JP4651987B2 - Cell disruption apparatus and temperature control method thereof - Google Patents

Description

  In order to analyze and fractionate samples such as microorganisms, plant tissues and seeds, animal tissues, and mineral materials, the present invention accommodates the samples in a crushing container together with a crushing medium, and vibrates the crushing container. The present invention relates to a cell crushing apparatus for crushing a sample and a temperature control method thereof.

  A crushing device is used to crush the sample, and by crushing the crushing container containing the sample and the crushing medium, the sample is crushed by compression caused by the crushing medium colliding with the sample and grinding by rotation. .

  As such a crushing apparatus, the applicant of the present application is a crushing apparatus that can efficiently and stably crush a sample by applying an 8-shaped vibration to a crushing container containing a sample and a crushing medium. (See Patent Document 1). Further, in order to improve crushing efficiency and stabilization, a crushing apparatus using a single crushing medium having a shape and size that maintains a posture along the axial center of the elongated crushing container and moves relative to the axial direction ( The applicant of the present application has proposed (see Patent Document 2).

FIG. 10 shows the configuration of the crushing device, wherein the rotary shaft 8 is provided with a tilted shaft body 11 whose axis is inclined with respect to the axis, and the annular body 15 is attached to the tilted shaft body 11 so as to be relatively rotatable. At the same time, the rotation of the annular body 15 is elastically constrained by the magnet 16 attached to the annular body 15 and the fixed magnet 18 opposite thereto, and the outer periphery of the annular holding body 20 attached to the annular body 15 In this configuration, the elongated crushing container 30 containing the crushing medium 32 and the sample in its part can be held in a posture parallel to the axis of the annular holder 20. When the rotary shaft 8 is rotationally driven by a motor (not shown), the crushing container 30 is combined with a main reciprocating movement with a relatively long stroke in the axial direction and a sub-reciprocating movement with a relatively short stroke in the direction orthogonal thereto. When the crushing medium 32 collides with the bottom of the crushing container 30 while rotating relatively, the crushing container 30 acts like a mortar and the crushing medium 32 acts like a pestle. Even plant tissues, animal tissues, plastic materials and mineral materials can be efficiently crushed.
JP 2000-023660 (pages 3 to 5, FIG. 1) Japanese Patent Laid-Open No. 2001-178444 (pages 3 to 5, FIG. 2)

  In the cell disruption apparatus, when the sample and the disruption medium are accommodated in the disruption container 30 and subjected to vibration, the heat is generated due to the disruption and the temperature of the apparatus is increased. However, there is a problem in that it is impossible to recover substances that are susceptible to thermal denaturation in the cell, and there are problems that interfere with work such as analysis, and there is a need for a cell disruption device that can perform cell disruption under predetermined temperature conditions. It was.

  An object of the present invention is to provide a cell disruption apparatus and a temperature control method thereof capable of performing cell disruption under a predetermined temperature condition.

In order to achieve the above object, the cell disruption apparatus according to the first invention of the present application is such that the refrigerant circulates between both cylinders in which the inner cylinder is arranged at a predetermined interval in the outer cylinder formed in the bottomed cylinder shape. the formed cooling vessel, an elongate crushing container was charged with the sample and the grinding media was retained in the inner cylinder, is vibrated to reciprocally vibrate in the axial direction and a direction intersecting that of the crushing container Niso is applied temperature When the temperature of the refrigerant detected by the detection means is equal to or higher than a predetermined value, the number of vibrations of the reciprocating vibration is changed .

  According to the above configuration, since the crushing container containing the sample is accommodated in the cooling container and cooled by the refrigerant, the temperature rise is suppressed regardless of the temperature rise due to crushing or the ambient temperature rise, and the sample is altered by heat. The crushing can be carried out without causing the.

  In the above configuration, the inner cylinder is formed to have an inner diameter that is spaced apart from the outer surface of the crushing container, and is configured to contact the crushing container via the heat conduction liquid. Since it is in a state of being thermally coupled to the cylinder, the temperature of the crushing container is quickly conducted to the inner cylinder and cooled. As the heat conduction liquid, water or antifreeze liquid is suitable for preventing the occurrence of contamination.

  In addition, a plurality of cooling containers are fixed on the circumference of an annular holder formed in an annular shape, and a refrigerant distribution unit that supplies the refrigerant supplied from the temperature control means in parallel to each cooling container is provided on the annular holder. By providing in, it can crush, cooling a some crushing container.

  Further, by providing temperature detecting means for detecting the temperature in the inner cylinder and temperature control means for controlling the temperature or circulation of the refrigerant into the outer cylinder based on the temperature detected by the temperature detecting means, Crushing can be performed by controlling the temperature to a set temperature.

  In addition, the rotary shaft that is rotationally driven is provided with an inclined shaft portion that is inclined with respect to the shaft center, and an annular body is fitted on the inclined shaft portion so as to be relatively rotatable, and the annular body is rotated. By providing a rotation restraining means for restraining and attaching an annular holding body holding a cooling container to the annular body, efficient crushing can be carried out.

Moreover, the temperature control method of the cell disruption apparatus according to the second invention of the present application is formed such that the refrigerant circulates between both cylinders in which the inner cylinder is arranged at a predetermined interval in the outer cylinder formed in the bottomed cylinder shape. A slender crushing container into which a sample and a crushing medium are charged is held in the inner cylinder, and when the temperature of the refrigerant reaches a predetermined value or more, a vibration speed of reciprocating vibration applied to the crushing container or The drive / stop is controlled. By this temperature control method, when the temperature of the crushing container rises due to crushing, it is possible to prevent the temperature from rising above a predetermined temperature by controlling to reduce the vibration speed for crushing or to stop the vibration. The stored sample is crushed without causing alteration due to the temperature rise accompanying crushing.

  The temperature of the crushing container in the above temperature control method is the difference between the temperature of the refrigerant supplied to the cooling container and the temperature of the refrigerant discharged from the cooling container, or heat conduction interposed between the inner cylinder and the crushing container. It can be detected from the temperature of the liquid or the temperature of the refrigerant.

  According to the present invention, since the crushing container containing the sample is accommodated in the cooling container and cooled by the refrigerant, the temperature rise is suppressed regardless of the temperature rise accompanying the crushing or the ambient temperature rise, and the sample is altered by the heat of the sample. The crushing can be carried out without causing the.

  Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings to provide an understanding of the present invention. The following embodiment is an example embodying the present invention, and does not limit the technical scope of the present invention. In addition, the same reference numerals are given to components common to the prior art.

  FIG. 1 shows an overall configuration of a cell disruption apparatus 1 according to the embodiment. A motor 5a or a motor 5a is attached to a base plate 40 suspended from a casing 29 arranged in an exterior body 2 via a vibration isolation coil 4. The drive means 5 comprising the speed reducer 5b is supported in a suspended state, and its output shaft is supported by a bearing portion 7 disposed on the base plate 40 via a shaft joint 6 so as to be rotatable around a vertical axis. The rotating shaft 8 is connected. Although details will be described later, a crushing function unit 50 is disposed in the casing 29 so as to be connected to the rotary shaft 8. Moreover, the opening part of the casing 29 opened above the exterior body 2 is closed by the door 9 so that opening and closing is possible. In addition, a control device 44, an operation unit 45, and a temperature control unit 46 (FIG. 5) are disposed in the exterior body 2, and the operation of the cell disruption device 1 is performed from an operation panel 45a disposed outside the operation unit 45. Can be operated.

  FIG. 2 shows the structure of the crushing function unit 50 as a cross-sectional view. The inclined shaft body 11 is fitted to the upper portion of the rotating shaft 8 with the axis inclined at an angle θ relative to the axis. Then, it is pressed and fixed by a nut 13 screwed into the upper end portion of the rotary shaft 8 via the inclined ring 12. An annular body 15 is attached to the outer periphery of the inclined shaft body 11 via a bearing 14 so as to be relatively rotatable. A magnet 16 is attached to the side surface of the annular body 15, and a counter electrode magnet 18 is attached to the bracket 17 so as to face the magnet 16. The magnet 16 and the counter electrode magnet 18 constrain the rotation of the annular body 15 by different-polar attraction so that the different poles face each other. Since the facing distance between the magnet 16 and the counter magnet 18 changes due to the swinging motion of the annular body 15, it is preferable to dispose the magnet 16 and the counter magnet 18 at a plurality of positions.

  An annular holding body 20 for holding the crushing container 30 (FIG. 4) is attached to the annular body 15 so as to be attachable / detachable. A plurality of types of annular holders 20 are prepared for each size of the crushing container 30. In the case of a crushing container 30 having a small size, a large number can be directly held on the circumference and simultaneously subjected to crushing treatment. The annular holder 20 is configured to hold the crushing container 30 via the cooling container 61 so that the crushing process can be performed while cooling the crushing container 30 having a relatively large size.

  As shown in FIG. 3, the annular holder 20 according to the present embodiment has cooling containers 61 attached to three locations, and refrigerant distribution portions 59 that distribute and circulate the refrigerant to each cooling container 61. It is attached to the annular body 15. As shown in FIG. 4, the cooling container 61 has an inner cylinder 63 fixed inside the outer cylinder 62 at a predetermined interval, and refrigerant is supplied to a closed space formed between the outer cylinder 62 and the inner cylinder 63. A refrigerant supply port 64 and a refrigerant discharge port 65 are provided in the outer cylinder 62 so as to be circulated. The refrigerant supply port 64 and the refrigerant discharge port 65 of each of the three cooling containers 61 are connected in parallel to the refrigerant distribution unit 59 by a refrigerant distribution pipe 66.

  As shown in FIGS. 1 and 2, the refrigerant supply pipe 70 a and the refrigerant discharge pipe 70 b drawn from the refrigerant distribution unit 59 are detachably relayed to a relay unit 69 attached to the bottom surface of the casing 29, and the exterior body 2 are connected to a temperature control unit 46 (not shown) through connection ports 80a and 80b attached to the port 2. As shown in FIG. 5, the temperature control unit 46 is provided in a refrigerant cooling device 46 a that cools the refrigerant to a predetermined temperature, a refrigerant pump 46 b that supplies the refrigerant to the refrigerant distribution unit 59, and a relay unit 69. And a temperature control device 46c that controls the refrigerant temperature to be a set temperature based on the temperature of the refrigerant detected by the temperature sensor 67 so that the cooling temperature set and inputted from the operation unit 45 can be obtained. Perform temperature control.

  The refrigerant supplied from the temperature control unit 46 is supplied from the connection port 80a to the refrigerant distribution unit 59 through the refrigerant supply pipe 70a and the relay unit 69, and is branched into three at the refrigerant distribution unit 59 and is cooled from the refrigerant distribution pipe 66. The refrigerant is supplied to the refrigerant supply port 64 of the container 61. The refrigerant whose temperature has been increased by exchanging heat in the cooling container 61 is returned from the refrigerant outlet 65 to the temperature controller 46 through the refrigerant distributor 59, the refrigerant outlet pipe 70b, and the relay 69, and is returned from the connection port 80b to the temperature controller 46a. Is cooled and recirculated. Since the relay unit 69 is provided with temperature sensors 67 at the connection part with the refrigerant supply pipe 70a and the connection part with the refrigerant discharge pipe 70b, respectively, the refrigerant temperature on the supply side and the discharge side can be detected. The cooling temperature can be controlled from the detected temperature difference of the refrigerant. The temperature control method will be described later.

  A heat conduction liquid made of water or antifreeze is injected into the inner cylinder 63 of the cooling container 61, and the crushing container 30 into which the sample and the crushing medium are put is accommodated therein. The opening of the cooling container 61 has a cap 60. Closed by.

  As shown in FIGS. 6 (a) and 6 (b), the crushing container 30 is formed of an elongated cylindrical container. A screw 30a is formed on the outer periphery of the opening, and the lid 31 can be screwed into the opening to be sealed. It is configured. The crushing container 30 has a capacity of 2 to 50 ml depending on the material and quantity of the sample, and the annular holder 20 is also configured so that a cooling container 61 corresponding to the size of the crushing container 30 can be mounted. .

  Further, as shown in FIGS. 7 and 8, the crushing medium 32 accommodated together with the sample in the crushing container 30 is composed of a single member having a length L larger than the inner diameter D of the crushing container 30. In addition, a protruding conical end portion 32a having a conical shape or a circular shape corresponding to the bottom shape of the crushing container 30 is formed at one end thereof. The other end portion is formed in the small diameter portion 32b so as not to interfere with or fit into the annular seal portion 31a on the inner periphery of the lid 31. Further, the outer diameter d of the crushing medium 32 is set to be 2 mm or less with respect to the inner diameter D of the crushing container 30, and about 1 mm or less when the inner diameter d is small. For example, when the capacity of the crushing container 30 is 2 ml, the inner diameter D is set to 8 mm, and the outer diameter d of the crushing medium 32 is set to 7 mm. Further, as shown in FIGS. 7B, 7C and 8B, 8C, one or more grooves 34 are radially or spirally formed in the protruding end portion 32a of the crushing medium 32 as necessary. When the formed material is used, it can be crushed while cutting a sample that is difficult to be crushed, such as a plant fiber.

  When the material to be crushed is a microorganism, the crushing medium 32 is not a single member as described above, and it is preferable to use many small spheres such as glass beads and zirconia beads, and the microorganisms and beads are efficiently mixed. And good crushing can be obtained.

  When the cell disruption apparatus 1 is operated by attaching the crushing container 30 containing the sample and the crushing medium 32 to the annular holder 20, and the rotating shaft 8 is rotated at a high speed of, for example, 1200 to 2800 r / min, the inclined shaft body rotates. The annular body 15 that is externally fitted to the rotary shaft 11 tries to rotate together with the inclined shaft body 11. However, since the corotation is constrained by the magnet 16 and the counter electrode magnet 18, every time the rotary shaft 8 rotates once. The annular body 15 swings in both axial directions.

  At this time, an arbitrary point of the annular body 15 moves in the shape of figure 8, as shown in FIGS. That is, as indicated by a solid line in FIG. 9A, the state of the annular body 15 inclined in the vertical direction on the paper surface is taken as a reference position, and the behavior of the point a position on the outer periphery of the annular body 15 at that time is examined. When the rotary shaft 8 rotates 90 degrees in the direction of the arrow from the solid line position, the annular body 15 shifts to a state inclined in the front and back direction of the paper surface as indicated by the phantom line, while the position corresponding to the point a is the path Move to point c via b. When the rotary shaft 8 further rotates 90 degrees, the annular body 15 shifts to a state inclined in the vertical direction and in the opposite direction on the paper surface as shown by the solid line in FIG. 9B, and the position corresponding to the point a. Returns from the point c through the path d to the original point a. When the rotating shaft 8 further rotates 90 degrees, the annular body 15 shifts to a state inclined in the opposite direction to the front and back of the paper surface as shown by the phantom line, and the position corresponding to the point a passes through the path e to the point f. When the rotary shaft 8 further rotates 90 degrees to the original rotational position, the position corresponding to the point a returns from the point f through the path g to the original point a.

  Accordingly, the crushing container 30 held by the annular holding body 20 attached to the annular body 15 is vibrated in the shape of an 8-shaped vibration with the rotation of the rotary shaft 8, and the sample is contained in the crushing container 30. The crushing medium 32 collides effectively, and the sample is crushed quickly and uniformly by the impact. Particularly, in the crushing medium 32 having a tip shape corresponding to the bottom shape of the crushing container 30 shown in FIGS. 7 and 8, the crushing container is rotated while being relatively rotated while maintaining the posture substantially along the axis of the crushing container 30. Since the crushing container 30 acts like a mortar and the crushing medium 32 acts like a pestle by repeating the movement of colliding with the bottom of the sample 30, even if the sample is a large plant cell or animal tissue, crushing is performed efficiently.

  When crushing is performed, the temperature of the sample rises due to friction and collision of the crushing medium 32, and further due to temperature rise accompanying operation of the apparatus. This is not necessary in the case of a sample in which temperature rise does not cause a problem. However, in the case of a sample that may be altered due to temperature rise, as shown in the configuration of this embodiment, the crushing container 30 is accommodated in a cooling container 61 and the sample is stored. Must be crushed under a certain temperature condition.

  Since the refrigerant circulates so as to be supplied to the cooling container 61 from the refrigerant supply port 64 and discharged from the refrigerant discharge port 65, the crushing container 30 is accommodated in the inner cylinder 63 and the inner cylinder 63 by adjusting the temperature of the refrigerant. Since it is cooled through the purified water, the crushing container 30 can be maintained at a constant temperature, and the temperature rise of the sample is suppressed. Here, ethanol having a concentration of 30% is used as the refrigerant, and the heat of the container 30 to be transferred to the inner cylinder 63 through pure water is efficiently exchanged.

  It is desirable to control the cooling temperature of the crushing container 30 so as to obtain a temperature condition of 4 ° C. or lower in order to prevent protein inactivation when crushing cells such as animal tissues. The temperature control in the above configuration is a method for controlling the operation of the cell crushing apparatus 1 that causes the temperature rise according to the detection output by the temperature sensor 67 that detects the temperature of the refrigerant supplied to and discharged from the cooling container 61. Can be applied.

  When the cooling temperature of the refrigerant by the refrigerant cooling device 46a is set to 0 ° C. by setting input from the operation unit 45 and the rotation speed of the rotary shaft 8 is set to 2500 r / min, the operation of the cell crushing device 1 is started. The temperature in the crushing container 30 rises due to the heat generated when the sample is crushed by the movement of the crushing medium 32 in the 30 and the increase in the ambient temperature accompanying the temperature rise of the apparatus. When this temperature rise is detected by the temperature sensor 67 and a predetermined upper limit temperature (for example, 4 ° C.) is detected, the operation of the cell disruption apparatus 1 is stopped. When the operation is stopped, the temperature in the crushing container 30 is lowered. Therefore, when a predetermined lower limit temperature (for example, 2 ° C.) is detected, the operation of the cell crushing apparatus 1 is started again. By this control, the inside of the crushing container 30 is maintained at 4 ° C. or lower, and crushing can be carried out without causing protein deactivation even when the sample contains protein.

  When controlling the operation of the cell disruption apparatus 1, instead of turning the operation on / off as described above, when the temperature rise reaches a predetermined upper limit temperature, the rotational speed of the rotary shaft 8 is decreased from the set rotational speed. It is also possible to control so that the temperature increase due to crushing is suppressed and the rotation speed of the rotary shaft 8 is returned to the set rotation speed when the temperature is lowered to the lower limit temperature.

  Further, the temperature rise of the crushing container 30 is detected from the temperature difference between the supply side and the discharge side of the refrigerant temperature detected by the temperature sensor 67, and control is performed so as to lower the temperature of the refrigerant supplied by the refrigerant cooling device 46a. it can.

  In addition, as a method for detecting the temperature in the crushing container 30, it is also possible to apply a method in which a temperature sensor is provided in the inner cylinder 63 to detect the temperature of the heat conduction liquid interposed between the inner cylinder 63 and the crushing container 30. it can.

  In the configuration described above, the refrigerant supplied to the cooling container 61 indirectly cools the crushing container 30 so that the refrigerant circulation path and the crushing container 30 are separated from each other. Even if a part of the sample adheres to the container 30 or the sample leaks from the crushing container 30, it does not scatter to the outside through the refrigerant. Even if the sample for cell disruption contains, for example, a BSE specimen or pathogen, they do not leak to the outside.

  In the cell disruption apparatus 1 configured as described above, the upper opening portion of the annular body 15 is closed by the sealing cap 43, the space between the annular body 15 and the bearing portion 7 is covered by the rubber cylinder 27, and further the casing 29. Since the opening formed for passing the rotating shaft 8 and the bearing portion 7 on the bottom surface of the shaft is closed by the shielding rubber 28, all the movable parts such as the bearing 14 located in the casing 29 are covered. Therefore, the inside of the casing 29 can be cleaned by steam cleaning, chemical cleaning, or the like. Of course, the inside of the casing 29 may be contaminated by dust or the like due to repetition of the crushing process, and the sample may be scattered. In particular, when the sample is harmful or contains pathogenic bacteria, the inside of the casing 29 needs to be sterilized and cleaned. Even at this time, even if steam or chemicals are injected, the movable part does not enter and can be completely cleaned. Further, as shown in FIGS. 1 and 2, the liquid accumulated in the casing 29 at the time of cleaning is discharged from the drain hose 54 to the outside when the drain plug 53 provided on the bottom surface of the casing 29 is opened. Can do.

  As described above, according to the present invention, since the crushing container can be maintained and maintained in a constant temperature state, even if the sample to be crushed is altered due to temperature rise, the required temperature condition is satisfied. It is possible to provide a cell crushing apparatus that enables crushing with a cell.

The side view which shows the whole structure of the cell crushing apparatus which concerns on embodiment. Sectional drawing which shows the structure of the crushing function part in a structure same as the above. (A) which shows the structure of a cyclic | annular holding body is a top view, (b) is a side view. Sectional drawing which shows the structure of a cooling container. The block diagram which shows a cooling structure. Sectional drawing which shows the structure of a crushing container. The side view which shows the structure of a crushing medium. The side view which shows the structure of a crushing medium. Explanatory drawing explaining a motion of an annular body. Sectional drawing which shows the principal part structure of the crushing apparatus which concerns on a prior art.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Cell crusher 8 Rotating shaft 11 Inclined shaft 15 Ring body 16 Magnet (rotation restraint means)
18 Counter magnet (rotation restraint means)
20 annular holder 30 crushing vessel 31 lid 32 crushing medium 46 temperature control unit 50 crushing function unit 59 refrigerant distribution unit 61 cooling vessel 62 outer cylinder 63 inner cylinder 64 refrigerant supply port 65 refrigerant discharge port 67 temperature sensor

Claims (11)

In the cooling vessel formed so that the refrigerant circulates between both cylinders arranged at a predetermined interval in the outer cylinder formed in the bottomed cylindrical shape,
Hold the elongated crushing container into which the sample and the crushing medium are put in the inner cylinder,
The crushing container is vibrated so that a reciprocating vibration is applied in an axial direction and a direction crossing the axial direction.
The cell disruption apparatus , wherein the number of vibrations of the reciprocating vibration is changed when the temperature of the refrigerant detected by the temperature detection means is equal to or higher than a predetermined value .   The cell disruption apparatus according to claim 1, wherein the inner cylinder is formed to have an inner diameter that is spaced from the outer surface of the disruption container by a predetermined distance, and is configured to come into contact with the disruption container via the heat conduction liquid.   A plurality of cooling containers are fixed on the circumference of an annular holding body formed in an annular shape, and a refrigerant distribution portion for supplying the coolant supplied from the temperature control means in parallel to each cooling container is provided on the annular holding body. The cell disruption apparatus according to claim 1 or 2, wherein Cell disruption device according to claim 1 any one of the temperature control means for controlling the temperature or circulation of the refrigerant thus provided on the basis of the temperature detected by said temperature detecting means.   A rotary shaft that is rotationally driven is provided with an inclined shaft portion that is inclined with respect to the shaft center, and an annular body is fitted on the inclined shaft portion so as to be relatively rotatable, and the rotation of the annular body is restricted. The cell disruption device according to any one of claims 1 to 4, wherein a rotation restraining means is provided, and an annular holding body holding a cooling container is attached to the annular body.   The cell disruption apparatus according to any one of claims 1 to 5, wherein the refrigerant is an aqueous solution of ethylene glycol, ethanol, or methanol.   The cell disruption apparatus according to claim 2, wherein the heat conduction liquid is water or an antifreeze liquid. An elongated crushing container in which a sample and a crushing medium are put into a cooling container formed so that a refrigerant circulates between both cylinders in which an inner cylinder is arranged at a predetermined interval in an outer cylinder formed into a bottomed cylinder. Is controlled in the inner cylinder, and when the temperature of the refrigerant reaches a predetermined value or more, the vibration speed or driving / stopping of reciprocating vibration applied to the crushing container is controlled. Method. The temperature control method for a cell disruption device according to claim 8, wherein the temperature of the crushing container is detected from a difference between a temperature of the refrigerant supplied to the cooling container and a temperature of the refrigerant discharged from the cooling container. The temperature control method for a cell disruption device according to claim 8, wherein the temperature of the crushing container detects the temperature of the heat transfer liquid interposed between the inner cylinder and the crushing container. The temperature control method for a cell disruption device according to claim 8, wherein the temperature of the disruption container is detected from the temperature of the refrigerant.

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