WO2005071057A1 - Dispositif de stimulation de champ magnetique ou electrique et procede permettant de favoriser, limiter ou entraver la croissance et la fonction d'une cellule ou d'un tissu vivant au moyen dudit dispositif - Google Patents

Dispositif de stimulation de champ magnetique ou electrique et procede permettant de favoriser, limiter ou entraver la croissance et la fonction d'une cellule ou d'un tissu vivant au moyen dudit dispositif Download PDF

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Publication number
WO2005071057A1
WO2005071057A1 PCT/JP2005/001164 JP2005001164W WO2005071057A1 WO 2005071057 A1 WO2005071057 A1 WO 2005071057A1 JP 2005001164 W JP2005001164 W JP 2005001164W WO 2005071057 A1 WO2005071057 A1 WO 2005071057A1
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WIPO (PCT)
Prior art keywords
electric field
magnetic
pulse
magnetic field
living
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PCT/JP2005/001164
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English (en)
Japanese (ja)
Inventor
Naofumi Tokutomi
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Japan Science And Technology Agency
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Publication date
Priority claimed from JP2004014108A external-priority patent/JP2004290180A/ja
Application filed by Japan Science And Technology Agency filed Critical Japan Science And Technology Agency
Publication of WO2005071057A1 publication Critical patent/WO2005071057A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/02—Electrical or electromagnetic means, e.g. for electroporation or for cell fusion
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0018—Culture media for cell or tissue culture

Definitions

  • the present invention relates to a magnetic or electric field stimulator for applying electrical stimulation to living cells, tissues, and the like, a method for promoting or suppressing the growth of living cells, tissues, and the like, and creation of a degenerative disease model for living cells, tissues, and the like.
  • the present invention is to promote or suppress the growth of living cells, tissues, etc. by applying a specific electrical stimulus to a living cell, tissue, etc. used in an experiment in a laboratory or the like via a magnetic field or an electric field.
  • Magnetic or electric field stimulating device, and a method of promoting or suppressing the growth of living cells and tissues using the magnetic or electric field stimulating device, and a model of a degenerative disease of living cells and tissues using the magnetic or electric field stimulating device About how to create.
  • the present invention relates to a method for transplanting a tissue in a living body.
  • the present invention relates to a method for accommodating the function of an organ or a tissue which constantly generates an electric signal and to which the constituent cells are receiving the electric signal with each other, or the function of a cell constituting the organ or the tissue, preferably the function of a myocardial tissue.
  • a culture vessel with a microchip electrode array bottom surface was developed to perform a long-term culture test under specified culture conditions, but depending on the type of cells, fixation in the culture vessel may occur.
  • the properties were not always good. Particularly when culturing neuronal cells, its poor fixation has been a problem.
  • some organs for example, cells constituting the tissue itself such as myocardium, nerves, and skeletal muscle have unique characteristics in vivo. It is hard to say that attempts to transplant myocardium, which is an organ in which cells are constantly receiving electrical input from the electrical environment specific to the organ by performing electrical activity, especially the heart, have been sufficiently successful. Disclosure of the invention
  • the present invention has been made in order to solve the above problems, and an object of the present invention is to provide a method for growing living cells or living tissues (hereinafter, also referred to as “living cells, etc.”). It is an object of the present invention to provide a magnetic or electric field stimulator which does not change the liquidity of a culture solution or decrease the degree of cell fixation even when electrical stimulation is applied to living cells or the like for a long period of time. Another object of the present invention is to provide a method for promoting, suppressing or impairing the growth and function of living cells and the like using the magnetic or electric field stimulator. Still another object of the present invention is to provide a method for creating a model of a degenerative disease such as a living cell using the magnetic or electric field stimulator.
  • the inventor of the present invention has intensively studied the importance of growth or suppression of living cells and the like, induction of degenerative pathologies, and particularly of excitatory reception when electrical stimulation is applied for a long period of time.
  • the present inventors have developed an apparatus and a method capable of applying an electrical stimulus while minimizing the amount of current supply as a result, and have completed the present invention.
  • a pulse current supply means for generating a pulse current and adjusting the current characteristics of the pulse current, and a pulse magnetic field or a pulse electric field from the pulse current supplied from the pulse current supply means
  • a pulse magnetic field or pulse electric field supply means for supplying the pulse magnetic field or pulse electric field from outside the culture vessel to living cells or living tissue in the culture vessel.
  • An electric field stimulator is provided.
  • the pulse current supply means is an electric circuit including at least a power supply, a switch, and a logic IC.
  • the pulse magnetic field or electric field supply means generates the pulse magnetic field or pulse electric field, and supplies a magnetic field or electric field stimulation probe for supplying the pulse magnetic field or electric field to a living cell or a living tissue; and A magnetic or electric field stimulating stage for storage.
  • the magnetic or electric field stimulating probe is a single or multiple magnetic field stimulating coils or electric field stimulating conductive plates.
  • the magnetic field or electric field stimulating probe generates an eddy current or a capacitive current in the culture vessel by supplying a pulsed magnetic field or an electric field into the culture vessel.
  • it further has an eddy current detection probe for detecting an eddy current in the culture vessel.
  • the culture container can be fitted in the pulse magnetic field or electric field supply means.
  • it is used to promote, suppress or impair the growth and function of living cells or living tissue.
  • the magnetic field or electric field stimulation probe selectively supplies a pulsed magnetic field to a predetermined cell group or a predetermined site of a living cell or a living tissue to promote or suppress the growth of the living cell or the living tissue. It is to make it.
  • the pulsed magnetic field or electric field stimulation probe determines the supply frequency of the stimulation pulse at a timing that mimics the appearance frequency of the main waveform of an electroencephalogram, an electrocardiogram, an electromyogram, a gastric electrogram, an electroretinogram, or a synaptic current.
  • a current noise with a frequency equal to or higher than the frequency of the high-frequency noise obtained by passing these bioelectric signals through a low cut filter (10 Hz) as a burst current cluster It is characterized in that it is included in a pulse and used for stimulation.
  • the pulse current supply means is an electric signal generator capable of generating a current and a voltage having a predetermined function waveform.
  • the magnetic or electric field stimulating probe has a shape that can correspond to the well arrangement of a multiwell plate.
  • the magnetic field or electric field stimulation probe generates a pulse magnetic field by changing a magnetic flux density of an energized pulse current.
  • said magnetic or electric field stimulation probe is said burst current cluster
  • a pulse electric field is generated by supplying a pulse current consisting of
  • the magnetic field or electric field stimulating device of the present invention has a structure having a pulse current supply means and a pulse magnetic field or pulse electric field supply means. Therefore, the magnetic or electric field stimulating apparatus of the present invention has a simple structure and is easy to manufacture.
  • the magnetic or electric field stimulator of the present invention can be easily attached and detached since the culture vessel containing the living cells and the like can be fitted into the pulsed magnetic field or pulse electric field supply means, and furthermore, the living cells and the like can be used.
  • an eddy current or a capacitive current (pulse signal) having a desired intensity and waveform is supplied to the living cells from outside the culture vessel without directly contacting the cells, so that the living cells grow in the culture process. And denaturation can be adjusted appropriately.
  • an eddy current or a capacitive current (pulse signal) having a desired intensity, waveform, or the like can be supplied from a magnetic or electric field stimulating probe to a specific cell group such as a living cell or the like. It can be selectively generated at a specific site to promote or suppress the growth of living cells or tissue cells.
  • Another object of the present invention is to provide a method for promoting or suppressing the growth of a living cell or a living tissue, a method for promoting or suppressing the function of a living cell or a living tissue, and a living cell using the magnetic or electric field stimulator.
  • it can be achieved by a method characterized by acclimatization of cell function to promote the function of living tissue. According to the method of this effort, the growth of living cysts, etc. is promoted without directly contacting the electrodes as in the conventional stimulator, or the growth of living cells and living tissues is suppressed by damaging the living cells, etc. can do.
  • Another object of the present invention is to create a biological cell or biological tissue degenerative disease model by degenerating normal biological cells or biological tissues, preferably neural cells or neural tissues using the magnetic or electric field stimulator. This is achieved by a method for creating a model of a degenerative disease of a living cell or living tissue, characterized by the above feature.
  • an organ or a tissue which constantly generates a bioelectric signal of a living body, and the constituent cells receive the electric signal with each other, or a cell constituting the same is used in a range of 0.5 to 500 Hz. , 1 O mV ⁇ : pulsed electric signal of 10 V, or 0.5 ⁇ 500 HZ, pulsed magnetic signal input of 0.1 mT ⁇ lO OmT, characterized by culturing with "F” Provides methods for acclimating the function of organs or tissues or the cells that compose them It is.
  • the organ is the heart.
  • the tissue is myocardium.
  • the functional acclimation is performed before transplanting the organ or tissue.
  • a specimen for drug efficacy evaluation and toxicity test comprising a tissue or a cell adapted to function by the above method.
  • FIG. 1 is a schematic diagram showing one embodiment of the magnetic or electric field stimulating device of the present invention.
  • FIG. 2 is an explanatory diagram showing a circuit arrangement of an electric circuit in the magnetic or electric field stimulating device of the present invention.
  • FIG. 3 is an explanatory diagram showing a circuit device using a logic IC in the magnetic or electric field stimulating device of the present invention.
  • FIG. 4 is an explanatory view showing a preferred embodiment of a magnetic or electric field stimulation probe and a magnetic or electric field stimulation stage in the present invention.
  • (Ii) A diagram showing different modes of the magnetic field or electric field stimulation probe and the magnetic field or electric field stimulation stage.
  • (II) Magnetic field or electric field It is a figure which shows the area
  • FIG. 5 is a diagram showing a state in which the magnetic field or electric field stimulator of the present invention is used to apply magnetic field stimulation to nerve cells to promote growth.
  • ( ⁇ ) is a diagram showing the results when culturing was performed by applying a magnetic field stimulation.
  • ( ⁇ ) is a diagram showing the results when culture was performed without applying magnetic field stimulation.
  • FIG. 6 is a diagram showing a state in which a magnetic field is applied to a nerve cell using the magnetic field or electric field stimulator of the present invention to suppress growth.
  • ⁇ is a diagram showing a state of a nerve cell before applying a magnetic field stimulation.
  • ⁇ is a diagram showing a state of a nerve cell after applying a magnetic field stimulation.
  • C is a diagram showing a state of a nerve cell after a magnetic field stimulation is applied by limiting a range of a magnetic field to a local area by a minimum coil and a metal shield.
  • FIG. 7 shows the eddy current generated in the culture vessel using the magnetic or electric field stimulator of the present invention.
  • FIG. 4 is an explanatory diagram showing an embodiment of a detection probe for measuring the measurement.
  • A It is a front view of the probe for eddy current measurement.
  • B It is the schematic explanatory drawing of the probe for eddy current measurement.
  • FIG. 8 shows the resting membrane potential of the cultured mouse sensory nerve measured under current clamp conditions using the patch clamp method.
  • Figure 9 shows prevention of cubsaicin-induced neuronal cell death by pulsed magnetic field stimulation pretreatment. You.
  • FIG. 10 shows the contraction frequency of each ventricular muscle cluster to electrical stimulation (upper photo) and the contraction pattern of the typical magnetic stimulation group cluster to 2 Hz and 5 Hz electrical stimulation (lower trace).
  • FIG. 11 shows the contraction frequency of ventricular muscle clusters cultured in the presence of pulsed magnetic field stimulation in response to electrical stimulation.
  • FIG. 12 is a schematic diagram showing an example of an apparatus used in the adaptation method of the present invention.
  • FIG. 13 is a photograph of one example of cardiomyocytes cultured by the adaptation method of the present invention.
  • a magnetic field or electric field stimulator of the present invention a method for promoting or suppressing the growth of living cells and the like using the magnetic field or electric field stimulator, and a method for preparing a degenerative disease model of a living cell or a living tissue Will be described in detail.
  • FIG. 1 is a schematic view showing a preferred embodiment of the magnetic or electric field stimulating device of the present invention.
  • reference numeral 1 denotes a magnetic field or electric field intensifying device main body
  • reference numeral 2 denotes a pulse current supply means
  • reference numeral 3 denotes a pulse magnetic field or pulse electric field supply means
  • reference numeral 4 denotes a magnetic field or stimulation probe
  • reference numeral 5 denotes a magnetic field or Electric field stimulation stage
  • 6 is a culture vessel
  • 7 is a power supply
  • 8 is a switch
  • 9 is a logic IC
  • 10 is a light emitting element
  • 11 is an electrolytic capacitor
  • 12 is a transistor
  • 13 is a transistor.
  • reference numeral 14 denotes a culture medium
  • reference numeral 15 denotes a living cell or a living tissue
  • reference numeral 16 denotes a power supply switching switch
  • reference numeral 17 denotes a magnetic field or electric field stimulation switching switch.
  • the pulse current supply means 2 has at least a power supply 7, a switch 8 and a logic IC 9, as shown in FIGS. 1 and 2, and preferably further includes a light emitting element 10, an electrolytic capacitor 11 and a transistor 12.
  • the pulse magnetic field or pulse electric field supply means 3 is composed of a magnetic or electric field stimulating probe 4 and a magnetic or electric field stimulating stage 5.
  • the magnetic field or electric field stimulating device of the present invention comprises a pulse current supply means 2 and a pulse magnetic field or pulse electric field supply means 3.
  • the pulse current supply means 2 is not particularly limited as long as it can generate a pulse current and can adjust the intensity and frequency of the pulse current.
  • the pulse current supply means 2 is preferably an electric signal generator capable of generating a current and a voltage having a predetermined function waveform. As shown in FIG. 1, at least a power supply 7, a switch 8 and a logic IC 9 are provided.
  • the present invention is achieved by magnetic field stimulation using a coil and (2) electric field stimulation between conductive plates sandwiching a culture vessel above and below, that is, capacitive current stimulation in which the space between two plates is regarded as a capacitor. Is done.
  • a conductive material such as a silver electrode is brought into monopolar contact with the culture medium instead of the conductive plate on the top of the culture vessel, and the cell and tissue growth layer and the bottom material of the culture vessel are combined into a single condenser.
  • the capacitive current stimulation of (2) the DC component of the current is completely removed, so that there is no change in liquidity due to electrolysis or the like. That is, the same effect as non-contact stimulation can be expected.
  • the waveform, intensity, cycle, energizing time, and the like of the pulse current supplied from the pulse current supply means 2 in the magnetic field or electric field stimulator of the present invention can be appropriately adjusted according to the magnetic field stimulation described later.
  • the power supply 7 may be either a power supply by an electric supply device or a power supply by batteries.
  • batteries a dry battery or a button battery is preferable.
  • the rechargeable battery such as a nickel-powered dominant battery, an electric double layer capacitor and a lithium battery may be used as well as a disposable battery such as a mercury battery or lithium.
  • the power supply 7 is an electricity supply
  • DC power supplies such as CZD C switching power supplies, A CZD C converters and various power transformer products are preferred.
  • the switch 8 is the same as the normal switch in the magnetic field or electric field stimulator 1. It is a device that controls the on / off control of the luster current.
  • the switch 8 may be a switch that is mechanically switched by a human operation, or may be a switch using a piezoelectric element, for example, in which current can be automatically turned on / off by pressure.
  • the pulse current supply means 2 may include a plurality of switches. For example, as shown in FIGS. 1 and 2, in addition to the toggle switch 8, a power supply switch 16 and a magnetic field or electric field stimulation switch May have 17.
  • the logic IC 9 may use an integrated circuit (IC) in which a series of work instructions is set or a series of work instructions can be given by a program, such as a general-purpose IC or a programmable IC. it can.
  • IC integrated circuit
  • the logic IC 9 has a role of changing the magnitude of the pulse current injected into the coil or the conductive plate on the time axis.
  • a large current pulse is to be injected into the coil to generate a high output magnetic field, or a very low or high frequency burst current cluster of 10 Hz or higher is applied to the conductive plate to stimulate with a capacitive current. If you want to inject in a pulse,
  • a power supply for injecting the large current pulse or the burst current cluster pulse is provided separately from the pulse current supply means 2, and a relay circuit driven by the pulse current obtained from the current supply means 2 supplies a current from the separate power supply to the coil.
  • the supply path may be opened and closed.
  • An example of a relay circuit is the PhotoMOS relay AQV-102 (maximum 60V (600mA)), and multiple relay circuits can be used in parallel.
  • a relay circuit It is opened and closed by a relay circuit, and the current supplied to the coil or conductive plate from the separate power supply is formed by a CR oscillation circuit, LC oscillation circuit, crystal oscillation circuit, crystal oscillation module, PLL synthesizer, digital synthesizer in addition to DC. 10 Hz
  • a relay circuit It is opened and closed by a relay circuit, and the current supplied to the coil or conductive plate from the separate power supply is formed by a CR oscillation circuit, LC oscillation circuit, crystal oscillation circuit, crystal oscillation module, PLL synthesizer, digital synthesizer in addition to DC. 10 Hz
  • the above-described rectangular wave or sine wave burst current can be used.
  • the logic IC 9 includes general-purpose logic ICs, PLDs (programmable logic devices), CPLDs (complex programmable logic devices), and FPGAs (field programmable gate arrays) custom ICs.
  • the general-purpose logic IC includes a CMOS type and a bipolar type, and is preferably a CMOS type.
  • Logic IC 9 is readily available on site.
  • the logic IC 5 may further include a peripheral element such as a capacitor.
  • the magnetic field or electric field stimulating apparatus of the present invention is set so that the pulse current adjusted by the logic IC 9 flows to the pulse magnetic field or pulse electric field supply means 3.
  • the pulse current is preferably such that the voltage (intensity), the frequency of occurrence (cycle), the generation pattern (waveform), and the like can be changed as a series of protocols by the logic IC 9 according to the purpose of the experiment.
  • One way to implement such a modifiable stimulus protocol is to write and erase the protocol in Logic IC9.
  • the method of forming the pulse current supply means 2 is not particularly limited, and examples thereof include a method of applying a conductive paint on a printed circuit board and printing the same on a printed circuit board, and a method of welding a thin conductive wire or the like. it can. Among them, a method of printing on a substrate is preferable from the viewpoint of making a circuit compact.
  • the pulse magnetic field or pulse electric field supply means 3 constituting the magnetic field or electric field stimulator of the present invention generates a pulse magnetic field or pulse electric field from the pulse current supplied from the pulse current supply means 2, and converts the pulse magnetic field or pulse electric field to It can be supplied to living cells or living tissues in the culture vessel from outside the culture vessel.
  • the present invention is achieved by magnetic field stimulation using a coil and (2) electric field stimulation between conductive plates sandwiching a culture vessel at the top and bottom, that is, capacitive current stimulation in which the space between two plates is regarded as a capacitor. Is done.
  • a conductive material such as a silver electrode is monopolarly contacted with the medium instead of the conductive plate on the top of the culture vessel, and the layer where cells and tissues grow and the bottom material of the culture vessel are placed. It is also possible to consider a single capacitor and perform stronger capacitive current stimulation. In either case, with the capacitive current stimulation of (2), the DC component of the current is completely removed, and there is no change in liquidity due to electrolysis or the like. That is, the same effect as non-contact stimulation can be expected.
  • the “pulse magnetic field” refers to a pulse-like magnetic field generated when the pulse current supplied from the pulse current supply unit 2 is changed, for example, in a coil by changing the magnetic flux density.
  • the “pulse electric field” refers to a pulse-like electric field generated by a pulse current supplied from the pulse / current supply means 2.
  • the pulsed magnetic field or pulsed electric field supply means 3 does not directly contact the electrode or the like with the medium outside the culture vessel, that is, outside the culture vessel, A pulse magnetic field or a pulsed electric field is supplied to the living cells in the culture vessel from the bottom of the cell to generate an eddy current or a capacity current in the culture vessel, and the eddy current or the capacity current is applied to the living cells as a pulse signal. Can be supplied.
  • the liquid properties of the culture medium in the culture vessel do not change even when a long-term electric stimulus is applied as compared with the conventional stimulator, and There is the merit that there is no influence of the electrode material and good adhesion of living cells can be obtained.
  • the pulse magnetic field or pulse electric field supplied from the pulse magnetic field or pulse electric field supply means 3 can be appropriately determined according to the waveform, intensity, cycle, duration, etc. of the pulse current supplied from the pulse current supply means 2. it can.
  • a pulse magnetic field or pulse electric field waveform a triangle, rectangle, or other various function waveforms (for example, sine waveform, exponential function waveform, etc.) can be selected according to the pulse current waveform. Preferably, there is.
  • the intensity of the pulsed magnetic field can be appropriately determined according to two culture purposes, that is, growth promotion and growth suppression (damage) of living cells and the like.
  • the intensity of the pulsed magnetic field is from 0.1 to 10 mT, preferably from 0.3 to 3 mT, and more preferably from 0.05 to 3 mT. 1.5 mT preferable.
  • the intensity of the pulse magnetic field is 0.05 to 500 mT, preferably 1 to 100 mT, and more preferably 3 to 3 OmT. Is more preferable.
  • the cycle of the pulsed magnetic field is, for example, from 0.001 to 1000 Hz, preferably from 0.005 to 100 Hz, and more preferably from 0.01 to LOHz.
  • the duration of the pulsed magnetic field is, for example, 1 microsecond to 10 seconds, preferably 5 microseconds to 1 second, and more preferably 0.1 millisecond to 0.1 second.
  • the intensity of the pulse electric field is, for example, 1 microvolt—100 volts, preferably 1 millivolt—10 volts, and more preferably 30 millivolts—3 volts.
  • the cycle of the pulsed electric field is, for example, 0.001 to 1000 Hz, preferably 0.005 to: L0OHz, and more preferably 0.011 to: LOHz.
  • the duration of the pulsed electric field is, for example, 1 microsecond to 10 seconds, preferably 5 microseconds to 1 second, and more preferably 0.1 millisecond to 0.1 second. Les ,.
  • Eddy current is an eddy current generated by electromagnetic induction in a direction that cancels the change in magnetic flux density due to the pulse magnetic field when a pulse magnetic field is supplied to the culture medium in the culture vessel.
  • the capacity current is the electric field stimulation between the conductive plates sandwiching the culture vessel from above and below, that is, the fluctuating current that passes through the capacity between the two plates, which is regarded as a capacitor.
  • a conductive material such as a silver electrode is brought into monopolar contact with the culture medium instead of the conductive plate on the top, and the layer where cells and tissues grow and the material on the bottom of the culture vessel are regarded as one capacitor, and pass through its capacity. It is a stronger fluctuating current.
  • the eddy current can be generated in the culture vessel 6 in accordance with the pulse of the pulse current supplied from the pulse current supply means 2.
  • the intensity of the eddy current is It can be appropriately determined according to the strength of the pulse magnetic field supplied from 3 and the resistance in the culture vessel.
  • the intensity of the eddy current can be measured, for example, using various eddy current detection probes including a conductive member 71 and a measuring lead wire 72 as shown in FIG.
  • the capacity current is a burst current cluster formed by a pulse current of the pulse current supplied from the pulse current supply means 2 directly or by another power source in the culture vessel 6 as a burst current cluster based on the duration and frequency of the pulse. Can be generated.
  • the intensity of the capacitance current can be directly measured with an oscilloscope or the like.
  • the pulse magnetic field or pulse electric field supply means 3 preferably includes a magnetic field or electric field stimulation probe 4 and a magnetic field or electric field stimulation stage 5 as shown in FIG.
  • the magnetic field or electric field stimulating probe 4 is not particularly limited as long as it can supply a pulsed magnetic field or a pulsed electric field to living cells or the like and generate an eddy current or a capacitive current in the culture vessel.
  • the magnetic field or electric field stimulation probe 4 preferably has a structure of a single or a plurality of magnetic field stimulation coils 13 or an electric field magnetic field stimulation coil as shown in FIG.
  • the two magnetic field stimulating coils 13, 13, 13 have a structure that allows the N pole and the S pole to contact the bottom of the culture vessel 6. That, even more preferred.
  • the magnetic field stimulation coil has an inductance of 1 H or less, preferably about 1 ⁇ m to 50 O mH.
  • the material of the magnetic field stimulating coil 13 examples include ferrite and magnetic particles.
  • the magnetic field stimulating coil 13 has a core coated with a magnetic material such as a ferrite type, an amorphous type, a metal compact, or a permalloy. It's coil.
  • the material of the electric field stimulation conductive plate is not particularly limited as long as it is conductive such as metal such as aluminum, copper, and iron, or conductive resin and conductive rubber.
  • the size, shape, number, and the like of the magnetic or electric field stimulation probes 4 can be appropriately determined according to the size, shape, and the like of the culture vessel 6 to be used.
  • the magnetic or electric field stimulating probe 4 preferably has a shape compatible with the well arrangement of the multiwell plate. Further, as shown in FIG. 4 (A), for example, a magnetic or electric field stimulation probe 4 for local stimulation using a small double coil and a wide area using a coil having a large diameter of 1 mm, as shown in FIG.
  • a probe 42 for stimulating a magnetic or electric field that can stimulate a magnetic or electric field can be formed.
  • the local double stimulus magnetic field or electric field stimulating probe 41 provides a local magnetic field or electric field.
  • the single coil type magnetic field or electric field stimulation probe 42 can supply a pulse magnetic field or an electric field over a wide range.
  • a pulsed magnetic field or a pulsed electric field can be selectively supplied to a predetermined cell group or a predetermined tissue site by the magnetic field or electric field stimulating probe 4, whereby local (for example, lxl 0 2 to 9 xl 0 m 2 , preferably 9 x 10 2 to 2.5 x 1
  • the range of “m 2 ” can promote or suppress the growth of living cells, etc.
  • the size, shape, thickness, etc. of the magnetic or electric field stimulation stage 5 are not particularly limited, It is preferable to have a size, shape, etc. that can accommodate, or preferably place, one or more culture vessels 6.
  • the material of the magnetic or electric field stimulating stage 5 is not particularly limited, but simplicity of processing and material Acrylic resin is preferred from the viewpoint of cost.
  • the size and shape of the culture vessel 6 can be freely selected according to the size and shape of the magnetic or electric field stimulation stage 5.
  • the culture vessel 6 is a commercially available 35 mm
  • Petri dishes of 6 mm, 90 mm, and 150 mm, a 6- to 4-84 mass well plate, a tissue culture tube having a diameter of 10 to 30 mm, and various square dishes can be used.
  • it is a multiwell plate.
  • the culture vessel 6 can be fitted in the pulse magnetic field supply means 3, preferably in the magnetic or electric field stimulating stage 4.
  • magnetic field stimulation or electric field stimulation can be independently supplied to the wells of the well plate.
  • the culture vessel is preferably made of a plastic that is excellent in transparency so that the cultured living cells and the like can be easily observed and is not easily damaged during handling, and is particularly preferably made of an acrylic resin having high transparency and excellent rigidity.
  • the culture vessel 6 can be filled with the medium 14 therein.
  • the medium 14 is usually prepared by adding various amino acids such as L-Arg, L-Cys, L-Gln, and L-His in physiological saline containing metal ions such as Na, Mg, and Ca.
  • a medium containing various vitamins, for example, folic acid, pantothenic acid, nicotinamide, pyridoxal, riboflavin and the like can be used as a liquid medium as it is, or as a gel medium to which collagen or the like is added.
  • Medium 14 contains various cytokines and growth factors conventionally proposed in tissue culture, such as interleukins, neurotropins, platelet-derived growth factor, epidermal growth factor, and fibroblast growth factor. It is preferable to add the compound in an amount ranging from Ong Zml to ⁇ ⁇ ⁇ g / m 1.
  • the medium 14 may be a gel medium or a liquid medium.
  • the original tissue various original tissues used as known cells or tissues can be used.
  • the “living body” such as a living cell to which an electric stimulus (eddy current) is given by the magnetic or electric field stimulating device of the present invention includes humans, mammals such as dogs, cats, horses, pigs, sheep, mice, rats, etc. In addition to birds, reptiles, amphibians, fish, bacteria, viruses, and other microorganisms and plants.
  • the “tissue” to be stimulated by the magnetic or electric field stimulating device of the present invention includes all tissues of living organisms, fl containers, and some of them.
  • central nervous system peripheral god Meridian, bone, cartilage, joints, lymph vessels, blood vessels, heart (myocardium, valves), lungs, liver, spleen, pancreas, esophagus, stomach, small intestine, large intestine, kidney, bladder, ovaries, ovaries, testes, diaphragm, Muscles, tendons, skin, eyes, nose, trachea, tongue, lips, nails, hair, etc.
  • the tissue used in the magnetic field or electric field stimulator of the present invention is a tissue in which excitatory electrical stimulation is resident in a living body, such as the heart, skeletal muscle, smooth muscle, peripheral nerve, and brain. Tissue such as the central nervous system.
  • the pulse current supply means 2 is a monitoring device for monitoring the supply state of the pulse current supplied to the magnetic field stimulation coil 13 immediately below the culture vessel 6 or the electric field stimulation conductive plate. Can be further provided.
  • a light emitting element (LED) 10 or the like is preferably provided as shown in a preferred embodiment of the present invention.
  • the light emitting element 10 preferably used in the magnetic field or electric field stimulating device of the present invention is an element capable of converting a pulse current into light when the pulse current flows in the pulse current supply means 2.
  • the intensity can be expressed as the intensity of light. Therefore, the light emitting element 10 plays a role in visually recognizing the supply state of the pulse magnetic field or the pulse electric field supplied to the culture medium in the culture vessel 6.
  • a light emitting element 10 for example, a light emitting diode or the like is preferably used.
  • the circuit arrangement of the logic IC 9, the power supply 7, the switch 8, the monitoring device (optical element 10), and the coil 13 of the pulse current supply means 2 in the magnetic or electric field stimulating device of the present invention is not particularly limited.
  • the magnetic field: X can be appropriately determined according to the shape, size, and the like of the electric field stimulation stage 5.
  • the magnetic or electric field stimulating device 1 of the present invention can have a circuit arrangement as shown in FIGS. 3 (A) to 3 (C).
  • FIG. 3A shows a circuit arrangement when the logic IC 9 is a CMOS IC (74HC).
  • the current generated from the power supply 7 is converted into a pulse current having a desired waveform in the CMOS-based IC, and is adjusted to a desired current intensity, cycle, and conduction time by an electrolytic capacitor or a resistor as a peripheral element.
  • the adjusted pulse current is The light is supplied to the coil while being monitored by the light emitting element (LED) 10.
  • FIG. 3B shows a circuit arrangement in a case where all peripheral elements are included in the logic IC 9.
  • the circuit arrangement shown in FIG. 3 (B) has an advantage that the space occupied by peripheral elements can be omitted, which can contribute to downsizing of the device.
  • FIG. 3 (C) shows a circuit arrangement in a case where all the peripheral elements are incorporated in the logic IC 9 and the switch 8 is a logic switch.
  • the use of the logic switch shown in FIG. 3 (C) is a feather touch switch, which is a light touch, which is particularly preferable.
  • the method for manufacturing the magnetic or electric field stimulating device of the present invention is not particularly limited.
  • a coil or conductive plate applied to a normal single vessel, a coil or conductive applied to a stimulation circuit and a multi-well plate is used.
  • the raw plate arrangement and the stimulating circuit can be integrally formed as a single microchip by a semiconductor technology as much as possible, and a coil or a conductive plate can be additionally constructed.
  • the magnetic or electric field stimulator of the present invention is preferably used to promote or suppress the growth of living cells and the like. More preferably, the magnetic or electric field stimulating probe in the magnetic or electric field stimulating device of the present invention is provided by selectively supplying a pulsed magnetic field of a predetermined intensity to a predetermined cell group or a predetermined site such as a living cell. It is used to promote or suppress the growth of living cells and the like.
  • a pulse magnetic field or a pulse electric field preset according to the purpose of an experiment is changed to an eddy current or a capacitive current (pulse signal).
  • the magnetic or electric field stimulator of the present invention can be used for a method of promoting or suppressing the function of a living cell or a living tissue, and a method characterized by acclimation of a cell function as the promotion of the function of a living cell or a living tissue. .
  • the magnetic or electric field stimulating device of the present invention can be used in a method of denaturing normal living cells or living tissues to create a degenerative disease model of living cells or the like.
  • This The biodegenerative disease model obtained by such a method can be used for the study of various disease models, and will be useful in future studies on pathogenesis.
  • the present invention relates to a method for acclimating the function of an organ or a tissue or cells constituting the same.
  • the function acclimatization method of the present invention will be described in detail.
  • Living organisms to be subjected to the method for acclimating the function of the present invention include humans, mammals such as dogs, cats, horses, pigs, sheep, mice, rats, etc., birds, reptiles, amphibians, fish, bacteria, viruses, etc.
  • This concept encompasses microorganisms and plants.
  • animals having nerves and hearts, preferably mammals, and particularly preferably humans are targeted.
  • the organs / tissues to be subjected to the function acclimation method in the present invention include all tissues and organs of a living body.
  • organs, tissues or tissues in which excitable electrical stimulation is resident in the living body such as the central nervous system such as the heart, skeletal muscle, smooth muscle, peripheral nerves, and the brain, especially the heart and the periphery Targets central nervous tissue.
  • the central nervous system such as the heart, skeletal muscle, smooth muscle, peripheral nerves, and the brain, especially the heart and the periphery Targets central nervous tissue.
  • the tissues to be functionally adapted for transplantation include, among the above-mentioned tissues and organs of the living body, cardiac muscles that form a part of an organ constantly exposed to a bioelectric signal specific to the organ, that is, special cardiac muscles Of particular interest is native myocardial tissue.
  • the organs, tissues, or cells that are adapted to function in the present invention are not limited to those extracted and collected from a living body.
  • differentiation from stem cells such as embryonic stem cells (ES cells), hematopoietic stem cells, and mesenchymal stem cells ⁇
  • stem cells such as embryonic stem cells (ES cells), hematopoietic stem cells, and mesenchymal stem cells ⁇
  • ES cells embryonic stem cells
  • hematopoietic stem cells hematopoietic stem cells
  • mesenchymal stem cells ⁇
  • fibrous tissue obtained by culturing it is also possible to use a fibrous tissue obtained by culturing.
  • An organ, tissue or cell that is adapted for transplantation according to the present invention is an organ, tissue, or tissue extracted from a part of a living body, for example, a mammalian organ such as a transgenic pig in which MHC is adapted to that of a human. Or it can be a cell.
  • the tissue to be functionally adapted in the present invention may be, for example, a tissue obtained by growing cells collected from a living body, for example, a mammal, for example, a human in inVitro.
  • the tissue obtained by the above method is subjected to functional adaptation under culture conditions.
  • This adaptation is performed by placing the organ, tissue or cell of interest in a container containing the medium.
  • the function acclimation means that an organ, tissue or cell cultured or proliferated or removed for transplantation is used as a source of electrical excitation of a living body in the same manner as an original organ or tissue. This refers to the operation of exposing to an electrically stimulated atmosphere for a certain period so that it can be adapted to the conditions.
  • functions in this project will be collectively referred to as “function acclimation”.
  • various amino acids such as L-arginine, L-cystine, and L-glutamine are added to physiological saline containing metal ions such as Na, Mg, and Ca. , L-histidine, and various vitamins such as folic acid, pantothenic acid, nicotinamide, pyridoxal, riboflavin, etc., preferably as an Eag1e MEM medium or Du1becco & Smodified medium.
  • a culture medium and the like available from Funorade can be used.
  • the temperature for acclimatizing the subject usually ranges from 10 ° C (degrees C) to 45 ° C (degrees 0, preferably 30 ° C (degrees C) to 37 (degrees C).
  • cytokines conventionally proposed for tissue regeneration in the above medium such as interleukins, neurotropins, platelet-derived growth factor, epidermal growth factor, and fibroblast growth factor, from 1 ng / m1 to 100 ng It is preferable to mix in an amount in the range of / m 1.
  • an electric signal is given to the object during the function acclimation.
  • the electrical signal is a pulsed electrical signal or a pulsed magnetic signal.
  • the electric signal may be given continuously or intermittently.
  • a pulse of 0.5 to 50 HZ, preferably 1 to 10 HZ, 10 mV to 10 V, preferably 100 mV to 1 V is applied by bringing the electrode into contact with the culture medium Z or the function acclimation target. It is necessary to provide an electrical signal.
  • the electric acupuncture stimulus is initially set to a low voltage, and the intensity is gradually increased.
  • the device for applying an electric current is preferably connected to a function generator capable of changing the current in a pulsed manner at regular intervals, that is, generating a pulsed current.
  • Another way to provide an electrical signal to the subject to be acclimated is to apply the electric signal to the subject to be acclimated, preferably the container containing the subject to be acclimated, by changing the magnetic field, preferably the magnetic flux density, as shown in Figure 12.
  • an electric wire is wound in a coil shape, as is known by an electromagnet, and a container containing an object to be transplanted, preferably an object to be functionalized, is placed in the center thereof.
  • a current preferably a pulse current
  • the intensity of the eddy current in the object to be functionalized can be changed in a pulse manner, that is, given as a pulse signal.
  • This latter method is more preferable because the stimulation can be performed without bringing a foreign object such as an electrode into contact with the object to be adapted.
  • the magnetic signal given to the object to be functionalized is 0.5 to 50 Hz, preferably:! It is necessary to use a pulse signal in the range of ⁇ 10 Hz, 0.1 mT to 10 mT, preferably 0.5 mT to 5 mT.
  • Another way to apply an electrical signal to the subject to be acclimated is to apply the object to be acclimated, preferably a container containing the subject to be acclimated, to an electric field, preferably a burst current cluster, as shown in Figure 12.
  • an electric field preferably a burst current cluster
  • a method of generating a capacitive current inside an object to be functionalized by placing it in an electric field that can be given in a state can also be mentioned.
  • the electric field that can apply the above-mentioned burst current cluster in a pulse form is, for example, a container containing an object to be transplanted, preferably an object to be functionalized, between the upper and lower two conductive plates, and the electric current between the plates.
  • a pulsed burst current cluster can be provided to provide a capacitive current within the subject to be functionally adapted.
  • the latter method is more preferable because stimulation can be performed without bringing a foreign object such as an electrode into contact with an object to be adapted.
  • a conductive material such as a silver electrode is brought into monopolar contact with the culture medium instead of the conductive plate at the top of the culture vessel, the DC component of the current can be completely removed. As if sex changes were eliminated as much as possible With the formula, the intensity of the capacitance current can be increased.
  • the frequency of the burst current is 10 Hz or more, the frequency of the current pulse is 5 to 50 H, preferably 1 to 10 Hz, and the intensity is preferably 1 microvolt to 100 volts, 5 millibonoreto to 10 volts, and 30 milliports. ⁇ 3 volts force S more preferred.
  • a mixed sample of sensory nerve cells and Schwann cells was cultured in the same manner as in Example 1 except that no magnetic field stimulation was given. The results are shown in FIG. 5 (B).
  • the specimen cultured with magnetic field stimulation (Fig. 5 (A) was compared with the specimen cultured without magnetic field stimulation (Fig. 5 (B)).
  • Significant neurite outgrowth and branching and proliferation of Schwann cells were observed.
  • the magnetic field stimulator of the present invention has good fixation of Schwann cells and nerve cells in the medium, greatly promotes growth of Schwann cells and nerve cells, and thereby enhances neurite outgrowth. It can be seen that branching can be promoted.
  • Example 2 Temperature 37 ° (, humidity 99%, under the conditions of C0 2 concentration of 5% by using a magnetic field stimulator of the present invention, for the purpose of growth inhibition (cytotoxicity grant) in a mixed sample of sensory neurons and Schwann cells
  • Figure 6 shows the results of applying the electrical stimulation under the conditions (3 mT, duration 20 ms ec, 50 Hz) for 3 days.
  • Fig. 8 shows the results.
  • Control group (control), group impaired by pulsed magnetic field stimulation (impared).
  • a of FIG. 8 shows the measurement result of the resting membrane potential of the cultured sensory nerve of the mouse under the condition of the membrane current fixation by the patch clamp technique.
  • FIG. 8 shows normal (control) and resting membrane potential 0 from the impaired sensory nerve (disorder) produced by applying pulse magnetic field stimulation (3 mT, .3 msec duration, 5 Hz) for 3 days.
  • MP control group
  • -59.7 ⁇ 2.5 mV mean and SEM
  • n 7
  • FIG. A of FIG. 9 shows the neuronal cell death (arrow head) of the control sensory nerve after 3 days in the medium containing 10 / zM capsaicin (CAPS).
  • 0 shows the survival rate of cultured sensory nerves on each day after capsaicin treatment.
  • Example 5 Frequency of contraction of each ventricular muscle cluster to 5 Hz electrical stimulation and contraction pattern of typical magnetic stimulation group cluster to 2 Hz and 5 Hz electrical stimulation
  • the purpose of the present invention is to promote growth of a mixed sample of ventricular myocytes and fibroblasts using the magnetic field stimulator of the present invention under the conditions of a temperature of 37 ° C (degree C), a humidity of 99% and a CO 2 concentration of 5%.
  • the cells were cultured while applying magnetic field stimulation (1-3 mT, 3 msec, 5-10 Hz) under the conditions described above for 7 days.
  • the results are shown in FIG.
  • changes in the response of the functionalized myocardium to electrical stimulation and the recovery of function by magnetic stimulation were evaluated using a video camera attached to a phase-contrast microscope. Seconds of video data were acquired and stored, and heart rate was measured and tabulated. (result)
  • Example 6 (Observation of function-conditioned cells) Contraction frequency of ventricular muscle clusters cultured in the presence of pulsed magnetic field stimulation in response to electrical stimulation
  • the heart rate of the activated myocardium decreases from the original heart rate of the mouse (48.6 to 738 times Z minutes, that is, 8.1 to 12.3 HZ).
  • the stimulus responsiveness to electrical stimulation ie, the frequency of stimuli that could be followed by cultured cardiomyocytes, was reduced to 2 to 5 Hz (control group (14d without magnetic stimulation)).
  • ventricular muscle clusters (magnetic stimulation 7d / 14d) cultured in the presence of pulsed magnetic field stimulation (l-3mT, 3msec, 5-10Hz) for 7 out of 14 days recovered responsiveness to high frequency stimulation And was able to follow the electrical stimulation of 7 HZ.
  • Figure 13 shows an enlarged photo of a cardiomyocyte cluster in culture.
  • the magnetic or electric field stimulating device of the present invention is capable of applying a desired pulsed magnetic field or pulsed electric field to living cells and the like in a culture vessel to generate an eddy current or a capacitive current in the culture vessel.
  • the stimulator that can be used is simple in structure, easy to manufacture, and inexpensive.
  • the magnetic or electric field stimulating device of the present invention can easily attach and detach a culture container to which magnetic or electric field stimulation is to be applied, and can locally apply magnetic or electric field stimulation to desired living cells and the like, and furthermore, cultivate A magnetic or electric field stimulator that does not come into direct contact with the medium in the container. Therefore, with the magnetic or electric field stimulating device of the present invention, it is possible to apply magnetic field or electric field stimulation to desired living cells for a long period of time without changing the characteristics of the culture medium in the culture vessel. Of the medium can be improved.
  • a method in which a pulsed magnetic field or a pulsed electric field is controlled to apply electric stimulation to a living cell or the like to promote or suppress the growth of the living cell or the like.
  • a pulse signal preferably an eddy current or a capacitance current preset according to the purpose of the experiment is applied to the original tissue or the like from the outside of the culture vessel without directly contacting the original tissue or the like.
  • a normal living cell or living tissue preferably a nerve cell or a nerve tissue is degenerated by the magnetic or electric field stimulating device of the present invention, so that a living cell or living tissue degenerative disease model can be easily created.
  • the organs or tissues accustomed to the excitatory input by the function acclimatization method of the present invention or the cells constituting them are adapted to the bioelectric signal, for example, they are transplanted immediately after transplantation and In this case, even if a bioelectric signal is received, excitatory conduction can be performed in cooperation with surrounding tissues.
  • myocardial tissue it has extremely higher adaptability than a transplant prepared by a conventional method, and can prevent the transplant from becoming a source of arrhythmia or the like as much as possible.
  • tissues or cells treated by the function acclimatization method of the present invention particularly myocardial tissues and myocardial cells, always maintain a stimulus tracking ability close to physiological conditions. As a result, it can be used as a more reliable sample for drug evaluation and toxicity testing of cardiovascular drugs.

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Abstract

L'invention concerne un dispositif de stimulation de champ magnétique ou électrique ne modifiant pas la liquidité de la solution de la culture et ne diminuant pas la vitesse de fixation des cellules, même si des cellules vivantes sont stimulées électriquement pendant une longue durée pendant le processus de croissance de celles-ci, un procédé permettant de favoriser ou de limiter la croissance de cellules vivantes au moyen du dispositif de stimulation de champ magnétique ou électrique, ainsi qu'un procédé permettant de créer un modèle de maladie dégénérative d'une cellule vivante. Le dispositif de stimulation de champ magnétique ou électrique est caractérisé en ce qu'il comprend des moyens d'alimentation de courant d'impulsions permettant de générer un courant d'impulsions et de régler la caractéristique du courant d'impulsions et des moyens d'alimentation de champ magnétique ou électrique permettant de produire un champ magnétique ou électrique d'impulsions, au moyen du courant d'impulsions alimenté à partir des moyens d'alimentation de courant d'impulsions et d'alimenter le champ magnétique ou électrique d'impulsions dans des cellules vivantes ou un tissu vivant dans un cultivateur à partir de l'extérieur de celui-ci.
PCT/JP2005/001164 2004-01-22 2005-01-21 Dispositif de stimulation de champ magnetique ou electrique et procede permettant de favoriser, limiter ou entraver la croissance et la fonction d'une cellule ou d'un tissu vivant au moyen dudit dispositif WO2005071057A1 (fr)

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US9694193B2 (en) 2009-03-03 2017-07-04 Technische Universiteit Eindhoven Device and method for treating cells
CN108554332A (zh) * 2018-05-15 2018-09-21 中亚民生科技发展有限公司 N-s矢量真量子牵引能量波植入设备
FR3090388A1 (fr) * 2018-12-20 2020-06-26 Centre National De La Recherche Scientifique Dispositif d’exposition d’échantillon biologique à au moins un champ électromagnétique et système correspondant.
CN115029238A (zh) * 2022-06-08 2022-09-09 哈尔滨医科大学 一种脉冲磁场生物学效应机理研究实验装置及方法
WO2023110063A1 (fr) * 2021-12-14 2023-06-22 Azyro Sa Appareil pour influencer in vitro un matériau cellulaire biologique, et son utilisation

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* Cited by examiner, † Cited by third party
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US9694193B2 (en) 2009-03-03 2017-07-04 Technische Universiteit Eindhoven Device and method for treating cells
CN108554332A (zh) * 2018-05-15 2018-09-21 中亚民生科技发展有限公司 N-s矢量真量子牵引能量波植入设备
CN108554332B (zh) * 2018-05-15 2024-03-08 中亚民生科技发展有限公司 N-s矢量真量子牵引能量波植入设备
FR3090388A1 (fr) * 2018-12-20 2020-06-26 Centre National De La Recherche Scientifique Dispositif d’exposition d’échantillon biologique à au moins un champ électromagnétique et système correspondant.
WO2023110063A1 (fr) * 2021-12-14 2023-06-22 Azyro Sa Appareil pour influencer in vitro un matériau cellulaire biologique, et son utilisation
CN115029238A (zh) * 2022-06-08 2022-09-09 哈尔滨医科大学 一种脉冲磁场生物学效应机理研究实验装置及方法

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