JP2004089018A - Electrical stimulator and method for promoting or suppressing growth of biological cell or biological tissue using the same electrical stimulator - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an electrical stimulator applying electrical stimulation to a biological cell or a biological tissue in a process for growing the biological cell or biological tissue and to provide a method for promoting or suppressing the growth of the biological cell or biological tissue using the stimulator. <P>SOLUTION: The electrical stimulator is equipped with a current-producing means 4 for producing a pulsed current and regulating the intensity, etc., of the pulsed current, a cap 1 for fixing the current-producing means 4 and a pair of electrodes 9 and 9' suspended from the cap 1 and further connected to both ends of the current-producing means 4 so as to apply the pulsed current as the electrical stimulation to a culture medium 11 in a culture vessel 2. <P>COPYRIGHT: (C)2004,JPO

Description

[0001]
[Field of the Invention]
The present invention relates to an electrical stimulator for living cells and tissues, and a method for promoting or suppressing the growth of living cells and tissues. More specifically, the present invention provides an electrical stimulation device capable of promoting or suppressing the growth of living cells, tissues, and the like by applying specific electrical stimulation to living cells, tissues, and the like used in experiments in a research laboratory and the like. The present invention relates to a method for promoting or suppressing the growth of living cells and tissues using an electric stimulator.
[0002]
[Prior art]
The development of an effective in vitro disease state model is important for the development of a therapeutic method for a degenerative disease in which hyperexcitation of nerve cells is directly caused, such as epileptic dementia and hypoxic encephalopathy.
[0003]
Conventionally, attempts have been made to administer a large amount of excitatory neurotransmitters to cultured cells and tissues of a living body, and electrical stimulation has been performed using a normal electrical stimulator. However, when excitatory neurotransmitters are administered in large doses, the types of receptors that are stimulated are limited to excitatory neurotransmitter receptors, and cellular responses to various signaling molecules that can occur during overexcitation in vivo. May be expressed in a manner biased on excitatory neurotransmitter receptor responses.
[0004]
On the other hand, the overexcitation induced by electrical stimulation is considered to be more physiologically close.However, with conventional electrical stimulation devices, it is complicated to attach electrodes and execute stimulation in a narrow incubator, and electrophysiology is difficult. It is difficult for many researchers, including biochemists and genomic scientists who do not specialize in experiments. Also, in other basic and applied research fields that require relatively long-term electrical stimulation in a culture environment, a simpler and more compact cultivation device with a stimulating function can be a powerful weapon in conducting research. .
[0005]
An ideal electrical stimulator for such simplicity and compactness could potentially bring enormous potential demands not only from physiology, pharmacology and biochemistry, but also from laboratories performing regenerative medicine and genomics. Have.
[0006]
[Problems to be solved by the invention]
The present invention has been made in order to solve the above-mentioned problems, and an object of the present invention is to provide electrical stimulation to a living cell or the like in a growth process of a living cell or a living tissue (hereinafter, also referred to as “living cell or the like”). It is an object of the present invention to provide an electric stimulator capable of providing the following.
Another object of the present invention is to provide a method for promoting or suppressing the growth of living cells and the like using the electric stimulator.
[0007]
[Means for Solving the Problems]
The inventors of the present invention have made intensive studies on the relationship between the growth of living cells and the like and the electrical stimulation, and have completed the present invention.
That is, an object of the present invention is to generate a pulse current, and a current generating means for adjusting the intensity or the like of the pulse current, a cap for fixing the current generating means, and a pulse suspended from the cap, and The present invention is achieved by an electric stimulator having a pair of electrodes connected to both ends of the current generating means for applying an electric current to a culture medium in a culture vessel as electric stimulation.
[0008]
The electrostimulator of the present invention has a structure in which a cap has a current generating means and a pair of electrodes. Therefore, with the electrical stimulation device of the present invention, the device can be easily manufactured, and the cap can be easily attached to and detached from the culture container. In addition, the electric stimulator of the present invention can easily give a pulse current having an arbitrary intensity and waveform to a living cell or the like simply by changing a cap, and can appropriately adjust the growth of the living cell or the like in a culture process. .
[0009]
Further, preferred embodiments of the electric stimulation device of the present invention are as follows.
(1) The electric stimulator in which the current generating means is an electric circuit including a logic IC, a microswitch, and a power supply.
(2) The electric stimulator in which the power source is a battery or an electric supply device.
(3) The electrostimulator according to (1) or (2), wherein the electric circuit is formed on a printed board, and the printed board and the cap are integrally mounted.
(4) The electric stimulator according to any one of (1) to (3), wherein the electric circuit further includes a monitoring device for monitoring a supply state of a pulse current supplied as electric stimulus to the culture medium in the culture vessel.
(5) The electrical stimulation device according to (4), wherein the monitoring device is a light emitting element.
(6) The electric stimulator according to any one of (1) to (5), wherein the cap is a cap that can correspond to a well arrangement in a multiwell plate.
(7) By fitting the cap to the culture vessel, the pulse current is applied as electrical stimulation from the electrode to the living cells or tissues loaded in the culture medium in the culture vessel (1) to ( The electrical stimulator according to any one of 6).
(8) The electrical stimulator according to any one of (1) to (7), which is used to promote or suppress the growth of living cells or living tissues.
(9) The electric stimulator according to any one of (1) to (8), wherein the pulse current has an intensity to promote or suppress the growth of living cells or tissue cells.
[0010]
Another object of the present invention is achieved by a method for promoting or suppressing the growth of living cells and the like using the electric stimulator.
According to the method of the present invention, not only cultivation to promote the growth of living cells and the like as in the conventional stimulator, but also damage to the living cells and the like to suppress the growth of living cells and living tissues. Can also be cultured.
[0011]
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Hereinafter, the electrical stimulator of the present invention and a method for promoting or suppressing the growth of living cells using the electrical stimulator will be described in detail.
[0012]
First, the configuration of the electrical stimulation device 10 of the present invention will be described below with reference to the drawings.
[0013]
FIG. 1 is a schematic diagram showing a preferred embodiment of the electrical stimulation device 10 of the present invention. In FIG. 1, reference numeral 1 denotes a cap for mounting an electrical stimulator (hereinafter, simply referred to as a “cap”), and reference numeral 2 denotes a culture vessel. As shown in FIG. 1A, a printed circuit board 3 is mounted on the cap 1 integrally with the cap 1. An electric circuit 4 as an electric supply means is formed on the printed circuit board 3.
[0014]
The electric circuit 4 in FIG. 1 includes a logic IC 5, a button battery 6, a microswitch 7, and a light emitting element (LED) 8. The electrodes 9 and 9 ′ are composed of an anode and a cathode, penetrate the cap 1, and are suspended from the cap 1.
[0015]
Next, the electrical stimulation device of the present invention will be specifically described.
The electric stimulator 10 of the present invention has a current generating means on the cap 1. The current generating means is not particularly limited as long as it can be fixed to the cap 1, generates a pulse current, and can adjust the intensity, cycle, and the like of the pulse current. The current generating means is preferably an electric circuit 4 including a logic IC 5, a button battery 6, a microswitch 7, and an LED 8 as shown in FIG.
[0016]
The waveform, intensity, cycle, and the like of the pulse current supplied from the current generating means can be appropriately adjusted according to the type of the living cell to which the electrical stimulation is applied and the purpose of culture (growth promotion or growth suppression). For example, as the waveform of the pulse current, a triangle, a rectangle, various other function waveforms (for example, a sine waveform, an exponential function waveform, and the like) can be given, and a rectangle is particularly preferable. In addition, the intensity of the pulse current can be appropriately adjusted according to two culture purposes, namely, promotion of growth of living cells and the like and suppression of growth (damage). For example, when culturing for the purpose of promoting the growth of living cells or the like, the intensity of the pulse current is 0.1 to 50 mV / mm, preferably 0.5 to 25 mV / mm, and more preferably 1 to 10 mV / mm. Is more preferable. On the other hand, when culturing for the purpose of suppressing the growth (damage) of living cells, the intensity of the pulse current is 50 to 250 mV / mm, preferably 75 to 200 mV / mm, and more preferably 100 to 150 mV / mm. Is more preferred. The cycle of the pulse current is, for example, 0.001 to 1000 Hz, preferably 0.005 to 100 Hz, and more preferably 0.01 to 10 Hz. Further, the energizing time of the pulse current is, for example, 1 μsec to 10 sec, preferably 5 μsec to 1 sec, and more preferably 10 μsec to 0.1 sec.
[0017]
The size and shape of the cap 1 can be freely selected according to the size and shape of the culture vessel 2 to be fitted. For example, the cap 1 is a commercially available 35 mm, 60 mm, 90 mm, 150 mm Petri dish, a 6-well to 384-well multi-well plate, a circular cap corresponding to a tissue culture tube having a diameter of 10 to 30 mm, and various square dishes. A corresponding square cap can be used. Preferably, the cap 1 is a cap corresponding to the well arrangement of the multi-well plate.
[0018]
As the material of the cap 1, various materials such as metal, glass, and plastic can be used, but it is preferable that the material is made of plastic that has excellent transparency so that the culture can be easily observed and that is not easily damaged during handling. In particular, it is preferably made of an acrylic resin having high transparency and excellent rigidity.
[0019]
The cap 1 is preferably attached integrally with the printed circuit board 3 in the electrostimulation device 10 of the present invention. The printed board 3 can be manufactured using a known material in which the electric circuit 4 is printed on a board made of heat-resistant plastic or a fiber-reinforced plastic such as glass fiber. Although the shape of the printed circuit board 3 is not particularly limited, it is preferable that the shape conforms to the shape of the electric circuit 4.
[0020]
The electric circuit 4 includes a logic IC 5, a power supply (6), and a microswitch 7, and preferably includes a light emitting element 8 described later. The method of forming the electric circuit 4 is not particularly limited. For example, a method of applying a conductive paint on a printed circuit board to print and print on a printed circuit board, or a method of welding a thin conductive wire or the like is used. Can be mentioned. Among them, a method of printing on a substrate is preferable from the viewpoint of making a circuit compact.
[0021]
As the logic IC 5 constituting the electric circuit 4, 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, can be used. In the electric stimulator of the present invention, the logic IC 5 has a role of changing the magnitude of the pulse current on the time axis.
[0022]
Examples of the logic IC 5 include well-known logic ICs, such as a general-purpose logic IC, a PLD (program logic device), a CPLD (complex program logic device), an FPGA (field program gate array), and a custom IC. Although general-purpose logic ICs include a CMOS type and a bipolar type, a CMOS type is preferable. The logic IC 5 can be easily obtained on the market. The logic IC 5 may further have a peripheral element such as a capacitor.
[0023]
In the electric stimulator of the present invention, the pulse current adjusted by the logic IC 5 is set to flow to the electric circuit 4. It is preferable that the voltage (intensity), the frequency (cycle) of generation, the generation pattern (waveform), and the like of the pulse current can be changed as a series of protocols by the logic IC according to the purpose of the experiment. As a method of realizing such a protocol of the stimulus which can be changed, there is a method of writing and erasing the protocol in the logic IC.
[0024]
The power source in the electrostimulation device of the present invention is preferably a battery or an electricity supply device for supplying electricity from outside. When the power source is a battery, the battery needs to be mounted on the cap 1, so that the battery is preferably as small as possible, and more preferably a button type. As the performance required for the battery, it is preferable that the battery has a voltage having a potential difference of 5 V or less, preferably about 1 to 3 V. In addition, it is desirable that the medium has a capacity to allow a pulse current of 10 μA on average to flow in the medium for 2 hours or more, preferably 5 hours or more, more preferably 24 hours or more.
[0025]
In the preferred embodiment of the electric stimulator of the present invention, a button battery 6 is used as a power source (see FIG. 1A). The button battery 6 may be a rechargeable storage battery such as a nickel-cadmium battery, an electric double layer capacitor, or a lithium storage battery, as well as a disposable battery such as a mercury battery or lithium.
[0026]
On the other hand, when the power supply in the electrostimulation device of the present invention is an electric supply device, the electric supply device may be a DC power supply such as an AC adapter, an AC / DC switching power supply, an AC / DC converter, and various power supply transformer products. .
[0027]
The microswitch 7 in the electric stimulator 10 of the present invention is a device that controls on / off of a pulse current in a circuit of the stimulator, like a normal switch. The microswitch 7 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. It is desirable that the microswitch 7 be as small and light as possible as long as operation is possible.
[0028]
In the electric stimulator of the present invention, the electric circuit 4 may further include a monitoring device for monitoring the supply status of the pulse current supplied to the culture medium 11 in the culture container 2. As the monitoring device, for example, a light emitting element (LED) 8 or the like is preferably provided as shown in a preferred embodiment of the present invention.
The light emitting element 8 preferably used in the electrostimulation device of the present invention is an element that can convert the pulse current into light when the pulse current flows in the electric circuit 4. Can be represented. For this reason, the light emitting element 8 has a role of visually recognizing the supply state of the pulse current supplied to the culture medium in the culture vessel. As such a light emitting element 8, for example, a light emitting diode or the like is preferably used.
[0029]
The electrodes 9, 9 'in the electric stimulator of the present invention are terminals for applying a pulse current to the culture medium in the culture vessel as electric stimulation. In FIG. 1, the electrodes 9, 9 ′ are connected to both ends of the electric circuit 4, penetrate the cap 1, and are suspended in the cap 1. The electrodes 9, 9 'are composed of a pair of an anode and a cathode. Although the shape of the electrodes 9, 9 'is T-shaped in FIG. 1, the electrodes in the electrostimulator of the present invention are not limited to this shape, and have other shapes (for example, a lattice or a concentric shape). It may be. Furthermore, the length of the terminals of the electrodes 9 and 9 ′ can be appropriately determined according to the depth (height) of the culture vessel 2. Further, the electrodes 9, 9 'may be suspended in parallel with each other, for example, may be suspended so as to form a C-shape or an inverted C-shape when facing each other. Furthermore, if the electrodes 9 and 9 ′ are connected to both ends of the electricity generating means (electric circuit 4), the positions where the electrodes 9 and 9 ′ are connected to the electricity generating means (electric circuit 4) match the shapes of the cap 1 and the culture vessel 2. It can be determined appropriately. Examples of the material for the electrodes 9 and 9 ′ include silver-silver chloride, platinum, titanium, titanium nitride, carbon, gold, and glass.
[0030]
As shown in FIG. 1 (B), the culture vessel 2 for fitting a cap in the electric stimulator 10 of the present invention can be filled with a culture medium 11 therein. The medium 11 is usually prepared by adding various amino acids, such as L-Arg, L-Cys, L-Gln, L-His, and various vitamins, such as folic acid, to physiological saline containing metal ions such as Na, Mg, and Ca. A medium containing pantothenic acid, nicotinamide, pyridoxal, riboflavin and the like can be used.
[0031]
The medium 11 contains 0.01 ng / kg of 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 mix in an amount ranging from ml to 100 mg / ml. The medium 11 may be a solid medium or a liquid medium.
[0032]
When cultivation is performed using the electrical stimulator 10 of the present invention so as to promote or suppress the growth of living cells or the like, the medium 11 contains living cells or living tissues 12 (hereinafter referred to as “original tissues”). Alternatively, charge the medium. As the original tissue, various original tissues used as known cells or tissues can be used. For example, cells (group) and stem cells (group) constituting the original tissue and the like, part of the tissue to be cultured with the original tissue and the like, tissues similar to the tissue to be cultured, embryo cells, ES cells, and the like Good.
[0033]
The “living body” such as a living cell to be stimulated by the electric stimulator of the present invention includes humans, mammals such as dogs, cats, horses, pigs, sheep, mice, rats, etc., as well as birds, reptiles, and amphibians This is a concept that also includes microorganisms such as fish, bacteria, viruses, and plants.
[0034]
The “tissue” to be stimulated by the electric stimulator of the present invention includes all tissues and organs of a living body and a part thereof. For example, central nervous system, peripheral nerve, bone, cartilage, joint, lymph vessel, blood vessel, heart (myocardium, valve), lung, liver, spleen, pancreas, esophagus, stomach, small intestine, large intestine, kidney, bladder, uterus, ovary, Testis, diaphragm, muscle, tendon, skin, eye, nose, trachea, tongue, lips, nails, hair, and a part thereof. Tissues used in the electric device of the present invention include, among these organs and tissues, tissues in which excitatory electrical stimulation is resident in vivo, such as the central nervous system such as heart, skeletal muscle, smooth muscle, peripheral nerve, and brain. Organizations can be the main target.
[0035]
The circuit arrangement of the logic IC 5, the power supply (6), the microswitch, the monitoring device (8) and the electrodes 9, 9 'of the electric circuit 4 in the electric stimulator of the present invention is not particularly limited. It can be appropriately determined according to the size and the like. For example, in the electrical stimulation device of the present invention, it is possible to adopt a circuit arrangement as shown in FIGS.
[0036]
FIG. 2A shows a circuit arrangement when the logic IC 5 is a CMOS IC (74HC). The current generated from the power source (6) is converted into a pulse current having a desired waveform in a CMOS IC, and is adjusted to a desired current intensity, cycle, and conduction time by a capacitor or a resistor as a peripheral element. The adjusted pulse current is supplied to the electrode while being monitored by the light emitting element (LED) 8.
FIG. 2B shows a circuit arrangement when all the peripheral elements are incorporated in the logic IC 5. The circuit arrangement shown in FIG. 2B has an advantage that the space occupied by the peripheral elements can be reduced, which can contribute to downsizing of the device.
FIG. 2C shows a circuit arrangement in which all the peripheral elements are incorporated in a logic IC and the microswitch 7 is a logic switch. When the logic switch shown in FIG. 2C is used, a feather touch switch is used, so that a light touch is obtained, which is particularly preferable.
[0037]
The electrical stimulator of the present invention is preferably used to promote or suppress the growth of living cells and the like. Furthermore, it is preferable that the electrical stimulator of the present invention is used in a method for promoting or suppressing growth of living cells. When the electrical stimulator of the present invention is used in a method for promoting or suppressing the growth of living cells, a pulse electrical signal preset according to the purpose of an experiment is applied to the original tissue as electrical stimulus, and intracellular cells that can be generated by an excitatory input It is possible to promote or suppress the growth of living cells and the like in consideration of the calcium ion concentration fluctuation.
[0038]
The method for manufacturing the electrical stimulator of the present invention is not particularly limited. For example, a general single-vessel application electrode, a stimulation circuit and a multi-well plate application electrode array, and a stimulation circuit Thereby, it can be integrally formed as one microchip, and can be constructed by adding a suspended electrode.
[0039]
【Example】
Hereinafter, preferred embodiments of the present invention will be described. The materials, amounts, ratios, procedures, and the like shown in the following examples can be appropriately changed without departing from the spirit of the present invention. Therefore, the scope of the present invention should not be construed as being limited by the specific examples described below.
[0040]
(Example 1)
Under the conditions of a temperature of 37 ° C., a humidity of 99%, and a CO ₂ concentration of 5%, the electric stimulator of the present invention was used to apply electric stimulation (5 mV) to a mixed sample of sensory nerve cells and Schwann cells under the condition of promoting growth. / Mm, duration 1 msec, 0.2 Hz) for 10 days. The results are shown in FIG.
[0041]
(Comparative Example 1)
A mixed sample of sensory nerve cells and Schwann cells was cultured in the same manner as in Example 1 except that no electrical stimulation was given. The results are shown in FIG.
[0042]
As shown in FIG. 3, the specimen cultured without electric stimulation (Example 1, FIG. 3 (B)) was compared with the specimen cultured without electric stimulation (Comparative Example 1, FIG. 3 (A)). In comparison, marked neurite outgrowth and branching and Schwann cell proliferation were observed.
[0043]
(Example 2)
Conditions for the purpose of suppressing growth (imparting cytotoxicity) of a mixed sample of sensory neurons and Schwann cells using the electrical stimulator of the present invention under the conditions of a temperature of 37 ° C., a humidity of 99%, and a CO ₂ concentration of 5%. Was applied for 5 hours (150 mV / mm, duration 5 msec, 2 Hz), and the cells were further cultured for 2 days. FIG. 4 shows the results.
[0044]
As shown in FIG. 4, delayed necrosis (necrosis) was observed around the neurites after the electrical stimulation as compared to the state before the electrical stimulation ((A) in FIG. 4). (B)).
[0045]
[Effects of the present invention]
As described above, the electric stimulator of the present invention has a simple structure, is easy to manufacture, and is inexpensive as a device for applying a desired pulse current to the culture medium in the culture vessel as an electric stimulus. Further, the cap of the electric stimulator of the present invention can be easily attached and detached.
Further, by using the electrical stimulation device of the present invention, it is possible to provide a method of promoting or suppressing the growth of living cells or the like by adjusting the pulse current given as the electrical stimulation.
[0046]
In particular, if a living cell or the like is grown using the electrical stimulator of the present invention, the conventional tissue culturing apparatus is allowed to stand still, at best, can be cultured under stirring, whereas the present invention provides a pulsed electrical signal. Preferably, a pulse electric signal preset according to the purpose of the experiment is given to the original tissue as electric stimulation to promote or suppress the growth of living cells and the like in consideration of intracellular calcium ion concentration fluctuations that can occur due to excitation input. be able to.
[Brief description of the drawings]
FIG. 1 is a schematic diagram showing an embodiment of the electrical stimulation device of the present invention.
FIG. 2 is an explanatory diagram showing a circuit arrangement of an electric circuit in the electric stimulation device of the present invention.
FIG. 3 is a photograph showing a state in which nerve stimulation is applied to nerve cells using the electrical stimulation device of the present invention to promote growth.
(A) Photographs showing the results when cultured without applying electrical stimulation.
(B) is a photograph showing a result in the case of culturing by applying electrical stimulation.
FIG. 4 is a photograph showing a state in which the electrical stimulation is applied to nerve cells using the electrical stimulation apparatus of the present invention to suppress the growth.
(A) is a photograph showing a state of a nerve cell before electric stimulation is applied.
(B) is a photograph showing a state of a nerve cell after applying electric stimulation.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 Cap for mounting electric stimulator 2 Culture container 3 Printed circuit board 4 Electric circuit (current generating means)
5 Logic IC
6. Button battery (power supply)
7 Micro switch 8 Light emitting element (monitoring device)
9, 9 'electrode 10 electric stimulator 11 medium 12 living cell or living tissue

Claims (11)

A current generating means for generating a pulse current and adjusting the intensity of the pulse current, a cap for fixing the current generating means, and a suspension from the cap, and the pulse current is used as an electrical stimulus in a culture vessel. An electrical stimulator for a living cell or a living tissue, comprising a pair of electrodes connected to both ends of the current generating means for supplying the current to the culture medium. The electric stimulator according to claim 1, wherein the current generating means is an electric circuit including a logic IC, a microswitch, and a power supply. The electrostimulator according to claim 2, wherein the power source is a battery or an electric supply device. The electrical stimulation device according to claim 2, wherein the electric circuit is formed on a printed board, and the printed board and the cap are integrally mounted. The electric stimulator according to any one of claims 2 to 4, wherein the electric circuit further includes a monitoring device for monitoring a supply state of a pulse current supplied as electric stimulus to the culture medium in the culture vessel. The electrical stimulation device according to claim 5, wherein the monitoring device is a light emitting element. The electrical stimulator according to any one of claims 1 to 6, wherein the cap is a cap that can correspond to a well arrangement of a multi-well plate. The pulse current is applied as electrical stimulation from the electrode to living cells or living tissue loaded in a culture medium in the culture container by fitting the cap to the culture container. An electrical stimulator according to claim 1. The electrical stimulator according to any one of claims 1 to 8, which is used for promoting or suppressing the growth of a living cell or a living tissue. The electrical stimulation device according to any one of claims 1 to 9, wherein the pulse current has an intensity that promotes or suppresses the growth of living cells or tissue cells. A method for promoting or suppressing the growth of living cells or living tissues using the electrical stimulator according to any one of claims 1 to 10.

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