JP4280690B2 - Sample container and crimping device for sample container - Google Patents

Description

  The present invention relates to a sample container and a crimping apparatus for a sample container. For example, a tubular sample container used for cryopreserving a biological sample such as semen or embryo, and an open end of the sample container are closed. It is effective when applied to a crimping device.

  When cells and tissues such as semen and embryos are frozen and stored in small quantities according to the amount used, a sample container made of straw-like tubules is often used as the container. FIG. 11 shows the configuration of a conventional sample container of this type. In this figure, (a) is a cross-sectional view, (b) is a cross-sectional view taken along arrow A in (a), and (c) is a cross-sectional view taken along line BB in (a).

  A sample container 10 ′ shown in the figure is formed by a straw-like tubule 11 having a uniform outer diameter φ 1, and a sample 100 as a container is filled in the tubule 11. The sample container 10 ′ is preliminarily filled with a moisture-sensitive sealing material 16 on one end (right end in the drawing) in an unused state (empty state) in which the sample 100 is not yet stored.

  The moisture-sensitive sealing material 16 is a cotton-like filler containing a polymer water retention agent in a dry state (powder), and has air permeability in an unused dry state, but gelates when exposed to moisture such as a sample. Thus, the sample container 10 ′ is hermetically closed. In other words, the solid sealing material has air permeability in the dry state but airtight in the wet state.

  By sucking air from one end of an unused sample container 10 ′ filled with a dry moisture-sensitive sealing material 16, the sample 100, which is the contents, is sucked from the other end (left end in the figure) of the sample container 10 ′. The sample container 10 'can be filled and accommodated.

  When the sample 100 is accommodated in the sample container 10 ′, the polymer water retention agent of the moisture-sensitive sealant 16 is gelled by the moisture of the sample 100, and one end side of the sample container 10 ′ is hermetically closed.

Thereafter, by crimping the other end of the sample container 10 ′, the other end is also closed as shown in FIG. Thereby, the both ends of sample container 10 'can be obstruct | occluded and the sample 100 can be confined in an airtight state. At the time of closing by this crimping, an air layer 17 is provided at the opening end of the sample container 10 ′ to prevent the sample container from being damaged due to expansion when the sample liquid part is frozen, as shown in FIG. This type of sample container 10 'is used in the freezing apparatus described in, for example, Patent Document 1 (Conventional Example 1) and Patent Document 2 (Conventional Example 2).
Japanese Patent Publication 8-4601 US Patent 5,873,254

  It has been found that the above-described conventional sample container 10 'has the following problems. That is, as shown in FIGS. 11 (b) and 11 (c), the other end portion of the sample container 10 ′ closed by the crimping is flattened at its crimping portion 11e, but this flat crimping portion 11e. The width w1 extends beyond the outer diameter (φ1) of the sample container 10 ′. That is, the sample container 10 ′ containing the sample 100 is not uniform in outer diameter (φ1), and has an irregular shape in which only the end portion is expanded in the width direction.

  For example, when the sample container 10 ′ having such an irregular shape is accommodated in an aggregate of a plurality of aggregates, the shape of the aggregate is difficult to determine, and when one or an arbitrary number is extracted from the aggregate, There is a problem that the wide crimped portion 11e gets caught in another sample container and only the target sample container cannot be taken out smoothly. That is, the deformed shape of the pressure-bonding portion 11e hinders the handling property (ease of handling) of the sample container 10 '.

  Furthermore, the present inventors cooled the sample container 10 ′ containing the sample 100 for cooling to a predetermined temperature or a predetermined temperature in order to perform the cooling process appropriately and with good reproducibility. It has been found that it is very effective to insert and insert one straw-like sample container 10 ′ into the through hole of the heat transfer block cooled at the cooling rate one by one. However, it has been found that when the cooling process is performed, the deformed shape of the crimping portion 11e becomes an obstacle.

  That is, in order to cool the sample container 10 'in the through hole of the heat transfer block appropriately and with good reproducibility, it is preferable to make the gap between the through hole and the sample container 10' as small as possible. However, in order to smoothly insert the sample container 10 'through the through hole, at least a gap larger than the protruding portion (w1-φ1) of the crimping portion 11e is required.

  The present invention has been made in view of the background and problems as described above, and the object thereof is formed by a straw-like tubule having a uniform outer diameter, and one or both open ends of the tubule are closed by pressure bonding. A sample container suitable for carrying out a cooling process for cryopreservation appropriately and with good reproducibility and effective for forming this sample container. The object is to provide a crimping device for a sample container.

The present invention is a sample container used for cryopreserving a biological sample, which solves the above problems,
Formed from a straw-like tubule made of a thermoplastic resin having a uniform outer diameter,
Freezing by passing through a tubular through-hole having an inner diameter slightly larger than the outer diameter formed in the cooled heat transfer block,
One or both open ends of the capillary are crimped and closed by ultrasonic vibration,
The closed opening end is crimped in a state in which the circular opening end is substantially divided into two in the circumferential direction, and one semicircular portion is overlapped in an arc shape inside the other semicircular portion, And in the said welding state, it is formed so that the protrusion part from the said outer diameter may not arise.

Moreover, it can also be set as the sample container by which the one open end part of the said straw-shaped thin tube is obstruct | occluded by filling with a sealing material, and the other open end part is obstruct | occluded by crimping | compression-bonding.

And it is more preferable if it is set as the sample container in which the embossing part which protrudes inside the said straw-shaped thin tube and latches and fixes the said sealing material is formed.

An apparatus for crimping the open end of the sample container according to any one of the above is also within the scope of the present invention,
  A jig for constraining the open end with a concave surface, and a convex surface for pairing with the concave surface and crimping the open end constrained by the jig in a circular arc shape. A pressure element, and an ultrasonic vibration source that applies ultrasonic vibration to the opening end that is pressure-formed by the concave surface and the convex surface,
  The jig is a crimping device for a sample container in which the inner diameter of the concave surface is slightly smaller than the outer diameter of the sample container, and the depth to the bottom and upper opening edge of the concave surface is equal to the outer diameter. .

  The sample container is formed by a straw-like tubule having a uniform outer diameter, and one or both open ends of the tubule are closed by crimping. Therefore, it is possible to provide a sample container that is suitable for performing the cooling process for the purpose and with good reproducibility.

  FIG. 1 shows an embodiment of a sample container to which the technique of the present invention is applied. In this figure, (a) is a partially omitted perspective view of a sample container 10 according to the present invention, (b) is a cross-sectional view thereof, (c) is a cross-sectional view along AA of (b), and (d) is (b) BB sectional drawing of) is shown.

  A sample container 10 shown in the figure is used to store and cryopreserve biological samples such as semen and embryos, and has a straw-like tubule 11 of a predetermined length having a uniform outer diameter φ1. It is formed using.

  A small tube 11 that forms the main part of the sample container 10 is filled with a sample 100 that is a container. One end (right end in the figure) of the sample container 10 is preliminarily filled with a moisture sensitive sealing material 16 in a dry state.

  This moisture-sensitive sealing material 16 has a dry state (powdered) polymer water retention agent 15 disposed between the cotton plug members 14 and 14, and is in an unused state (empty) before the sample 100 is accommodated. In the state), the water retaining agent 15 is gelled by moisture absorption or water absorption, and one end of the sample container 10 is hermetically closed.

  By performing air suction from one end of the sample container 10 filled with the moisture-sensitive sealing material 16, the sample 100 as the contents is sucked from the other end (left end in the figure) of the sample container 10 and the sample container 10 is inhaled. 10 can be accommodated.

  When the sample 100 is accommodated in the sample container 10, the polymer water retention agent 15 of the moisture-sensitive sealing material 16 is gelled by the moisture of the sample 100, and one end of the sample container 10 is closed. Although the cotton plug member 14 and the water retention agent 15 are arranged separately in the illustrated example, the water retention agent 15 may be mixed into the cotton plug member 14 so as to be integrated.

  Thereafter, by crimping the other end of the sample container 10, the other end is closed and the sample container 10 is hermetically closed as shown in FIG. Thereby, the sample 100 of the sample container 10 is hermetically sealed. At the time of closing by this crimping, an air layer 17 is provided at the opening end of the sample container 10 to prevent breakage of the sample container due to expansion when the sample liquid part is frozen, as shown in FIG.

  As described above, in the sample container 10 according to the present invention, the end portion of the thin tube 11 is substantially divided into two in the circumferential direction, and one semicircular portion 12 is circularly formed inside the other semicircular portion 13. They are pressure-bonded to each other in an arcuate state. Thereby, as shown in (c) of the figure in particular, the end portion is closed without causing a protruding portion from the outer diameter φ1.

  Since the sample container 10 configured as described above does not have an irregular protruding portion, for example, when the sample container 10 is accommodated in an aggregate of a plurality of samples, the shape of the aggregate can be determined stably. Further, when one or an arbitrary number is extracted from the aggregate, only the target sample or the target number can be smoothly extracted. That is, the handling property of the sample container 10 is good.

  Furthermore, when the sample container 10 containing the sample 100 is subjected to a cooling process for cryopreservation, the cooling process can be performed appropriately and with good reproducibility by a biological sample freezing apparatus described later.

  As a material for forming the straw-shaped tubule 11, a thermoplastic resin suitable for deformation processing by pressure bonding is used. Use of a transparent resin is preferable in order to easily check the presence or absence of contents.

  In addition, as shown in FIG. 1 (b) with a partially enlarged view, the straw-like tubule 11 is formed with an embossed portion 18 that protrudes inward to lock and fix the sealing material 16. When the other end side of the sample container 10 is sucked in order to suck the sample 100 into the sample container 10, the embossed part 18 sucks the sealing material 16 out by the suction, or This has the effect of reliably preventing the position from moving.

  FIG. 2 shows a main part of a crimping apparatus particularly effective for use in crimping and closing the end of the sample container 10. In the same figure, (a) is the state before setting the open end of the sample container 10 to the apparatus, (b) is the state where the open end of the sample container 10 is crimped, and (c) is the open end. Shows the sample containers 10 that are closed by pressure bonding.

  The crimping device for a sample container shown in the figure includes a jig 71 for restraining the open end of the straw-shaped tubule 11 with a semicircular concave surface 71a corresponding to the outer shape of the thin tube 11, and the concave surface 71a. And a pressing element 72 having a convex surface 72a that is paired with each other, and the opening end is pressed and closed while being pressure-formed in an arc shape with the concave surface 71a and the convex surface 72a.

  In the case of the illustrated embodiment, the bottom of the concave surface 71a is formed in an arcuate curved surface having a width w (twice the radius of curvature) w slightly smaller than the outer diameter φ1 of the sample container 10. Further, the depth h from the upper opening edge to the bottom of the concave surface 71a is formed to be substantially the same as the outer diameter φ1 of the sample container 10. As a specific dimension example, when the outer diameter φ1 of the sample container 10 is 3 mm, the width w may be 2.7 mm and the depth h may be 3 mm.

  As a result, the ends of the thin tubes 11 are substantially bisected in the circumferential direction, and one semicircular portion 12 is crimped to each other in an arcuate manner inside the other semicircular portion 13. The maximum diameter of the crimped portion can be reliably accommodated within the outer diameter φ1.

  FIG. 3 is an abbreviated side view showing an example of the overall configuration of the sample container crimping apparatus according to process steps. In the same figure, (a) is the state before setting the opening end of the sample container 10 to the crimping device, b) is the state where the opening end of the sample container 10 is crimped, and (c) is the opening end. The state where the crimping process of the part is completed is shown.

  As shown in the drawing, in the sample container crimping apparatus, a jig 71 is fixed to a mount 73, and the presser 72 is mounted above the jig 71 so as to be movable up and down. An ultrasonic vibration source 74 is attached to the presser 72 so that ultrasonic vibration is applied to the open end of the sample container 10 that is pressure-formed by the concave surface 71a and the convex surface 72a. It is. Thereby, the opening edge part of the sample container 10 can be crimped-closed more reliably and more reliably.

  The pressing element 72 is driven to move up and down with a predetermined driving torque by the movement driving device 75 together with the ultrasonic vibration source 74. The movement drive device 75 is configured using, for example, a stepping motor.

  By using an electric drive means such as a stepping motor, the pressing torque during the crimping process can be set to a predetermined value by controlling the drive current. Further, when a stepping motor is used, the movement stroke can be managed and controlled by managing the number of drive pulses of the stepping motor.

  In addition, the pressing end surface (lower end surface) of the pressing element 72 is gently formed on one end side of the concave surface 71a that press-deforms the thin tube 11 in an arc shape as shown in a partially enlarged view in FIG. The slope curved surface 72b is formed in order to form a gentle buffer curved surface at the boundary between the crimping closed portion and the non-crimping closed portion of the thin tube 11 (sample container 10).

  In the above-described embodiment, the ultrasonic vibration is applied to the opening end portion that is pressure-formed by the concave surface 71a and the convex surface 72a. However, instead of the ultrasonic vibration, a high-frequency current is applied. Even if it is made to supply electricity, the same effect can be acquired even if it uses the heating apparatus by an electric heater. Further, if necessary, ultrasonic vibration and high frequency current may be applied in combination.

  FIG. 4 shows a preferred application example of the sample container crimping apparatus. The apparatus shown in FIG. 1 is a hopper-shaped supply unit 81 that stocks the sample containers 10 one by one, and the sample container 10 supplied from the supply unit 71 is crimped by a jig 71 in a predetermined alignment state. Conveying means 82 for sequentially conveying to the processing position is provided.

  The conveying means 82 is formed in a drum shape or a disk shape, and a large number of sample container holder portions 83 are arranged along the outer circumference of the circle at equiangular intervals, and in a certain direction by a stepping motor (not shown). Step rotation is driven. The sample containers 10 stocked in the supply means 71 are transported between the jig 71 and the presser 72 while being held in the holder portion 83 one by one.

  The jig 71 is driven to move up and down by the movement drive device 76, and when the sample container 10 is positioned on the jig 71, the jig 71 is driven up to the crimping position of the sample container 10. In synchronism with this, the pressing member 72 is pressed downward by the movement driving means 75, whereby the sample container 10 is crimped. Thereby, the opening edge part of the sample container 10 is crimped-closed.

  The crimped sample container 10 is conveyed to the collection container 85 by the conveying means 82. Reference numeral 84 denotes a detachment jig (rejector), which is installed so as to detach the sample container 10 from the conveying means 82.

  Next, a biological sample freezing apparatus particularly suitable for cooling the above-described sample container 10 for cryopreservation will be described.

  5 to 7 show an embodiment of a biological sample freezing apparatus to which the technology of the present invention is applied. FIG. 5 shows an outline of the entire apparatus in a side view (a) and a top view (b). 6 is a perspective view of the heat transfer block and the guide table portion of the apparatus shown in FIG. FIG. 7 shows a side section (a), a transverse section and a partially enlarged section (b) of the heat transfer block.

  The biological sample freezing apparatus shown in the figure is used for cooling cells and tissues such as semen and embryos for cryopreservation, and its main part is transmitted as shown in FIG. The heat block 20, the guide table 30, the movement drive means 40, and the control drive unit 50 are configured. The biological sample is stored in a tubular sample container (also called a straw) 10 and subjected to a cooling process for cryopreservation.

  The heat transfer block 20 is a rectangular plate-shaped solid block as shown in FIGS. 5 to 7 and is cooled by the cooling unit (cooling means) 25 at a predetermined temperature and / or a predetermined cooling rate. It has become. As a material of the heat transfer block 20, a highly heat conductive metal or ceramic is used. As the metal material, for example, aluminum or an aluminum alloy, or a copper alloy such as copper or brass is suitable. As the ceramic, for example, aluminum nitride is suitable.

  The heat transfer block 20 is provided with a plurality of independent through holes 21 extending from one end to the other end. The plurality of through holes 21 are arranged in parallel in the heat transfer block 20 at a predetermined interval. Each through hole 21 is formed to have a minimum inner diameter (φ2) necessary to insert and pass the sample containers 10 one by one.

  As shown in FIG. 7B, the through hole 21 is formed to have an inner diameter φ2 that is slightly larger than the outer diameter φ1 of the sample container 10. As a result, the sample container 10 moves and passes through the through hole 21 without being formed a useless gap and is closely fitted in the through hole 21 in a substantially fitted state without any gap. Yes.

  As for the through hole 21, if the gap formed between the inner surface of the through hole 21 and the sample container 10 is formed with an inner diameter φ2 that is at least smaller than the outer diameter φ1 of the sample container, Conventional technical problems can be solved.

  That is, for example, in the technique of Conventional Example 2 disclosed in Patent Document 2, a gap space (tunnel) that is significantly larger than the outer diameter of the sample container is formed between the sample container and the heat transfer block. Therefore, the air layer in this large gap space causes a delay in the cooling rate, adversely affects the cells in the sample container, and prevents the sample container from being cooled properly and reproducibly. On the other hand, in the apparatus according to the present invention, the gap space can be made smaller than at least the outer diameter φ1 of the sample container 10 to perform a proper and reproducible cooling process.

  Further, in the present invention, for example, as shown in a partially enlarged view in FIG. Thereby, the cooling process of the plurality of sample containers 10 can be performed more appropriately. Specifically, the gap formed between the inner surface of the through hole 21 and the sample container 10 may be 5% or less of the outer diameter φ1 of the sample container or 0.1 mm or less.

  For example, when the outer diameter φ1 of the sample container is 3 mm, the difference between the outer diameter φ2 and the inner diameter φ1 (φ2−φ1) is 0.15 mm (5% of φ1) or less, so that the heat transfer block 20 and the sample A stable and good heat transfer state can be ensured between the container 10 and an appropriate and reproducible cooling process.

  When the outer diameter φ1 of the sample container is 2 mm, the difference between the outer diameter φ2 and the inner diameter φ1 (φ2−φ1) is set to 0.1 mm or less between the heat transfer block 20 and the sample container 10. A stable and good heat transfer state can be ensured.

  That is, the difference (φ2−φ1) between the outer diameter φ2 and the inner diameter φ1 is preferably defined as a ratio (5% or less) to the outer diameter φ1 when the outer diameter φ1 of the sample container 10 is large. In addition, when the outer diameter φ1 of the sample container 10 is small, it may be specified by an absolute dimension (0.1 mm or less).

  The heat transfer block 20 having the through-hole 21 may be integrally formed from the beginning using a single block material, but a so-called divided structure in which a plurality of divided blocks are combined is also possible. In the case of a divided configuration, the through-hole 21 can also be divided and formed together. For example, in the case of a two-divided configuration, the through-hole 21 can be formed by combining grooves formed in two divided blocks.

  The cooling unit 25 is configured using a Peltier element, and the cooling side (heat absorption side) surface thereof is in close contact with the entire lower surface of the heat transfer block 20. The Peltier elements can be cooled to the required cooling temperature (low temperature) by stacking a plurality of stages in series.

  With this cooling unit 25, the heat transfer block 20 can be cooled to a predetermined cooling temperature, or can be cooled at a predetermined cooling rate. In addition, although illustration is abbreviate | omitted, the cooling side of the said heat-transfer block 20 and the cooling unit 25 is thermally insulated from the external temperature with the heat insulating material using foamed resin etc.

  As shown in FIG. 6, the guide table 30 is installed on one end side of the heat transfer block 20. A plurality of (many) guide grooves 31 are formed in parallel on the upper surface of the guide table 30. Each guide groove 31 is provided in a one-to-one correspondence on the extension shaft of the through hole 21 so as to move and guide the sample container 10 into the through hole 21 while positioning and holding the sample containers 10 one by one. Is formed.

  Although a slight heat insulation gap is provided between the guide table 30 and the heat transfer block 20, the thin tubular sample container 10 is guided to move into the through hole 21 of the heat transfer block 20 across the heat insulation gap. It is like that.

  The movement driving means 40 is configured by using a plurality of propulsion rods 42, a rod guide 43, a movable member 44, a linear motion guide (linear guide) 45, a stepping motor 46, a ball screw 47, and the like. The movement driving means 40 is installed on a common base 41 together with the heat transfer block 20, the guide table 30, and the like.

  The propulsion rods 42 are arranged on the extension shaft of the through-hole 21 one by one while being positioned parallel to each other, and each sample container 10 held in a positioning state in the guide groove 31 of the guide table 30 is placed in the through-hole. It is comprised so that it may push and move to 21 axial directions.

  The rod guide 43 is installed in front of the guide table 30. The rod guide 43 performs positioning and movement guidance of the rod 42 so that the tip of each propulsion rod 42 can smoothly enter the through hole 21 while precisely matching the tip of the propulsion rod 42 with the central axis of the through hole 21. . For this reason, although not shown, the rod guide 43 has a guide hole (or a guide groove) for slidingly guiding the propelling rods 42 inserted one by one.

  The proximal end side of the propulsion rod 42 is held and fixed to the movable member 44. The movable member 44 is provided so as to reciprocate in parallel with the extension axis of the through hole 21 guided by the linear guide 45. The movable member 44 is driven to move while being guided by the linear motion guide 45 by a ball screw 47 that is rotationally driven by a stepping motor 46.

  By the movement of the movable member 44, the propulsion rods 42 are simultaneously moved forward to move the sample container 10 on the guide table 30 into the through hole 21 in the heat transfer block 20. As the propulsion rod 42 advances, the sample container 10 passes through the through hole 21 and is finally pushed out onto the air-permeable base in the final freezing container 60. In the final freezing container 60, a predetermined amount of liquid nitrogen is filled under the breathable gantry, and the current flowing through the electric heater immersed therein is adjusted so that the final freezing container has a predetermined temperature, for example, −100 The temperature is maintained at a temperature of from-° C to -196 ° C.

  The stepping motor 46 and the ball screw 47 constitute propulsion drive means for moving the plurality of propulsion rods 42 in the axial direction of the through hole 21 at a predetermined speed. The moving speed and the moving amount of the propulsion rod 42 are controlled easily and with high accuracy without using complicated feedback control based on position detection by managing the drive pulse period and the cumulative number of pulses of the stepping motor 46. be able to.

  The control drive unit 50 includes a temperature adjustment circuit 51, a motor drive control circuit 52, an operation / setting unit 53, a main control unit 54, and the like. The temperature adjustment circuit 51 detects the temperature of the heat transfer block 20 with the temperature sensor 26 installed in the block 20, and the energization current of the Peltier element and the like so that the temperature observed by this detection becomes a predetermined cooling temperature. To control.

  The motor drive control circuit 52 manages (monitors) the period, number of pulses (number of rotation steps), and rotation direction of the rotation drive pulse of the stepping motor 46, while moving (moving) the propulsion rod 42, moving amount, moving direction, Control the moving position.

  The operation / setting unit 53 is configured using an input device such as a keyboard, and is responsible for setting input such as each operation condition and operation procedure method (sequence) of the temperature adjustment circuit 51 and the motor drive control circuit 52. The main control unit 54 is configured by using a micro-circuited computer (so-called microcomputer), and based on various operation conditions and operation procedure methods set by the operation / setting unit 53, each of the units (51, 52) The operation is controlled by taking time elements into account.

  In addition, the control drive unit 50 described above can perform centralized processing of the functions of the temperature adjustment circuit 51 and the motor drive control circuit 52 by software in the main control unit 54, for example. On the contrary, the temperature control circuit 51 and the motor drive control circuit 52 may have some or all of the functions of the main control unit 54. Thus, the hardware configuration of the control drive unit 50 is not particularly limited.

  FIG. 8 shows a main part of another embodiment of the biological sample freezing apparatus according to the present invention. If it demonstrates paying attention to the difference with embodiment mentioned above, while the apparatus shown to the figure will install two (several) heat-transfer blocks 20A and 20B in series in the moving direction of the sample container 10, each heat-transfer Two systems (a plurality of systems) of cooling units (cooling means) 25A and 25B for cooling the blocks 20A and 20B to a predetermined temperature or cooling at a predetermined cooling rate are provided.

  Each of the heat transfer blocks 20A and 20B is provided with a through hole 21 coaxially for allowing the sample container 10 to be inserted and passed in a substantially fitted state. A heat insulating gap is placed between the two heat transfer blocks 20A and 20B. Moreover, although illustration is abbreviate | omitted, each heat-transfer block 20A, 20B and cooling unit 25A, 25B are each thermally insulated from external temperature.

  In this embodiment, by setting the temperature conditions of the two (plural) heat transfer blocks 20A and 20B independently, a much more diverse and complex cooling process is possible than when only one heat transfer block is provided. become. Thereby, the optimal cooling process according to the kind of biological sample etc. can be set more precisely.

  As shown in FIG. 1, the sample container 10 is substantially bisected at the end in the circumferential direction, and one semicircular portion 12 is overlapped inside the other semicircular portion 13 in an arc shape, By crimping the arcuate overlapping portions to each other, the end portion of the container 10 is crimped and closed without causing a protruding portion from the outer diameter φ1 of the container 10.

  As a result, as shown in FIG. 7, even if the gap (φ2−φ1) between the sample container 10 and the through hole 21 is reduced, the sample container 10 can be smoothly passed through the through hole 21. . Therefore, the effect of preventing the variation in cooling by smoothly transferring the cold heat of the heat transfer block into the sample container 10 can be obtained more remarkably.

Next, the operation and use method of the biological sample freezing apparatus described above will be described.
In the apparatus described above, as shown in FIGS. 5 and 6, the unprocessed sample container 10 is placed on the guide table 30. Since the guide table 30 has a guide groove 31, the sample containers 10 are guided to the guide groove 31 and held one by one in a positioning state.

  When the sample container 10 is set, the stepping motor 46 is rotated to advance the propelling rod 42 all at once. Thereby, the sample container 10 is moved from the guide table 30 into the through hole 21 of the heat transfer block 20. At this time, the heat transfer block 20 is preliminarily adjusted to a predetermined temperature.

  In the process of passing through the through-hole 21, the sample container 10 is cooled by cold heat transfer from the heat transfer block 20. By this cooling process, the sample in the container 10 is frozen into a state where it can be cryopreserved. The sample container 10 for which the cooling process has been completed is pushed out of the through hole 21 by the propulsion rod 42 and collected in the final freezing container 60.

In the above operation, the sample container 10 can be cooled by a process including the following steps (1) to (3), for example.
(1) A step of passing the sample container 10 through the through hole 21 of the heat transfer block 20 cooled to a predetermined temperature at a predetermined speed.
(2) A step of allowing the sample container 10 to stay in the through hole 21 of the heat transfer block 20 cooled to a predetermined temperature for a predetermined time.
(3) A step of allowing the sample container 10 to stay in the through hole 21 of the heat transfer block 20 cooled at a predetermined cooling rate for a predetermined time.
The above steps (1) to (3) can be selected in any combination depending on the type of sample, and various conditions (moving speed, staying time, cooling temperature, cooling rate, etc.) in each step are also arbitrary. Can be set.

  Further, in an apparatus having a plurality of heat transfer blocks 20A and 20B (FIG. 8), the sample container 10 can be cooled sequentially using two or more heat transfer blocks 20A and 20B having different temperature setting conditions. it can. Thereby, more various and complicated cooling processes are possible, and more optimal cooling can be performed with high accuracy in accordance with the type of sample.

  In order to make the cooling conditions in each through-hole more uniform, it is desirable that the plurality of through-holes 21 are arranged in parallel at predetermined intervals in the heat transfer block 20.

  FIG. 9 is a graph showing the total motor sperm rate (motility rate) and the most active motor sperm rate of semen (cow) frozen with the heat transfer block 20 using one device, according to the cooling conditions during freezing. As shown in the figure, the motor total sperm rate and the most active motor sperm rate of the semen that was once frozen and thawed were compared with the freezing method using the above-described apparatus of the present invention compared to the freezing method of Conventional Example 1. , Clearly getting good results.

  In addition, as shown in the figure, sperm motility varies significantly depending on conditions such as the temperature of the heat transfer block, the moving speed of the sample container, and the holding time in the heat transfer block (through hole). According to the apparatus of the present invention, it is possible to set optimum conditions with high reproducibility and high accuracy.

  FIG. 10 shows cooling of sperm motility (motor sperm rate and maximum active sperm rate) of semen (cow) frozen using a device (FIG. 8) having two heat transfer blocks 20A and 20B during freezing. It is the graph shown according to conditions. As shown in the figure, also in this example, the sperm motility of the semen that was once cryopreserved and then thawed was clearly better in the freezing method using the apparatus of the present invention than in the freezing method of Conventional Example 1. Have gained.

  Further, as shown in the figure, the sperm motility varies depending on conditions such as the temperature in each of the heat transfer blocks 20A and 20B, the moving speed of the sample container, and the holding time in the heat transfer block (through hole). According to the apparatus of the present invention, optimum conditions can be set in various ways with good reproducibility.

  As mentioned above, although this invention was demonstrated based on the typical Example, this invention can have various aspects other than having mentioned above.

  For example, in FIG. 4, the jig 71 that performs the crimping process of the sample container 10 and the transport unit 82 that transports the sample containers 10 one by one are configured as independent devices, but a part of the transport unit 82, Specifically, the holder part 83 may be used also as the function of the concave surface 71 a of the jig 71.

  Further, the through hole 21 of the heat transfer block 20 can be variously changed according to the outer shape of the sample container 10. The heat transfer block 20 can have various shapes other than the rectangular plate shape. The arrangement of the through holes 21 is not limited to one row, and for example, two or more rows may be arranged. Alternatively, a circle or other arrangement may be used. The direction of the through hole 21 may be a direction other than horizontal.

  As the cooling means, the cooling unit 25 using a Peltier element is optimal in terms of downsizing and simplification of the apparatus, controllability, etc., but use of other cooling means (freezing means) is not hindered. Further, the heat transfer block may be provided with a plurality of three or more.

  The sample container is formed by a straw-like tubule having a uniform outer diameter, and one or both open ends of the tubule are closed by crimping. Therefore, it is possible to provide a sample container that is suitable for performing the cooling process for the purpose and with good reproducibility.

It is a partially-omission perspective view and principal part sectional drawing which show one Embodiment of the sample container which concerns on this invention. It is the abbreviated perspective view and sectional drawing which show the principal part of the crimping | compression-bonding apparatus for sample containers which concerns on this invention. FIG. 3 is an abbreviated side view showing an example of the overall configuration of the sample container crimping apparatus shown in FIG. It is an abbreviated side view showing a preferred application example of the crimping device for a sample container according to the present invention. It is the side view (a) and top view (a) which show the outline | summary of the biological sample freezing apparatus especially effective when applied to this invention. It is a perspective view which shows the part of the heat-transfer block and guide table of the apparatus shown in FIG. It is a figure which shows the side cross section (a) of a heat-transfer block, a cross section, and a partially expanded cross section (b). It is principal part sectional drawing which shows another embodiment of the biological sample freezing apparatus especially effective when applied to this invention. It is a graph which shows the state after the thaw | melting about the sample frozen by 1st Example of the biological sample freezing method using the sample container of this invention compared with the prior art example 1. FIG. It is a graph which shows the state after the thawing | decompression compared with the prior art example 1 about the sample frozen by 2nd Example of the biological sample freezing method using the sample container of this invention. It is the top view and sectional drawing which show the structure of the conventional sample container.

Explanation of symbols

10 Sample container (present invention) 10 'Sample container (conventional)
DESCRIPTION OF SYMBOLS 11 Straw-shaped thin tube 12 One semicircular part 13 The other semicircular part 14 Cotton-like plug member 15 Polymer water retention agent 16 Moisture-sensitive sealing material 17 Air layer 18 Embossed part 20, 20A, 20B Heat-transfer block 21 Through-hole 25, 25A, 25B Cooling unit (cooling means)
26 temperature sensor 30 guide table 31 guide groove 41 base 40 movement drive means 42 propulsion rod 43 rod guide 44 movable member 45 linear motion guide (linear guide)
46 Stepping Motor 47 Ball Screw 50 Control Drive Unit 51 Temperature Control Circuit 52 Motor Drive Control Circuit 53 Operation / Setting Unit 54 Main Control Unit 60 Final Freezing Container 71 Jig 71a Concave Surface 72 Presser 72a Convex Surface 72b Slope Curved Surface 73 Mount 74 Ultrasonic vibration source 75 Movement drive device (presser) 76 Movement drive device (jig)
81 Hopper-shaped supply means 82 Conveyance means 83 Sample container holder 84 Detachment jig (rejector)
85 Collection container 100 Biological sample φ1 Outer diameter of capillary tube (sample container)

Claims (4)

A sample container used for cryopreserving biological samples,
  Formed from straw-like tubules made of thermoplastic resin having a uniform outer diameter,
  Freezing by passing through a tubular through-hole having an inner diameter slightly larger than the outer diameter formed in the cooled heat transfer block,
  One or both open ends of the capillary are crimped and closed by ultrasonic vibration,
  The closed opening end is substantially bisected in the circumferential direction of the circular opening end, and is crimped in a state where one semicircular portion is overlapped in an arc shape inside the other semicircular portion, And in the said crimping | compression-bonding state, it forms so that the protrusion part from the said outer diameter may not arise. 2. The sample container according to claim 1, wherein one open end of the straw-like tubule is closed by filling with a sealing material, and the other open end is closed by pressure bonding. The sample container according to claim 2, wherein an embossed portion is formed to protrude to the inside of the straw-like tubule and to lock and fix the sealing material. An apparatus for crimping the open end of the sample container according to claim 1,
  A jig for constraining the open end with a concave surface, and a convex surface for pairing with the concave surface and crimping the open end constrained by the jig in a circular arc shape. And a ultrasonic vibration source that applies ultrasonic vibration to the opening end that is pressure-formed by the concave surface and the convex surface,
  The jig has a concave surface whose inner diameter is slightly smaller than the outer diameter of the sample container, and the depth to the bottom and upper opening edge of the concave surface is equal to the outer diameter.
  A crimping apparatus for a sample container, characterized in that.

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