere measurement by quantitative PCR. Nucleic Acids Res 30, e47 (2002). This technique represents the most frequently used method for assessing repeat number, which reflects telomere length.

[0242] In this experiment, it was also observed that different stocks of keratinocytes from the same donor and passage can respond differently to 432 / 7.83 Hz MF in Phase 1. Without being bound by theory, it is believed that there may be some initial variation in responsiveness to MF related to the initial thawing of the sample.

[0243] To further assess the effect of 432 / 7.83 HzMF on telomere length, early time points (passage 1) were compared with late time points (passage 6). Using methods similar to those described in Example 1, pooled young donor keratinocytes were lysed and DNA isolated after passages 1 and 6 in phase 1. After six passages, the control (off) cells were characterized by a greater than 50% shortening of telomere length compared to the first passage. In contrast, the shortening of telomere length in 432 / 7.83 HzMF-treated keratinocytes was not significant (Figure 13).

[0244] These results suggest that 432 / 7.83 Hz MF treatment provides a protective effect on telomere length.

[0245] Example 8: Effect of MF on cell stress Young donor primary keratinocytes were seeded and then cultured in either normal medium (CnT-07) or starvation medium (25% CnT-07, 75% PBS) and either coiled off or exposed to 432 / 7.83 Hz MF.

[0246] All samples were collected for in-cell Western analysis and stained for heat shock proteins 47 (Hsp47) and 90 (Hsp90), both of which are stress markers (Scieglinska et al., Heat shock proteins in the physiology and pathophysiology of epidermal keratinocytes; Cell Stress Chaperones 24, 1027-1044 (2019)).

[0247] The group cultured in 25% medium without exposure to MF (Off) showed an approximately three-fold increase in relative Hsp47 expression levels compared to cells cultured in 100% medium. Hsp90 expression also increased compared to 100% medium. In contrast, the expression of Hsp47 (Figure 14A) and Hsp90 (Figure 14B) in the 432 / 7.83 Hz MF-exposed group was significantly reduced compared to the unexposed control (Coil Off). In Figure 14, the relative Hsp47 and Hsp90 expression was normalized to β-tubulin, which is conventionally used as a housekeeping protein control to normalize the detected protein levels.

[0248] Summary of Examples 1 to 8 These studies demonstrated that 432 / 7.83 Hz amplitude-modulated magnetic fields have significant effects on the proliferation and survival of human epidermal stem and progenitor cells, and the effects on human epidermal stem and progenitor cells were superior to those achieved with 7.83 Hz alone, 432 Hz alone, various amplitude-modulated frequencies, various driving frequencies, and various average magnetic field strengths.

[0249] Furthermore, 432 / 7.83 Hz amplitude modulated MF showed additional benefits such as increased cell survival across all phases of epidermal maturation.

[0250] The use of 432 / 7.83 Hz amplitude modulated MF, rather than 7.83 Hz alone, further demonstrated prevention of telomere shortening. Telomere maintenance is a characteristic feature of stem cell-mediated tissue regeneration.

[0251] Example 9: EEG studies To assess the effects of modulated 432 / 7.83 Hz MF on EEG entrainment, a one-blinded crossover pilot study was conducted. In the study, human brain states were examined by measuring subjects' electroencephalogram (EEG) activity before and after a 15-minute exposure to sham (no MF) or real (432 / 7.83 Hz MF) in a chair. Here, subjects were unaware of the exposure conditions and sat in a cavity between Helmholtz coils positioned on the side walls of the chair so that the subject's head and almost the entire torso were exposed to MF (Figure 15). A magnetic field strength of 30 μT was used to comply with ICNIRP Recommendations 2010.

[0252] A total of four healthy subjects (three females, one male) were measured in a study to assess the potential of the 432 / 7.83 Hz modulation to systematically alter brain functional states (i.e., anxiety, stress, and arousal) and the potential of using multi-channel resting-state EEG to reliably detect these changes (Cantonal Ethics Committee Bern, approval Req-2020-00895).

[0253] Subjects were seated comfortably in an isolated room with adequate lighting and background noise. Multiple blind crossover sessions (n = 18) were conducted under either a sham (no MF) or true (432 / 7.83 Hz MF) condition. Effects on EEG activity were examined by comparing 5 min before and 5 min after exposure, with eyes open and eyes closed, at 30 s intervals. Specifically, each EEG was recorded using 32 electrodes positioned according to the international 10-10 system. All recordings were performed using a commercially available, safety-certified system (BrainVision Analyzer, Ver. 2.2.0, Brain Products GmbH, Gilching, Germany; recording reference FCz, 0.1 Hz to 200 Hz analog bandpass filter, 500 Hz sampling rate).

[0254] The EEG data were then processed in Brain Vision Analyzer. First, channels with poor signal quality were filtered out due to a high signal-to-noise ratio. Preprocessing of the EEG data included bandpass filtering with a low cutoff of 0.1 Hz and a high cutoff of 30 Hz. Independent component analysis (ICA) was then used to identify and eliminate eye movement components. The EEG was then visually inspected for residual artifacts and re-referenced to the mean baseline. Artifact-free 2-second epochs were then extracted and submitted to FFT, separately for the pre- and post-periods and for the eyes open and closed. These single-epoch FFT results were averaged within predefined frequency bands (Delta: 0.5–3.5 Hz, Theta 1: 3.5–5.5 Hz, Theta 2: 5.5–8.5 Hz, Alpha 1: 8.5–10.5 Hz, Alpha 2: 10.5–12.5 Hz, Beta 1: 12.5–18 Hz, Beta 2: 18–21 Hz, and Beta 3: 21–30 Hz). Results were averaged within conditions, i.e., separately for eyes-open and eyes-closed, both in the pre-stimulus and post-stimulus periods, and before and after sham or true subtraction. The standard deviations of these means were also retained.

[0255] After one 15-minute session of 432 / 7.83 Hz MF exposure, but not after sham, subjects described an increased state of relaxation, an effect that persisted after each subsequent session.

[0256] Figure 16A shows the average power spectrum of "before" minus "after" for different frequency bands for each participant in the sham and mid-eyes-open and eyes-closed conditions. There is a clear increase in the upper alpha frequency band during the true (solid curve) condition compared to the sham condition.

[0257] To summarize the EEG data, t maps averaged across all subjects for alpha1, alpha2, and beta1 are shown in Figure 16B. These average t maps were calculated as the "before" minus the "after" for each recording and were then used to compare conditions: from left to right, eyes closed (EC), sham eyes closed, eyes opened (EO), and sham eyes open.

[0258] Alpha 1 waves were increased mid-way in EC compared to sham, and this was also true for EO. Alpha 2 waves were also increased mid-way in both EC and EO compared to sham. Increased alpha 1 and alpha 2 activity in true both indicate decreased anxiety and stress. Beta 1 waves were increased in true EO but not EC, indicating increased attention and focus.

[0259] Since EO suppresses alpha wave activity, alpha waves are usually present during EC. Interestingly, EO increases alpha wave activity even in the presence of EC, where alpha waves are normally undetectable. This indicates that alpha waves persist after 432 / 7.83 Hz MF to such a high degree that even eye opening does not suppress alpha wave activity.

[0260] Summarizing the results of the EEG study, when comparing multiple sessions over multiple days, modulated MF exposure significantly increased both alpha and beta wave activity (Figure 16), which is expected to decrease anxiety and stress as well as increase attention and focus in subjects exposed to modulated 432 / 7.83 Hz MF.

[0261] The study was carried out at a magnetic field strength of 30 μT (in accordance with ICNIRP Recommendation 2010).

Claims

1. 1. A magnetic field exposure system for use in exposing at least a portion of an organic cell, cellular tissue, or subject to a low frequency magnetic field, the system comprising: a magnetic field generator comprising one or more coils configured to generate a magnetic field that varies in accordance with an amplitude modulated signal having a carrier frequency of 360 Hz to 450 Hz, wherein the modulation frequency is between 0.5 Hz and 100 Hz, and the magnetic field has a field strength of between 0.5 μT and 250 μT.

2. The magnetic field exposure system of claim 1 , wherein the one or more coils are configured to at least partially surround the subject being exposed to the low frequency magnetic field.

3. 3. The magnetic field exposure system of claim 1 or 2, wherein the system comprises a chair or bed configured to expose at least a portion of a subject sitting in the chair or lying in bed to the low frequency magnetic field.

4. 4. The magnetic field exposure system of claim 3, wherein the chair comprises a seat (1801) disposed within a cavity formed between two side walls (1802).

5. 5. The magnetic field exposure system of claim 4, wherein a circular coil (1803) of the same diameter is disposed on each of said side walls (1802), said coils (1803) being arranged coaxially, thereby forming a Helmholtz coil.

6. A magnetic field exposure system according to any one of claims 1 to 3, wherein the system comprises a bed and one or more coils surrounding the bed or positioned above and / or below the bed.

7. 7. The magnetic field exposure system of claim 6, wherein one or more coils surrounding the bed are arranged coaxially along a horizontal axis of the bed.

8. 8. The magnetic field exposure system of claim 6 or 7, wherein the one or more coils comprise Helmholtz coils.

9. 10. The magnetic field exposure system of claim 1, wherein the system comprises a housing having one or more coils disposed therein, the housing configured to position a particular body part or tissue of a subject to be exposed to the low frequency magnetic field.

10. The magnetic field exposure system of claim 9 , wherein the one or more coils comprise Helmholtz coils.

11. 10. The magnetic field exposure system of claim 1, wherein the system is in the form of a cell culture system, the system comprising: a cell culture vessel defining a cell culture cavity therein; and a magnetic field generator having one or more coils configured to generate a magnetic field within the cell culture cavity that varies according to an amplitude modulated signal having a carrier frequency between 360 Hz and 450 Hz, wherein the modulation frequency is between 0.5 Hz and 100 Hz, and the magnetic field has a field strength between 0.5 μT and 250 μT.

12. 1. A method of operating a magnetic field exposure system for treating a disorder associated with skin stem cells or skin progenitor cells in a subject, comprising: the magnetic field exposure system comprises a magnetic field generator comprising one or more coils configured to generate a low frequency magnetic field that varies according to an amplitude modulated signal having a carrier frequency between 360 Hz and 450 Hz, the one or more coils configured to expose at least a portion of tissue of the subject to the low frequency magnetic field; The method includes the magnetic field generator generating the low frequency magnetic field having a modulation frequency between 0.5 Hz and 100 Hz and a field strength between 0.5 μT and 250 μT.

13. 13. The method of claim 12, wherein the disease associated with the skin stem cells or skin progenitor cells is selected from maintaining skin homeostasis, reducing skin aging, promoting skin rejuvenation, preventing or reducing skin wrinkles, increasing skin elasticity, preventing or reducing skin stretch marks, preventing or reducing cellulitis, increasing skin hydration, reducing skin roughness, reducing skin pore size, inducing hair growth, preventing or reducing hair loss, inhibiting hair graying, inducing hair darkening, maintaining skin pigmentation, and inducing nail growth.

14. 1. A method of operating a magnetic field exposure system for treating a disease associated with stem or progenitor cells in a subject, comprising: the magnetic field exposure system comprises a magnetic field generator comprising one or more coils configured to generate a low frequency magnetic field that varies according to an amplitude modulated signal having a carrier frequency between 360 Hz and 450 Hz, the one or more coils configured to expose at least a portion of tissue of the subject to the low frequency magnetic field; The method includes the magnetic field generator generating the low frequency magnetic field having a modulation frequency between 0.5 Hz and 100 Hz, the magnetic field having a field strength between 0.5 μT and 250 μT.

15. 15. The method of claim 14, wherein the disease is selected from skin regeneration, wound healing, bone regeneration, inflammatory diseases, cardiovascular diseases, neurodegenerative diseases, cognitive disorders, autoimmune diseases, osteoarthritis, tissue fibrosis, periodontal disease, or skin diseases.

16. 15. The method of claim 14, wherein the condition is the healing of wounds, abrasions, burns, blisters, ulcers, or the prevention or minimization of scarring.

17. A method of operating a magnetic field exposure system for promoting the growth of organic cells, comprising: the magnetic field exposure system comprises a magnetic field generator comprising one or more coils configured to generate a low frequency magnetic field that varies according to an amplitude modulated signal having a carrier frequency between 360 Hz and 450 Hz, the one or more coils being configured to expose the low frequency magnetic field; The method includes the magnetic field generator generating the low frequency magnetic field having a modulation frequency between 0.5 Hz and 100 Hz, the magnetic field having a field strength between 0.5 μT and 250 μT.

18. 20. The method of claim 17, wherein the magnetic field exposure system is for promoting tissue regeneration in a subject.

19. the magnetic field exposure system is for promoting cell proliferation; (i) the cells are stem cells or progenitor cells; (ii) the cell is a human stem cell or a human progenitor cell; (iii) the cells are human epidermal keratinocyte stem cells or epidermal keratinocyte precursor cells, or (iv) the cells are terminally differentiated primary cells.

20. 1. A method of operating a magnetic field exposure system for rejuvenating skin of a subject, comprising: the magnetic field exposure system comprises a magnetic field generator comprising one or more coils configured to generate a low frequency magnetic field that varies according to an amplitude modulated signal having a carrier frequency between 360 Hz and 450 Hz, the one or more coils configured to expose one or more in vitro cultured stem or progenitor cells from the subject to the low frequency magnetic field; 10. The method of claim 1, wherein the magnetic field generator generates the low frequency magnetic field having a modulation frequency of 0.5 Hz to 100 Hz and a field strength of 0.5 μT to 250 μT, thereby expanding the population of stem cells or progenitor cells.

21. 1. A method of operating a magnetic field exposure system for inhibiting cellular senescence, comprising: the magnetic field exposure system comprises a magnetic field generator comprising one or more coils configured to generate LF-MF that varies according to an amplitude modulated signal having a carrier frequency between 360 Hz and 450 Hz, the one or more coils configured to expose the organic cells to the LF-MF; The method includes the magnetic field generator generating the LF-MF having a modulation frequency between 0.5 Hz and 100 Hz and a magnetic field strength between 0.5 μT and 250 μT.

22. 22. The method of claim 21, wherein the organic cells are stem or progenitor cells.

23. The method according to any one of claims 17 to 20 or 22, wherein the stem cells are human adult stem cells selected from muscle stem cells, hematopoietic stem cells, epithelial stem cells, neural stem cells, mesenchymal stem cells, mammary stem cells, intestinal stem cells, mesodermal stem cells, endothelial stem cells, skin stem cells, melanocyte stem cells, hair follicle stem cells, olfactory stem cells, neural crest stem cells, and dental pulp stem cells.

24. 23. The method according to any one of claims 17 to 20 or 22, wherein the stem cells or progenitor cells are human keratinocyte stem cells or human keratinocyte progenitor cells.

25. 1. A method of operating a magnetic field exposure system for treating an age-related disorder in a subject, comprising: the magnetic field exposure system comprises a magnetic field generator comprising one or more coils configured to generate a low frequency magnetic field that varies according to an amplitude modulated signal having a carrier frequency between 360 Hz and 450 Hz, the one or more coils configured to expose at least a portion of tissue of the subject to the low frequency magnetic field; The method includes the magnetic field generator generating the low frequency magnetic field having a modulation frequency between 0.5 Hz and 100 Hz and a field strength between 0.5 μT and 250 μT.

26. 26. The method of claim 25, wherein the aging-related disease is selected from cardiovascular disease, pulmonary disease, osteoarthritis, aging-related skin disease or disorder, neurodegenerative disease, eye disease, diabetes, liver disease, sarcopenia, and benign prostatic hyperplasia.

27. 1. A method of operating a magnetic field exposure system for improving well-being, reducing stress, promoting focus, and / or reducing anxiety in a subject, comprising: the magnetic field exposure system comprises a magnetic field generator comprising one or more coils configured to generate a low frequency magnetic field that varies according to an amplitude modulated signal having a carrier frequency between 360 Hz and 450 Hz, the one or more coils configured to expose at least a portion of the subject to the low frequency magnetic field; The method includes the magnetic field generator generating the low frequency magnetic field having a modulation frequency between 0.5 Hz and 100 Hz and a field strength between 0.5 μT and 250 μT.

28. 28. The method of claim 27, wherein the magnetic field exposure system is for reducing psychological stress, inducing relaxation, improving concentration, increasing focus, increasing attention, enhancing cognition, and / or reducing anxiety.

29. A system according to any one of the preceding claims, wherein the magnetic field is varied according to an amplitude modulated signal having a carrier frequency within 400 Hz to 450 Hz and a modulation frequency between 3 Hz and 30 Hz.

30. The system of any one of claims 1 to 11, wherein the carrier frequency is 432 Hz.

31. A system according to any one of claims 1 to 11, wherein the resonant frequency is between 3 Hz and 28 Hz.

32. The system of any one of claims 1 to 11, wherein the modulation frequency corresponds to a Schumann resonance mode.

33. The system of any one of claims 1 to 11, wherein the modulation frequency is in the range of 7.50 Hz to 8.00 Hz, such as 7.83 Hz.

34. 12. The system of any one of claims 1 to 11, wherein the modulation frequency is in the range of 14.0 Hz to 14.5 Hz, such as 14.1 Hz, or in the range of 20.0 Hz to 21.0 Hz, such as 20.3 Hz, or in the range of 26.0 Hz to 27.5 Hz, such as 26.4 Hz.

35. A system according to any preceding claim, wherein the carrier frequency is 432 Hz and the modulation frequency is 7.83 Hz.

36. The maximum magnetic field strength is 200 μT, Optionally, (i) the maximum magnetic field strength is 30 μT; or (ii) The system of any one of claims 1 to 11, wherein the maximum magnetic field strength is 11 μT.

37. A system according to any preceding claim, wherein the modulation frequency is sinusoidal.

38. The system of any one of claims 1 to 11, wherein the low frequency magnetic field is non-pulsed.

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