JP2007209233A - Electro-stimulator generating morbid electric signal and/or bio-electric signal - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an electro-stimulator equipped with simplicity and compactness, simply feeding the electric phenomena of a living body in its morbid state and/or normal state to cultured cells. <P>SOLUTION: This electro-stimulating device for generating the morbid electric signals and/or bio-electric signals, at least containing a digital IC preserving the electric phenomenal information of the living body in its morbid and/or normal state consisting of the combination of pulse electric signals as an electro-stimulation protocol prepared as a database is provided by having (1) an electric current-generator for outputting the pulse electric signals as the electro-stimulating protocol based on the electric phenomenal information from the digital IC into the medium of a culture, (2) a cap for fixing the electric current-generator and (3) a pair of electrodes hung from the cap and joined with the electric current-generator in order to output the pulse electric current as the electro-stimulation to the medium in a culturing container. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to an electrical stimulation apparatus for living cells or tissues, and a method for creating a model cell using the electrical stimulation apparatus. More specifically, the present invention can form a degenerative disease model or modify the function or form of a cell by giving a specific electrical stimulus to a living cell or tissue used in an experiment in a laboratory or the like. The present invention relates to an electrical stimulation device that can be used.

  Development of an effective in vitro pathologic model is important in the development of therapeutic methods for degenerative diseases in which neuronal hyperexcitability is directly caused, such as epileptic dementia and hypoxic encephalopathy. Similarly, it is important to develop an in vitro model that can easily examine long-term changes that occur in the heart muscle of arrhythmia patients, especially the expression of genes involved in the synthesis of biochemical components.

  Conventionally, attempts have been made to administer a large amount of excitatory neurotransmitters to cultured cells or tissues of a living body, and electrical stimulation using a normal electrical stimulation device has been performed. However, in large doses of excitatory neurotransmitters, the types of receptors that are stimulated are limited to excitatory neurotransmitter receptors, and cellular responses to various signaling molecules that can occur during hyperexcitation in vivo May be expressed in a manner biased towards excitatory neurotransmitter receptor responses.

  On the other hand, the excessive excitement caused by electrical stimulation is considered to be more physiologically close. However, in conventional electrical stimulation devices, the mounting of electrodes and the execution of stimulation in a narrow incubator are complicated, and electrophysiology It was difficult for many researchers, including biochemists and genomic scientists who do not specialize in experiments. In other basic and applied research fields that require relatively long periods of electrical stimulation in a culture environment, a simpler and more compact culture device with electrical stimulation functions is a powerful tool for conducting research. Can be a weapon.

  An object of the present invention is to provide an electrical stimulation device that has simplicity and compactness and can easily supply an electrical phenomenon during a disease state and / or normal state of a living body to cultured cells. Furthermore, the present invention provides a degenerative disease model by applying electrical stimulation to living cells and the like in the growth process of living cells or tissues (hereinafter also referred to as “living cells and the like”) to modify the functions and forms of the cells and tissues. Providing an electrical stimulation device that can be manufactured was an issue to be solved.

  As a result of diligent investigations to solve the above problems, the present inventors have created a database of electrical phenomena in the pathological state and / or normal state of a living body created by combining pulse electrical signals as an electrical stimulation protocol, and digitally An in vitro demyelinating disease model and high frequency stimulation responsiveness were maintained for a long time by storing in an IC and outputting an electrical signal into the culture medium as the electrical stimulation protocol based on the information from the digital IC Succeeded in creating myocardial tissue. The present invention has been completed based on these findings.

  That is, according to the present invention, (1) at least a digital IC in which electrical phenomenon information at the time of pathological condition and / or normal state of a living body composed of a combination of pulsed electrical signals is stored as a database as an electrical stimulation protocol is stored. Current generating means for outputting a pulse electric signal into the culture medium as the electrical stimulation protocol based on the electrical phenomenon information from the digital IC; and (2) fixing the current generating means. And (3) a pathological electrical signal that is suspended from the cap and connected to the current generating means for outputting a pulsed current to the medium in the culture vessel as an electrical stimulus. An electrical stimulation device that generates bioelectric signals and / or is provided.

Preferably, the digital IC includes a logic IC, a memory IC, and a microprocessor.
Preferably, the electrical phenomenon during the pathological condition of the living body is an electrical phenomenon of arrhythmia, epilepsy, or abnormal electromyogram.

Preferably, the current generating means includes a digital IC, a micro switch, and a power source.
Preferably, different electrical phenomenon information is stored in the digital IC as different electrical stimulation protocols, and a plurality of output terminals of the current generating means are connected in parallel, thereby combining a plurality of electrical stimulation patterns in the culture medium. Can be output.

Preferably, the current generating means is integrally formed with the cap.
Preferably, the cap has a form corresponding to the form of the culture dish.
Preferably, the cap has a structure corresponding to the well arrangement of the multi-well plate.

  Preferably, a pulse current is applied as an electrical stimulus from the electrode to a living cell or a living tissue inserted in a culture medium in the culture container by fitting a cap to a specimen arrangement portion of the culture container.

  According to another aspect of the present invention, the above-described electrical stimulation device of the present invention is used to supply the cells cultured in the culture vessel with an electrical phenomenon during normal and / or normal conditions of the living body, And / or a method for producing a model cell, comprising producing a model cell at normal time.

Preferably, an in vitro demyelinating disease model is created by selectively degenerating cultured myelinated myelinating cells.
Preferably, an epileptic focus neural tissue model specimen is created by culturing the cultured central nerve cells while outputting electrical stimulation having a pattern similar to the electroencephalogram and synaptic activity during the epileptic seizure.

Preferably, a cultured myocardial cell or myocardial tissue is cultured while outputting an electrical stimulus having a pattern similar to the pacing electrical activity of the arrhythmia, thereby creating a myocardial model specimen at the time of occurrence of the arrhythmia.
Preferably, culturing myocardial tissue or myocardial tissue while outputting electrical stimulation having a pattern similar to normal pacing electrical activity to produce myocardial tissue that maintains high-frequency stimulation responsiveness over a long period of time.

  The electrical stimulation apparatus of the present invention has a structure in which a container cap, which is a main circuit mounting portion of a culture container, has a current generating means including a digital IC and a pair of electrodes. For this reason, according to the electrical stimulation device of the present invention, the device can be easily manufactured and the cap can be easily detached from the culture vessel. In addition, the electrical stimulation device of the present invention can simply set and give a pulse current having an arbitrary intensity and waveform to a living cell in a culture vessel by simply exchanging the cap. The function and form can be adjusted as appropriate. That is, the electrical stimulation device of the present invention is simple in structure, easy to manufacture, and inexpensive as a device that applies a desired pulse current to the culture medium in the culture vessel as electrical stimulation. Moreover, the cap of the electrical stimulation apparatus of the present invention can be easily detached.

  In addition, by using the electrical stimulation device of the present invention, it is possible to generate a pathological electrical signal and / or a bioelectric signal, thereby making it possible to modify the function and form of biological cells and the like or to create a degenerative disease model. is there. The cell electrical stimulation device of the present invention is useful not only in the fields of physiology, pharmacology, and biochemistry, but also in the fields of regenerative medicine and genomic science.

  Below, the electrical stimulation apparatus of this invention and the preparation method of the model cell using this electrical stimulation apparatus are demonstrated in detail.

  The electrical stimulation device of the present invention is a device capable of generating a pathological electrical signal and / or a bioelectric signal, and specifically, (1) during a pathological condition of a living body consisting of a combination of pulse electrical signals and / or Electricity generation means including at least a digital IC in which electrical phenomenon information at normal time is stored as a database as an electrical stimulation protocol, and pulse electrical power is generated as the electrical stimulation protocol based on the electrical phenomenon information from the digital IC. Current generating means for outputting a signal into the culture medium; (2) a cap for fixing the current generating means; and (3) suspended from the cap and using a pulsed current as an electrical stimulus in the culture vessel. And a pair of electrodes connected to the current generating means for outputting to the culture medium. First, the configuration of the electrical stimulation device 10 of the present invention will be described below with reference to the drawings.

  FIG. 1 is a schematic view showing a preferred embodiment of the electrical stimulation device of the present invention. In FIG. 1, reference numeral 1 denotes a cap for fixing a current generating means including a digital IC (hereinafter simply referred to as “cap”), and reference numeral 2 denotes a culture vessel specimen arrangement portion. 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 is formed on the printed circuit board 3.

  The electric circuit 4 in FIG. 1 includes a digital IC 5 including a logic IC, a memory IC, a microprocessor, and the like, a button battery 6, a micro switch 7, and a light emitting element (LED) 8. The electrodes 9, 9 ′ are composed of an anode and a cathode, and are arranged suspended through the cap 1 and suspended in the cap 1.

  FIG. 2 is an example of the electrical stimulation device of the present invention manufactured by adapting to the shape of a commercially available 35 mm Petri dish.

  Next, the electrical stimulation apparatus of the present invention will be specifically described.

  The electrical stimulation device 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 adhere to the cap 1 and can generate an electrical stimulation pulse and adjust the intensity, cycle, etc. of the electrical stimulation pulse. The current generating means is preferably an electric circuit 4 including a button battery 6, a microswitch 7 and an LED 8 as shown in FIG.

  Regarding the waveform, intensity, cycle, etc. of the electrical stimulation pulse supplied from the current generating means, the type of biological cells to which electrical stimulation is applied, the purpose of culture (growth promotion or growth suppression, and further, the creation of degenerative disease model, function and form It can be adjusted as appropriate according to the modification. For example, regarding the waveform of the electrical stimulation pulse, a triangle, a rectangle, and other various function waveforms (for example, a sine waveform, an exponential function waveform, etc.) can be exemplified, and among them, a rectangle is preferable. In addition, the intensity of the electrical stimulation pulse can be appropriately adjusted according to the two culture purposes of growth promotion and growth inhibition (damage) of living cells and the like. For example, when culturing for the purpose of promoting growth of biological cells or the like and modifying the function and form, the intensity of the electrical stimulation pulse is 0.1 to 100 mV / mm, preferably 0.5 to 50 mV / mm, More preferably, it is 1-25 mV / mm. On the other hand, when culturing for the purpose of inhibiting growth or denaturation of living cells, etc., the intensity of the electrical stimulation pulse is 5 to 500 mV / mm, preferably 75 to 250 mV / mm, and 50 to 150 mV / mm. Is more preferable. The cycle of the electrical stimulation pulse is, for example, 0.001 to 1000 Hz, preferably 0.005 to 100 Hz, and more preferably 0.01 to 50 Hz. The energization time of the electrical stimulation pulse is, for example, 0.1 μsec to 10 seconds, preferably 0.5 μsec to 1 sec, and more preferably 1 μsec to 0.1 sec.

  The cap 1 can be freely selected in size and shape in accordance with the size and shape of the culture vessel specimen placement portion 2 to be fitted. For example, the cap 1 can be a commercially available 35 mm, 60 mm, 90 mm, 150 mm Petri dish, a 6- to 384-well multiwell 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.

  The material of the cap 1 can be various materials such as metal, glass, plastic, etc., but is preferably made of plastic that is excellent in transparency so that the culture can be easily observed and is not easily damaged during handling. In particular, it is preferably made of an acrylic resin that is highly transparent and excellent in rigidity.

  The cap 1 is preferably mounted integrally with the printed circuit board 3 in the electrical stimulation device 10 of the present invention. The printed circuit board 3 can be manufactured using a known material in which the electric circuit 4 is printed on a substrate made of heat-resistant plastic or fiber reinforced plastic such as glass fiber. The shape of the printed circuit board 3 is not particularly limited, but it is preferable to match the shape of the electric circuit 4.

  The electric circuit 4 includes a digital IC 5 including a logic IC, a memory IC, a microprocessor, and the like, a power supply 6 and a microswitch 7, and preferably has a light emitting element 8 which will be described later. The method of forming the electric circuit 4 is not particularly limited. For example, according to a conventional method, a method of forming a printed circuit board by etching a copper thin film, a conductive paint is applied on the printed circuit board, and the printed circuit board is coated. And a method of printing and printing a thin wire or the like. Of these, the etching method and the method of printing on the substrate are preferable from the viewpoint of making the circuit compact.

  The digital IC 5 that constitutes the electric circuit 4 is an integrated circuit in which a series of work instructions are set or a series of work instructions can be given by writing a program, such as a general-purpose logic IC and a kind of programmable IC or microprocessor of a memory IC. Can be used. In the electrical stimulation device of the present invention, the digital IC 5 has a role of changing the magnitude, frequency, and pattern of the pulse current on the time axis.

  Examples of the logic ICs include general-purpose logic ICs, PLDs (programmable logic devices), CPLDs (complex program logic devices), FPGAs (field programmable gate arrays), custom ICs and various microprocessors. . The general-purpose logic IC includes a CMOS type and a bipolar type, but is preferably a CMOS type. The digital IC 5 is easily available on the market. The digital IC 5 may further include peripheral elements such as a capacitor.

  Memory ICs include various ROM (read-only memory) including mask ROM, PROM, EROM, and EEPROM, random access memory such as RAM (read-write memory) including DRAM and SRAM, and CCD (charge coupled devices). A publicly known one such as a sequential access memory can be listed.

  In the digital IC used in the present invention, electrical phenomenon information at the time of pathological condition and / or normal condition consisting of a combination of pulsed electrical signals is stored in a database as an electrical stimulation protocol. In the present invention, it is preferable that the electrical phenomenon information during the pathological condition and / or normal state of the living body, which is a combination of pulse electrical signals, is stored in the memory IC.

  Examples of the electrical phenomenon information in the pathological state and / or normal state of a living body composed of a combination of pulsed electrical signals include arrhythmia, epilepsy, or electrical phenomenon of abnormal electromyogram. Specifically, as an example, an ectopic trigger activity and a tremor electromyogram shown in FIG. 4 that show a characteristic appearance pattern as a pathological condition, or a tachycardia (100 ~ 250 / min), coarse movement (250-350 / min), fibrillation (350 / min or more).

“The electrical phenomenon information at the time of a disease state and / or normal state is stored as a database as an electrical stimulation protocol” indicates the following two operations.
(1) When the electrical signal at the time of disease state and / or normal has the appearance frequency and appearance pattern of a constant known specific basic waveform, the frequency and pattern are made into a database as a standard output pattern of current pulses. save.
(2) When the electrical signal at the time of disease state and / or normal is not a known specific basic waveform but takes various forms within a certain range, the source bioelectric signal is once stored in the data recorder, The main waveform of the data is subjected to wave height selection using a trigger function of a commercially available electrical stimulation device or electrical stimulation experiment software, the appearance frequency and appearance pattern of the main waveform are determined, and the database is stored as in (1). In this case, the main waveform is easily discriminated by monitor observation by humans, but for those that are difficult to discriminate, the source bioelectric signal is subjected to frequency analysis after Fourier transform, and the power spectrum has the highest power density. In some cases, a large waveform is used as the main waveform. Further, all the basic waveforms constituting the source bioelectric signal may be picked up, and each basic waveform may be stored in a database as a standard output pattern, or may be stored in a database as an exhaustive combination of basic waveforms.

  FIG. 4 shows an example of a known pathological electrical signal and an example of a pathological electrical signal generated by the electrical stimulation device of the present invention based on the example. That is, FIG. 4 shows an example of pathological electrical signals generated using the electrical stimulation device of the present invention. Ectopic trigger activity related to pacing rhythm of arrhythmia and abnormal myoelectricity during Parkinsonism tremor and Cronus tremor The figure similar signal is shown. In addition, extrasystoles, tachycardia, coarse motion, fibrillation, epileptiform electrical signals, and the like can be reproduced based on a known original waveform pattern.

  In the electrical stimulation device of the present invention, the pulse current adjusted by the digital IC 5 including a logic IC, a memory IC, a microprocessor, and the like is set to flow through the electrical circuit 4. It is preferable that the voltage (intensity), frequency of occurrence (cycle), generation pattern (waveform), and the like of this pulse current can be changed as a series of protocols according to the purpose of the experiment using a digital IC. As a method for realizing such a changeable stimulation protocol, there is writing and erasing of a protocol and a program in the digital IC, and the protocol and the program can be made into a database.

  The power source in the electrical stimulation device of the present invention is preferably an electricity supply device for supplying electricity from a battery or the outside. When the power source is a battery, since the battery needs to be mounted on the cap 1, it 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 potential difference voltage of 10 V or less, preferably about 1.5 to 6 V. Further, it is desirable to have a capacity capable of flowing an average 10 μA pulse current through the medium for 2 hours or more, preferably 5 hours or more, more preferably 24 hours or more.

  In the preferred embodiment of the electrical stimulation device of the present invention, the button battery 6 is used as the 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, a lithium storage battery, as well as a disposable battery such as a mercury battery or lithium.

  On the other hand, when the power supply in the electrical stimulation apparatus of the present invention is an electrical supply apparatus, the electrical supply apparatus may be a direct current power supply such as an AC adapter, an AC / DC switching power supply, an AC / DC converter, and various power transformer products. .

  The microswitch 7 in the electrical stimulation device 10 of the present invention is a device that controls on / off of the pulse current in the circuit of the stimulation device, like a normal switch. The micro switch 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. The microswitch 7 is desirably as small and light as possible as long as it can be operated.

  In the electrical stimulation device of the present invention, the electrical circuit 4 can further include a monitoring device for monitoring the supply status of the pulse current supplied to the culture medium 11 in the culture vessel 2. As the monitoring device, for example, a light emitting element (LED) 8 is preferably provided as shown in a preferred embodiment of the present invention.

  The electrodes 9, 9 'in the electrical stimulation device of the present invention are terminals for applying a pulse current to the culture medium in the culture vessel as electrical stimulation. In FIG. 1, the electrodes 9 and 9 ′ are connected to both ends of the electric circuit 4, pass through the cap 1, and are suspended in the cap 1. The electrodes 9, 9 'are composed of a pair of anode and cathode. The shape of the electrodes 9 and 9 'is a T shape in FIG. 1, but the electrodes in the electrical stimulation device of the present invention are not limited to this shape, and other shapes (for example, a lattice, concentric circles or semicircles). Shape). Furthermore, the lengths 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 have 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) are matched to the shapes of the cap 1 and the culture vessel 2. It can be determined as appropriate. Examples of the material for the electrodes 9 and 9 'include silver, silver-silver chloride, platinum, titanium, titanium nitride, carbon, gold, and glass.

  As shown in FIG. 1 (B), the culture container specimen placement portion 2 for fitting the cap in the electrical stimulation device 10 of the present invention can be filled with the 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 in a 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.

  The medium 11 contains 0.01 ng / ml 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. To 100 μg / ml is preferable. The medium 11 may be a solid medium or a liquid medium.

  In the case of culturing so as to promote or suppress the growth of living cells using the electrical stimulation device 10 of the present invention, and further realize the modification of function and form and the creation of a degenerative disease model, the medium 11 includes living cells or living tissues. 12 (hereinafter referred to as “original tissue or the like”) is placed on the surface of the medium or in the medium. As this original tissue, various kinds of original tissues used as known cells or tissues can be used. For example, cells (group) constituting the original tissue and the stem cells (group) thereof, a part of the tissue to be cultured, the tissue similar to the tissue to be cultured, embryonic cells, ES cells, etc. Good.

  The “living body” such as living cells to be stimulated by the electrical stimulation device of the present invention includes humans, mammals such as dogs, cats, horses, pigs, sheep, mice, rats, birds, reptiles, and amphibians. It is also a concept including microorganisms such as fish, bacteria and viruses, and plants.

  In addition, the “tissue” to which stimulation is applied by the electrical stimulation apparatus of the present invention includes all tissues and organs of the living body and parts thereof. For example, central nerve, peripheral nerve, bone, cartilage, joint, lymphatic vessel, blood vessel, heart (myocardium, valve), lung, liver, spleen, pancreas, esophagus, stomach, small intestine, large intestine, kidney, bladder, uterus, ovary, The testis, diaphragm, muscles, tendons, skin, eyes, nose, trachea, tongue, lips, nails, hair, etc. The tissue used in the electrical apparatus of the present invention is a tissue in which excitatory electrical stimulation is resident in a living body among these organs or tissues, for example, a central nerve such as heart, skeletal muscle, smooth muscle, peripheral nerve, brain, etc. Can be the main target.

  The circuit arrangement of the digital IC 5 of the electric circuit 4, the power supply (6), the micro switch, the monitoring device (8) and the electrodes 9 and 9 ′ in the electric stimulation device of the present invention is not particularly limited, and the shape of the cap 1, It can be determined appropriately according to the size and the like. For example, in the electrical stimulation device of the present invention, it is possible to take a circuit arrangement as shown in FIGS.

  FIG. 3A shows a circuit arrangement when the digital IC 5 is a CMOS logic IC (74HC). The current generated from the power source (6) is converted into a pulse current having a desired waveform in the CMOS IC, and further adjusted to a desired current intensity, cycle, and energization time by a capacitor and a resistor as peripheral elements. The adjusted pulse current is supplied to the electrode while being monitored by the light emitting element (LED) 8.

  FIG. 3B shows a circuit arrangement in the case where all the peripheral elements are incorporated in the digital IC 5 by further integration. The circuit arrangement shown in FIG. 3B has an advantage that it can contribute to the downsizing of the device because the space occupied by the peripheral elements can be omitted.

  FIG. 3B also shows a circuit arrangement for writing and outputting all peripheral element functions as a program in a programmable logic device together with stimulus signals.

  FIG. 3C shows an example of a circuit arrangement for writing and outputting a program for forming an arbitrary stimulus pattern in the microprocessor.

  The method of manufacturing the electrical stimulation device of the present invention is not particularly limited. For example, an ordinary single-container electrode, an electrode array for a stimulation circuit and a multiwell plate, and a stimulation circuit are integrated into a semiconductor technology. Thus, it can be integrally formed as one microchip and can be constructed by adding a suspended electrode.

  The preferred embodiments of the present invention are shown below. In addition, the material, usage-amount, ratio, procedure, etc. which are shown by the following example can be suitably changed unless it deviates from the meaning of this invention. Therefore, the scope of the present invention should not be construed as being limited by the specific examples shown below.

Example 1
Using the electrical stimulation apparatus described in FIGS. 1 and 2 of the present invention under conditions of a temperature of 37 ° C., a humidity of 99%, and a CO ₂ concentration of 5%, growth inhibition (cells) was performed on a mixed specimen of sensory nerve cells and Schwann cells. Electrical stimulation (150 mV / mm, duration 5 msec, 2 Hz) under the condition for the purpose of imparting damage was applied for 5 hours, and further cultured for 2 days. The results are shown in FIG.

  As shown in FIG. 5, compared with the state before electrical stimulation ((A) of FIG. 5), delayed necrosis (necrosis) centered on neurites was observed after electrical stimulation (FIG. 5). (B)). In addition, when stimulation is performed for 3 days with reduced electrical stimulation intensity (10 mV / mm, duration 20 μsec, 10 Hz), only Schwann cells that have formed myelin sheaths can selectively cause necrosis. It was possible and succeeded in causing the neurite to be exposed, that is, demyelination (FIG. 5C).

(Example 2)
<Observation of functionally conditioned cells>
Regarding the functional acclimation of the functionalized myocardium in the following and the functional recovery by electrical stimulation, 35 seconds of moving image data is acquired and stored for each case using a digital movie camera attached to a phase contrast microscope, and the heart rate Measurement and aggregation were performed.

<< Extraction / Activation of Myocardial Tissue >>
Hearts were removed from BALB / c mice (3-4 weeks old) and washed 2-3 times with calcium-free phosphate buffered saline (PBS (-)) containing kanamycin (100 mg / l). The perfusion treatment was performed with the same PBS (-) containing mg / ml collagenase (type1, Sigma) at 37 ° C. for 30 min by the Langendorf method.

The collagenase-treated heart sections were washed 2-3 times with the same PBS (−), and the cells were isolated by pipetting using a Pasteur pipette whose tip diameter was reduced with a burner flame (200 to 400 μm). The cell suspension thus prepared is centrifuged, the sedimented cell mass is suspended in normal PBS containing calcium, and an appropriate amount is placed in a pre-incubated culture dish at a cell number of 2000 to 3000 / dish. The culture was started under conditions of seeding temperature 37 ° C., humidity 99%, CO ₂ concentration 5%. 24 hours after the start of the culture, the EAGLEs medium was changed and the culture was continued. The preincubation was carried out for 0.5 to 1 hour by placing 1.5 ml of EAGLEs medium (Nihon Suisan) containing 10% fetal bovine serum in a 35 mm culture dish (FALCON, 3801).

《Function acclimatization result》
As the culture progresses, the heart rate of the activated myocardium decreases from the original heart rate of the mouse (486-738 beats / minute, that is, 8.1-12. 3 Hz). , Sustained 3 msec), i.e., the frequency of stimulation of cultured cardiomyocytes can be reduced to 2-5 Hz.

<< Function habituation operation before transplantation >>
Using the electrical stimulation apparatus described in FIGS. 1 and 2 of the present invention, 150 mV / mm electrical stimulation is applied to the tissue composed of cardiomyocytes at the second week of activation according to the strength of response. It was given for 3 days at a frequency of 1-10 Hz. The stimulus responsiveness of all 12 cases recovered, and it was possible to follow the electrical stimulation of 7 Hz (FIGS. 6C and D).

  As a comparative example, cardiomyocytes were cultured for 2 weeks in the same manner as above except that no electrical stimulation was given. As a result of examining the stimulus responsiveness, the frequency of follow-up to electrical stimulation (intensity 3 V, sustained 3 msec) of myocardial tissue on the 14th day of culture is only 2 to 3 Hz in all the examined cases (11 cases), 7 Hz or more There was no recovery of responsiveness to any stimulation (Fig. 6D).

FIG. 1 is a schematic diagram showing an embodiment of the electrical stimulation device of the present invention. FIG. 2 shows an example of the electrical stimulation device of the present invention manufactured by adapting to the shape of a commercially available 35 mm Petri dish. FIG. 3 is an explanatory diagram showing a circuit arrangement of an electric circuit in the electric stimulation apparatus of the present invention. FIG. 4 shows an example of pathological electrical signals generated using the electrical stimulation device of the present invention, ectopic trigger activity related to pacing rhythm of arrhythmia, and abnormal electromyogram similar to Parkinsonism tremor and Cronus tremor Signals are shown. FIG. 5 is a photograph showing a state in which growth is suppressed by applying electrical stimulation to nerve cells using the electrical stimulation apparatus of the present invention. (A) It is a photograph which shows the state of the nerve cell before giving electrical stimulation. (B) It is a photograph which shows the state of the nerve cell after giving electrical stimulation. (C) It is a photograph showing the state of a nerve cell that has caused a demyelination phenomenon by reducing the intensity of electrical stimulation and performing stimulation for 3 days. FIG. 6 shows the recovery of the high frequency stimulation responsiveness of cultured mouse ventricular muscle when pacing stimulation of 1 to 10 Hz was given for 3 days using the electrical stimulation apparatus of the present invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Cap 2 Culture container sample arrangement | positioning part 3 Printed circuit board 4 Electric circuit (electric current generation means)
5 Digital IC
6 Button battery (power)
7 Micro switch 8 Light-emitting element (monitoring device)
9, 9 'electrode 10 electrical stimulator 11 medium 12 biological cell or biological tissue

Claims (14)

(1) Current generation means including at least a digital IC in which electrical phenomenon information at the time of pathological condition and / or normal state of a living body consisting of a combination of pulsed electrical signals is stored as a database as an electrical stimulation protocol, Current generating means for outputting a pulsed electric signal into the culture medium as the electrical stimulation protocol based on the electrical phenomenon information from the digital IC; (2) a cap for fixing the current generating means; (3) A pathological electrical signal and / or a bioelectric signal is generated that has a pair of electrodes suspended from the cap and connected to the current generating means for outputting a pulsed current to the medium in the culture vessel as an electrical stimulus. Electrical stimulation device. 2. The electrical stimulation device for generating a pathological electrical signal and / or a bioelectric signal according to claim 1, wherein the digital IC comprises a logic IC, a memory IC, and a microprocessor. The electrical stimulation device for generating a pathological electrical signal and / or a bioelectric signal according to claim 1 or 2, wherein the electrical phenomenon during the pathological condition of the living body is an electrical phenomenon of arrhythmia, epilepsy, or abnormal electromyogram. The electrical stimulation device for generating a pathological electrical signal and / or a bioelectric signal according to any one of claims 1 to 3, wherein the current generating means includes a digital IC, a microswitch, and a power source. Different electrical phenomenon information is stored in the digital IC as different electrical stimulation protocols, and a plurality of electrical stimulation pattern combinations are output into the culture medium by connecting a plurality of output terminals of the current generating means in parallel. An electrical stimulation device capable of generating a pathological electrical signal and / or a bioelectric signal according to any one of claims 1 to 4. The electrical stimulation device for generating a pathological electrical signal and / or a bioelectric signal according to any one of claims 1 to 5, wherein the current generating means is integrally formed with the cap. The electrical stimulation device for generating a pathological electrical signal and / or a bioelectric signal according to any one of claims 1 to 6, wherein the cap has a form corresponding to a form of a culture dish. The electrical stimulation device for generating a pathological electrical signal and / or a bioelectric signal according to any one of claims 1 to 6, wherein the cap has a structure corresponding to a well arrangement of a multi-well plate. The pulse current is applied as an electrical stimulus from the electrode to a living cell or a living tissue inserted in the culture medium in the culture container by fitting the cap to the specimen arrangement portion of the culture container. An electrical stimulation device that generates the electrical pathological signal and / or the bioelectric signal according to any one of the above. Using the electrical stimulation apparatus according to any one of claims 1 to 9, an electrical phenomenon at the time of pathological condition and / or normal state of a living body is supplied to cells cultured in a culture container, and at the time of pathological condition and / or normal condition A method of creating a model cell, comprising creating a model cell of time. The method according to claim 10, wherein an in vitro demyelinating disease model is created by selectively degenerating cultured myelinating cells of myelinated nerves. 11. The method according to claim 10, wherein a neural tissue model specimen of epilepsy focus is created by culturing the cultured central nerve cells while outputting electrical stimulation having a pattern similar to an electroencephalogram or synaptic activity during an epileptic seizure. The method according to claim 10, wherein a pathologic myocardial model specimen at the time of occurrence of arrhythmia is prepared by culturing the cultured cardiomyocytes or myocardial tissue while outputting electrical stimulation having a pattern similar to pacing electrical activity of arrhythmia. The myocardial tissue that maintains high frequency stimulation responsiveness over a long period of time is created by culturing the cultured cardiomyocytes or myocardial tissue while outputting electrical stimulation in a pattern similar to normal pacing electrical activity. The method described.