JP3425626B2 - Single cell tension measurement system - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a single cell tension measuring system for inspecting functions such as contraction force of a single muscle cell, and in particular, micro single carbon cells of the heart muscle using a carbon rod. The present invention relates to a single-cell tension measuring system capable of reliably adsorbing and holding cells and easily measuring functions such as contractile force.

[0002]

2. Description of the Related Art Conventionally, in order to examine the biological function of muscles of animals such as human beings and to examine the effect of drugs on muscles, the function of muscles is measured by measuring the contraction force of the muscles. There are times when you are told. There are various kinds of muscles. For example, regarding human muscles, there are various kinds of muscles everywhere from the head to the legs of the body, and there are muscles in various organs in the body. Many of these muscles have a few cm of single muscle cells
There is a length of.

When examining functions such as the contractile force of muscles as described above, a single muscle is separated from a mass of muscles, and both ends of this single muscle cell are made of a metal or glass rod. It is fixed by pulling it with a predetermined force and measuring the amount of movement of the rod as a change in the length of the muscle at that time, or by measuring the force for maintaining a constant length Functions such as contraction force are measured. In order to perform such a measurement, a force is applied to the part that fixes both ends of a single muscle cell to a rod or the like, so it is necessary to securely fix this part.
In the case of a muscle having a length of several cm as described above, the single muscle cell itself can be tied to the rod,
Even if it is not possible, it can be fixed by binding with a wire or a synthetic fiber thread.

[0004]

However, the heart muscle, which is the most important muscle among various muscles such as humans as described above, has a single muscle cell, that is, a single cardiomyocyte has a length of Only about 100 μm. Therefore, in order to fix the single cardiomyocyte to the rod when measuring functions such as the contractile force of the heart muscle, the cardiomyocyte itself is bound to the rod as described above, and the cardiomyocyte is bound by a thread. I can't do it either.

Further, it is conceivable to fix the both with an adhesive, but it is difficult to surely adhere to a microscopic portion, and depending on the material of the adhesive, it is possible to rapidly chemically react with minute cardiomyocytes as a living body. However, it cannot be an appropriate fixing means. Therefore, in order to inspect the function of the cardiomyocyte, it is inevitable to inspect the function indirectly by other inspection means such as visually confirming the morphology of the muscle, and accurately inspect the function such as the contractile force of the cardiomyocyte. Was extremely difficult.

As a countermeasure, it is possible to select a cardiomyocyte having a predetermined number of beads connected to each other, such as 3 cells, and tie both ends thereof to a rod with a thread or the like. However, when collecting cardiomyocytes, a thin tube like an injection needle is inserted into the heart of a living body to collect a part of the muscle, and a large number of cardiomyocytes are combined to form a lump of the collected minute muscle. The clumps are placed in a solution containing the enzyme to facilitate separation of cell-cell connections. Separate the connections between muscle cells by using a sonicator to crush the cells with ultrasonic waves, and select a predetermined number of cardiomyocytes that have not been completely separated from the numerous cardiomyocytes thus obtained. It was necessary to carry out a test, and there was no certainty in selecting such an appropriate cardiomyocyte, and the test was difficult.

On the other hand, the heart may undergo organ transplantation in the same manner as other organs, and at the time of transplantation, it is necessary to take out the heart from a person who no longer needs the heart and perform transplantation. The function is often incomplete. Since transplanting a heart with incomplete function in this way is dangerous to the life of the transplant recipient, it is necessary to accurately test the function of the transplanted heart immediately before performing a heart transplant. Further, in developing a drug for the heart, it is necessary to know the contractile force of the heart muscle before and after giving the drug in order to confirm the effect of the drug. Need to know.

Therefore, it is extremely important to securely fix the single muscle cell of about 100 μm as described above to the rod as described above, but it is extremely difficult with the above conventional technique. In particular, the rod itself for fixing such fine muscles needs to be thin and small in proportion to the size of the muscles, and it is extremely difficult to fix thin cardiomyocytes to such thin rods with sufficient adhesive force. It has to be difficult.

Further, the rod is hollowed into a tubular shape, the muscle is placed in the opening at the tip of the rod, and the tubular portion is evacuated to adsorb the muscle like a pipette. It has also been proposed to hold. However, when the force generated by such a vacuum is applied to the muscle, the cells of the muscle may be damaged, and accurate function may not be measured.

Even if research and development has developed means for securely fixing the single cardiomyocyte to a thin rod as described above, a technique for accurately measuring the contraction force of such a minute muscle has been established. Not not. That is, when measuring the contraction force of a conventional single muscle cell, since a relatively large muscle cell was handled, for example, a small reflecting mirror was attached to a muscle or a movable rod to detect a change in the reflection of light projected onto it. The measurement was performed using a means such as.

However, since it is extremely difficult to provide such a reflecting mirror for equipment having a size of about 100 μm, this measuring means cannot be used for measuring the above-mentioned minute muscles. . Therefore, also in this respect, it has been one factor that makes it impossible to carry out the above-described examination of a single cardiomyocyte.

Further, even if the measurement can be performed by some means, an actuator that operates accurately enough to accurately measure the tension of a single fine muscle cell is required. Although the conventional device can measure the tension of a long muscle relatively roughly, it is accurate enough to test the function of an extremely small and important muscle cell like the function test of myocardial cells. It is impossible to measure it, and some solution is needed also in this point.

Such short single muscle cells are not limited to the above-mentioned cardiomyocytes, but also exist in various muscles of various animals including small experimental animals, and when measuring their functions The establishment of technology is desired.

Therefore, according to the present invention, both ends of the muscle are securely fixed to the rod by a simple means in order to inspect the function such as the contractile force of the muscle when inspecting the function such as the contractile force of the minute muscle. In addition, it is an object of the present invention to provide a single-cell tension measuring system capable of accurately measuring the tension of the muscle thus fixed.

[0015]

In order to solve the above-mentioned problems, the present invention according to claim 1 provides a carbon member for adsorbing and holding a part of a single cell and another end of the single cell. Corresponding to the displacement of the micro carbon rod by a micro carbon rod that holds a part of it by suction and the other end is fixed to a piezo element, a position sensor that projects an image of the micro carbon rod, and an output signal of the position sensor. The displacement signal output means for outputting the signal, and the microcarbon rod position control means for actuating the piezo element by the displacement signal to control the microcarbon rod to a predetermined position. This is a tension measurement system.

The invention according to claim 2 is the microcarbon rod, wherein the carbon member is shorter than the microcarbon rod, one end of which adsorbs and holds a part of the single cell and the other end of which is fixed. Claim 1 characterized by the above-mentioned.
This is the described single cell tension measurement system.

The invention according to claim 3 is characterized in that the position sensor is a 4-division CCD, and the displacement signal output means detects and outputs a difference between output signals from a plurality of CCD elements. The single-cell tension measurement system according to Item 1.

The invention according to claim 4 provides the tension measuring system for a single cell according to claim 1, wherein the single cell is a cardiomyocyte.

[0019]

Embodiments of the present invention will be described with reference to the drawings. FIG. 1 shows an example of an apparatus for measuring the tension of a single cardiomyocyte, which is a minute single cell, as a single cell tension measuring system according to the present invention. First, the overall configuration will be described. The cardiomyocyte 10 includes two micro carbon rods 12, 1 as described later.
3 is attracted and held by electrostatic force. Since the length of the microcarbon rod 13 is longer than that of the microcarbon rod 12, the longer microcarbon rod 12 bends more when the cells contract.

The longer microcarbon rod 13 is fixed to the piezo element 14, and the tension applied to the myocardial cells is controlled by controlling the voltage applied to the piezo element 14.
The image of the micro carbon rod 13 illuminated by the lamp 16 passes through the lens 18 and the mirror 20 and is formed on the position sensor 22 composed of a four-division photodiode. Four
A signal from each region of the position sensor 22 composed of divided photodiodes is amplified by the differential amplifier 24, and the piezo element 14
By feeding back to, the contractile force is measured under the condition that the length of the cardiomyocytes is controlled to be constant. The position sensor 22 composed of the four-division photodiode can accurately measure a minute length in nanometer units,
It is suitable for the above-mentioned functional examination of the myocardium.

The above system will be described in more detail. For example, at the time of heart transplantation, a donor's heart is determined immediately before transplantation as to whether or not the heart is suitable for transplantation. Therefore, as a means of the determination, the functional test of the heart muscle of the transplanted heart according to the present invention is performed. To do. At that time, a tube such as an injection needle is inserted into a part of the heart, and a minute mass of myocardium is collected in the tube. As mentioned above, since the myocardium is as small as 100 μm,
It is easily collected without straining the heart of the donor.

The collected mass of myocardium is soaked in an appropriate enzyme to loosen the bond between single cardiomyocytes and separate them. Such separating means can be performed by well-known means that have been conventionally used.

The thus obtained single cardiomyocytes having a size of about 100 μm are two microcarbons composed of microcarbon rods containing glassy carbon and crystalline carbon invented in connection with the present invention. Rods 12 and 13
Hold both ends by suction. The micro carbon rod containing the glassy carbon and the crystalline carbon is separately patented and described in detail there, but the outline is shown at the end of the text, and the single cell according to the present invention is shown. The tension measurement system of was developed as a result of being able to reliably hold the minute single cardiomyocytes as described above by adsorbing to the surface by this micro carbon rod by electrostatic force. is there.

Of the two microrods that adsorb myocardial cells as described above, in the illustrated embodiment, the second microcarbon rod 13 is formed longer than the first microcarbon rod 12, and the short first microcarbon rod is formed. The base portion of the carbon rod 12 is fixed to the device by the fixing means 11, and the long second micro carbon rod 13 is fixed.
The base of is supported by a piezo element (PZT) 14.
It should be noted that the actuating portion of the first microcarbon rod 12 by this piezo element is different from the one that conventionally operates large muscles, and in the present invention, minute things actuate muscles. It has become possible.

The light from the lamp 16 projects a part of the second micro carbon rod 13 which adsorbs and fixes one end of the myocardial cell 10, and the image thereof passes through the lens 18 and the mirror 20 as described above and is divided into four CCDs. Position sensor 22 consisting of
Is projected and imaged. For example, in a four-divided CCD as in the illustrated embodiment, at least output signals of CCD elements facing each other are taken out, and signals from respective voltage detectors 23 are input to a differential amplifier 24.

As a result, when the second microcarbon rod 13 is moved by the piezo element as described later, the movement of the rod moves the image of the rod on the four-division CCD, so that the four-division CCD faces each other. The amount of light received by the CCD element relatively changes, and the change is detected by the differential amplifier 24 as a voltage difference between the signals of the two voltage detectors 23, 23 and output. By appropriately amplifying this signal and adding / subtracting it to and from the offset input, which is the signal of the offset value to be input separately, the second microcarbon rod 13 is moved by the piezo element 14 to the side that relaxes the myocardial cells, or conversely, the tension is applied. A feedback value indicating whether to move to the side to be moved and how much to move is output. By obtaining this signal value, it is possible to obtain data as a function of the contractile force of the cardiomyocytes to be examined.

In this way, the length of the single cardiomyocyte 10 whose both ends are adsorbed and fixed by the two microcarbon rods 12 and 13 changes according to the tension of the cells, and the length is constant. The tension applied to the inspected cardiomyocytes is measured by the current applied to the piezo element to be tested, and the biological function test of the transplanted heart can be performed.

On the other hand, the microcarbon rod used in the above system is capable of surely adsorbing the minute muscle as described above. Specifically, this microcarbon rod is as follows. In addition, it is manufactured as follows. A separate patent application has been filed for this technology as described above.

The characteristic feature of this micro carbon rod is that it contains glassy carbon and crystalline carbon, and it is desirable that the crystalline carbon be oriented substantially in one direction. Further, in order to obtain a sufficient holding force, it is desirable that at least 20% by mass, and more desirably 60% by mass or more of crystalline carbon be contained in this microcarbon rod, and a microscopic object The diameter of the cross section is 100 to enable the application to objects.
It is also desirable that the thickness is μm or less.

The feature of this method for producing a micro carbon rod is that graphite powder is mixed with an organic substance that leaves glassy carbon after firing, the mixture is molded into a desired shape, and the molded product is baked. It is preferable that the method comprises a step, and in the forming step, extrusion molding is performed so that the graphite crystals are substantially oriented in one direction.

In this microcarbon rod, a large number of functional groups derived from carbon atoms in the crystal end face and / or basal plane defect portion are present on the surface, and sufficient adhesion is achieved by chemisorption (adsorption by electrostatic force) due to this. Power is gained. By performing extrusion molding so that the graphite crystals are substantially oriented in one direction during molding, this ratio is further increased, and further higher adhesive strength is obtained.

The organic material that leaves the glassy carbon after firing is an organic resin material having three-dimensional cross-linking, a natural organic material that solid-phase carbonizes, and specifically, an organic polymer material and
The monomer / oligomer, tar / pitch, dry-distilled pitch, thermoplastic resin, prepolymer of thermosetting resin, etc., or a mixture of two or more thereof.

Here, the organic polymer substance is a substance other than a thermoplastic resin and a thermosetting resin which will be described later, and includes lignin, cellulose, tragacanth gum, gum arabic, natural gum and its derivatives, sugars, chitin, chitosan and the like. Such as compounds having condensed polycyclic aromatics in the basic structure of the molecule, and formalin condensate of naphthalene sulfonic acid, dinitronaphthalene, pyrene, pyranthrone, violanthrone, benzanthrone, etc. It is an intermediate.

As the thermoplastic resin, polyvinyl chloride, polyacrylonitrile, polyvinylidene chloride, post-chlorinated polyvinyl chloride, polyvinyl acetate, polyvinyl alcohol, polyvinylpyrrolidone, ethyl cellulose, carboxymethyl cellulose, polyvinyl chloride / vinyl acetate As a carbon precursor treatment using an ordinary thermoplastic resin such as polymer and polyphenylene oxide, polyparaxylene, polysulfone, polyimide, polyamideimide, polybenzimidazole, polyoxadiazole, etc. , Oxidatively crosslinked.

As the thermosetting resin, phenol resin,
Furan resin, epoxy resin, xylene resin, Kopuna resin, etc. are used, and they flow when heated and undergo intermolecular cross-linking to three-dimensionally harden, resulting in high carbon residue collection without special carbon precursor treatment. It shows the rate.

Regarding the graphite powder, in order to increase the adhesive force, it is important to prepare a composite carbon material in which the crystal end faces (edge faces) of highly developed graphite are aligned vertically to the surface. Is. Therefore, graphite whiskers, highly oriented vapor phase pyrolytic graphite (HOPG), quiche graphite, and crystalline natural graphite are preferably used. The particle size of the graphite fine powder varies depending on the diameter of the target rod, but the maximum diameter is preferably several μm or less.

When the organic substance and the graphite powder are mixed, the powder is sufficiently dispersed with a Henschel mixer or the like. Furthermore, using a kneader such as a pressure kneader or a two-roll machine to which a high shearing force is applied, thoroughly mix and disperse, granulate with a pelletizer, and then use a screw type or plunger type extruder to obtain the desired mixture. At high speed by extrusion molding
A molded product can be obtained by performing an orientation operation so that the graphite crystals are well aligned in the extrusion direction.

If necessary, at the time of firing, a precursor (carbon precursor) is first treated for about 10 hours in an air oven heated to about 180 ° C. while stretching the molded body. Use as material. Further, the temperature is controlled gradually in an inert atmosphere to gradually heat up to about 1500 ° C. to complete the carbonization and obtain the desired microcarbon rod.

Specific examples will be described. In order to confirm the adsorption force due to the electrostatic force of the micro carbon rod, the fact that the amount of the functional residue on the carbon surface can be semiquantified by electrochemical measurement is used to evaluate the adsorption amount due to the electrostatic force. Specifically, first, using an electrochemical measuring device, a 1 M (mol dm-3) potassium chloride aqueous solution is put into an electrolytic cell, and a certain length of a microrod with a lead wire is dipped to flow a blank. The electric current is measured by the cyclic voltammetry (CV) method, and the adsorption force is judged from the magnitude of the electric current value.

Furthermore, it is considered that the presence of many graphite edges on the carbon surface after the rod is manufactured is an index of having many surface functional groups closely related to the adsorption force by electrostatic force. Therefore, the charge transfer characteristic of the present rod is measured by utilizing the fact that the carbon surface with a large number of edges easily moves electrons and is closely related to the good electrode characteristics. Specifically, an electrochemical measuring device is used to add 5 mM of ferri-ferrocyanine ion to the potassium chloride solution, and the redox (oxidation / reduction) reaction of the ion is measured by the CV method. The electrochemical characteristic of the rod surface is judged from the potential difference ΔEp between the oxidation peak and reduction peak of the CV curve-shaped redox reaction.

(Example 1) 65% by mass of chlorinated vinyl chloride resin (T-741 manufactured by Nippon Carbide Co., Ltd.) as a matrix carbon raw material of a micro carbon rod, and graphite fine powder (average particle size 1 μm manufactured by Hitachi Powder Metallurgy Co., Ltd.) Three
25% by mass of a diallyl phthalate monomer was added as a plasticizer to a composite composition of 5% by mass, and the mixture was dispersed using a Henschel mixer.
Kneading was sufficiently repeated using a two-roll mixing roll kept at ℃ to obtain a composition, which was pelletized by a pelletizer to obtain a molding composition.

The pellets were extruded at a speed of 0.1 m / min at 140 ° C. at a rate of 0.1 m / min while deaeration using a ceramic die having a diameter of 50 μm with a screw type extruder, fixed to a frame and heated to 180 ° C. Precursor (carbon precursor) after 10 hours treatment in an air oven
It was a wire rod. Then, this is heated to 500 ° C in nitrogen gas for 2
The temperature is raised at a heating rate of 5 ° C / hour, and then 1500 ° C up to 1500 ° C.
The temperature was raised at 00 ° C./hour, and the temperature was maintained at 1500 ° C. for 3 hours, and then naturally cooled to complete firing.

The obtained micro carbon rod has a diameter of 3
The bending strength was 210 MPa at 8 μm. The graphite contained in this is about 65 mass% of the whole. In order to confirm the adsorption force of the micro carbon rod by the electrostatic force, the electrochemical response was measured and judged.

The amount of adsorption of the obtained microcarbon rod by electrostatic force was determined as follows according to a predetermined evaluation method. First, in order to determine the amount of adsorption by the electrostatic force of the rod, a Yanagimoto bolographic analyzer (P-1100) was used as an electrochemical measuring device.
In 1M potassium chloride solution, a blank current was applied, then 5
The CV curve of mM ferro-ferricyan ion was measured by the three-electrode method using a silver-silver chloride reference electrode. As a result, the blank current of the carbon microrod obtained in this example was extremely large, and the redox peak potential difference ΔEp of the redox ion was 70 mV, which was close to the theoretical value (60 mV) of the peak potential difference of the reversible redox ion. Was determined to have sufficient suction force to be used for the purpose of measuring myocardium.

Cardiomyocytes were chemically adsorbed to this microcarbon rod to give stress, and the adhesive force was calculated from the deflection amount of the rod and the spring constant when the adhesive was released. The obtained adhesive force is 2.42 μN, which is 1 to 2 μN of the contraction force of cardiomyocytes.
It was a sufficiently large value.

(Example 2) As a matrix carbon raw material for a micro carbon rod, a mixture of 50 mass% of vinyl chloride resin (Nipolite MQ manufactured by Chisso Co.) and 15 mass% of furan resin (Hitafuran VF302 manufactured by Hitachi Chemical Co., Ltd.), Fine graphite powder (made by Nippon Graphite Co., Ltd. average particle size 1 μ
m) 2 mass% of diallyl phthalate monomer was added as a plasticizer to 35 mass% of the composite composition, and the mixture was dispersed using a Henschel mixer, and then two surfaces for mixing were kept at a surface temperature of 110 ° C. Kneading was sufficiently repeated using a roll to obtain a composition, which was pelletized by a pelletizer to obtain a molding composition.

The pellets were extruded by a screw type extruder with a ceramic die having a diameter of 50 μm at 0.1 ° C./min at 130 ° C., fixed to a frame and heated to 180 ° C. Precursor (carbon precursor) after 10 hours treatment in an air oven
It was a wire rod. Then, this is heated to 500 ° C in nitrogen gas for 2
The temperature is raised at a heating rate of 5 ° C / hour, and then 1500 ° C up to 1500 ° C.
The temperature was raised at 00 ° C./hour, and the temperature was maintained at 1500 ° C. for 3 hours, and then naturally cooled to complete firing.

The obtained micro carbon rod has a diameter of 4
The bending strength was 180 MPa at 0 μm. The graphite contained in this is about 65 mass% of the whole.

The adsorption amount of the obtained micro carbon rod was determined by electrostatic force in the same manner as in Example 1. As a result, the blank current of the carbon microrod obtained in this example is extremely large, and the redox peak potential difference ΔEp of the redox ion is 70 mV, which is close to the theoretical value (60 mV) of the peak potential difference of the reversible redox ion. Was determined to have sufficient suction force to be used for the purpose of measuring myocardium.

Cardiomyocytes were chemically adsorbed on the microcarbon rods to give stress, and the adhesive force was calculated from the deflection amount of the rods when the adhesion was released and the spring constant. The obtained adhesive force was 2.4 μN, which was sufficiently larger than the contractile force of cardiomyocytes of 1 to 2 μN.

Although the single cell tension measuring system according to the present invention is described in the embodiment shown in FIG. 1, the single cardiomyocyte tension measuring system has been described, but it can be effectively used for other fine muscle cells. You can In addition, an example using two micro carbon rods for adsorbing and holding a single cell has been shown. For example, a plate made of the above-mentioned carbon fixed to the body instead of the first micro carbon rod 12 is used. Even if various fixing members such as the above are used, the same operation as described above can be performed by detecting the displacement of the second carbon rod 13.

Further, in the above embodiment, the four-divided CCD was used as the CCD, but it is sufficient that at least two CCD pixels facing each other are provided, and a line sensor in which CCD elements are arranged in line is used as necessary. However, the displacement of the micro carbon rod can be detected.

[0053]

Since the present invention is configured as described above, it is possible to easily and accurately measure the functions such as the contraction force of minute muscles.

[Brief description of drawings]

FIG. 1 is a system configuration diagram showing an overall configuration of a single cell tension measuring system according to the present invention.

[Explanation of symbols]

10 cardiomyocytes 12 First micro carbon rod 13 Second Micro Carbon Rod 14 Piezo element 15 Feedback amplifier 16 lamps 18 lenses 20 mirror 22 4-division CCD 24 differential amplifier

Front page continued (72) Inventor Kiyohisa Sugiura 7-3-1 Hongo, Bunkyo-ku, Tokyo Inside the School of Medicine, The University of Tokyo (72) Inventor Soichiro Yasuda 7-3-1 Hongo, Bunkyo-ku, Tokyo Inside the School of Medicine (72) Inventor Yasutoshi Saegaki 2-1-3 Tsurumi, Tsurumi-ku, Yokohama-shi, Kanagawa Prefecture, Faculty of Dentistry, Tsurumi University (72) Haruo Sugi, 2-11-1, Kaga, Itabashi-ku, Tokyo Tokyo, Faculty of Medicine, Teikyo University (58) Fields investigated (Int .Cl. ⁷ , DB name) G01N 33/483 A61B 5/22 G01L 5/04

Claims (4)

(57) [Claims] 1. A carbon member that adsorbs and holds a part of a single cell; a microcarbon rod that adsorbs and holds another part of the single cell at one end and is fixed to a piezo element at the other end; A position sensor on which the image of the micro carbon rod is projected, a displacement signal output means for outputting a signal corresponding to the displacement of the micro carbon rod by the output signal of the position sensor, and the piezo element is operated by the displacement signal, A tension measurement system for a single cell, comprising: a microcarbon rod position control means for controlling the microcarbon rod to a predetermined position. 2. The micro carbon rod, wherein the carbon member is shorter than the micro carbon rod, adsorbs and holds a part of the single cell at one end, and is fixed at the other end. Single cell tension measurement system. 3. The position sensor is a 4-division CCD,
2. The tension measuring system for a single cell according to claim 1, wherein the displacement signal output means detects and outputs a difference between output signals from a plurality of CCD elements. 4. The single cell is a cardiomyocyte.
A single cell tension measurement system as described.