JPS6359465B2 - - Google Patents

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to an apparatus for disrupting cells using ultrasonic waves. The present invention relates to an improvement in a device that applies ultrasonic pressure to cells in the container to disrupt cell membranes.

[Conventional technology]

In recent years, in medicine, in addition to urine and serum tests during clinical examinations, intracellular biochemical analysis has become necessary. Mechanical crushing is required. For this purpose, an apparatus for disrupting cells using ultrasonic waves has been used, and in the past, an apparatus such as the one illustrated in FIG. 7 was frequently used.

That is, the horn 3' is made up of a high frequency oscillator 1', a vibrator 2', and a horn 3', and the horn 3' is immersed in a beaker 4' containing a liquid containing red blood cells obtained by centrifuging blood, for example. A method of exciting a vibrator 2' with the output of a high-frequency oscillator 1' and applying ultrasonic vibration to red blood cells in a beaker 4' through a horn 3' to cause a cavitation phenomenon etc. to disrupt cell membranes of red blood cells, etc. It is a device.

This ultrasonic disruption device has the feature of storing a large amount of the same solution containing the cells to be disrupted in the beaker 4' and disrupting a large amount of the same cells, etc. at once. It has several drawbacks.

That is, since one beaker 4' that can contain the liquid of the object to be crushed corresponds to one horn 3', only one object can be crushed by one crushing device, and at the same time Cannot crush multiple items.

Therefore, it is not suitable for simultaneously disrupting a plurality of microscopic amounts of cells, each of which is different from the other. In addition, in the process of disrupting cell membranes of red blood cells etc. by ultrasonic action, the cells become atomized, but in the conventional case, the horn 3'
must be inserted into beaker 4' from above,
Since the beaker 4' remains open, there is a risk that atomized particles may scatter from the beaker 4', which is dangerous for inspectors and researchers. In particular, in order to crush a large number of cells of the same type, the crushing time is relatively long, and this causes the cells to undergo changes in temperature.
susceptible to other changes over time.

Therefore, the present applicant has developed an ultrasonic wave that emits ultrasonic waves into the liquid in the tank by driving a vibrator attached to the bottom surface of the tank, and the ultrasonic waves are applied to cells in a plurality of containers with lids placed in the liquid. Crushing the cells by applying pressure, i.e.
We developed and put into practical use an ultrasonic disruption device designed to disrupt cell membranes.

An example of the container mentioned above is composed of a container body, a lid placed on the container body, and a processing rod made of a metal rod to which a metal ring is attached to the upper part of the container body, and which is inserted into the container body. The diameter of the metal ring of the processing rod can be set so that an extremely small gap is formed between the side surface of the metal ring and the inner peripheral surface of the container.

In addition, there are containers that consist of a container body, a lid that is placed on the container, and a metal rod with a tapered tip that is suspended from the lid and inserted into the container body, or a container that consists of only the container and the lid. There are some that have a tapered lower part. In any case, applying ultrasonic pressure to the cells housed in the container,
The cells may be violently hit against the walls of the container, or the metal rods or rings inside the container may be caused to resonate and violently vibrate, and the vibrations may shock the cells, or the cells may be exposed to the liquid inside the container. This method creates cavitation and applies a violent impact to disrupt cell membranes.

In this case, in conventional devices of this type, a plurality of containers are each fixed and set on the lid plate of the tank. That is, when focusing on one container, the container is fixedly disposed vertically with the container body immersed in the liquid in the tank.

[Problem to be solved by the invention]

According to the above-mentioned conventional technology, the drawbacks of the previous technology shown in FIG. 7 can be solved, but in other words, the cell membranes of multiple cells can be disrupted at the same time, and contamination does not reach the outside, and cells are also prevented from being contaminated from the outside. However, as the present applicant continued to pursue this technology, several demands arose.

The first is when multiple cells are disrupted at the same time.
Multiple cells need to be homogeneously disrupted. This is because when a crushed specimen is to be tested in a subsequent process or used for other purposes, it needs to be homogeneous in order to improve the accuracy of the testing work. However, in conventional crushing equipment, a plurality of containers are fixedly suspended in the same location in the liquid in the tank, so the ultrasonic pressure may vary slightly depending on the location of each container. Therefore, there is a inherent possibility that the state of crushing becomes non-uniform.

Second, when cells are placed in a container and ultrasonic pressure is applied, they usually generate heat. This will cause heat to build up inside the container. For this reason, there is a risk that the cells to be crushed may be deactivated. In the case of the prior art, since each container is fixedly provided, it is necessary to devise ways to facilitate heat dissipation after the heat is generated.

〔the purpose〕

Therefore, it is an object of the present invention to provide a means for homogeneously disrupting each cell in a plurality of containers, while allowing cells to be housed in a plurality of containers and simultaneously disrupting them. be. This provides a means for improving the accuracy and efficiency of inspections after cell disruption. In addition, by making it easier to radiate the heat generated in the container during cell crushing by ultrasonic pressure, it is possible to provide a means to prevent cells from being deactivated as much as possible.

[Means to solve problems]

In order to achieve the above object, the present invention has the following technical means. That is, to explain this using the reference numerals in the attached drawings corresponding to the embodiments, the present invention applies ultrasonic waves to the liquid W in the tank 4 by driving the vibrator 2 attached to the bottom surface of the tank 4. radiate the above liquid W
In a crushing device that applies ultrasonic pressure to cells in a plurality of containers 8a to 8f with lids disposed therein to crush the cell membranes of the cells; approximately in the center of the lid plate 22 of the tank 4; A gear plate 23 is rotatably arranged, and a plurality of lidded containers 8 are mounted on the gear plate 23.
A plurality of through holes 24a and 24b for holding each of the containers 8a to 8f in a vertical manner are arranged in a circumferential manner at a predetermined interval.
a, 24b, the gear 25 that meshes with the gear plate 23 is driven by the motor 26, and the gear plate 2
3 slowly rotate the container 8a, 8b.
This is an ultrasonic cell crushing device characterized by slowly rotating the...... at the same time.

[Effect]

Based on the above configuration, first, a cell solution to be disrupted is poured into the main body of each container 8a to 8f, and the container is covered with a lid. Thereafter, these containers 8a to 8f are inserted into the through holes 24a, 24b, . . . of the gear plate 23 in the cover plate 22. As a result, each container has its main body connected to the liquid W in tank 4.
It is immersed inside and held in a vertical position.

Here, ultrasonic pressure is applied to the cells in the container, and the motor 26 is driven to slowly rotate the gear plate 23 via the gear 25. By rotating slowly in the liquid W in the tank 4, each of the containers 8a to 8f leaves an equal rotation locus, so that each container receives uniform ultrasonic radiation for a certain period of time.

That is, the cells in each of the containers 8a to 8f are crushed by the same ultrasonic pressure. Therefore, the state of disruption of the cell membrane becomes uniform. In addition, even if heat is generated in each of the containers 8a to 8f, the heat is actively radiated into the liquid W during the process of slowly rotating the liquid W in the tank 4, so the heat is not trapped in each container. It is possible to prevent cell deactivation, etc. without causing any problems.

〔Example〕

Next, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings.

1 is a high frequency oscillator, 2 is a vibrator such as a ferrite magnetostrictive vibrator equipped with a vibration coil 3 driven by the high frequency oscillator 1, and 4 is a tank filled with liquid W, which is housed in a case 5. This is a conventionally known ultrasonic generator. The present invention is characterized in the following points.

That is, a lid plate 22 is mounted on the tank 4, a gear plate 23 is rotatably arranged in the center of this lid plate 22, and the gear plate 2
3, a plurality of through holes 24a, 24b... are arranged circumferentially at predetermined intervals, and the gear plate 2
A gear 25 meshing with the gear plate 23 is disposed adjacent to the gear plate 23 and driven by the motor 26.
slowly rotate the through hole 2 of the gear plate 23.
Containers 8a, 8b... held in 4a, 24b...
It rotates slowly. In this embodiment, a through hole is also formed in the center of the gear plate 23. An example of the plurality of containers 8a to 8f that can accommodate the cells T described above is shown below.

That is, containers 8a, 8 made of stainless steel, glass, etc. are placed in each of the through holes 24a, 24b...
b, 8c... are inserted into the rack, and containers 8a, 8b, 8c...
... are immersed in the liquid W in the tank 4 in a vertical manner. In this case, a container may or may not be inserted into the central through hole of the gear plate 23.

Each of the containers 8a, 8b, 8c, .
By engaging with the through-holes 24a and 24b, it is held in a hanging manner. And each container 8a, 8
For example, b, 8c, . . . , a processing rod 11 consisting of a metal rod 9 and a metal ring 10 attached to the top of the metal rod 9 is inserted into each of the containers. In the inserted state, the tip 12 of the processing rod 11 sits on the bottom 13 of the container, and in the inserted state, there is a slight gap S between the outer surface 14 of the metal ring 10 and the inner surface 15 of the container. The diameter of the metal ring 10 is set to be slightly smaller than the inner diameter of the container so that the container is partitioned. And each of the containers 8a, 8b,
The upper opening 16 of 8c... is covered with a rubber lid 17 made of, for example, silicone rubber, and when crushing objects, usually the process inserted into the containers 8a, 8b, 8c... A liquid T containing objects to be crushed is dripped onto the upper surface 18 of the metal ring 10 of the rod 11, and then a rubber lid 17 is placed on top.

The processing rod 11 is caused to resonate by the longitudinal vibration generated by the vibrator 2. The outer shape of the processing rod 11 is determined so as to have a natural frequency that satisfies the above-mentioned better resonance conditions.

The vibrator 2 preferably has a good mechanical conversion rate, good directivity, and a large amplitude, and the shape of the tank 4 is such that the bottom part has a good shape in order to make full use of the characteristics of the vibrator 2. The vibrating surface should be as thin as possible. However, when all the vibrators are driven, mechanical and acoustic coupling occurs between each vibrator, which may cause erosion due to the waving phenomenon in which the bottom vibration surface bends, so it is natural to withstand this. On the other hand, the side wall surface is made anti-resonant.
It is desirable to prevent turbulent vibrations from occurring as much as possible.

Based on the above-described configuration, the operation of crushing objects to be crushed, such as red blood cells, will now be described.

First, the processing rod 11 is inserted into each of the containers 8a, 8b...8f. The object to be crushed is dripped onto the upper surface 18 of the ring 10 of the processing rod 11, and then the rubber lid 1 is
7 to each opening 1 of each container 8a, 8b...8f.
Crowned with 6.

Thereafter, each container 8a, 8b, . In this state, the cell liquid T in each container 8a, 8b...8f is affected by the surface tension effect due to the extremely small gap S between the side surface 14 of the metal ring 10 and the inner surface 15 of the container. Therefore, the particles remain near the upper surface of the metal ring 10. FIG. 3 shows this state.

Next, the oscillator 1 is driven to vibrate the vibrator 2 through excitation of the vibrating coil 3, thereby generating ultrasonic waves. The generated ultrasonic vibrations propagate through the liquid W in the tank 4 and are transmitted to the cell liquid T and the processing rod 11 in each container via the walls of each container 8a, 8b, . . . 8f. As a result, the surface tension effect of the cell liquid T on the metal ring 10 or the inner surface 15 of the container is weakened by the ultrasonic action, and the liquid T flows downward through the extremely small gap S. FIG. 4 shows this state. And at this time,
Since the processing rod 11 vibrates in sharp resonance with the vibration of the vibrator 2, when the cell liquid T passes through the gap S,
Intense ultrasonic pressure is applied by the metal ring 10, and the pressure of ultrasonic waves transmitted through the wall of the container is applied, so that the cells in the liquid T are primarily crushed. Next, the liquid T flowing down through the gap S flows downward along the inner surfaces of the containers 8a, 8b, . . . and accumulates at the bottom 13 of the containers. and,
At the bottom, ultrasonic pressure is again applied to the liquid T by the tip 12 of the processing rod 11, which vibrates in sharp resonance, thereby causing secondary crushing.

Now, the characteristic action of the present invention in such a crushing process is as follows. That is, it is possible to correct the uneven radiation pressure of ultrasonic vibrations on the container due to the arrangement of the vibrators. More specifically, each container rotates slowly due to the slow rotation of the gear plate 23, and each container is positioned at the same location on average during a certain period of time. The radiation pressure is also transmitted uniformly to each container other than the container inserted into the through hole in the center of the plate 23. Therefore, each cell in a plurality of containers can be homogeneously disrupted.

Therefore, the accuracy and efficiency of the inspection after the cell membrane is disrupted can be improved. In addition, even if heat is generated in each container 8a to 8f due to the application of ultrasonic pressure, the heat is radiated from each container into the liquid in the tank while the gear plate 23 is rotating, that is, while each container is rotating. Therefore, there is no risk of cell deactivation.

〔effect〕

Therefore, according to the present invention, cells can be housed in a plurality of containers and the cells can be simultaneously crushed, while each cell in the plurality of containers can be homogeneously crushed. This makes it possible to improve the accuracy and efficiency of inspections after cell disruption. In addition, by making it easier to dissipate the heat generated in the container while the cells are being disrupted by ultrasonic pressure, it is possible to provide a means to prevent the cells from being deactivated as much as possible.

[Brief explanation of the drawing]

The accompanying drawings show embodiments of the present invention, in which Fig. 1 is a longitudinal side view, Fig. 2 is a top view, and Fig. 3 is a cross-sectional view of a container showing the place where a cell solution is poured onto the upper surface of a metal ring of a processing rod. , FIG. 4 is a cross-sectional view of the container showing the place where the water flows down through the gap S, FIG.
FIG. 7 is a diagram of a conventional example, in which 1 is an oscillator, 2 is a vibrator, 3 is an excitation coil, 4 is a tank, 5 is a case,
8a to 8f are containers, 9 is a metal rod, 10 is a metal ring, 11 is a processing rod, 12 is a tip, 13 is a bottom, 1
4 is an outer surface, 15 is an inner surface, 16 is an opening, 17 is a lid, 18 is an upper surface, 21 is an upper surface, 22 is a lid plate, 23
24 is a gear plate, 24a, 24b, . . . are through holes, 25 is a gear, 28 is a motor, S is a gap, T is a cell liquid, and W is a liquid in a tank.

Claims (1)

[Claims] 1. Ultrasonic waves are emitted into the liquid W in the tank 4 by driving the vibrator 2 attached to the bottom surface of the tank 4, and a plurality of lidded Container 8a~
In a crushing device that applies ultrasonic pressure to the cells in 8f to crush the cell membranes of the cells; a gear plate 23 is rotatably disposed approximately in the center of the lid plate 22 of the tank 4; Plural containers 8 with lids are mounted on the plate 23
A plurality of through holes 24a, 24b for holding each of the containers 8a to 8f in a vertical manner are arranged circumferentially at a predetermined interval, and the containers 8a to 8f are held in the through holes 24.
a, 24b, the gear 25 that meshes with the gear plate 23 is driven by the motor 26, and the gear plate 2
3 slowly rotate the container 8a, 8b.
An ultrasonic cell disruption device characterized by slowly rotating the...