WO2016158479A1 - Freezer and freezing method - Google Patents

Freezer and freezing method Download PDF

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Publication number
WO2016158479A1
WO2016158479A1 PCT/JP2016/058634 JP2016058634W WO2016158479A1 WO 2016158479 A1 WO2016158479 A1 WO 2016158479A1 JP 2016058634 W JP2016058634 W JP 2016058634W WO 2016158479 A1 WO2016158479 A1 WO 2016158479A1
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Prior art keywords
spray
freezing
fine solid
cryogenic
cryopreservation container
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PCT/JP2016/058634
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French (fr)
Japanese (ja)
Inventor
淳 石本
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国立大学法人東北大学
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Publication of WO2016158479A1 publication Critical patent/WO2016158479A1/en

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
    • A23L3/36—Freezing; Subsequent thawing; Cooling
    • A23L3/37—Freezing; Subsequent thawing; Cooling with addition of or treatment with chemicals
    • A23L3/375—Freezing; Subsequent thawing; Cooling with addition of or treatment with chemicals with direct contact between the food and the chemical, e.g. liquid nitrogen, at cryogenic temperature
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L17/00—Food-from-the-sea products; Fish products; Fish meal; Fish-egg substitutes; Preparation or treatment thereof
    • A23L17/30—Fish eggs, e.g. caviar; Fish-egg substitutes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/04—Preserving or maintaining viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/04—Plant cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to a freezing apparatus and a freezing method.
  • Stem cells which have a self-replicating function and a property of differentiating into other cells, are expected to make great industrial contributions, for example, because they serve as a source of human cells.
  • Known methods for cryopreserving cells such as stem cells include slow freezing and vitrification freezing.
  • a general slow freezing method cells are suspended in a storage solution to which a cryoprotectant is added, and the temperature is slowly lowered and stored at a liquid nitrogen temperature.
  • the cooling rate it is necessary to appropriately control the cooling rate. If the cooling rate is not appropriate, a large amount of ice crystals may be formed on the cells at a certain timing, and the cells may be physically damaged.
  • the slow freezing method is not suitable for cryopreservation of primate embryonic stem cells (ES cells: Embryonic Stem Cells), iPS cells (induced pluripotent stem cells), germ cells, etc., and cell viability upon thawing Is 1% or less, indicating a very low value. Vitrification is used for these cells (see, for example, Patent Document 1 and Patent Document 2).
  • vitrification freezing method cells are suspended in a vitrification preservation solution containing a special cryoprotective solution with a high concentration, and the cells are accommodated in a container, and within about 15 seconds from the start of cell suspension.
  • This is a method of immersing in liquid nitrogen, rapidly cooling to below the glass transition point, and solidifying and freezing in an amorphous glass state without crystallizing intracellular and external moisture.
  • this vitrification freezing method there is no volume expansion of water and there is little damage to cells. It should be noted that when thawing the vitrified cells, it is necessary to add a warm medium and rapidly thaw to avoid recrystallization of water.
  • vitrification freezing method cells are suspended in a cryoprotective solution and frozen by spraying liquid nitrogen into a container containing the cells, a method in which a container containing the suspension is directly poured into liquid nitrogen and frozen, Etc. are known (see, for example, Patent Document 2).
  • the conventional general vitrification freezing method requires a cryopreservation solution having a high solute concentration, and thus has a problem that the cytotoxicity due to the solute is high.
  • the cooling rate is about ⁇ 72 ° C./min, and a container containing cells is directly put into liquid nitrogen.
  • the cooling rate is about ⁇ 300 ° C./min, and the temperature drop rate is relatively small.
  • the present invention has been made in view of the above-described problems, and provides a freezing apparatus that vitrifies and freezes cells rapidly with a spray flow containing cryogenic fine solid particles at the time of freezing so that a high cell viability can be obtained upon thawing.
  • a freezing device for vitrifying cells with a small amount of cryopreservation solution to provide a freezing device with a high temperature drop rate, to provide a freezing method for the freezing device, etc. .
  • the freezing apparatus of the present invention has at least the following configuration.
  • a freezing device for elastic membrane capsules containing moisture as a freezing object A cryopreservation container containing the object to be frozen;
  • the freezing method of the present invention comprises at least the following configuration.
  • a freezing method of a freezing apparatus for elastic membrane capsules containing water as a freezing object The freezing device includes a cryopreservation container that houses the object to be frozen, Spraying a spray stream containing cryogenic fine solid particles in the cryopreservation container, Support the cryopreservation container containing the object to be frozen on the downstream side of the spray flow by the support, The spray unit continuously sprays a spray flow containing cryogenic fine solid particles on the cryopreservation container to vitrify and freeze the object to be frozen stored in the cryopreservation container.
  • the freezing apparatus which vitrifies and freezes freezing objects, such as a cell, rapidly can be provided by the spray flow containing a cryogenic fine solid particle at the time of freezing so that it may become a high cell viability at the time of thawing
  • the freezing apparatus with a large temperature fall rate can be provided.
  • a method for freezing the freezing apparatus can be provided.
  • the front view which shows an example of the freezing apparatus which concerns on embodiment of this invention.
  • generation apparatus which provided the spiral nozzle at the front-end
  • generation apparatus (a) is a figure which shows an example when not applying an ultrasonic wave to a Laval nozzle part,
  • FIG. 4B is a diagram illustrating an example when ultrasonic waves from an ultrasonic vibrator are applied to a Laval nozzle portion.
  • generation apparatus (a) is an example when not applying the ultrasonic wave by an ultrasonic transducer to a Laval nozzle part
  • the conceptual diagram which shows an example of the freezing apparatus which concerns on embodiment of this invention.
  • a freezing apparatus includes a cryopreservation container that contains a subject to be frozen, such as cells, and a cryopreservation container that continuously sprays a spray flow containing cryogenic fine solid particles at a high speed to form a cryopreservation container. It has a cryogenic fine solid particle continuous generation device as a spraying section for vitrifying and freezing objects to be frozen such as contained cells.
  • the object to be frozen is not particularly limited as long as it can be vitrified and frozen by the freezing apparatus according to the embodiment of the present invention.
  • the elastic membrane sac containing water such as cells, specifically iPS cells, ES Examples thereof include cells, germ cells, living tissues, blood, plant cells, and foods (frozen foods).
  • the cryogenic fine solid particle continuous generator may be a one-component cryogenic fine solid particle continuous generator or a two-component cryogenic fine solid particle continuous generator.
  • the one-component cryogenic fine solid particle continuous production apparatus continuously sprays a spray stream containing, for example, a cryogenic fine solid nitrogen particle at a high speed.
  • generation apparatus may spray the spray flow of cryogenic fine solid particles, such as a carbon dioxide, argon, and hydrogen, instead of nitrogen.
  • a two-component cryogenic fine solid particle continuous production device is a spray flow containing cryogenic fine solid nitrogen particles produced by mixing two fluids such as cryogenic helium gas as a cryogen and supercooled liquid nitrogen in a nozzle. Spray continuously at high speed.
  • the cryogenic fine solid particles produced by the two-component cryogenic fine solid particle continuous production apparatus may be composed of any one of nitrogen, carbon dioxide, argon, hydrogen, or a combination of two or more. Good.
  • the embodiment of the present invention includes the contents shown in the drawings, but is not limited to this.
  • generation apparatus is employ
  • FIG. 1 is a front view showing an example of a cryogenic fine solid particle continuous generation device 100 (fine particle generation device) as a cryopreservation container 60 and a spray section of a freezing device 200 according to an embodiment of the present invention.
  • FIG. 2 is a diagram illustrating an example of a main part of the fine particle generation apparatus 100.
  • FIG. 3 is an enlarged view of a main part of the fine particle generating apparatus 100 shown in FIG.
  • FIG. 4 is an enlarged cross-sectional view of the vicinity of the nozzle of the fine particle generating apparatus 100.
  • the cryopreservation container 60 accommodates objects to be frozen such as cells 61.
  • the support device 70 supports the cryopreservation container 60 on the downstream side of the spray flow sprayed by the cryogenic fine solid particle continuous production device 100.
  • the fine particle generator 100 continuously generates one-component cryogenic fine solid particles by using a cryogenic supercooled liquid and a cryogenic gas composed of the same element as the supercooled liquid.
  • the fine particle generating apparatus 100 is provided downstream of the mixing unit 10, the mixing unit 10 that generates a one-component mixed phase flow by mixing a supercooled liquid and a high-speed flow of a cryogenic gas, and the mixing thereof.
  • a Laval nozzle unit 11 that is a nozzle 1 that generates a spray flow including cryogenic fine solid particles from the one-component mixed phase flow generated in the unit 10.
  • cryogenic gas nitrogen is employed as the cryogenic gas composed of the same elements as the supercooled liquid nitrogen.
  • a two-phase flow (LN 2 -GN 2 ) of supercooled nitrogen liquid (LN 2 ) and cryogenic nitrogen gas (GN 2 ) is discharged from the reduced diameter portion 11b (throat portion) to the injection portion 11c (expanded portion). passes through the opening portion), by solid phase formation based on adiabatic expansion is performed, is generated fine solid nitrogen particles (SN 2), the spray flow is injected containing fine solid nitrogen particles (SN 2) .
  • the frame 210 is provided with the fine particle generating device 100 in the vicinity of the upper portion thereof, and the support device 70 for supporting the cryopreservation container 60 that stores the cells 61 and the like is provided in the substantially central portion. It has been.
  • the cryopreservation container 60 is supported by the support device 70 so as to be movable in a predetermined direction and / or to be rotatable.
  • the nozzle 1 for ejecting a spray flow containing cryogenic fine solid particles is disposed below the fine particle generating apparatus 100.
  • the nozzle 1 is configured to inject a spray flow toward the cryopreservation container 60.
  • the nozzle 1 is provided with a liquid nitrogen conduit 3, a nitrogen gas conduit 4, and the like.
  • the liquid nitrogen conduit 3 supplies liquid nozzle (LN 2 ) supercooled to a cryogenic temperature to the nozzle 1 from, for example, a liquid nitrogen tank (not shown).
  • the liquid nitrogen conduit 3 includes a valve 3a, and the supply amount and pressure of the liquid nitrogen can be controlled by the valve 3a.
  • the nitrogen gas conduit 4 supplies cryogenic nitrogen gas (GN 2 ) to the nozzle 1 from a nitrogen gas tank (not shown), for example.
  • the nitrogen gas conduit 4 includes a valve 4a, and the supply amount of nitrogen gas (GN 2 ) can be controlled by the valve 4a.
  • the nozzle 1 is configured such that all or part of the nozzle 1 is accommodated in the heat insulating portion 5, and the tip portion of the nozzle 1 protrudes outside the heat insulating portion 5.
  • the heat insulation part 5 has a vacuum heat insulation structure so as to insulate the nozzle 1 from the outside air.
  • the vicinity of the tip of the nozzle 1, the liquid nitrogen conduit 3, and the nitrogen gas conduit 4 is accommodated in the vacuum heat insulating portion 5, and the temperature rise of the nozzle 1, the liquid nitrogen conduit 3, and the nitrogen gas conduit 4 is reduced. Yes.
  • the movable dish portion 7 is provided below the nozzle 1.
  • the movable dish part 7 is disposed between the nozzle 1 and the cryopreservation container 60, the spray flow from the nozzle 1 to the cryopreservation container 60 and the radiation of cold air are prevented, and the movable dish part 7 is disposed at other positions.
  • the movable dish portion 7 is configured so that the spray flow from the nozzle 1 is sprayed onto the cryopreservation container 60.
  • the cryogenic fine solid particle continuous generation apparatus 100 employs a concentric mixing type high-speed two-fluid nozzle.
  • the nozzle 1 includes a mixing unit 10, a laval nozzle unit 11, and the like.
  • the mixing unit 10 of the nozzle 1 has an inner tube 14 and an outer tube 15 that are concentrically combined.
  • the outer diameter of the inner tube 14 is configured to be smaller than the inner diameter of the outer tube 15.
  • a gap 45 is formed between the inner tube 14 and the outer tube 15.
  • the inner tube 14 communicates with the nitrogen gas conduit 4, and the distal end portion 14 a of the inner tube 14 is formed in a tapered shape.
  • a communication portion 15 a communicating with the liquid nitrogen conduit 3 is formed on the side wall of the outer tube 15, and the communication portion 15 a is connected to an opening portion 15 b formed on the inner wall of the outer tube 15.
  • the opening 15b is configured to be located near the side surface in the vicinity of the distal end portion 14a of the inner tube 14.
  • cryogenic nitrogen gas (GN 2 ) is jetted at high speed from the distal end portion 14 a of the inner tube 14.
  • the supercooled liquid (LN 2 ) is supplied to the gap 45 between the inner tube 14 and the outer tube 15 through the liquid nitrogen conduit 3, the communication portion 15a, and the opening portion 15b.
  • the supercooled liquid nitrogen (LN 2 ) and the high-speed flow of the cryogenic gas (GN 2 ) are mixed in the mixing section 10 near the downstream side of the distal end portion 14 a of the inner tube 14, and a one-component mixed phase is obtained.
  • a stream (LN 2 -GN 2 ) is generated.
  • This one-component mixed phase flow (LN 2 -GN 2 ) is introduced into a Laval nozzle unit 11 provided downstream of the mixing unit 10.
  • the Laval nozzle portion 11 is provided on the downstream side of the mixing portion 10 and in the vicinity of the distal end portion of the outer tube 15.
  • the Laval nozzle part 11 has the introduction part 11a, the diameter reducing part 11b (throat part), and the injection part 11c (expansion part).
  • the introduction part 11 a is formed so that the inner diameter on the upstream side is substantially the same as the inner diameter of the outer tube 15 of the mixing part 10.
  • a high-speed one-component mixed phase flow (LN 2 -GN 2 ) generated in the mixing unit 10 is introduced into the introduction unit 11a.
  • the reduced diameter portion 11b is provided on the downstream side of the introduction portion 11a, and is formed to have an opening cross-sectional area smaller than the opening cross-sectional area of the introduction portion 11a. Specifically, the reduced diameter portion 11b is formed in a shape in which the inner diameter decreases as it approaches the minimum inner diameter portion of the reduced diameter portion 11b from the introduction portion 11a.
  • the injection part 11c (expanded part) is provided on the downstream side of the reduced diameter part 11b, and is formed to have an opening sectional area larger than the opening sectional area of the reduced diameter part 11b. Specifically, the injection portion 11c is formed in an expanded shape in which the opening cross-sectional area increases from the reduced diameter portion 11b toward the downstream side.
  • the high-pressure / high-speed one-component mixed phase flow has a velocity equal to or lower than the sound velocity, and the velocity increases as the inner diameter decreases from upstream to downstream.
  • the one-component mixed phase flow is substantially sonic.
  • the opening cross-sectional area increases from the reduced diameter part 11b to the downstream end part of the injection part 11c, the adiabatic expansion of the one-component multiphase flow causes the flow to exceed the speed of sound, and the ice nucleus grows.
  • One-component cryogenic fine solid particles are generated, and a spray flow containing the one-component cryogenic fine solid particles is continuously ejected from the ejection unit 11c. Further, the high-pressure / high-speed one-component mixed phase flow is extremely low temperature, and the one-component mixed phase flow adiabatically expands from the reduced diameter portion 11b of the Laval nozzle portion 11 to the downstream end portion of the injection portion 11c. Becomes a speed exceeding the speed of sound, and the temperature is significantly lowered as compared with the introduction part 11a, and the generation of one-component cryogenic fine solid particles is promoted.
  • the inner diameter of the outer tube 15 of the mixing unit 10 described above is about 2.5 mm
  • the outer diameter of the inner tube 14 is about 1.4 mm
  • the inner diameter of the inner tube 14 is about 0.5 mm
  • the introduction portion 11a of the Laval nozzle unit 11 The inner diameter of the upstream end portion is about 2.5 mm
  • the inner diameter of the reduced diameter portion 11b is about 1.0 mm
  • the inner diameter of the tip portion of the injection port of the injection portion 11c is about 2.2 mm.
  • Each size of the mixing part 10 and the Laval nozzle part 11 is not restricted to the said form, It is preferable to set suitably.
  • the cryogenic fine solid particle continuous generation apparatus 100 includes the ultrasonic transducer 6.
  • the ultrasonic transducer 6 applies ultrasonic waves to the Laval nozzle unit 11 as shown in FIGS.
  • the ultrasonic wave generated by the ultrasonic vibrator 6 applies ultrasonic waves to the Laval nozzle unit 11 as shown in FIGS.
  • cavitation is generated in the one-component mixed phase flow in the Laval nozzle part 11, and ice of one-component cryogenic fine solid particles (SN 2 particles) is generated.
  • Nucleation can be promoted, and refinement of a substantially spherical one-component cryogenic fine solid particle (SN 2 particle) having a fine uniform particle diameter can be promoted.
  • Cavitation is a physical phenomenon in which bubbles are generated and disappeared in a short time due to a pressure difference in a fluid.
  • a short-lived high-temperature, high-pressure local field hot spot
  • the refined SN 2 particles are formed in a fine substantially spherical shape.
  • the ultrasonic transducer 6 includes an ultrasonic vibration generation unit 6a and an ultrasonic transmission unit 6b.
  • the ultrasonic vibration generation unit 6a generates ultrasonic waves having a specified frequency and a specified amplitude under the control of a control device (not shown).
  • the ultrasonic transmission unit 6 b is configured by a substantially rod-shaped metal member, and transmits the ultrasonic wave generated by the ultrasonic vibration generation unit 6 a to the Laval nozzle unit 11.
  • the ultrasonic wave applied from the ultrasonic transducer 6 to the Laval nozzle unit 11 has, for example, a frequency of about 30 kHz to 2 MHz and an amplitude of about 10 ⁇ m to 50 ⁇ m, preferably a frequency of about 40 kHz to 950 kHz and an amplitude. It is about 20 ⁇ m to 40 ⁇ m, and optimally, 45 kHz and an amplitude of about 30 ⁇ m.
  • the frequency and amplitude of the ultrasonic wave applied to the Laval nozzle unit 11 are appropriately set according to the particle size, number, etc. of the one-component cryogenic fine solid particles generated by the Laval nozzle unit 11.
  • ultrasonic waves applied from the ultrasonic transducer 6 to the Laval nozzle unit 11 are ultrasonic waves having a high frequency (megasonic) of about 1 MHz to several tens of MHz, or several tens of MHz to several hundreds of MHz, for example, extremely low temperature
  • a high frequency megasonic
  • the ice nucleation and atomization promoting characteristics of fine solid particles are further improved.
  • the position where the ultrasonic wave generated by the ultrasonic transducer 6 is applied is preferably near the position where ice nucleation of one-component cryogenic fine solid particles of the Laval nozzle unit 11 is performed. A position slightly downstream from the diameter portion 11b is preferable. Further, the position where the ultrasonic wave generated by the ultrasonic transducer 6 is applied may be an arbitrary position of the injection portion 11c on the downstream side from the reduced diameter portion 11b of the Laval nozzle portion 11, or in the entire Laval nozzle portion 11. It may also be set as appropriate according to the particle diameter, the number, etc. of the one-component cryogenic fine solid particles produced by the Laval nozzle unit 11.
  • the ultrasonic transducer 6 may apply ultrasonic waves directly or indirectly to the Laval nozzle unit 11 or may apply the ultrasonic wave to the Laval nozzle unit 11 from within the heat insulating unit 5 as a vacuum heat insulating unit.
  • Cryogenic supercooled liquid nitrogen (LN 2 ) is introduced from a liquid nitrogen tank (not shown) into the mixing unit 10 in the outer tube 15 through the liquid nitrogen conduit 3, the communicating portion 15a of the outer tube 15, and the opening 15b.
  • the High-pressure and high-speed cryogenic nitrogen gas (LN 2 ) is introduced from a nitrogen gas tank (not shown) into the mixing unit 10 through the nitrogen gas conduit 4 and the inner tube 14.
  • the pressure of the cryogenic nitrogen gas (LN 2 ) is, for example, about 0.1 MPa to 1.0 MPa, and in this embodiment is about 0.4 MPa.
  • the pressure of the cryogenic nitrogen gas (LN 2 ) may be about 0.5 MPa to 1000 MPa or about 1.0 to 10 MPa.
  • high-pressure and high-speed cryogenic nitrogen gas (GN 2 ) is jetted at a high speed from the distal end portion 14 a of the inner tube 14, and supercooled liquid (LN 2 ) is discharged from the inner tube 14. It is introduced into the mixing unit 10 through the gap 45 between the outer tubes 15, and in the mixing unit 10, the supercooled liquid nitrogen (LN 2 ) and the high-speed flow of cryogenic gas (GN 2 ) are mixed to form one component. A multiphase flow (LN 2 -GN 2 ) is generated.
  • the high-pressure, high-speed, low-temperature, one-component mixed phase flow (LN 2 -GN 2 ) generated in the mixing unit 10 is introduced into the introduction unit 11a of the Laval nozzle unit 11, and at the minimum inner diameter part of the reduced diameter unit 11b.
  • the one-component multiphase flow becomes substantially sonic velocity, and the flow crosses the sonic velocity due to adiabatic expansion of the one-component multiphase flow as the opening cross-sectional area increases from the reduced diameter portion 11b to the downstream end portion of the injection portion 11c.
  • the one-component multiphase flow adiabatically expands in a state exceeding the speed of sound, and a spray flow including one-component cryogenic fine solid particles (SN 2 particles) is continuously generated.
  • ultrasonic waves about 45 kHz, amplitude of about 30 ⁇ m
  • cavitation is generated in the one-component mixed phase flow in the Laval nozzle unit 11
  • one-component cryogenic temperature is generated.
  • Formation of ice nuclei of fine solid particles (SN 2 particles) can be promoted, and miniaturization of one-component cryogenic fine solid particles (SN 2 particles) having a substantially uniform spherical particle shape can be promoted.
  • FIG. 5 shows a cooling heat flow of cryogenic fine solid nitrogen particles (SN 2 ) generated by cryogenic nitrogen gas (GN 2 ) and supercooled liquid nitrogen (LN 2 ) by the cryogenic fine solid particle continuous production apparatus 100.
  • SN 2 cryogenic fine solid nitrogen particles
  • LN 2 supercooled liquid nitrogen
  • the cooling heat flow flux value q w is rapidly increases in a short time reach the maximum cooling heat flux value, then decreased gradually.
  • the spray flow containing the cryogenic fine solid particles ejected from the cryogenic fine solid particle continuous production apparatus has a heat flux of 10 5 W / m 2 level and has a very high cooling ability.
  • FIG. 6 is a diagram showing an example of a spray collision pressure value of a one-component cryogenic fine solid nitrogen particle (SN 2 ) and a spray collision pressure value by simulation.
  • the horizontal axis (x axis) represents the dimensionless time t *
  • the vertical axis (y axis) represents the pressure p * obtained by making the collision pressure dimensionless.
  • one-component cryogenic fine solid nitrogen particles (SN 2 particles) are caused to collide with a piezoelectric piezoelectric pressure sensor, and the spray collision pressure value of the SN 2 particles is indicated by a dotted line.
  • the pressure of the nitrogen gas (GN 2 ) tank was set to 0.4 MPa, and the pressure of the liquid nitrogen (LN 2 ) tank was set to 0.2 MPa.
  • the collision pressure value obtained by single particle collision numerical calculation (CFD) is shown by a solid line.
  • the collision pressure p * increased rapidly and then decreased.
  • the numerical calculation result shows that the collision pressure p * tends to rise and decrease immediately after the collision.
  • FIG. 7 is a diagram illustrating an example of the nozzle 1 of the cryogenic fine solid particle continuous generation apparatus 100 including the spiral nozzle 18 at the tip of the Laval nozzle.
  • a spiral nozzle 18 as shown in Japanese Patent Application Laid-Open No. 2011-171691 is provided at the tip of the Laval nozzle portion 11 of the nozzle 1.
  • SN 2 particles one-component cryogenic fine solid nitrogen particles
  • PIA Physical Imaging Techniques.
  • PIA for example, as shown in FIG. 7, in a defined region CR (Control region), SN 2 particles flying are magnified using a microscope lens and the particle size is analyzed by image analysis. And measure the speed.
  • a two-color laser for example, a dual pulse YAG laser, a dye laser for depth of field check, a high-resolution color camera for PIA, PIA image analysis software, or the like
  • a high-load distributed computing (Grid Computing) method based on massively parallel computation using a cluster-type high-speed workstation, and the atomization characteristics of the nozzle, for example, the particle size distribution
  • the number density distribution, flow velocity / temperature distribution, etc. can be appropriately and quantitatively evaluated.
  • a PTV (Particle Tracking Velocimetry) algorithm can be used for quantifying the particle velocity.
  • the PTV algorithm to be used considers a distribution pattern of a particle image group composed of a target particle image and a particle image in the vicinity thereof, and performs particle tracking using the similarity.
  • FIG. 8 is a diagram showing an example of the particle size distribution of one-component cryogenic fine solid particles produced by the cryogenic fine solid particle continuous production apparatus 100.
  • FIG. 8A is a diagram illustrating an example in which ultrasonic waves are not applied to the Laval nozzle unit 11
  • FIG. 8B is an example in which ultrasonic waves from an ultrasonic transducer are applied to the Laval nozzle unit 11.
  • FIG. 7 the analysis by the PIA-PTV was performed in a specified region (horizontal 0.92 mm, vertical 0.7 mm) as a visual field from a position below 4.5 mm from just below the nozzle hole.
  • the horizontal axis (x axis) represents the particle diameter d p [ ⁇ m]
  • the left vertical axis (y 1 axis) represents the frequency f f [%]
  • the right the vertical axis (y 2 axis) shows the cumulative f a [%].
  • the average particle size was reduced by about 2.5% in the case where the ultrasonic transducer was installed in the nozzle (ULA) compared to the case where no ultrasonic wave was applied (non-ULA). Specifically, when the ultrasonic wave by the ultrasonic vibrator is not applied to the Laval nozzle part (non-ULA), the average particle diameter is 4.1 ⁇ m, and when the ultrasonic wave by the ultrasonic vibrator is applied to the Laval nozzle part (ULA) ), And the average particle size was 3.9 ⁇ m.
  • FIG. 9 is a diagram illustrating an example of a particle velocity distribution of one-component cryogenic fine solid particles generated by the cryogenic fine solid particle continuous production apparatus 100.
  • FIG. 9A is a diagram showing an example when ultrasonic waves from an ultrasonic transducer are not applied to the Laval nozzle portion 11
  • FIG. 9B is a diagram showing an example when ultrasonic waves are applied to the Laval nozzle portion.
  • the horizontal axis (x axis) indicates the particle velocity V p [m / s]
  • the left vertical axis (y 1 axis) indicates the frequency f f [%].
  • FIG. 7 shows the cumulative f a [%] to the right vertical axis (y 2 axes).
  • the analysis by the PIA-PTV was performed in a specified region (0.92 mm in width, 0.7 mm in length) as a visual field from a position below 4.5 mm from right below the nozzle hole.
  • the particle velocity is lower when the ultrasonic wave from the ultrasonic transducer is applied to the Laval nozzle (ULA) than when the ultrasonic wave is not applied (non-ULA).
  • UUA Laval nozzle
  • the particle size becomes smaller, and “air resistance acting on the particles> inertial force acting on the particles”, the flow velocity decreases.
  • the particle diameter is reduced, it is easy to evaporate, and it is difficult to form a film boiling state due to particle aggregation, and latent heat cooling using high-speed vapor phase change of solid particles is promoted. That is, under the ultrasonic wave application condition, the forced convection cooling effect is slightly reduced due to the decrease in the flow velocity, but the contact heat transfer and latent heat transfer characteristics are increased and the cooling effect is increased.
  • FIG. 10 is a conceptual diagram showing an example of the freezing apparatus 200 according to the embodiment of the present invention.
  • the freezing device 200 includes a support device 70 that supports the cryopreservation container 60.
  • the support device 70 includes a support portion 71, a spray position changing portion 72 (spray position changing means), and the like.
  • the support part 71 is detachable from the cryopreservation container 60 on the downstream side of the spray flow containing the cryogenic fine solid particles sprayed from the nozzle 1 (spiral nozzle 18) of the cryogenic fine solid particle continuous production apparatus as the spraying part.
  • the support portion 71 has a gripping portion 71a for gripping the cryopreservation container 60 at the tip portion.
  • the shape of the gripping portion 71a may be any shape as long as the cryopreservation container 60 can be detachably supported.
  • the gripping portion 71a has a substantially C-shaped cross section.
  • the cryopreservation container 60 may have an arbitrary shape such as a cylindrical shape.
  • the cryopreservation container 60 is formed in a round bottomed cylindrical shape so that it can be cooled with high efficiency without freezing unevenness, and a suspension in which cells 61 are suspended in a cryopreservation solution. And has a structure that can be sealed by a lid.
  • the spray position changing unit 72 changes the spray position of the spray flow with respect to the cryopreservation container 60 supported by the support part 71 in order to reduce freezing unevenness for the cryopreservation container and shorten the freezing time.
  • a drive unit 81 such as a motor is connected to the spray position changing unit 72, and the cryopreservation container 60 supported by the support unit 71 is controlled by the control unit 82 in the vertical direction, the horizontal direction, It is configured to be movable in a predetermined direction such as the front-rear direction and the axial direction of the cylindrical cryopreservation container.
  • the spray position changing unit 72 is configured to be able to rotate the cryopreservation container 60 supported by the support unit 71 with a predetermined rotation direction, for example, the axial direction of the cylindrical cryopreservation container as the rotation axis.
  • the spray position changing part 72 may change the spray position to the cryopreservation container by changing the spray angle from the nozzle of the cryogenic fine solid particle continuous generation device.
  • the control unit 82 comprehensively controls each component of the freezing apparatus 200.
  • the control unit 82 is configured to be able to control the spray flow speed, pressure, spray time, and the like of the cryogenic fine solid particle continuous generation device of the freezing device 200.
  • the control part 82 controls the spray position to a cryopreservation container suitably by the spray position change part 72.
  • the freezing device includes a temperature detection unit such as a temperature sensor for detecting the temperature of the cryopreservation container and an infrared camera, and sprays the control unit 82 according to the temperature of the cryopreservation container detected by the temperature detection unit. You may be comprised so that the spraying time by the control of the spray position by the position change part 72, the cryogenic fine solid particle continuous production
  • A549 cells are human alveolar basal epithelial adenocarcinoma cells.
  • FIG. 11 is a diagram showing an example of a micrograph of a cell (A549 cell).
  • A549 cells are prepared (harvested) as cells to be frozen.
  • the concentration is 1 ⁇ 10 6 cells / mL with 5.08 ⁇ 10 6 cells and 4 ml of RPMI 1640 medium.
  • This cryopreservation container is a round-bottomed cylindrical container having a diameter of about 10 mm, a capacity of 1.8 mL, and an outer cap type.
  • the cryopreservation container may be formed of a resin material or metal material for cryopreservation having high thermal conductivity and high low-temperature strength.
  • a cryopreservation container containing a suspension obtained by directly suspending the above cells in a small amount of cryopreservation solution is supported by a support part of a freezing device, and a cryogenic fine solid particle is continuously produced by a cryogenic fine solid particle continuous production device.
  • a high-speed spray flow containing is continuously sprayed on a cryopreservation container containing cells and the like, and the cells in the cryopreservation container are rapidly vitrified and frozen.
  • CELLBANKER 1 plus from Nippon Zenyaku Kogyo Co., Ltd. is used as the cryopreservation solution, and the amount of the cryopreservation solution used is 1.5 mL per ampoule.
  • the components of the cryopreservation solution are defined as shown in Table 1.
  • a suspension in which cells are directly suspended in a cryopreservation solution at a pressure of about 0.252 to 0.253 MPa in the liquid nitrogen N 2 tank and a pressure of about 0.33 to 0.44 MPa in the vicinity of the injection nozzle A high-speed spray flow containing cryogenic fine solid nitrogen particles is continuously sprayed to each stored cryopreservation container at a spraying time of 10, 20, 30, 45, 50, 60, 90, and 120 seconds.
  • the cells were vitrified and frozen rapidly, and then immersed in liquid nitrogen and stored.
  • a cryogenic fine solid nitrogen particle was stored in a cryopreservation container by being immersed in liquid nitrogen without spraying the cryopreservation container (spray time 0 second).
  • the cryotube (cryopreservation container) containing the vitrified cryopreserved cells is thawed in a water bath at a temperature of 37 ° C. Then, after pipetting 5 times, each is diluted with 9 mL of RPMI 1640 medium. Next, the centrifuge is centrifuged at 1500 rpm for 2 minutes. Then, the medium is removed by suction and suspended in 2 ml of RPMI 1640 medium. 10 ⁇ L of this suspension and 10 ⁇ L of trypan blue (BioRad) as a staining agent were mixed by pipetting, and the number of cells was counted with a cell counter. Table 2 shows an example of the experimental results of the spraying time and cell viability (%) of the cryogenic fine solid nitrogen particles.
  • FIG. 12 is a diagram showing an example of spraying time and cell survival rate (%) of cryogenic fine solid nitrogen particles.
  • the horizontal axis represents the spray time (sec) of the cryogenic fine solid nitrogen particles
  • the vertical axis represents the cell viability.
  • FIG. 12 and Table 2 the cell viability when the container containing the cells was cooled by simply immersing in liquid nitrogen without spraying a high-speed spray flow containing cryogenic fine solid nitrogen particles is shown in FIG. Shown as a value.
  • a spray flow containing cryogenic fine solid nitrogen particles is continuously sprayed for a predetermined number of seconds by the cryogenic fine solid particle continuous production apparatus 100 as a spraying section according to an embodiment of the present invention, and the cells in the container are vitrified.
  • the freezing method of the freezing apparatus increased the cell viability by about 23% (spray time 90 seconds).
  • the longer the spray time the higher the cell viability. It is considered that the longer the spray time, the higher the cooling rate, and the cells vitrify and freeze in high quality in a short time.
  • the freezing apparatus 200 freezes an elastic body membrane containing water such as the cells 61 as a freezing object.
  • This freezing apparatus includes a cryopreservation container 60 for storing a target to be frozen, and a cryopreservation object stored in the cryopreservation container 60 by continuously spraying the cryopreservation container 60 with a spray flow containing cryogenic fine solid particles at a high speed. And a cryogenic fine solid particle continuous production apparatus 100 as a spraying section for vitrification and freezing.
  • the cryogenic fine solid particle continuous generation apparatus 100 applies the nebulized stream containing the cryogenic fine solid particles to the cryopreservation container 60 containing the object to be frozen such as cells and the nebulized stream containing the cryogenic fine solid particles.
  • the object to be frozen such as the cells 61 housed in the cryopreservation container 60 is put into a glass frozen state by a synergistic effect of impact heat transfer, convection heat transfer, and latent heat of vaporization heat transfer.
  • the freezing device suppresses ice nucleation in the cell and rapidly freezes the cell in a glass state.
  • the maximum value of the freezing rate by continuous spraying of the high-speed spray flow containing the cryogenic fine solid nitrogen particles in the freezing apparatus according to the embodiment of the present invention is about ⁇ 25.8 K / Sec (a 25.8 ° C. drop per second). And can be rapidly cooled to about 63 K ( ⁇ 21.15 ° C.). Since new cryogenic fine solid particles always collide with the cryopreservation container, the freezing rate is maintained at a high speed, and the vitrification freezing of the cells is completed in a short time. Thereafter, the cryopreservation container containing the vitrified and frozen cells is immersed in liquid nitrogen and stored for a relatively long time.
  • the cells can be thawed with a high quality while suppressing renucleation by rapidly warming the cells with the thawing solution contained in the cryopreservation container 60. As described above, high cells Survival rate.
  • freezing is achieved by the synergistic effect of collision heat transfer, convection heat transfer, and latent heat of vaporization heat transfer. It is possible to provide a freezing device 200 that rapidly vitrifies and freezes objects such as cells in a storage container. Further, since the cells to be frozen are vitrified and frozen in a state of being stored in a cryopreservation container, no impurities are mixed during freezing and there is no damage due to a high-speed spray flow. As a comparative example, for example, there is a possibility that impurities are mixed in a method of cooling by directly immersing cells in liquid nitrogen.
  • cryopreservation liquid even when only a very small amount of the cryopreservation liquid is added to the cells to be frozen contained in the cryopreservation container 60, a high-speed spray flow containing cryogenic fine solid particles is continuously sprayed onto the cryopreservation container 60.
  • a high-speed spray flow containing cryogenic fine solid particles is continuously sprayed onto the cryopreservation container 60.
  • the particle number density of the cryogenic fine solid nitrogen particles (SN 2 particles) is increased, the particle speed is increased, etc. Improvement of the cooling capacity, specifically, the temperature drop rate can be increased, and the cells can be vitrified and frozen in a short time.
  • the freezing apparatus 200 is supported on the downstream side of the spray flow including the cryogenic fine solid particles ejected from the cryogenic fine solid particle continuous production apparatus 100 using the cryopreservation container 60 as a spraying unit.
  • a spray position changing portion 72 spray position changing means for changing the spray position of the spray flow with respect to the cryopreservation container 60 supported by the support portion 71.
  • the cryopreservation container 60 containing the cells to be frozen is supported by the support unit 71 on the downstream side of the spray flow containing the cryogenic fine solid particles ejected from the cryogenic fine solid particle continuous generation device 100, and the spray position
  • the changing portion 72 changes the spray position of the spray flow with respect to the cryopreservation container 60 so as to reduce the freezing unevenness with respect to the object to be frozen in the cryopreservation container 60.
  • the object to be frozen such as cells in the cryopreservation container is vitrified and frozen in a short time without unevenness of freezing. can do.
  • the spray position changing unit 72 is configured such that the cryopreservation container supported by the support unit 71 can be moved in a predetermined direction such as an up-down direction, a left-right direction, and a front-rear direction, and can be rotated in a predetermined rotation axis direction.
  • the spray position with respect to the cryopreservation container can be easily shifted.
  • the axis of the elongated cylindrical container may be rotated about the rotation axis.
  • the jet flow from the cryogenic fine solid particle continuous generation device 100 is inclined with respect to the side surface of the container.
  • the freezing speed increases and the freezing time can be shortened.
  • the jet flow from the cryogenic fine solid particle continuous production apparatus 100 is continuously sprayed on the side surface of the elongated cylindrical cryopreservation container obliquely at a high speed obliquely to the side surface of the container, and the elongated cylindrical shape is formed.
  • the freezing speed can be further increased and the freezing time can be further shortened.
  • cryogenic fine solid particles used in the freezing apparatus are configured by any one of nitrogen, carbon dioxide, argon, hydrogen, or a combination of two or more.
  • nitrogen as the cryogenic fine solid particles, cells and the like can be vitrified and frozen with low cooling cost and high efficiency.
  • the spray flow of cryogenic fine solid nitrogen particles has a very high cold enthalpy (cooling ability) among existing refrigerants, is harmless to living tissues, and is suitable as a refrigerant for freezing glass by high-speed rapid cooling .
  • the freezing object of the freezing apparatus according to the embodiment of the present invention can include iPS cells, ES cells, blood, plant cells, food (eg, fish eggs), and the like, and the application range of the freezing object is wide.
  • cryopreserving foods such as frozen food with the freezing apparatus according to the embodiment of the present invention, the cryopreservation solution can be frozen in a trace amount or not at all.
  • the spray part of the freezing apparatus which concerns on embodiment of this invention is a one-component cryogenic fine solid particle continuous production
  • generation apparatus mixes a cryogenic supercooled liquid with a high-speed flow of a cryogenic gas composed of the same elements as the supercooled liquid, thereby producing a one-component mixed phase flow.
  • a Laval nozzle unit 11 that is provided downstream of the mixing unit 10 and generates a spray flow including cryogenic fine solid particles from a one-component mixed phase flow generated in the mixing unit.
  • the Laval nozzle portion 11 is provided on the downstream side of the introduction portion 11a and the introduction portion 11a for introducing the one-component mixed phase flow generated by the mixing portion 10, and has a smaller opening cross-sectional area than the opening cross-sectional area of the introduction portion 11a.
  • a diameter portion 11b, and an injection portion 11c that is provided on the downstream side of the reduced diameter portion 11b, has an opening cross-sectional area that is larger than the opening cross-sectional area than the reduced diameter portion 11b, and has a shape that expands toward the downstream side.
  • the one-component mixed phase flow is adiabatically expanded in a state exceeding the speed of sound, and a spray flow including one-component cryogenic fine solid particles is continuously generated.
  • generation apparatus 100 can produce
  • the fine solid nitrogen spray generated by the one-component method (LN 2 -GN 2 ) is SN compared to the fine solid nitrogen spray generated by the two-component method (LN 2 -GHe) using cryogenic helium gas. Since the number density of two particles increases, the cooling effect is large.
  • the one-component (LN 2 -GN 2 ) cryogenic fine solid particle continuous generator is simple and inexpensive, and does not use cryogenic helium as a cryogen. Can be continuously generated.
  • the cryogenic fine solid particle continuous generation apparatus 100 of the freezing apparatus 200 may include a spiral nozzle 18 at the tip of the Laval nozzle section 11.
  • the cryogenic fine solid particles can be further refined.
  • the cryogenic fine solid particle continuous generation apparatus 100 of the freezing apparatus 200 includes an ultrasonic transducer 6 that applies ultrasonic waves to the Laval nozzle unit 11.
  • an ultrasonic transducer 6 that applies ultrasonic waves to the Laval nozzle unit 11.
  • cavitation is generated in the one-component mixed phase flow in the Laval nozzle unit 11, promoting the ice nucleus generation of the cryogenic fine solid particles, and Further, it is possible to promote the miniaturization of one-component cryogenic fine solid particles having a substantially spherical shape with a fine uniform particle diameter.
  • the particle size of the cryogenic fine solid particles is reduced and the flow velocity is reduced.
  • the particle diameter is reduced, it is easy to evaporate, and it is difficult to form a film boiling state due to particle aggregation, and latent heat cooling using high-speed vapor phase change of solid particles is promoted. That is, under the ultrasonic wave application condition, the forced convection cooling effect is slightly reduced due to the decrease in the flow velocity, but the contact heat transfer and latent heat transfer characteristics are increased and the cooling effect is increased.
  • the cryogenic fine solid particle continuous production apparatus 100 of the freezing apparatus 200 has a heat insulating part 5 that vacuum-insulates all or part of the Laval nozzle part 11 with respect to the outside air. Specifically, the vicinity of the tip of the nozzle 1, the liquid nitrogen conduit 3, and the nitrogen gas conduit 4 is accommodated in the vacuum heat insulating portion 5, and the temperature rise of the nozzle 1, the liquid nitrogen conduit 3, and the nitrogen gas conduit 4 is reduced. It has a structure. Therefore, a high-speed spray flow containing a cryogenic fine solid particle can be stably ejected from the Laval nozzle portion 11 of the nozzle 1 with a simple structure for a relatively long time.
  • the spray unit of the freezing device is a one-component cryogenic fine solid particle production continuous device, but is not limited to this form, and the spraying unit of the freezing device is a two-component cryogenic fine particle It may be a solid particle continuous production apparatus.
  • the two-component cryogenic fine solid particle continuous production apparatus may produce a high-speed spray flow containing cryogenic fine solid particles by cooling supercooled liquid nitrogen or the like with a refrigerant such as liquid helium, for example.
  • the cryopreservation container containing the object to be frozen is supported downstream of the spray flow by the support unit, and the cryogenic fine solid particle continuous generation apparatus 100 as the spray unit is frozen.
  • the spray container containing cryogenic fine solid particles continuously into the storage container 60 and vitrifying and freezing the object to be frozen stored in the cryopreservation container it is easy to achieve a high cell viability upon thawing. Furthermore, it is possible to rapidly vitrify an object to be frozen such as a cell.
  • the freezing apparatus continuously sprays the cryogenic container containing the cryogenic fine solid particles onto the cryopreservation container by the spraying unit to vitrify and freeze the object to be frozen contained in the cryopreservation container.
  • the freezing apparatus may spray the object to be frozen by vitrification by continuously spraying the object to be frozen directly with the spray flow containing the cryogenic fine solid particles by the spray unit.
  • the nitrogen (N 2) cryogenic fine solid particles continuously generating apparatus 100 using the may not in this form.
  • hydrogen (H 2 ), oxygen (O 2 ), argon (Ar), or the like may be employed.
  • a one-component mixed phase flow of cryogenic supercooled liquid hydrogen (GH 2 ) and cryogenic hydrogen gas (LH 2 ) is introduced into the Laval nozzle at a high speed, and the Laval nozzle is injected.
  • the one-component multiphase flow is adiabatically expanded in a state exceeding the speed of sound (the speed of sound of the multiphase flow) to continuously generate a spray flow containing one-component cryogenic fine solid particles.
  • a one-component mixed phase flow of supercooled liquid oxygen (GO 2 ) and cryogenic oxygen gas (LO 2 ) at a low temperature is introduced into the Laval nozzle at a high speed, and injected into the injection part of the Laval nozzle.
  • the one-component multiphase flow is adiabatically expanded in a state exceeding the speed of sound (sound speed of the multiphase flow) to continuously generate a spray flow containing one-component cryogenic fine solid particles.
  • argon (Ar) When argon (Ar) is used, a one-component mixed phase flow of supercooled liquid argon and cryogenic argon gas at a low temperature is introduced into the Laval nozzle portion at high speed, and the one-component mixed phase flow is sonic ( Adiabatic expansion is performed in a state exceeding the sound velocity of the multiphase flow, and a spray flow containing one-component cryogenic fine solid particles is continuously generated.
  • the cryogenic temperature is a temperature lower than a general low temperature (0 ° C.) and a temperature equal to or lower than the boiling point at a standard pressure such as nitrogen, hydrogen, helium, or argon.
  • a standard pressure such as nitrogen, hydrogen, helium, or argon.
  • nitrogen (N 2 ) the temperature is about 77.36 K ( ⁇ 195.79 ° C.), which is the boiling point of nitrogen at standard pressure
  • hydrogen (H 2 ) is used.
  • the temperature is about 20.28 K ( ⁇ 252.87 ° C.) or less, which is the boiling point of hydrogen at the standard pressure
  • oxygen (O 2 ) oxygen
  • the boiling point of oxygen at the standard pressure is 90.2 K.
  • the temperature is about ( ⁇ 182.96 ° C.) or less, and when argon (Ar) is used, the temperature is about 83.80 K ( ⁇ 189.35 ° C.) or less.
  • a cryogenic supercooled liquid is used.
  • the present invention is not limited to this mode.
  • a cryogenic liquid that is not in a supercooled state may be used.
  • a cryogenic supercooled liquid it is possible to easily produce one-component cryogenic fine solid particles in a short time.
  • vitrification freezing control of the cell by a super-high heat flow rate can be performed by utilization of the high-speed spray flow containing a cryogenic fine solid particle.
  • various cells such as iPS cells and ES cells with high survival rate can be frozen at high quality and rapidly frozen. Vitrification and cryopreservation technology can be established.
  • Ultra-high-speed glass freezing method that does not require cryoprotection solution and suppresses ice nucleation as much as possible with conventional iPS cell freezing method due to ultra-high heat flux cooling effect of micro / nano level cryogenic fine solid particles And can provide this new technology to the biochemical field, medical engineering field, and various medical industries.
  • this technology has a high contribution not only in the medical, medical engineering, and life science fields but also in a wide range of different industrial fields.

Abstract

Provided are: a freezer for quickly vitrifying a subject to be frozen such as cells by using a spray flow containing ultralow-temperature solid microparticles in freezing so as to achieve a high cell survival rate at thawing; and a freezing method using same. The freezer for freezing a subject to be frozen such as cells, said freezer comprising: a cryopreservation container 60 that houses the subject to be frozen such as cells; and a spray part that continuously sprays a high-speed spray flow containing ultralow-temperature solid microparticles to the cryopreservation container 60 so as to freeze the subject to be frozen that is housed in the cryopreservation container. This freezer is provided with a support part 71 that supports the cryopreservation container 60 and a spray position-changing part 72 (spray position-changing means) that changes a spray position of the high-seed spray flow to the cryopreservation container supported by the support part 71.

Description

凍結装置、凍結方法Freezing device, freezing method
　本発明は、凍結装置、凍結方法に関する。 The present invention relates to a freezing apparatus and a freezing method.
　自己複製機能と他の細胞へ分化する特性を兼ね備えた幹細胞は、例えば人間の細胞の供給源となることから多大な産業貢献が期待されている。幹細胞などの細胞の凍結保存方法としては、緩慢凍結法、ガラス化凍結法などが知られている。 Stem cells, which have a self-replicating function and a property of differentiating into other cells, are expected to make great industrial contributions, for example, because they serve as a source of human cells. Known methods for cryopreserving cells such as stem cells include slow freezing and vitrification freezing.
　一般的な緩慢凍結法は、凍結保護剤を加えた保存液に細胞を懸濁し、緩やかに温度を低下させ、液体窒素温度で保存する。しかし、冷却速度を適切にコントロールする必要があり、冷却速度が適切でないと、あるタイミングで細胞に氷晶が多量に形成され、細胞が物理的に損傷を受ける場合がある。また、緩慢凍結法は、霊長類の胚性幹細胞（ＥＳ細胞：Embryonic Stem Cells）、ｉＰＳ細胞（induced pluripotent stem Cells）、生殖細胞などの細胞の凍結保存には適さず、解凍時の細胞生存率が１％以下となり非常に低い値を示す。
　これらの細胞にはガラス化凍結法（Vitrification）が用いられる（例えば、特許文献１、特許文献２参照）。 In a general slow freezing method, cells are suspended in a storage solution to which a cryoprotectant is added, and the temperature is slowly lowered and stored at a liquid nitrogen temperature. However, it is necessary to appropriately control the cooling rate. If the cooling rate is not appropriate, a large amount of ice crystals may be formed on the cells at a certain timing, and the cells may be physically damaged. In addition, the slow freezing method is not suitable for cryopreservation of primate embryonic stem cells (ES cells: Embryonic Stem Cells), iPS cells (induced pluripotent stem cells), germ cells, etc., and cell viability upon thawing Is 1% or less, indicating a very low value.
Vitrification is used for these cells (see, for example, Patent Document 1 and Patent Document 2).
　従来の一般的なガラス化凍結法は、高濃度の特殊な凍結保護液を含むガラス化保存液に細胞を懸濁し、それを容器に収容し、細胞の懸濁をはじめてから約１５秒以内に液体窒素に浸して、ガラス転移点以下まで急速に冷却し、細胞内外の水分を結晶化させることなく、非晶質のガラス状態で固化、凍結させる方法である。このガラス化凍結法によれば、水の体積膨張がなく、細胞へのダメージが少ない。尚、ガラス化凍結された細胞を解凍する時、水の再結晶化を避けるために、温培地を添加して急速に解凍することを要する。 In the conventional general vitrification freezing method, cells are suspended in a vitrification preservation solution containing a special cryoprotective solution with a high concentration, and the cells are accommodated in a container, and within about 15 seconds from the start of cell suspension. This is a method of immersing in liquid nitrogen, rapidly cooling to below the glass transition point, and solidifying and freezing in an amorphous glass state without crystallizing intracellular and external moisture. According to this vitrification freezing method, there is no volume expansion of water and there is little damage to cells. It should be noted that when thawing the vitrified cells, it is necessary to add a warm medium and rapidly thaw to avoid recrystallization of water.
　ガラス化凍結方法としては、細胞を凍結保護液に懸濁し、それを収容した容器に液体窒素を吹き付けて凍結する方法、懸濁液を収容した容器を液体窒素に直接投入して凍結する方法、などが知られている（例えば、特許文献２参照）。 As a vitrification freezing method, cells are suspended in a cryoprotective solution and frozen by spraying liquid nitrogen into a container containing the cells, a method in which a container containing the suspension is directly poured into liquid nitrogen and frozen, Etc. are known (see, for example, Patent Document 2).
特許第４７０５４７３号公報Japanese Patent No. 4705473 特開２０１２－２１７３４２号公報JP 2012-217342 A
　しかしながら、従来の一般的なガラス化凍結法では、高い溶質濃度の凍結保存液を必要とするため、溶質による細胞毒性が高いという問題がある。 However, the conventional general vitrification freezing method requires a cryopreservation solution having a high solute concentration, and thus has a problem that the cytotoxicity due to the solute is high.
　また、特許文献２に記載された凍結方法として挙げられている、液体窒素を容器に吹き付ける方法では冷却速度が約－７２℃／ｍｉｎ程度であり、細胞を収容した容器を液体窒素に直接投入する方法では冷却速度が約－３００℃／ｍｉｎ程度であり、それぞれ温度降下速度が比較的小さい。 Also, in the method of spraying liquid nitrogen onto a container, which is cited as a freezing method described in Patent Document 2, the cooling rate is about −72 ° C./min, and a container containing cells is directly put into liquid nitrogen. In the method, the cooling rate is about −300 ° C./min, and the temperature drop rate is relatively small.
　本発明は、上述した問題に鑑みてなされたもので、解凍時に高い細胞生存率となるように、凍結時に極低温微細固体粒子を含む噴霧流により急速に細胞をガラス化凍結する凍結装置を提供すること、少量の凍結保存液で細胞をガラス化凍結する凍結装置を提供すること、温度降下速度の大きい凍結装置を提供すること、その凍結装置の凍結方法を提供すること、などを目的とする。 The present invention has been made in view of the above-described problems, and provides a freezing apparatus that vitrifies and freezes cells rapidly with a spray flow containing cryogenic fine solid particles at the time of freezing so that a high cell viability can be obtained upon thawing. To provide a freezing device for vitrifying cells with a small amount of cryopreservation solution, to provide a freezing device with a high temperature drop rate, to provide a freezing method for the freezing device, etc. .
　このような目的を達成するために、本発明の凍結装置は、以下の構成を少なくとも具備するものである。
　凍結対象として水分を含む弾性体膜包の凍結装置であって、
　前記凍結対象を収容する凍結保存容器と、
　前記凍結保存容器に極低温微細固体粒子を含む噴霧流を連続して噴霧して、前記凍結保存容器に収容された凍結対象をガラス化凍結させる噴霧部と、を有することを特徴とする。 In order to achieve such an object, the freezing apparatus of the present invention has at least the following configuration.
A freezing device for elastic membrane capsules containing moisture as a freezing object,
A cryopreservation container containing the object to be frozen;
A spray unit that continuously sprays a cryogenic fine solid particle-containing spray stream on the cryopreservation container to vitrify and freeze the object to be frozen contained in the cryopreservation container.
　また、本発明の凍結方法は、以下の構成を少なくとも具備するものである。
　凍結対象として水分を含む弾性体膜包の凍結装置の凍結方法であって、
　前記凍結装置は、前記凍結対象を収容する凍結保存容器と、
　前記凍結保存容器に極低温微細固体粒子を含む噴霧流を噴霧する噴霧部と、を有し、
　前記凍結対象を収容する凍結保存容器を支持部により噴霧流の下流側に支持し、
　前記噴霧部が、前記凍結保存容器に極低温微細固体粒子を含む噴霧流を連続して噴霧して、前記凍結保存容器に収容された凍結対象をガラス化凍結させることを特徴とする。 The freezing method of the present invention comprises at least the following configuration.
A freezing method of a freezing apparatus for elastic membrane capsules containing water as a freezing object,
The freezing device includes a cryopreservation container that houses the object to be frozen,
Spraying a spray stream containing cryogenic fine solid particles in the cryopreservation container,
Support the cryopreservation container containing the object to be frozen on the downstream side of the spray flow by the support,
The spray unit continuously sprays a spray flow containing cryogenic fine solid particles on the cryopreservation container to vitrify and freeze the object to be frozen stored in the cryopreservation container.
　本発明によれば、解凍時に高い細胞生存率となるように、凍結時に極低温微細固体粒子を含む噴霧流により、細胞などの凍結対象を急速にガラス化凍結する凍結装置を提供することができる。
　また、本発明によれば、少量の凍結保存液で細胞をガラス化凍結する凍結装置を提供することができる。
　また、本発明によれば、温度降下速度の大きい凍結装置を提供することができる。
　また、本発明によれば、その凍結装置の凍結方法を提供することができる。 ADVANTAGE OF THE INVENTION According to this invention, the freezing apparatus which vitrifies and freezes freezing objects, such as a cell, rapidly can be provided by the spray flow containing a cryogenic fine solid particle at the time of freezing so that it may become a high cell viability at the time of thawing | decompression. .
In addition, according to the present invention, it is possible to provide a freezing apparatus that vitrifies and freezes cells with a small amount of a cryopreservation solution.
Moreover, according to this invention, the freezing apparatus with a large temperature fall rate can be provided.
Further, according to the present invention, a method for freezing the freezing apparatus can be provided.
本発明の実施形態に係る凍結装置の一例を示す正面図。The front view which shows an example of the freezing apparatus which concerns on embodiment of this invention. 本発明の実施形態に係る凍結装置の極低温微細固体粒子連続生成装置の要部の一例を示す図。The figure which shows an example of the principal part of the cryogenic fine solid particle continuous production | generation apparatus of the freezing apparatus which concerns on embodiment of this invention. 極低温微細固体粒子連続生成装置の要部の拡大図。The enlarged view of the principal part of a cryogenic fine solid particle continuous production | generation apparatus. 極低温微細固体粒子連続生成装置のノズル部付近の拡大断面図。The expanded sectional view of nozzle part vicinity of a cryogenic fine solid particle continuous production | generation apparatus. 極低温微細固体窒素粒子の冷却熱流束値の一例を示す図。The figure which shows an example of the cooling heat flux value of a cryogenic fine solid nitrogen particle. 一成分極低温微細固体窒素粒子の噴霧衝突圧力値およびシミュレーションによる噴霧衝突圧力値の一例を示す図。The figure which shows an example of the spray collision pressure value of one-component cryogenic fine solid nitrogen particle, and the spray collision pressure value by simulation. ラバルノズル部の先端部にスパイラルノズルを備えた極低温微細固体粒子連続生成装置の一例を示す図。The figure which shows an example of the cryogenic fine solid particle continuous production | generation apparatus which provided the spiral nozzle at the front-end | tip part of the Laval nozzle part. 極低温微細固体粒子連続生成装置により生成される一成分極低温微細固体粒子の粒子径の分布の一例を示す図、（ａ）は超音波をラバルノズル部に印加しない場合の一例を示す図、（ｂ）は超音波振動子による超音波をラバルノズル部に印加した場合の一例を示す図。The figure which shows an example of distribution of the particle diameter of the one-component cryogenic fine solid particle produced | generated by the cryogenic fine solid particle continuous production | generation apparatus, (a) is a figure which shows an example when not applying an ultrasonic wave to a Laval nozzle part, FIG. 4B is a diagram illustrating an example when ultrasonic waves from an ultrasonic vibrator are applied to a Laval nozzle portion. 極低温微細固体粒子連続生成装置により生成される一成分極低温微細固体粒子の粒子速度分布の一例を示す図、（ａ）は超音波振動子による超音波をラバルノズル部に印加しない場合の一例を示す図、（ｂ）はラバルノズル部に超音波を印加した場合の一例を示す図。The figure which shows an example of the particle velocity distribution of the one component cryogenic fine solid particle produced | generated by the cryogenic fine solid particle continuous production | generation apparatus, (a) is an example when not applying the ultrasonic wave by an ultrasonic transducer to a Laval nozzle part The figure shown, (b) is a figure which shows an example at the time of applying an ultrasonic wave to a Laval nozzle part. 本発明の実施形態に係る凍結装置の一例を示す概念図。The conceptual diagram which shows an example of the freezing apparatus which concerns on embodiment of this invention. 細胞の顕微鏡写真の一例を示す図。The figure which shows an example of the microscope picture of a cell. 極低温微細固体窒素粒子の噴霧時間と細胞生存率の一例を示す図。The figure which shows an example of the spraying time of a cryogenic fine solid nitrogen particle, and a cell viability.
　本発明の実施形態に係る凍結装置は、細胞などの凍結対象を収容する凍結保存容器と、凍結保存容器に極低温微細固体粒子を含む噴霧流を連続して高速噴霧して、凍結保存容器に収容された細胞などの凍結対象をガラス化凍結させる噴霧部としての極低温微細固体粒子連続生成装置を有する。凍結対象としては、本発明の実施形態に係る凍結装置によりガラス化凍結することができれば特に限定されるものではなく、細胞などの水分を含有する弾性体膜包、詳細には、ｉＰＳ細胞、ＥＳ細胞、生殖細胞、生体組織、血液、植物細胞、食品（冷凍食品）などを挙げることができる。 A freezing apparatus according to an embodiment of the present invention includes a cryopreservation container that contains a subject to be frozen, such as cells, and a cryopreservation container that continuously sprays a spray flow containing cryogenic fine solid particles at a high speed to form a cryopreservation container. It has a cryogenic fine solid particle continuous generation device as a spraying section for vitrifying and freezing objects to be frozen such as contained cells. The object to be frozen is not particularly limited as long as it can be vitrified and frozen by the freezing apparatus according to the embodiment of the present invention. The elastic membrane sac containing water such as cells, specifically iPS cells, ES Examples thereof include cells, germ cells, living tissues, blood, plant cells, and foods (frozen foods).
　極低温微細固体粒子連続生成装置としては、一成分極低温微細固体粒子連続生成装置であってもよいし、二成分極低温微細固体粒子連続生成装置であってもよい。
　一成分極低温微細固体粒子連続生成装置は、例えば、極低温微細固体窒素粒子を含む噴霧流を連続して高速噴霧する。尚、一成分極低温微細固体粒子連続生成装置は、窒素の代わりに、二酸化炭素、アルゴン、水素などの極低温微細固体粒子の噴霧流を噴霧してもよい。
　二成分極低温微細固体粒子連続生成装置は、例えば、寒剤としての極低温のヘリウムガス及び過冷却液体窒素などの二流体をノズル内で混合して生成した極低温微細固体窒素粒子を含む噴霧流を連続的に高速噴霧する。尚、二成分極低温微細固体粒子連続生成装置により生成される極低温微細固体粒子は、窒素、二酸化炭素、アルゴン、水素のいずれか１つ、又は、２つ以上の組み合わせにより構成されていてもよい。 The cryogenic fine solid particle continuous generator may be a one-component cryogenic fine solid particle continuous generator or a two-component cryogenic fine solid particle continuous generator.
The one-component cryogenic fine solid particle continuous production apparatus continuously sprays a spray stream containing, for example, a cryogenic fine solid nitrogen particle at a high speed. In addition, the one-component cryogenic fine solid particle continuous production | generation apparatus may spray the spray flow of cryogenic fine solid particles, such as a carbon dioxide, argon, and hydrogen, instead of nitrogen.
A two-component cryogenic fine solid particle continuous production device is a spray flow containing cryogenic fine solid nitrogen particles produced by mixing two fluids such as cryogenic helium gas as a cryogen and supercooled liquid nitrogen in a nozzle. Spray continuously at high speed. Note that the cryogenic fine solid particles produced by the two-component cryogenic fine solid particle continuous production apparatus may be composed of any one of nitrogen, carbon dioxide, argon, hydrogen, or a combination of two or more. Good.
　以下、本発明の実施形態に係る凍結装置を図面を参照しながら説明する。本発明の実施形態は図示の内容を含むが、これのみに限定されるものではない。なお、以後の各図の説明で、既に説明した部位と共通する部分は同一符号を付して重複説明を一部省略する。
　本実施形態では、噴射部の極低温微細固体粒子連続生成装置として一成分極低温微細固体粒子連続生成装置を採用している。 Hereinafter, a freezing apparatus according to an embodiment of the present invention will be described with reference to the drawings. The embodiment of the present invention includes the contents shown in the drawings, but is not limited to this. In the following description of each drawing, parts that are common to the parts that have already been described are assigned the same reference numerals, and duplicate descriptions are partially omitted.
In this embodiment, the one-component cryogenic fine solid particle continuous production | generation apparatus is employ | adopted as a cryogenic fine solid particle continuous production | generation apparatus of an injection part.
　図１は、本発明の実施形態に係る凍結装置２００の凍結保存容器６０と噴霧部としての極低温微細固体粒子連続生成装置１００（微細粒子生成装置）の一例を示す正面図である。図２は微細粒子生成装置１００の要部の一例を示す図である。図３は図２に示した微細粒子生成装置１００の要部の拡大図である。図４は微細粒子生成装置１００のノズル付近の拡大断面図である。 1 is a front view showing an example of a cryogenic fine solid particle continuous generation device 100 (fine particle generation device) as a cryopreservation container 60 and a spray section of a freezing device 200 according to an embodiment of the present invention. FIG. 2 is a diagram illustrating an example of a main part of the fine particle generation apparatus 100. FIG. 3 is an enlarged view of a main part of the fine particle generating apparatus 100 shown in FIG. FIG. 4 is an enlarged cross-sectional view of the vicinity of the nozzle of the fine particle generating apparatus 100.
　凍結保存容器６０は、細胞６１などの凍結対象を収容する。支持装置７０は凍結保存容器６０を極低温微細固体粒子連続生成装置１００により噴霧される噴霧流の下流側に支持する。 The cryopreservation container 60 accommodates objects to be frozen such as cells 61. The support device 70 supports the cryopreservation container 60 on the downstream side of the spray flow sprayed by the cryogenic fine solid particle continuous production device 100.
　微細粒子生成装置１００は、極低温の過冷却液体と、その過冷却液体と同一の元素で構成される極低温気体とにより、一成分極低温微細固体粒子を連続して生成する。詳細には、微細粒子生成装置１００は、過冷却液体と、極低温気体の高速流とを混合して一成分混相流を生成する混合部１０と、混合部１０の下流に設けられ、その混合部１０で生成された一成分混相流から極低温微細固体粒子を含む噴霧流を生成するノズル１であるラバルノズル部１１と、を有する。 The fine particle generator 100 continuously generates one-component cryogenic fine solid particles by using a cryogenic supercooled liquid and a cryogenic gas composed of the same element as the supercooled liquid. Specifically, the fine particle generating apparatus 100 is provided downstream of the mixing unit 10, the mixing unit 10 that generates a one-component mixed phase flow by mixing a supercooled liquid and a high-speed flow of a cryogenic gas, and the mixing thereof. A Laval nozzle unit 11 that is a nozzle 1 that generates a spray flow including cryogenic fine solid particles from the one-component mixed phase flow generated in the unit 10.
　本実施形態では、過冷却液体として過冷却液体窒素（ＬＮ2）を採用し、過冷却液体窒素と同一の元素で構成される極低温気体として極低温気体窒素（ＧＮ2）を採用する。 In this embodiment, supercooled liquid nitrogen (LN 2 ) is employed as the supercooled liquid, and cryogenic gas nitrogen (GN 2 ) is employed as the cryogenic gas composed of the same elements as the supercooled liquid nitrogen.
　上記ラバルノズル部１１では、過冷却窒素液体（ＬＮ2）と極低温窒素ガス（ＧＮ2）とによる二相流（ＬＮ2－ＧＮ2）が縮径部１１ｂ（スロート部）から噴射部１１ｃ（拡開部）を通過して、断熱膨張に基づく固相形成が行われることにより、微細固体窒素粒子（ＳＮ2）が生成されて、微細固体窒素粒子（ＳＮ2）を含む噴霧流が噴射される。 In the Laval nozzle portion 11, a two-phase flow (LN 2 -GN 2 ) of supercooled nitrogen liquid (LN 2 ) and cryogenic nitrogen gas (GN 2 ) is discharged from the reduced diameter portion 11b (throat portion) to the injection portion 11c (expanded portion). passes through the opening portion), by solid phase formation based on adiabatic expansion is performed, is generated fine solid nitrogen particles (SN 2), the spray flow is injected containing fine solid nitrogen particles (SN 2) .
　次に、図１～図４を参照しながら、極低温微細固体粒子連続生成装置１００（微細粒子生成装置）を採用した凍結装置２００を詳細に説明する。 Next, the freezing apparatus 200 employing the cryogenic fine solid particle continuous production apparatus 100 (fine particle production apparatus) will be described in detail with reference to FIGS.
　図１に示したように、枠体２１０には、その上部付近に微細粒子生成装置１００が設けられ、略中央部に、細胞６１などを収容する凍結保存容器６０を支持する支持装置７０が設けられている。凍結保存容器６０は、支持装置７０により所定方向に移動自在に、および／または回転自在に支持されている。 As shown in FIG. 1, the frame 210 is provided with the fine particle generating device 100 in the vicinity of the upper portion thereof, and the support device 70 for supporting the cryopreservation container 60 that stores the cells 61 and the like is provided in the substantially central portion. It has been. The cryopreservation container 60 is supported by the support device 70 so as to be movable in a predetermined direction and / or to be rotatable.
　微細粒子生成装置１００は、その下部に極低温微細固体粒子を含む噴霧流を噴出するノズル１が配置されている。ノズル１は、凍結保存容器６０に向けて噴霧流を噴射するように構成されている。 The nozzle 1 for ejecting a spray flow containing cryogenic fine solid particles is disposed below the fine particle generating apparatus 100. The nozzle 1 is configured to inject a spray flow toward the cryopreservation container 60.
　ノズル１には、液体窒素導管３、窒素ガス導管４などが配設されている。
　液体窒素導管３は、例えば、液体窒素タンク（不図示）などから、極低温に過冷却された液体窒素（ＬＮ2）をノズル１に供給する。液体窒素導管３は、バルブ３ａを備え、そのバルブ３ａにより液体窒素の供給量や圧力などを制御可能に構成されている。 The nozzle 1 is provided with a liquid nitrogen conduit 3, a nitrogen gas conduit 4, and the like.
The liquid nitrogen conduit 3 supplies liquid nozzle (LN 2 ) supercooled to a cryogenic temperature to the nozzle 1 from, for example, a liquid nitrogen tank (not shown). The liquid nitrogen conduit 3 includes a valve 3a, and the supply amount and pressure of the liquid nitrogen can be controlled by the valve 3a.
　窒素ガス導管４は、例えば、窒素ガスタンク（不図示）などから、極低温の窒素ガス（ＧＮ2）をノズル１に供給する。窒素ガス導管４は、バルブ４ａを備え、そのバルブ４ａにより窒素ガス（ＧＮ2）の供給量を制御可能に構成されている。 The nitrogen gas conduit 4 supplies cryogenic nitrogen gas (GN 2 ) to the nozzle 1 from a nitrogen gas tank (not shown), for example. The nitrogen gas conduit 4 includes a valve 4a, and the supply amount of nitrogen gas (GN 2 ) can be controlled by the valve 4a.
＜断熱部５＞
　ノズル１は、全部または一部分が断熱部５内に収容され、ノズル１の先端部が断熱部５の外部に突出するように構成されている。本実施形態では、断熱部５は、ノズル１と外気とを断熱するように真空断熱構造となっている。ノズル１、液体窒素導管３、窒素ガス導管４の先端部付近が真空断熱部５内に収容されており、ノズル１や液体窒素導管３、窒素ガス導管４の温度上昇を低減する構造となっている。 <Insulation part 5>
The nozzle 1 is configured such that all or part of the nozzle 1 is accommodated in the heat insulating portion 5, and the tip portion of the nozzle 1 protrudes outside the heat insulating portion 5. In this embodiment, the heat insulation part 5 has a vacuum heat insulation structure so as to insulate the nozzle 1 from the outside air. The vicinity of the tip of the nozzle 1, the liquid nitrogen conduit 3, and the nitrogen gas conduit 4 is accommodated in the vacuum heat insulating portion 5, and the temperature rise of the nozzle 1, the liquid nitrogen conduit 3, and the nitrogen gas conduit 4 is reduced. Yes.
＜可動皿部７＞
　本実施形態では、図２に示したように、ノズル１の下方に、可動皿部７が設けられている。可動皿部７がノズル１と凍結保存容器６０の間に配置された場合、ノズル１から凍結保存容器６０への噴霧流や冷気の放射を防止し、可動皿部７がそれ以外の位置に配置された場合、ノズル１からの噴霧流が凍結保存容器６０に噴霧するように、可動皿部７が構成されている。 <Moving plate part 7>
In the present embodiment, as shown in FIG. 2, the movable dish portion 7 is provided below the nozzle 1. When the movable dish part 7 is disposed between the nozzle 1 and the cryopreservation container 60, the spray flow from the nozzle 1 to the cryopreservation container 60 and the radiation of cold air are prevented, and the movable dish part 7 is disposed at other positions. In such a case, the movable dish portion 7 is configured so that the spray flow from the nozzle 1 is sprayed onto the cryopreservation container 60.
　図４に示したように、極低温微細固体粒子連続生成装置１００は、同心混合型高速二流体ノズルを採用している。ノズル１は、混合部１０と、ラバルノズル部１１と、などを有する。 As shown in FIG. 4, the cryogenic fine solid particle continuous generation apparatus 100 employs a concentric mixing type high-speed two-fluid nozzle. The nozzle 1 includes a mixing unit 10, a laval nozzle unit 11, and the like.
＜混合部１０＞
　図４に示したように、ノズル１の混合部１０は、同心状に組み合わされた内管１４と外管１５とを有する。内管１４の外径は、外管１５の内径よりも小さく構成されている。内管１４と外管１５の間には、隙間４５が形成されている。 <Mixing unit 10>
As shown in FIG. 4, the mixing unit 10 of the nozzle 1 has an inner tube 14 and an outer tube 15 that are concentrically combined. The outer diameter of the inner tube 14 is configured to be smaller than the inner diameter of the outer tube 15. A gap 45 is formed between the inner tube 14 and the outer tube 15.
　内管１４は、窒素ガス導管４に連通しており、内管１４の先端部１４ａは、先細り形状に形成されている。
　外管１５の側壁には、液体窒素導管３に連通する連通部１５ａが形成され、外管１５の内壁に形成された開口部１５ｂに連通部１５ａが接続されている。この開口部１５ｂは、内管１４の先端部１４ａ近傍の側面付近に位置するように構成されている。 The inner tube 14 communicates with the nitrogen gas conduit 4, and the distal end portion 14 a of the inner tube 14 is formed in a tapered shape.
A communication portion 15 a communicating with the liquid nitrogen conduit 3 is formed on the side wall of the outer tube 15, and the communication portion 15 a is connected to an opening portion 15 b formed on the inner wall of the outer tube 15. The opening 15b is configured to be located near the side surface in the vicinity of the distal end portion 14a of the inner tube 14.
　図４に示したように、高圧、極低温の窒素ガス（ＧＮ2）は、内管１４の先端部１４ａから高速に噴射される。過冷却液体（ＬＮ2）は、液体窒素導管３、連通部１５ａ、開口部１５ｂを介して、内管１４と外管１５の間の隙間４５に供給される。このため、内管１４の先端部１４ａの下流側付近の混合部１０にて、過冷却液体窒素（ＬＮ2）と、極低温気体（ＧＮ2）の高速流とが混合して、一成分混相流（ＬＮ2－ＧＮ2）が生成される。
　この一成分混相流（ＬＮ2－ＧＮ2）は、混合部１０の下流に設けられたラバルノズル部１１に導入される。 As shown in FIG. 4, high-pressure, cryogenic nitrogen gas (GN 2 ) is jetted at high speed from the distal end portion 14 a of the inner tube 14. The supercooled liquid (LN 2 ) is supplied to the gap 45 between the inner tube 14 and the outer tube 15 through the liquid nitrogen conduit 3, the communication portion 15a, and the opening portion 15b. For this reason, the supercooled liquid nitrogen (LN 2 ) and the high-speed flow of the cryogenic gas (GN 2 ) are mixed in the mixing section 10 near the downstream side of the distal end portion 14 a of the inner tube 14, and a one-component mixed phase is obtained. A stream (LN 2 -GN 2 ) is generated.
This one-component mixed phase flow (LN 2 -GN 2 ) is introduced into a Laval nozzle unit 11 provided downstream of the mixing unit 10.
＜ラバルノズル部１１＞
　ラバルノズル部１１は、本実施形態では、混合部１０の下流側、且つ、外管１５の先端部付近に設けられている。詳細には、ラバルノズル部１１は、導入部１１ａと、縮径部１１ｂ（スロート部）と、噴射部１１ｃ（拡開部）と、を有する。 <Laval nozzle part 11>
In this embodiment, the Laval nozzle portion 11 is provided on the downstream side of the mixing portion 10 and in the vicinity of the distal end portion of the outer tube 15. In detail, the Laval nozzle part 11 has the introduction part 11a, the diameter reducing part 11b (throat part), and the injection part 11c (expansion part).
　導入部１１ａは、上流側の内径が、混合部１０の外管１５の内径と略同じとなるように形成されている。この導入部１１ａには、混合部１０で生成された高速の一成分混相流（ＬＮ2－ＧＮ2）が導入される。 The introduction part 11 a is formed so that the inner diameter on the upstream side is substantially the same as the inner diameter of the outer tube 15 of the mixing part 10. A high-speed one-component mixed phase flow (LN 2 -GN 2 ) generated in the mixing unit 10 is introduced into the introduction unit 11a.
　縮径部１１ｂは、導入部１１ａの下流側に設けられ、その導入部１１ａの開口断面積よりも小さい開口断面積となるように形成されている。詳細には、縮径部１１ｂは、導入部１１ａから縮径部１１ｂの最小の内径部分に近づくほど、内径が小さくなる形状に形成されている。 The reduced diameter portion 11b is provided on the downstream side of the introduction portion 11a, and is formed to have an opening cross-sectional area smaller than the opening cross-sectional area of the introduction portion 11a. Specifically, the reduced diameter portion 11b is formed in a shape in which the inner diameter decreases as it approaches the minimum inner diameter portion of the reduced diameter portion 11b from the introduction portion 11a.
　噴射部１１ｃ（拡開部）は、縮径部１１ｂの下流側に設けられ、その縮径部１１ｂの開口断面積よりも大きい開口断面積に形成されている。詳細には、噴射部１１ｃは、縮径部１１ｂから下流側に向かって開口断面積が大きくなる拡開した形状に形成されている。 The injection part 11c (expanded part) is provided on the downstream side of the reduced diameter part 11b, and is formed to have an opening sectional area larger than the opening sectional area of the reduced diameter part 11b. Specifically, the injection portion 11c is formed in an expanded shape in which the opening cross-sectional area increases from the reduced diameter portion 11b toward the downstream side.
　導入部１１ａおよび縮径部１１ｂの最小内径部分より上流の範囲では、高圧・高速の一成分混相流は、音速以下の速度であり、上流から下流へ内径が小さくなるほど速度が増加する。そして、縮径部１１ｂの最小の内径部分では、一成分混相流が略音速となる。
　そして、縮径部１１ｂから噴射部１１ｃの下流側先端部にかけて、開口断面積が大きくなるほど、一成分混相流の断熱膨張により、その流れが音速を超えた状態となり、氷核が成長して、一成分極低温微細固体粒子が生成され、一成分極低温微細固体粒子を含む噴霧流が連続して噴射部１１ｃから噴射される。
　また、高圧・高速の一成分混相流は極低温となっており、ラバルノズル部１１の縮径部１１ｂから噴射部１１ｃの下流側先端部では、一成分混相流が断熱膨張することで、その流れが音速を超えた速度となり、且つ、導入部１１ａと比較して温度が著しく低下した状態となり、一成分極低温微細固体粒子の生成が促進される。 In a range upstream from the minimum inner diameter portion of the introduction portion 11a and the reduced diameter portion 11b, the high-pressure / high-speed one-component mixed phase flow has a velocity equal to or lower than the sound velocity, and the velocity increases as the inner diameter decreases from upstream to downstream. In the minimum inner diameter portion of the reduced diameter portion 11b, the one-component mixed phase flow is substantially sonic.
And as the opening cross-sectional area increases from the reduced diameter part 11b to the downstream end part of the injection part 11c, the adiabatic expansion of the one-component multiphase flow causes the flow to exceed the speed of sound, and the ice nucleus grows. One-component cryogenic fine solid particles are generated, and a spray flow containing the one-component cryogenic fine solid particles is continuously ejected from the ejection unit 11c.
Further, the high-pressure / high-speed one-component mixed phase flow is extremely low temperature, and the one-component mixed phase flow adiabatically expands from the reduced diameter portion 11b of the Laval nozzle portion 11 to the downstream end portion of the injection portion 11c. Becomes a speed exceeding the speed of sound, and the temperature is significantly lowered as compared with the introduction part 11a, and the generation of one-component cryogenic fine solid particles is promoted.
　例えば、上述した混合部１０の外管１５の内径は２．５ｍｍ程度、内管１４の外径は１．４ｍｍ程度、内管１４の内径は０．５ｍｍ程度、ラバルノズル部１１の導入部１１ａの上流側端部の内径は２．５ｍｍ程度、縮径部１１ｂの内径は１．０ｍｍ程度、噴射部１１ｃの射出口の先端部分の内径は、２．２ｍｍ程度である。
　混合部１０、ラバルノズル部１１の各サイズは、上記形態に限られるものではなく、適宜、設定することが好ましい。 For example, the inner diameter of the outer tube 15 of the mixing unit 10 described above is about 2.5 mm, the outer diameter of the inner tube 14 is about 1.4 mm, the inner diameter of the inner tube 14 is about 0.5 mm, and the introduction portion 11a of the Laval nozzle unit 11 The inner diameter of the upstream end portion is about 2.5 mm, the inner diameter of the reduced diameter portion 11b is about 1.0 mm, and the inner diameter of the tip portion of the injection port of the injection portion 11c is about 2.2 mm.
Each size of the mixing part 10 and the Laval nozzle part 11 is not restricted to the said form, It is preferable to set suitably.
＜超音波振動子６＞
　本実施形態では、極低温微細固体粒子連続生成装置１００は、超音波振動子６を有する。
　超音波振動子６は、図４、図１に示したように、ラバルノズル部１１に超音波を印加する。超音波振動子６により生成された超音波をラバルノズル部１１に印加することで、ラバルノズル部１１内の一成分混相流にキャビテーションを発生させ、一成分極低温微細固体粒子（ＳＮ2粒子）の氷核生成を促進し、且つ、微小均一粒子径の略球形状の一成分極低温微細固体粒子（ＳＮ2粒子）の微細化を促進することができる。キャビテーションとは、流体の中で、圧力差により短時間に泡の発生と消滅が起きる物理現象である。キャビテーションによる気泡の崩壊時に、短寿命の高温・高圧の局所場（ホットスポット）が形成される。これを利用することにより、ＳＮ2粒子の氷核生成の促進、ＳＮ2粒子の微細化の促進などを行うことができる。微細化されたＳＮ2粒子は、微細な略球形状に形成されている。 <Ultrasonic vibrator 6>
In the present embodiment, the cryogenic fine solid particle continuous generation apparatus 100 includes the ultrasonic transducer 6.
The ultrasonic transducer 6 applies ultrasonic waves to the Laval nozzle unit 11 as shown in FIGS. By applying the ultrasonic wave generated by the ultrasonic vibrator 6 to the Laval nozzle part 11, cavitation is generated in the one-component mixed phase flow in the Laval nozzle part 11, and ice of one-component cryogenic fine solid particles (SN 2 particles) is generated. Nucleation can be promoted, and refinement of a substantially spherical one-component cryogenic fine solid particle (SN 2 particle) having a fine uniform particle diameter can be promoted. Cavitation is a physical phenomenon in which bubbles are generated and disappeared in a short time due to a pressure difference in a fluid. When bubbles collapse due to cavitation, a short-lived high-temperature, high-pressure local field (hot spot) is formed. By utilizing this, the promotion of the ice nucleation SN 2 particles, it is possible to perform such promotion of miniaturization of the SN 2 particles. The refined SN 2 particles are formed in a fine substantially spherical shape.
　超音波振動子６は、詳細には、超音波振動生成部６ａと超音波伝達部６ｂとを有する。
　超音波振動生成部６ａは、制御装置（不図示）の制御により、規定の振動数、規定の振幅の超音波を生成する。超音波伝達部６ｂは、略棒形状の金属部材で構成され、超音波振動生成部６ａで生成された超音波をラバルノズル部１１に伝達する。 Specifically, the ultrasonic transducer 6 includes an ultrasonic vibration generation unit 6a and an ultrasonic transmission unit 6b.
The ultrasonic vibration generation unit 6a generates ultrasonic waves having a specified frequency and a specified amplitude under the control of a control device (not shown). The ultrasonic transmission unit 6 b is configured by a substantially rod-shaped metal member, and transmits the ultrasonic wave generated by the ultrasonic vibration generation unit 6 a to the Laval nozzle unit 11.
　超音波振動子６からラバルノズル部１１に印加される超音波は、具体的には、例えば、振動数３０ｋＨｚ～２ＭＨｚ程度、振幅１０μｍ～５０μｍ程度であり、好ましくは、振動数４０ｋＨｚ～９５０ｋＨｚ程度、振幅２０μｍ～４０μｍ程度であり、最適には、４５ｋＨｚ、振幅３０μｍ程度である。このラバルノズル部１１に印加される超音波の振動数や振幅については、ラバルノズル部１１で生成される一成分極低温微細固体粒子の粒径、個数などに応じて適宜、設定する。
　尚、超音波振動子６からラバルノズル部１１に印加される超音波として、例えば１ＭＨｚ～数１０ＭＨｚ、または、数１０ＭＨｚ～数１００ＭＨｚ程度の高周波数（メガソニック）の超音波を採用した場合、極低温微細固体粒子に関する、氷核生成や微粒化促進特性がさらに向上する。 Specifically, the ultrasonic wave applied from the ultrasonic transducer 6 to the Laval nozzle unit 11 has, for example, a frequency of about 30 kHz to 2 MHz and an amplitude of about 10 μm to 50 μm, preferably a frequency of about 40 kHz to 950 kHz and an amplitude. It is about 20 μm to 40 μm, and optimally, 45 kHz and an amplitude of about 30 μm. The frequency and amplitude of the ultrasonic wave applied to the Laval nozzle unit 11 are appropriately set according to the particle size, number, etc. of the one-component cryogenic fine solid particles generated by the Laval nozzle unit 11.
When ultrasonic waves applied from the ultrasonic transducer 6 to the Laval nozzle unit 11 are ultrasonic waves having a high frequency (megasonic) of about 1 MHz to several tens of MHz, or several tens of MHz to several hundreds of MHz, for example, extremely low temperature The ice nucleation and atomization promoting characteristics of fine solid particles are further improved.
　超音波振動子６で生成した超音波を印加する位置は、例えば、ラバルノズル部１１の一成分極低温微細固体粒子の氷核形成が行われる位置付近が好ましく、詳細には、ラバルノズル部１１の縮径部１１ｂから僅かに下流側の位置が好ましい。また、超音波振動子６で生成した超音波を印加する位置は、ラバルノズル部１１の縮径部１１ｂから下流側の噴射部１１ｃの任意の位置であってもよく、また、ラバルノズル部１１全体であってもよく、ラバルノズル部１１で生成する一成分極低温微細固体粒子の粒径、個数などに応じて適宜、設定してもよい。
　また、超音波振動子６は超音波を直接または間接的にラバルノズル部１１に印加してもよいし、真空断熱部としての断熱部５内からラバルノズル部１１に印加してもよい。 For example, the position where the ultrasonic wave generated by the ultrasonic transducer 6 is applied is preferably near the position where ice nucleation of one-component cryogenic fine solid particles of the Laval nozzle unit 11 is performed. A position slightly downstream from the diameter portion 11b is preferable. Further, the position where the ultrasonic wave generated by the ultrasonic transducer 6 is applied may be an arbitrary position of the injection portion 11c on the downstream side from the reduced diameter portion 11b of the Laval nozzle portion 11, or in the entire Laval nozzle portion 11. It may also be set as appropriate according to the particle diameter, the number, etc. of the one-component cryogenic fine solid particles produced by the Laval nozzle unit 11.
The ultrasonic transducer 6 may apply ultrasonic waves directly or indirectly to the Laval nozzle unit 11 or may apply the ultrasonic wave to the Laval nozzle unit 11 from within the heat insulating unit 5 as a vacuum heat insulating unit.
＜極低温微細固体粒子連続生成装置１００の動作＞
　次に、極低温微細固体粒子連続生成装置１００の動作を説明する。 <Operation of Cryogenic Solid Particle Continuous Generation Device 100>
Next, operation | movement of the cryogenic fine solid particle continuous production | generation apparatus 100 is demonstrated.
　液体窒素タンク（不図示）から極低温の過冷却液体窒素（ＬＮ2）が液体窒素導管３、外管１５の連通部１５ａ、開口部１５ｂを介して外管１５内の混合部１０に導入される。
　窒素ガスタンク（不図示）から高圧・高速の極低温の窒素ガス（ＬＮ2）が窒素ガス導管４、内管１４を介して混合部１０に導入される。極低温の窒素ガス（ＬＮ2）の圧力は、例えば、０．１ＭＰａ～１．０ＭＰａ程度であり、本実施形態では０．４ＭＰａ程度である。極低温の窒素ガス（ＬＮ2）の圧力が高いほど、混合部１０での流速が高速となり、好ましい。尚、この極低温の窒素ガス（ＬＮ2）の圧力は、０．５ＭＰａ～１０００ＭＰａ程度であってもよいし、１．０～１０ＭＰａ程度であってもよい。 Cryogenic supercooled liquid nitrogen (LN 2 ) is introduced from a liquid nitrogen tank (not shown) into the mixing unit 10 in the outer tube 15 through the liquid nitrogen conduit 3, the communicating portion 15a of the outer tube 15, and the opening 15b. The
High-pressure and high-speed cryogenic nitrogen gas (LN 2 ) is introduced from a nitrogen gas tank (not shown) into the mixing unit 10 through the nitrogen gas conduit 4 and the inner tube 14. The pressure of the cryogenic nitrogen gas (LN 2 ) is, for example, about 0.1 MPa to 1.0 MPa, and in this embodiment is about 0.4 MPa. The higher the pressure of the cryogenic nitrogen gas (LN 2 ), the higher the flow rate in the mixing unit 10, which is preferable. The pressure of the cryogenic nitrogen gas (LN 2 ) may be about 0.5 MPa to 1000 MPa or about 1.0 to 10 MPa.
　そして、図４に示したように、高圧・高速の極低温の窒素ガス（ＧＮ2）が、内管１４の先端部１４ａから高速に噴射され、過冷却液体（ＬＮ2）が内管１４と外管１５の間の隙間４５から混合部１０に導入されて、混合部１０にて、過冷却液体窒素（ＬＮ2）と極低温気体（ＧＮ2）の高速流とが混合して、一成分混相流（ＬＮ2－ＧＮ2）が生成される。 Then, as shown in FIG. 4, high-pressure and high-speed cryogenic nitrogen gas (GN 2 ) is jetted at a high speed from the distal end portion 14 a of the inner tube 14, and supercooled liquid (LN 2 ) is discharged from the inner tube 14. It is introduced into the mixing unit 10 through the gap 45 between the outer tubes 15, and in the mixing unit 10, the supercooled liquid nitrogen (LN 2 ) and the high-speed flow of cryogenic gas (GN 2 ) are mixed to form one component. A multiphase flow (LN 2 -GN 2 ) is generated.
　そして、混合部１０で生成された高圧・高速の極低温の一成分混相流（ＬＮ2－ＧＮ2）が、ラバルノズル部１１の導入部１１ａに導入され、縮径部１１ｂの最小の内径部分では、一成分混相流が略音速となり、縮径部１１ｂから噴射部１１ｃの下流側先端部にかけて、開口断面積が大きくなるほど、一成分混相流の断熱膨張により、その流れが音速を超えた状態となり、噴射部１１ｃにて、一成分混相流が音速を超えた状態で断熱膨張して、一成分極低温微細固体粒子（ＳＮ2粒子）を含む噴霧流が連続して生成される。 Then, the high-pressure, high-speed, low-temperature, one-component mixed phase flow (LN 2 -GN 2 ) generated in the mixing unit 10 is introduced into the introduction unit 11a of the Laval nozzle unit 11, and at the minimum inner diameter part of the reduced diameter unit 11b. The one-component multiphase flow becomes substantially sonic velocity, and the flow crosses the sonic velocity due to adiabatic expansion of the one-component multiphase flow as the opening cross-sectional area increases from the reduced diameter portion 11b to the downstream end portion of the injection portion 11c. In the injection unit 11c, the one-component multiphase flow adiabatically expands in a state exceeding the speed of sound, and a spray flow including one-component cryogenic fine solid particles (SN 2 particles) is continuously generated.
　また、超音波振動子６により生成された超音波（４５ｋＨｚ程度、振幅３０μｍ程度）をラバルノズル部１１に印加することで、ラバルノズル部１１内の一成分混相流にキャビテーションを発生させ、一成分極低温微細固体粒子（ＳＮ2粒子）の氷核生成を促進し、且つ、微小均一粒子径の略球形状の一成分極低温微細固体粒子（ＳＮ2粒子）の微細化を促進することができる。 Further, by applying ultrasonic waves (about 45 kHz, amplitude of about 30 μm) generated by the ultrasonic transducer 6 to the Laval nozzle unit 11, cavitation is generated in the one-component mixed phase flow in the Laval nozzle unit 11, and one-component cryogenic temperature is generated. Formation of ice nuclei of fine solid particles (SN 2 particles) can be promoted, and miniaturization of one-component cryogenic fine solid particles (SN 2 particles) having a substantially uniform spherical particle shape can be promoted.
＜冷却熱流束値＞
　図５は極低温微細固体粒子連続生成装置１００により、極低温の窒素ガス（ＧＮ2）と過冷却した液体窒素（ＬＮ2）とで生成した極低温微細固体窒素粒子（ＳＮ2）の冷却熱流束値ｑwを示す図である。
　図５においては、縦軸に冷却熱流束値〔Ｗ／ｍ2〕を示し、横軸に時間ｔ（ｓｅｃ）を示す。 <Cooling heat flux value>
FIG. 5 shows a cooling heat flow of cryogenic fine solid nitrogen particles (SN 2 ) generated by cryogenic nitrogen gas (GN 2 ) and supercooled liquid nitrogen (LN 2 ) by the cryogenic fine solid particle continuous production apparatus 100. is a diagram showing the flux value q w.
In FIG. 5, the vertical axis represents the cooling heat flux value [W / m 2 ], and the horizontal axis represents time t (sec).
　図５に示したように、噴霧を開始すると、冷却熱流束値ｑwは、急激に大きくなり、短時間で最大冷却熱流束値に達し、その後、緩やかに低下する。 As shown in FIG. 5, when starting the spraying, the cooling heat flow flux value q w is rapidly increases in a short time reach the maximum cooling heat flux value, then decreased gradually.
　極低温の窒素ガス（ＧＮ2）と過冷却状態の液体窒素（ＬＮ2）とにより生成した微細固体窒素粒子（ＳＮ2粒子）を含む噴霧流中のＳＮ2粒子の数密度が比較的大きいので、冷却熱流束値の減衰率が小さい。
　また、上述したように、極低温微細固体粒子連続生成装置から噴射される極低温微細固体粒子を含む噴霧流は、１０5Ｗ／ｍ2レベルの熱流束であり、冷却能が非常に高い。 Because the number density of SN 2 particles in the spray flow containing fine solid nitrogen particles (SN 2 particles) generated by cryogenic nitrogen gas (GN 2 ) and supercooled liquid nitrogen (LN 2 ) is relatively large The decay rate of the cooling heat flux value is small.
Further, as described above, the spray flow containing the cryogenic fine solid particles ejected from the cryogenic fine solid particle continuous production apparatus has a heat flux of 10 5 W / m 2 level and has a very high cooling ability.
＜ＳＮ2粒子の衝突圧力＞
　図６は一成分極低温微細固体窒素粒子（ＳＮ2）の噴霧衝突圧力値およびシミュレーションによる噴霧衝突圧力値の一例を示す図である。図６において、横軸（ｘ軸）に無次元時間ｔ*を示し、縦軸（ｙ軸）に衝突圧力を無次元化した圧力ｐ*を示す。
　詳細には、一成分極低温微細固体窒素粒子（ＳＮ2粒子）をピエゾ圧電型圧力センサに衝突させて、そのＳＮ2粒子の噴霧衝突圧力値を点線で示す。実験において窒素ガス（ＧＮ2）タンクの圧力を０．４ＭＰａ、液体窒素（ＬＮ2）タンクの圧力を０．２ＭＰａに設定した。
　シミュレーションとして単一粒子衝突数値計算（ＣＦＤ）によって得られた衝突圧力値を実線で示す。図６に示したように、ＳＮ2粒子が圧力センサに衝突した直後、衝突圧力ｐ*が急激に増加した後、減少した。また、数値計算結果でも同様に、衝突圧力ｐ*が衝突直後に立ち上がり、減少する傾向を有していることが分かる。 <SN 2 particle collision pressure>
FIG. 6 is a diagram showing an example of a spray collision pressure value of a one-component cryogenic fine solid nitrogen particle (SN 2 ) and a spray collision pressure value by simulation. In FIG. 6, the horizontal axis (x axis) represents the dimensionless time t * , and the vertical axis (y axis) represents the pressure p * obtained by making the collision pressure dimensionless.
Specifically, one-component cryogenic fine solid nitrogen particles (SN 2 particles) are caused to collide with a piezoelectric piezoelectric pressure sensor, and the spray collision pressure value of the SN 2 particles is indicated by a dotted line. In the experiment, the pressure of the nitrogen gas (GN 2 ) tank was set to 0.4 MPa, and the pressure of the liquid nitrogen (LN 2 ) tank was set to 0.2 MPa.
As a simulation, the collision pressure value obtained by single particle collision numerical calculation (CFD) is shown by a solid line. As shown in FIG. 6, immediately after the SN 2 particles collided with the pressure sensor, the collision pressure p * increased rapidly and then decreased. Similarly, the numerical calculation result shows that the collision pressure p * tends to rise and decrease immediately after the collision.
　図７は、ラバルノズル部の先端部にスパイラルノズル１８を備えた極低温微細固体粒子連続生成装置１００のノズル１の一例を示す図である。
　図７に示したように、例えば、特開２０１１－１７１６９１号に示したようなスパイラルノズル１８を、ノズル１のラバルノズル部１１の先端部に設ける。スパイラルノズル１８に一成分極低温微細固体窒素粒子（ＳＮ2粒子）の高速ジェット流が通過することにより、適度な乱流を生じさせ、ＳＮ2粒子をより細粒子化させることができ、且つ、ＳＮ2粒子がノズルに付着するのを防止して、連続的にＳＮ2粒子を含む噴霧流を生成することができる。 FIG. 7 is a diagram illustrating an example of the nozzle 1 of the cryogenic fine solid particle continuous generation apparatus 100 including the spiral nozzle 18 at the tip of the Laval nozzle.
As shown in FIG. 7, for example, a spiral nozzle 18 as shown in Japanese Patent Application Laid-Open No. 2011-171691 is provided at the tip of the Laval nozzle portion 11 of the nozzle 1. When a high-speed jet flow of one-component cryogenic fine solid nitrogen particles (SN 2 particles) passes through the spiral nozzle 18, an appropriate turbulent flow is generated, and the SN 2 particles can be made finer, and It is possible to prevent the SN 2 particles from adhering to the nozzle and to continuously generate a spray flow including SN 2 particles.
＜一成分極低温微細固体粒子（ＳＮ2）の粒子径分布および粒子速度分布＞
　極低温微細固体粒子連続生成装置１００により生成された一成分極低温微細固体粒子（ＳＮ2）の粒子径分布、粒子速度分布に関しては、粒子の撮影画像の解析、例えば、直接撮影法（Direct-Imaging Techniques）による粒子径分布・数密度分布計測を行う二色レーザーＰＩＡ（Particle Image Analyzer）光学計測システムを使用した二次元化可視化画像計測などにより解析できる。ＰＩＡでは、例えば、図７に示したように規定領域ＣＲ（Control region）にて、飛翔しているＳＮ2粒子を顕微鏡レンズ等を用いて拡大撮影し、粒子像を画像解析することで粒子径と速度を測定する。例えば、二色レーザーＰＩＡ光学計測システムでは、二色レーザー、例えば、デュアルパルスＹＡＧレーザー、被写界深度チェック用色素レーザーなど、ＰＩＡ用高解像度カラーカメラ、ＰＩＡ画像解析ソフトウェアなどを使用できる。また、数値解析などの解析に、クラスタ型高速ワークステーションを使用した超並列計算による高負荷分散型コンピューティング（Grid Computing）手法を使用することが好ましく、ノズルの微粒化特性、例えば、粒径分布、数密度分布、流速・温度分布等を適切に定量的に評価することができる。
　また、粒子速度の定量化には、ＰＴＶ（Particle Tracking Velocimetry）アルゴリズムを使用することができる。使用するＰＴＶアルゴリズムは、着目粒子像とその近傍の粒子像とから構成される粒子像群の分布パターンを考え、その類似性を利用して粒子追跡（Particle Tracking）を行うものである。 <Particle size distribution and particle velocity distribution of one-component cryogenic fine solid particles (SN 2 )>
Regarding the particle size distribution and particle velocity distribution of the one-component cryogenic fine solid particles (SN 2 ) produced by the cryogenic fine solid particle continuous production apparatus 100, analysis of the photographed image of the particle, for example, a direct photographing method (Direct- It can be analyzed by two-dimensional visualization image measurement using a two-color laser PIA (Particle Image Analyzer) optical measurement system that measures particle size distribution and number density distribution by Imaging Techniques. In PIA, for example, as shown in FIG. 7, in a defined region CR (Control region), SN 2 particles flying are magnified using a microscope lens and the particle size is analyzed by image analysis. And measure the speed. For example, in a two-color laser PIA optical measurement system, a two-color laser, for example, a dual pulse YAG laser, a dye laser for depth of field check, a high-resolution color camera for PIA, PIA image analysis software, or the like can be used. For analysis such as numerical analysis, it is preferable to use a high-load distributed computing (Grid Computing) method based on massively parallel computation using a cluster-type high-speed workstation, and the atomization characteristics of the nozzle, for example, the particle size distribution The number density distribution, flow velocity / temperature distribution, etc. can be appropriately and quantitatively evaluated.
In addition, a PTV (Particle Tracking Velocimetry) algorithm can be used for quantifying the particle velocity. The PTV algorithm to be used considers a distribution pattern of a particle image group composed of a target particle image and a particle image in the vicinity thereof, and performs particle tracking using the similarity.
＜粒子径分布＞
　図８は、極低温微細固体粒子連続生成装置１００により生成される一成分極低温微細固体粒子の粒子径の分布の一例を示す図である。詳細には、図８（ａ）は超音波をラバルノズル部１１に印加しない場合の一例を示す図、図８（ｂ）は超音波振動子による超音波をラバルノズル部１１に印加した場合の一例を示す図である。
　ここでは、図７に示したように、噴孔直下から４．５ｍｍだけ下方位置から視野としての規定領域（横０．９２ｍｍ、縦０．７ｍｍ）で上記ＰＩＡ－ＰＴＶによる解析を行った。
　図８（ａ）、図８（ｂ）においては、横軸（ｘ軸）に粒子直径ｄp〔μｍ〕を示し、左縦軸（ｙ1軸）に頻度ｆf〔％〕を示し、右縦軸（ｙ2軸）に累積ｆa〔％〕を示している。 <Particle size distribution>
FIG. 8 is a diagram showing an example of the particle size distribution of one-component cryogenic fine solid particles produced by the cryogenic fine solid particle continuous production apparatus 100. Specifically, FIG. 8A is a diagram illustrating an example in which ultrasonic waves are not applied to the Laval nozzle unit 11, and FIG. 8B is an example in which ultrasonic waves from an ultrasonic transducer are applied to the Laval nozzle unit 11. FIG.
Here, as shown in FIG. 7, the analysis by the PIA-PTV was performed in a specified region (horizontal 0.92 mm, vertical 0.7 mm) as a visual field from a position below 4.5 mm from just below the nozzle hole.
8 (a) and 8 (b), the horizontal axis (x axis) represents the particle diameter d p [μm], the left vertical axis (y 1 axis) represents the frequency f f [%], and the right the vertical axis (y 2 axis) shows the cumulative f a [%].
　超音波を印加しない場合と比較して、超音波振動子による超音波をラバルノズル部に加えて微粒化促進させた場合、最小粒子径の占める分布割合が大きいことが分かった。 It was found that the distribution ratio occupied by the minimum particle size was larger when the atomization was promoted by applying ultrasonic waves from the ultrasonic vibrator to the Laval nozzle as compared with the case where no ultrasonic wave was applied.
　超音波を印加しない場合（ｎｏｎ－ＵＬＡ）と比較して、超音波振動子をノズルに設置した場合（ＵＬＡ）では、平均粒子径が約２．５％程度減少していることが分かった。
　詳細には、超音波振動子による超音波をラバルノズル部に印加させない場合（ｎｏｎ－ＵＬＡ）、平均粒子径は４．１μｍであり、超音波振動子による超音波をラバルノズル部に印加した場合（ＵＬＡ）、平均粒子径が３．９μｍであった。 It was found that the average particle size was reduced by about 2.5% in the case where the ultrasonic transducer was installed in the nozzle (ULA) compared to the case where no ultrasonic wave was applied (non-ULA).
Specifically, when the ultrasonic wave by the ultrasonic vibrator is not applied to the Laval nozzle part (non-ULA), the average particle diameter is 4.1 μm, and when the ultrasonic wave by the ultrasonic vibrator is applied to the Laval nozzle part (ULA) ), And the average particle size was 3.9 μm.
＜粒子速度分布＞
　図９は、極低温微細固体粒子連続生成装置１００により生成される一成分極低温微細固体粒子の粒子速度分布の一例を示す図である。詳細には図９（ａ）は超音波振動子による超音波をラバルノズル部１１に印加しない場合の一例を示す図、図９（ｂ）はラバルノズル部に超音波を印加した場合の一例を示す図である。
　図９（ａ）、図９（ｂ）においては、横軸（ｘ軸）に粒子速度Ｖp〔ｍ／ｓ〕を示し、左縦軸（ｙ1軸）に頻度ｆf〔％〕を示し、右縦軸（ｙ2軸）に累積ｆa〔％〕を示している。
　同様に、図７に示したように、噴孔直下から４．５ｍｍだけ下方位置から視野としての規定領域（横０．９２ｍｍ、縦０．７ｍｍ）で上記ＰＩＡ－ＰＴＶによる解析を行った。 <Particle velocity distribution>
FIG. 9 is a diagram illustrating an example of a particle velocity distribution of one-component cryogenic fine solid particles generated by the cryogenic fine solid particle continuous production apparatus 100. Specifically, FIG. 9A is a diagram showing an example when ultrasonic waves from an ultrasonic transducer are not applied to the Laval nozzle portion 11, and FIG. 9B is a diagram showing an example when ultrasonic waves are applied to the Laval nozzle portion. It is.
9 (a) and 9 (b), the horizontal axis (x axis) indicates the particle velocity V p [m / s], and the left vertical axis (y 1 axis) indicates the frequency f f [%]. shows the cumulative f a [%] to the right vertical axis (y 2 axes).
Similarly, as shown in FIG. 7, the analysis by the PIA-PTV was performed in a specified region (0.92 mm in width, 0.7 mm in length) as a visual field from a position below 4.5 mm from right below the nozzle hole.
　超音波を印加しない場合（ｎｏｎ－ＵＬＡ）と比較して、超音波振動子による超音波をラバルノズル部に印加した場合（ＵＬＡ）の粒子速度は小さいことが分かる。詳細には、超音波を付加すると粒径は小さくなり、“粒子に作用する空気抵抗力＞粒子に作用する慣性力”となるので、流速は減少する。しかし、超音波付加条件下では、氷核生成促進により凍結粒子を形成しやすくなり、微粒化促進効果も加わるので粒子数密度が増大し、噴霧照射部におけるトータルの接触伝熱面積が増大し、結果的に冷却効果が増大する。また、粒子径が小さくなったことで蒸発しやすくなり、粒子凝集による膜沸騰状態をさらに形成しにくくし、固体粒子の高速蒸気相変化を利用した潜熱冷却が促進される。つまり、超音波付加条件下では、流速が小さくなったことで強制対流冷却効果は若干減少するが、接触熱伝達と潜熱熱伝達特性は増加し、冷却効果が増大する。 It can be seen that the particle velocity is lower when the ultrasonic wave from the ultrasonic transducer is applied to the Laval nozzle (ULA) than when the ultrasonic wave is not applied (non-ULA). Specifically, when ultrasonic waves are added, the particle size becomes smaller, and “air resistance acting on the particles> inertial force acting on the particles”, the flow velocity decreases. However, under ultrasonic addition conditions, it becomes easier to form frozen particles due to the promotion of ice nucleation, and the effect of atomization is also added, so the particle number density increases, and the total contact heat transfer area in the spray irradiation part increases, As a result, the cooling effect increases. In addition, since the particle diameter is reduced, it is easy to evaporate, and it is difficult to form a film boiling state due to particle aggregation, and latent heat cooling using high-speed vapor phase change of solid particles is promoted. That is, under the ultrasonic wave application condition, the forced convection cooling effect is slightly reduced due to the decrease in the flow velocity, but the contact heat transfer and latent heat transfer characteristics are increased and the cooling effect is increased.
　図１０は、本発明の実施形態に係る凍結装置２００の一例を示す概念図である。図１０に示したように、凍結装置２００は、凍結保存容器６０を支持する支持装置７０を有する。支持装置７０は、支持部７１、噴霧位置変更部７２（噴霧位置変更手段）、などを有する。
　支持部７１は、噴霧部としての極低温微細固体粒子連続生成装置のノズル１（スパイラルノズル１８）から噴霧される極低温微細固体粒子を含む噴霧流の下流側に、凍結保存容器６０を着脱自在に支持する。
　本実施形態では、支持部７１は先端部に凍結保存容器６０を把持する把持部７１ａを有する。把持部７１ａの形状は凍結保存容器６０を着脱自在に支持できれば任意の形状でよく、本実施形態では、断面略Ｃ字形状に形成されている。
　凍結保存容器６０は、筒形状など任意の形状であってよい。本実施形態では、凍結保存容器６０は、凍結ムラなく高効率で冷却可能させるために、丸底の有底筒形状に形成されており、凍結保存液に細胞６１を懸濁させた懸濁液を収容し、蓋部により密閉可能な構造を有する。 FIG. 10 is a conceptual diagram showing an example of the freezing apparatus 200 according to the embodiment of the present invention. As shown in FIG. 10, the freezing device 200 includes a support device 70 that supports the cryopreservation container 60. The support device 70 includes a support portion 71, a spray position changing portion 72 (spray position changing means), and the like.
The support part 71 is detachable from the cryopreservation container 60 on the downstream side of the spray flow containing the cryogenic fine solid particles sprayed from the nozzle 1 (spiral nozzle 18) of the cryogenic fine solid particle continuous production apparatus as the spraying part. To support.
In the present embodiment, the support portion 71 has a gripping portion 71a for gripping the cryopreservation container 60 at the tip portion. The shape of the gripping portion 71a may be any shape as long as the cryopreservation container 60 can be detachably supported. In the present embodiment, the gripping portion 71a has a substantially C-shaped cross section.
The cryopreservation container 60 may have an arbitrary shape such as a cylindrical shape. In this embodiment, the cryopreservation container 60 is formed in a round bottomed cylindrical shape so that it can be cooled with high efficiency without freezing unevenness, and a suspension in which cells 61 are suspended in a cryopreservation solution. And has a structure that can be sealed by a lid.
　噴霧位置変更部７２（噴霧位置変更手段）は、凍結保存容器に対する凍結ムラの低減や、凍結時間の短縮のために、支持部７１に支持された凍結保存容器６０に対する噴霧流の噴霧位置を変更する。詳細には、噴霧位置変更部７２には、モータなどの駆動部８１が接続されており、制御部８２の制御により、支持部７１により支持された凍結保存容器６０を、上下方向、左右方向、前後方向、筒状の凍結保存容器の軸方向など所定の方向に移動可能に構成されている。また、噴霧位置変更部７２は、所定の回転方向、例えば、筒状の凍結保存容器の軸方向を回転軸として、支持部７１により支持された凍結保存容器６０を回転可能に構成されている。尚、噴霧位置変更部７２（噴霧位置変更手段）は、極低温微細固体粒子連続生成装置のノズルからの噴霧角度を変更することで、凍結保存容器への噴霧位置を変更してもよい。 The spray position changing unit 72 (spray position changing means) changes the spray position of the spray flow with respect to the cryopreservation container 60 supported by the support part 71 in order to reduce freezing unevenness for the cryopreservation container and shorten the freezing time. To do. Specifically, a drive unit 81 such as a motor is connected to the spray position changing unit 72, and the cryopreservation container 60 supported by the support unit 71 is controlled by the control unit 82 in the vertical direction, the horizontal direction, It is configured to be movable in a predetermined direction such as the front-rear direction and the axial direction of the cylindrical cryopreservation container. Further, the spray position changing unit 72 is configured to be able to rotate the cryopreservation container 60 supported by the support unit 71 with a predetermined rotation direction, for example, the axial direction of the cylindrical cryopreservation container as the rotation axis. In addition, the spray position changing part 72 (spray position changing means) may change the spray position to the cryopreservation container by changing the spray angle from the nozzle of the cryogenic fine solid particle continuous generation device.
　制御部８２は、凍結装置２００の各構成要素を統括的に制御する。詳細には、制御部８２は、凍結装置２００の極低温微細固体粒子連続生成装置の噴霧流の速度、圧力、噴霧時間などを制御可能に構成されている。また、制御部８２は、噴霧位置変更部７２により凍結保存容器への噴霧位置を適宜制御する。尚、凍結装置は、例えば、凍結保存容器の温度を検出する温度センサや赤外線カメラなどの温度検出部を備え、制御部８２が温度検出部により検出された凍結保存容器の温度に応じて、噴霧位置変更部７２による噴霧位置の制御や極低温微細固体粒子連続生成装置による噴霧時間などを制御するように構成されていてもよい。 The control unit 82 comprehensively controls each component of the freezing apparatus 200. Specifically, the control unit 82 is configured to be able to control the spray flow speed, pressure, spray time, and the like of the cryogenic fine solid particle continuous generation device of the freezing device 200. Moreover, the control part 82 controls the spray position to a cryopreservation container suitably by the spray position change part 72. FIG. The freezing device includes a temperature detection unit such as a temperature sensor for detecting the temperature of the cryopreservation container and an infrared camera, and sprays the control unit 82 according to the temperature of the cryopreservation container detected by the temperature detection unit. You may be comprised so that the spraying time by the control of the spray position by the position change part 72, the cryogenic fine solid particle continuous production | generation apparatus, etc. may be controlled.
　本願発明者は、本願発明に係る凍結装置の効果を確認するために、実際に、凍結対象としてＡ５４９細胞に対して凍結、および、解凍を行った。Ａ５４９細胞は、ヒト肺胞基底上皮腺癌細胞である。図１１は細胞（Ａ５４９細胞）の顕微鏡写真の一例を示す図である。 In order to confirm the effect of the freezing apparatus according to the present invention, the inventor of the present application actually frozen and thawed A549 cells as a target for freezing. A549 cells are human alveolar basal epithelial adenocarcinoma cells. FIG. 11 is a diagram showing an example of a micrograph of a cell (A549 cell).
＜凍結前準備工程＞
　先ず、凍結対象の細胞としてＡ５４９細胞を準備する（収穫する）。
　次に、５．０８×１０6ｃｅｌｌｓの細胞とＲＰＭＩ１６４０培地４ｍｌにより、濃度１×１０6ｃｅｌｌｓ／ｍＬとする。これを、凍結保存容器としての各クライオチューブに１５００μＬ分注し、２本のチューブを予備として保管し、１０本のチューブ（凍結保存容器）を凍結対象とした。
　この凍結保存容器は、丸底の有底筒状の容器であり、直径約１０ｍｍ、容量１．８ｍＬ、アウターキャップ型のものを用いた。尚、凍結保存容器は、高い熱伝導性と低温強度の高い、凍結保存用の樹脂材料や金属材料で形成されたものであってもよい。 <Preparation process before freezing>
First, A549 cells are prepared (harvested) as cells to be frozen.
Next, the concentration is 1 × 10 6 cells / mL with 5.08 × 10 6 cells and 4 ml of RPMI 1640 medium. This was dispensed in 1500 μL into each cryotube as a cryopreservation container, two tubes were stored as reserves, and 10 tubes (cryopreservation containers) were to be frozen.
This cryopreservation container is a round-bottomed cylindrical container having a diameter of about 10 mm, a capacity of 1.8 mL, and an outer cap type. The cryopreservation container may be formed of a resin material or metal material for cryopreservation having high thermal conductivity and high low-temperature strength.
＜凍結工程＞
　微量の凍結保存液に上記細胞を直接懸濁させた懸濁液を収容した凍結保存容器を、凍結装置の支持部により支持し、極低温微細固体粒子連続生成装置により、極低温微細固体窒素粒子を含む高速噴霧流を、細胞等を収容した凍結保存容器に連続噴霧して、凍結保存容器内の細胞を急速にガラス化凍結する。本具体例では、凍結保存液としては、日本全薬工業株式会社のCELLBANKER 1 plusを用い、凍結保存液の使用量は１アンプルあたり１．５ｍＬである。凍結保存液の成分は表１に示したように規定されている。 <Freezing process>
A cryopreservation container containing a suspension obtained by directly suspending the above cells in a small amount of cryopreservation solution is supported by a support part of a freezing device, and a cryogenic fine solid particle is continuously produced by a cryogenic fine solid particle continuous production device. A high-speed spray flow containing is continuously sprayed on a cryopreservation container containing cells and the like, and the cells in the cryopreservation container are rapidly vitrified and frozen. In this specific example, CELLBANKER 1 plus from Nippon Zenyaku Kogyo Co., Ltd. is used as the cryopreservation solution, and the amount of the cryopreservation solution used is 1.5 mL per ampoule. The components of the cryopreservation solution are defined as shown in Table 1.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　詳細には、液体窒素Ｎ2タンクの圧力０．２５２～０．２５３ＭＰａ程度、噴射ノズル付近の圧力０．３３～０．４４ＭＰａ程度で、凍結保存液に直接細胞を懸濁させた懸濁液を収容した各凍結保存容器に、噴霧時間１０、２０、３０、４５、５０、６０、９０、１２０秒で、極低温微細固体窒素粒子を含む高速噴霧流を連続噴霧して、凍結保存容器内の細胞を急速にガラス化凍結した後、液体窒素に浸けて保存した。比較例として、極低温微細固体窒素粒子を凍結保存容器に噴霧せずに（噴霧時間０秒）、液体窒素に浸けて凍結保存したものを用意した。 Specifically, a suspension in which cells are directly suspended in a cryopreservation solution at a pressure of about 0.252 to 0.253 MPa in the liquid nitrogen N 2 tank and a pressure of about 0.33 to 0.44 MPa in the vicinity of the injection nozzle. A high-speed spray flow containing cryogenic fine solid nitrogen particles is continuously sprayed to each stored cryopreservation container at a spraying time of 10, 20, 30, 45, 50, 60, 90, and 120 seconds. The cells were vitrified and frozen rapidly, and then immersed in liquid nitrogen and stored. As a comparative example, a cryogenic fine solid nitrogen particle was stored in a cryopreservation container by being immersed in liquid nitrogen without spraying the cryopreservation container (spray time 0 second).
＜解凍工程＞
　ガラス化凍結保存した細胞を収容するクライオチューブ（凍結保存容器）を、温度３７℃水浴で解凍する。そして、それぞれ５回ピペッティングした後、ＲＰＭＩ１６４０培地９ｍＬに希釈する。次に、遠心分離器にて１５００ｒｐｍで２分間、遠心分離処理を行う。そして、培地を吸引除去し、ＲＰＭＩ１６４０培地２ｍｌに懸濁する。この懸濁液１０μＬと染色剤としてのトリパンブルー（ＢｉｏＲａｄ）１０μＬをピペッティングにより混和させ、セルカウンターで細胞数を計測した。
　表２は極低温微細固体窒素粒子の噴霧時間と細胞生存率（％）の実験結果の一例を示す。 <Thawing process>
The cryotube (cryopreservation container) containing the vitrified cryopreserved cells is thawed in a water bath at a temperature of 37 ° C. Then, after pipetting 5 times, each is diluted with 9 mL of RPMI 1640 medium. Next, the centrifuge is centrifuged at 1500 rpm for 2 minutes. Then, the medium is removed by suction and suspended in 2 ml of RPMI 1640 medium. 10 μL of this suspension and 10 μL of trypan blue (BioRad) as a staining agent were mixed by pipetting, and the number of cells was counted with a cell counter.
Table 2 shows an example of the experimental results of the spraying time and cell viability (%) of the cryogenic fine solid nitrogen particles.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　図１２は極低温微細固体窒素粒子の噴霧時間と細胞生存率（％）の一例を示す図である。図１２において横軸に極低温微細固体窒素粒子の噴霧時間（sec）を示し、縦軸に細胞の生存率を示す。
　図１２および表２において、極低温微細固体窒素粒子を含む高速噴霧流を噴霧せずに、細胞を収容した容器を単に液体窒素に浸けて冷却した場合の細胞生存率を、噴霧時間０秒の値として示している。
　本発明の実施形態に係る噴霧部としての極低温微細固体粒子連続生成装置１００による、極低温微細固体窒素粒子を含む噴霧流を連続して所定秒数噴霧して、容器内の細胞をガラス化凍結させたものは、いずれも解凍時に高い細胞生存率となった。具体的には、液体窒素浸漬のみによる凍結法と比較して、本発明の実施形態に係る凍結装置の凍結方法では、細胞生存率が２３％程度増加した（噴霧時間９０秒）。
　また、噴霧時間が長いほど、細胞生存率が高くなる傾向となった。これは噴霧時間を長くするほど、冷却速度が高くなり、細胞が短時間に高品質でガラス化凍結すると考えられる。 FIG. 12 is a diagram showing an example of spraying time and cell survival rate (%) of cryogenic fine solid nitrogen particles. In FIG. 12, the horizontal axis represents the spray time (sec) of the cryogenic fine solid nitrogen particles, and the vertical axis represents the cell viability.
In FIG. 12 and Table 2, the cell viability when the container containing the cells was cooled by simply immersing in liquid nitrogen without spraying a high-speed spray flow containing cryogenic fine solid nitrogen particles is shown in FIG. Shown as a value.
A spray flow containing cryogenic fine solid nitrogen particles is continuously sprayed for a predetermined number of seconds by the cryogenic fine solid particle continuous production apparatus 100 as a spraying section according to an embodiment of the present invention, and the cells in the container are vitrified. All frozen samples had high cell viability upon thawing. Specifically, in comparison with the freezing method using only liquid nitrogen immersion, the freezing method of the freezing apparatus according to the embodiment of the present invention increased the cell viability by about 23% (spray time 90 seconds).
In addition, the longer the spray time, the higher the cell viability. It is considered that the longer the spray time, the higher the cooling rate, and the cells vitrify and freeze in high quality in a short time.
　以上、説明したように、本発明の実施形態に係る凍結装置２００は、凍結対象として、細胞６１などの水分を含む弾性体膜包を凍結する。この凍結装置は、凍結対象を収容する凍結保存容器６０と、凍結保存容器６０に極低温微細固体粒子を含む噴霧流を連続して高速噴霧して、凍結保存容器６０に収容された凍結対象をガラス化凍結させる噴霧部としての極低温微細固体粒子連続生成装置１００とを有する。
　このように、極低温微細固体粒子連続生成装置１００により、極低温微細固体粒子を含む噴霧流を、細胞などの凍結対象を収容した凍結保存容器６０に、極低温微細固体粒子を含む噴霧流を連続して高速噴霧し、衝突熱伝達・対流熱伝達・蒸発潜熱熱伝達の相乗効果により、凍結保存容器６０に収容した細胞６１など凍結対象をガラス凍結状態にする。詳細には、凍結装置は、細胞内の氷核形成を抑えて、細胞をガラス状態で急速凍結させる。
　本発明の実施形態に係る凍結装置の極低温微細固体窒素粒子を含む高速噴霧流の連続噴霧による凍結速度の最大値は、約－２５．８Ｋ／Ｓｅｃ程度（１秒間に２５．８℃降下）であり、約６３Ｋ（－２１０．１５℃）まで急速に冷却することができる。常に新しい極低温微細固体粒子が冷凍保存容器に衝突するので、凍結速度が速いまま維持され、細胞のガラス化凍結が短時間で完了する。
　その後、ガラス化凍結した細胞を収容する凍結保存容器を液体窒素に浸けて、比較的長時間保存する。そして、所定時間後の解凍時には、凍結保存容器６０に収容されていた解凍液により細胞を急速に温めることで再核形成を抑えつつ高品質に解凍することができ、上述したように、高い細胞生存率となる。 As described above, the freezing apparatus 200 according to the embodiment of the present invention freezes an elastic body membrane containing water such as the cells 61 as a freezing object. This freezing apparatus includes a cryopreservation container 60 for storing a target to be frozen, and a cryopreservation object stored in the cryopreservation container 60 by continuously spraying the cryopreservation container 60 with a spray flow containing cryogenic fine solid particles at a high speed. And a cryogenic fine solid particle continuous production apparatus 100 as a spraying section for vitrification and freezing.
Thus, the cryogenic fine solid particle continuous generation apparatus 100 applies the nebulized stream containing the cryogenic fine solid particles to the cryopreservation container 60 containing the object to be frozen such as cells and the nebulized stream containing the cryogenic fine solid particles. By spraying continuously at high speed, the object to be frozen such as the cells 61 housed in the cryopreservation container 60 is put into a glass frozen state by a synergistic effect of impact heat transfer, convection heat transfer, and latent heat of vaporization heat transfer. Specifically, the freezing device suppresses ice nucleation in the cell and rapidly freezes the cell in a glass state.
The maximum value of the freezing rate by continuous spraying of the high-speed spray flow containing the cryogenic fine solid nitrogen particles in the freezing apparatus according to the embodiment of the present invention is about −25.8 K / Sec (a 25.8 ° C. drop per second). And can be rapidly cooled to about 63 K (−21.15 ° C.). Since new cryogenic fine solid particles always collide with the cryopreservation container, the freezing rate is maintained at a high speed, and the vitrification freezing of the cells is completed in a short time.
Thereafter, the cryopreservation container containing the vitrified and frozen cells is immersed in liquid nitrogen and stored for a relatively long time. At the time of thawing after a predetermined time, the cells can be thawed with a high quality while suppressing renucleation by rapidly warming the cells with the thawing solution contained in the cryopreservation container 60. As described above, high cells Survival rate.
　このように、凍結時に、極低温微細固体粒子を含む高速噴霧流を次々に連続的に凍結保存容器に噴霧することにより、衝突熱伝達・対流熱伝達・蒸発潜熱熱伝達の相乗効果により、凍結保存容器内の細胞などの凍結対象を急速にガラス化凍結する凍結装置２００を提供することができる。
　また、凍結対象の細胞などを凍結保存容器内に収容した状態でガラス化凍結させるので、凍結時に不純物の混入がなく、高速噴霧流による損傷がない。比較例として、例えば、液体窒素に細胞を直接浸けて冷却する方法では不純物混入の虞がある。 In this way, during freezing, by spraying a high-speed spray flow containing cryogenic fine solid particles one after another onto a cryopreservation container, freezing is achieved by the synergistic effect of collision heat transfer, convection heat transfer, and latent heat of vaporization heat transfer. It is possible to provide a freezing device 200 that rapidly vitrifies and freezes objects such as cells in a storage container.
Further, since the cells to be frozen are vitrified and frozen in a state of being stored in a cryopreservation container, no impurities are mixed during freezing and there is no damage due to a high-speed spray flow. As a comparative example, for example, there is a possibility that impurities are mixed in a method of cooling by directly immersing cells in liquid nitrogen.
　また、凍結保存容器６０に収容される凍結対象の細胞に凍結保存液を極少量だけ添加した場合であっても、極低温微細固体粒子を含む高速噴霧流を連続して凍結保存容器６０に噴霧させて極めて短時間にガラス化凍結させることで、解凍時の細胞生存率が高くなる。 Further, even when only a very small amount of the cryopreservation liquid is added to the cells to be frozen contained in the cryopreservation container 60, a high-speed spray flow containing cryogenic fine solid particles is continuously sprayed onto the cryopreservation container 60. By vitrifying and freezing in a very short time, the cell survival rate upon thawing is increased.
　本発明の実施形態に係る極低温微細固体粒子による噴霧による急速ガラス化凍結について、詳細に説明する。
　極低温微細固体粒子による噴霧の場合、潜熱熱伝達による冷却の際、固体から液体に相変化する際の融解潜熱が付加される。 The rapid vitrification freezing by spraying with the cryogenic fine solid particles according to the embodiment of the present invention will be described in detail.
In the case of spraying with cryogenic fine solid particles, the latent heat of fusion at the time of phase change from solid to liquid is added during cooling by latent heat transfer.
　融解潜熱と潜熱熱伝達の相違点を説明する。液体（液滴）の場合、液相から気相へ相変化する際の蒸発潜熱のみとなる（液体窒素だと２１４．０ｋＪ／ｋｇの蒸発潜熱を有する）。
　極低温微細固体粒子の場合、固相から液相への融解潜熱（固体窒素だと２５．５６ｋＪ／ｋｇの融解潜熱を有する）と、上記の液相から気相変化時の蒸発潜熱の相乗効果となる。固体窒素を使用した場合、液体窒素に比較して１２％程度潜熱熱伝達による冷却特性が上昇する。実際、固体窒素が相変化を行う場合、固体から気体へ高速度で相変化を行うため、昇華に近い現象となる。固体粒子の場合、昇華潜熱熱伝達による冷却が行われるともいえる。
　極低温固体粒子の噴霧の場合、物体に衝突した極低温固体粒子は蒸気相へ高速で相変化を行うため、伝熱面（バイアル表面）が殆どぬれることなく、冷却が完了する。相変化速度は粒子径が小さいほど高速となる。 Differences between latent heat of fusion and latent heat transfer will be described. In the case of a liquid (droplet), only the latent heat of vaporization when the phase changes from the liquid phase to the gas phase (liquid nitrogen has a latent heat of vaporization of 214.0 kJ / kg).
In the case of ultra-low-temperature fine solid particles, the synergistic effect of the latent heat of fusion from the solid phase to the liquid phase (solid nitrogen has a latent heat of fusion of 25.56 kJ / kg) and the latent heat of vaporization when the gas phase changes from the above liquid phase It becomes. When solid nitrogen is used, the cooling characteristic by latent heat transfer is increased by about 12% compared to liquid nitrogen. In fact, when solid nitrogen undergoes a phase change, it undergoes a phase change at a high speed from a solid to a gas. In the case of solid particles, it can be said that cooling by sublimation latent heat transfer is performed.
In the case of spraying of cryogenic solid particles, the cryogenic solid particles that collide with the object undergo a phase change at high speed to the vapor phase, so that the cooling is completed with almost no heat transfer surface (vial surface) getting wet. The phase change rate increases as the particle size decreases.
　比較例として、極低温液体を直接接触させて冷却する方式の場合、極低温液体が伝熱面に接触した瞬間、伝熱面近傍（細胞近傍）で局所的な沸騰が発生し冷媒である極低温液体と伝熱面との間に蒸気膜が発生し、蒸気膜（気体）の熱伝導率は小さいことから、伝熱面から極低温液体への冷却熱伝達特性は著しく低下する。つまり、伝導冷却のみでは時間とともに冷却速度が遅くなる。
　伝熱面近傍において局所的に蒸気膜が生じる現象を遷移沸騰から膜沸騰状態と呼び、遷移沸騰から膜沸騰状態においては熱伝達特性が劣化することが知られている。特に、極低温液体の場合、冷却対象物との温度差が大きくなりやすいことから膜沸騰状態が生じやすいので液体を用いた冷却の欠点となり得る。また、液体窒素は表面張力が小さいので噴霧状態を作り出すことが困難である。
　また、水の場合、適度な表面張力を有するため少量では球形を維持しやすく、ノズル等の噴孔から噴出させた場合には容易に微小液滴を形成することが可能であるが、液体窒素の場合表面張力が水の１／６程度であり、また粘度が小さいため細長い噴流は形成可能でも液滴群からなる噴霧状態を作り出すことはきわめて困難である。
　また、水の場合、高圧を利用して噴霧を形成するが、極低温液体の場合高圧にすると液温が上昇し液相状態を維持できなくなるという困難さも有している。
　従って、本発明の実施形態に係る凍結装置による微細固体粒子の噴霧冷却と同様の冷却性能を、比較例としての液体状態による冷却で得ることは極めて困難といえる。 As a comparative example, in the case of a system in which a cryogenic liquid is directly contacted and cooled, the local boiling occurs near the heat transfer surface (near the cell) at the moment when the cryogenic liquid contacts the heat transfer surface. A vapor film is generated between the low-temperature liquid and the heat transfer surface, and since the heat conductivity of the vapor film (gas) is small, the cooling heat transfer characteristics from the heat transfer surface to the cryogenic liquid are significantly deteriorated. That is, with only conduction cooling, the cooling rate decreases with time.
A phenomenon in which a vapor film is locally generated in the vicinity of the heat transfer surface is called a transition boiling to a film boiling state, and it is known that heat transfer characteristics deteriorate in a transition boiling to a film boiling state. In particular, in the case of a cryogenic liquid, since the temperature difference from the object to be cooled tends to be large, a film boiling state is likely to occur, which can be a drawback of cooling using the liquid. Moreover, since liquid nitrogen has a small surface tension, it is difficult to produce a spray state.
In addition, in the case of water, since it has an appropriate surface tension, it is easy to maintain a spherical shape with a small amount, and when it is ejected from an injection hole such as a nozzle, it is possible to easily form fine droplets. In this case, since the surface tension is about 1/6 of water and the viscosity is small, it is very difficult to create a spray state composed of droplets even though a long and narrow jet can be formed.
Further, in the case of water, spraying is formed using high pressure. However, in the case of a cryogenic liquid, if the pressure is increased, the liquid temperature rises and it is difficult to maintain the liquid phase state.
Therefore, it can be said that it is extremely difficult to obtain the cooling performance similar to the spray cooling of fine solid particles by the freezing apparatus according to the embodiment of the present invention by cooling in the liquid state as a comparative example.
　また、極低温窒素ガス（ＧＮ2）の圧力を上げるなどの処理を行うことにより、極低温微細固体窒素粒子（ＳＮ2粒子）の粒子数密度を大きくする、高い粒子速度とする、などにより、冷却能力の向上、詳細には、温度降下速度を大きくすることができ、短時間で細胞をガラス化凍結させることができる。 In addition, by increasing the pressure of the cryogenic nitrogen gas (GN 2 ), the particle number density of the cryogenic fine solid nitrogen particles (SN 2 particles) is increased, the particle speed is increased, etc. Improvement of the cooling capacity, specifically, the temperature drop rate can be increased, and the cells can be vitrified and frozen in a short time.
　また、本発明の実施形態に係る凍結装置２００は、凍結保存容器６０を噴霧部としての極低温微細固体粒子連続生成装置１００から噴射される極低温微細固体粒子を含む噴霧流の下流側に支持する支持部７１と、支持部７１に支持された凍結保存容器６０に対する噴霧流の噴霧位置を変更する噴霧位置変更部７２（噴霧位置変更手段）を有する。
　支持部７１により、凍結対象の細胞などを収容した凍結保存容器６０を、極低温微細固体粒子連続生成装置１００から噴射される極低温微細固体粒子を含む噴霧流の下流側に支持し、噴霧位置変更部７２（噴霧位置変更手段）により、凍結保存容器６０内の凍結対象に対する凍結ムラを低減するように、凍結保存容器６０に対する噴霧流の噴霧位置を変更する。このように、凍結保存容器に対する噴霧位置をずらしながら、極低温微細固体粒子を含む噴霧流を噴霧することで、凍結保存容器内の細胞などの凍結対象を凍結ムラなく、短時間にガラス化凍結することができる。 Further, the freezing apparatus 200 according to the embodiment of the present invention is supported on the downstream side of the spray flow including the cryogenic fine solid particles ejected from the cryogenic fine solid particle continuous production apparatus 100 using the cryopreservation container 60 as a spraying unit. And a spray position changing portion 72 (spray position changing means) for changing the spray position of the spray flow with respect to the cryopreservation container 60 supported by the support portion 71.
The cryopreservation container 60 containing the cells to be frozen is supported by the support unit 71 on the downstream side of the spray flow containing the cryogenic fine solid particles ejected from the cryogenic fine solid particle continuous generation device 100, and the spray position The changing portion 72 (spray position changing means) changes the spray position of the spray flow with respect to the cryopreservation container 60 so as to reduce the freezing unevenness with respect to the object to be frozen in the cryopreservation container 60. In this way, by spraying the spray flow containing cryogenic fine solid particles while shifting the spray position with respect to the cryopreservation container, the object to be frozen such as cells in the cryopreservation container is vitrified and frozen in a short time without unevenness of freezing. can do.
　例えば、噴霧位置変更部７２は、支持部７１により支持された凍結保存容器を、上下方向、左右方向、前後方向など所定の方向に移動可能、所定の回転軸方向に回転可能に構成することで、凍結保存容器に対する噴霧位置を容易にずらすことができる。
　具体的には、底円筒状の細長の凍結保存容器を回転させる場合、例えば、細長円筒状の容器の軸を回転軸として回転させてもよい。また、底円筒状の細長の凍結保存容器の側面に対して、垂直に噴霧流を当てる場合と比較して、極低温微細固体粒子連続生成装置１００から噴射流を容器の側面に対して斜めに連続的に高速噴霧することで凍結速度が大きくなり、凍結時間を短くすることができる。
　また、底円筒状の細長の凍結保存容器の側面に対して、極低温微細固体粒子連続生成装置１００から噴射流を容器の側面に対して斜めに連続的に高速噴霧し、且つ、細長円筒状の凍結保存容器の軸を回転軸として回転させることで、凍結速度がさらに大きくなり、凍結時間をさらに短くすることができる。 For example, the spray position changing unit 72 is configured such that the cryopreservation container supported by the support unit 71 can be moved in a predetermined direction such as an up-down direction, a left-right direction, and a front-rear direction, and can be rotated in a predetermined rotation axis direction. The spray position with respect to the cryopreservation container can be easily shifted.
Specifically, when rotating the bottom cylindrical elongated cryopreservation container, for example, the axis of the elongated cylindrical container may be rotated about the rotation axis. Moreover, compared with the case where a spray flow is applied perpendicularly to the side surface of an elongated cryopreservation container having a bottom cylindrical shape, the jet flow from the cryogenic fine solid particle continuous generation device 100 is inclined with respect to the side surface of the container. By continuously spraying at a high speed, the freezing speed increases and the freezing time can be shortened.
In addition, the jet flow from the cryogenic fine solid particle continuous production apparatus 100 is continuously sprayed on the side surface of the elongated cylindrical cryopreservation container obliquely at a high speed obliquely to the side surface of the container, and the elongated cylindrical shape is formed. By rotating about the axis of the cryopreservation container as a rotation axis, the freezing speed can be further increased and the freezing time can be further shortened.
　また、本発明の実施形態に係る凍結装置に用いられる極低温微細固体粒子は、窒素、二酸化炭素、アルゴン、水素のいずれか１つ、又は、２つ以上の組み合わせにより構成されている。特に、極低温微細固体粒子として窒素を用いることで、低い冷却コスト、高い効率で細胞などをガラス化凍結することができる。
　極低温微細固体窒素粒子の噴霧流は、現存する冷媒の中でも非常に高い寒冷エンタルピ（冷却能）を有し、生体組織に対して無害であり、高速急冷によるガラス凍結用の冷媒として適している。 Moreover, the cryogenic fine solid particles used in the freezing apparatus according to the embodiment of the present invention are configured by any one of nitrogen, carbon dioxide, argon, hydrogen, or a combination of two or more. In particular, by using nitrogen as the cryogenic fine solid particles, cells and the like can be vitrified and frozen with low cooling cost and high efficiency.
The spray flow of cryogenic fine solid nitrogen particles has a very high cold enthalpy (cooling ability) among existing refrigerants, is harmless to living tissues, and is suitable as a refrigerant for freezing glass by high-speed rapid cooling .
　本発明の実施形態に係る凍結装置の凍結対象は、ｉＰＳ細胞、ＥＳ細胞、血液、植物細胞、食品（魚卵等）、などを挙げることができ、凍結対象の適用範囲が広い。冷凍食品などの食品に対して、本発明の実施形態に係る凍結装置により凍結保存することにより、凍結保存液を極微量、または、全く使用せずに凍結することができる。 The freezing object of the freezing apparatus according to the embodiment of the present invention can include iPS cells, ES cells, blood, plant cells, food (eg, fish eggs), and the like, and the application range of the freezing object is wide. By cryopreserving foods such as frozen food with the freezing apparatus according to the embodiment of the present invention, the cryopreservation solution can be frozen in a trace amount or not at all.
　また、本発明の実施形態に係る凍結装置の噴霧部は、一成分極低温微細固体粒子連続生成装置である。詳細には、極低温微細固体粒子連続生成装置１００は、極低温の過冷却液体と、その過冷却液体と同一の元素で構成される極低温気体の高速流とを混合して一成分混相流を生成する混合部１０と、混合部１０の下流に設けられ、その混合部で生成された一成分混相流から極低温微細固体粒子を含む噴霧流を生成するラバルノズル部１１を有する。このラバルノズル部１１は、混合部１０で生成された一成分混相流を導入する導入部１１ａと、導入部１１ａの下流側に設けられ、導入部１１ａの開口断面積よりも小さい開口断面積の縮径部１１ｂと、縮径部１１ｂの下流側に設けられ、縮径部１１ｂより開口断面積よりも大きい開口断面積に形成され、且つ、下流側に向かって拡開した形状の噴射部１１ｃと、を有する。噴射部１１ｃにて、一成分混相流を音速を超えた状態で断熱膨張させて、一成分極低温微細固体粒子を含む噴霧流を連続して生成する。
　このため、極低温微細固体粒子連続生成装置１００は、簡単な構造で、窒素などを用いた一成分極低温微細固体粒子を含む噴霧流を連続して生成することができる。
　また、一成分方式（ＬＮ2－ＧＮ2）によって生成された微細固体窒素噴霧では、極低温ヘリウムガスを使用する二成分方式（ＬＮ2－ＧＨｅ）によって生成された微細固体窒素噴霧に比べるとＳＮ2粒子の数密度が増大するため、冷却効果が大きい。
　また、一成分方式（ＬＮ2－ＧＮ2）の極低温微細固体粒子連続生成装置は、寒剤として極低温ヘリウムを用いることなく、簡単な構成で、安価に、一成分で極低温の微細固体粒子を連続生成することができる。 Moreover, the spray part of the freezing apparatus which concerns on embodiment of this invention is a one-component cryogenic fine solid particle continuous production | generation apparatus. Specifically, the cryogenic fine solid particle continuous generation apparatus 100 mixes a cryogenic supercooled liquid with a high-speed flow of a cryogenic gas composed of the same elements as the supercooled liquid, thereby producing a one-component mixed phase flow. And a Laval nozzle unit 11 that is provided downstream of the mixing unit 10 and generates a spray flow including cryogenic fine solid particles from a one-component mixed phase flow generated in the mixing unit. The Laval nozzle portion 11 is provided on the downstream side of the introduction portion 11a and the introduction portion 11a for introducing the one-component mixed phase flow generated by the mixing portion 10, and has a smaller opening cross-sectional area than the opening cross-sectional area of the introduction portion 11a. A diameter portion 11b, and an injection portion 11c that is provided on the downstream side of the reduced diameter portion 11b, has an opening cross-sectional area that is larger than the opening cross-sectional area than the reduced diameter portion 11b, and has a shape that expands toward the downstream side. Have. In the injection unit 11c, the one-component mixed phase flow is adiabatically expanded in a state exceeding the speed of sound, and a spray flow including one-component cryogenic fine solid particles is continuously generated.
For this reason, the cryogenic fine solid particle continuous production | generation apparatus 100 can produce | generate the spray flow containing the one-component cryogenic fine solid particle using nitrogen etc. continuously with a simple structure.
In addition, the fine solid nitrogen spray generated by the one-component method (LN 2 -GN 2 ) is SN compared to the fine solid nitrogen spray generated by the two-component method (LN 2 -GHe) using cryogenic helium gas. Since the number density of two particles increases, the cooling effect is large.
In addition, the one-component (LN 2 -GN 2 ) cryogenic fine solid particle continuous generator is simple and inexpensive, and does not use cryogenic helium as a cryogen. Can be continuously generated.
　また、本発明の実施形態に係る凍結装置２００の極低温微細固体粒子連続生成装置１００は、ラバルノズル部１１の先端部にスパイラルノズル１８を備えていてもよい。ラバルノズル部１１の先端部にスパイラルノズル１８を設けることにより、さらに、極低温微細固体粒子を微細化させることができる。 Moreover, the cryogenic fine solid particle continuous generation apparatus 100 of the freezing apparatus 200 according to the embodiment of the present invention may include a spiral nozzle 18 at the tip of the Laval nozzle section 11. By providing the spiral nozzle 18 at the tip of the Laval nozzle portion 11, the cryogenic fine solid particles can be further refined.
　また、本発明の実施形態に係る凍結装置２００の極低温微細固体粒子連続生成装置１００はラバルノズル部１１に超音波を印加する超音波振動子６を有する。
　この超音波振動子６で生成した超音波をラバルノズル部１１に印加することで、ラバルノズル部１１内の一成分混相流にキャビテーションを発生させ、極低温微細固体粒子の氷核生成を促進し、且つ、微小均一粒子径の略球形状の一成分極低温微細固体粒子の微細化を促進することができる。
　また、上述したように、超音波振動子による超音波をラバルノズル部に印加した場合、極低温微細固体粒子の粒径が小さくなり、流速は減少が、超音波付加条件下では、氷核生成促進により凍結粒子を形成しやすくなり、微粒化促進効果も加わるので粒子数密度が増大し、噴霧照射部におけるトータルの接触伝熱面積が増大し、結果的に冷却効果が増大する。また、粒子径が小さくなったことで蒸発しやすくなり、粒子凝集による膜沸騰状態をさらに形成しにくくし、固体粒子の高速蒸気相変化を利用した潜熱冷却が促進される。つまり、超音波付加条件下では、流速が小さくなったことで強制対流冷却効果は若干減少するが、接触熱伝達と潜熱熱伝達特性は増加し、冷却効果が増大する。 Moreover, the cryogenic fine solid particle continuous generation apparatus 100 of the freezing apparatus 200 according to the embodiment of the present invention includes an ultrasonic transducer 6 that applies ultrasonic waves to the Laval nozzle unit 11.
By applying the ultrasonic wave generated by the ultrasonic vibrator 6 to the Laval nozzle unit 11, cavitation is generated in the one-component mixed phase flow in the Laval nozzle unit 11, promoting the ice nucleus generation of the cryogenic fine solid particles, and Further, it is possible to promote the miniaturization of one-component cryogenic fine solid particles having a substantially spherical shape with a fine uniform particle diameter.
In addition, as described above, when ultrasonic waves from an ultrasonic transducer are applied to the Laval nozzle, the particle size of the cryogenic fine solid particles is reduced and the flow velocity is reduced. As a result, it becomes easier to form frozen particles and the effect of promoting atomization is added, so that the number density of particles increases, the total contact heat transfer area in the spray irradiation section increases, and the cooling effect increases as a result. In addition, since the particle diameter is reduced, it is easy to evaporate, and it is difficult to form a film boiling state due to particle aggregation, and latent heat cooling using high-speed vapor phase change of solid particles is promoted. That is, under the ultrasonic wave application condition, the forced convection cooling effect is slightly reduced due to the decrease in the flow velocity, but the contact heat transfer and latent heat transfer characteristics are increased and the cooling effect is increased.
　また、本発明の実施形態に係る凍結装置２００の極低温微細固体粒子連続生成装置１００は、ラバルノズル部１１の全部または一部を外気に対して真空断熱する断熱部５を有する。詳細には、ノズル１、液体窒素導管３、窒素ガス導管４の先端部付近が真空断熱部５内に収容されており、ノズル１や液体窒素導管３、窒素ガス導管４の温度上昇を低減した構造となっている。このため簡単な構造で、比較的長時間、安定して極低温微細固体粒子を含む高速噴霧流をノズル１のラバルノズル部１１から噴射させることができる。 Also, the cryogenic fine solid particle continuous production apparatus 100 of the freezing apparatus 200 according to the embodiment of the present invention has a heat insulating part 5 that vacuum-insulates all or part of the Laval nozzle part 11 with respect to the outside air. Specifically, the vicinity of the tip of the nozzle 1, the liquid nitrogen conduit 3, and the nitrogen gas conduit 4 is accommodated in the vacuum heat insulating portion 5, and the temperature rise of the nozzle 1, the liquid nitrogen conduit 3, and the nitrogen gas conduit 4 is reduced. It has a structure. Therefore, a high-speed spray flow containing a cryogenic fine solid particle can be stably ejected from the Laval nozzle portion 11 of the nozzle 1 with a simple structure for a relatively long time.
　また、上記実施形態では、凍結装置の噴霧部は、一成分極低温微細固体粒子生成連続装置であったが、この形態に限られるものではなく、凍結装置の噴霧部は、二成分極低温微細固体粒子連続生成装置であってもよい。二成分極低温微細固体粒子連続生成装置は、例えば、過冷却状態の液体窒素などを液体ヘリウムなどの冷媒で冷却することで、極低温微細固体粒子を含む高速噴霧流を生成してもよい。 Further, in the above embodiment, the spray unit of the freezing device is a one-component cryogenic fine solid particle production continuous device, but is not limited to this form, and the spraying unit of the freezing device is a two-component cryogenic fine particle It may be a solid particle continuous production apparatus. The two-component cryogenic fine solid particle continuous production apparatus may produce a high-speed spray flow containing cryogenic fine solid particles by cooling supercooled liquid nitrogen or the like with a refrigerant such as liquid helium, for example.
　また、本発明の実施形態に係る凍結方法は、凍結対象を収容する凍結保存容器を支持部により噴霧流の下流側に支持し、噴霧部としての極低温微細固体粒子連続生成装置１００が、凍結保存容器６０に極低温微細固体粒子を含む噴霧流を連続して噴霧して、凍結保存容器に収容された凍結対象をガラス化凍結させることで、解凍時に高い細胞生存率となるように、簡単に、細胞などの凍結対象を急速にガラス化凍結することができる。 Further, in the freezing method according to the embodiment of the present invention, the cryopreservation container containing the object to be frozen is supported downstream of the spray flow by the support unit, and the cryogenic fine solid particle continuous generation apparatus 100 as the spray unit is frozen. By spraying the spray container containing cryogenic fine solid particles continuously into the storage container 60 and vitrifying and freezing the object to be frozen stored in the cryopreservation container, it is easy to achieve a high cell viability upon thawing. Furthermore, it is possible to rapidly vitrify an object to be frozen such as a cell.
　尚、上記実施形態では、凍結装置は、噴霧部により、凍結保存容器に極低温微細固体粒子を含む噴霧流を連続して噴霧して、凍結保存容器に収容された凍結対象をガラス化凍結させたが、この形態に限られるものではない。例えば、凍結装置は、噴霧部により、凍結対象に対して直接、極低温微細固体粒子を含む噴霧流を連続して噴霧して、凍結対象をガラス化凍結させてもよい。 In the above embodiment, the freezing apparatus continuously sprays the cryogenic container containing the cryogenic fine solid particles onto the cryopreservation container by the spraying unit to vitrify and freeze the object to be frozen contained in the cryopreservation container. However, it is not limited to this form. For example, the freezing apparatus may spray the object to be frozen by vitrification by continuously spraying the object to be frozen directly with the spray flow containing the cryogenic fine solid particles by the spray unit.
　尚、上記実施形態では、窒素（Ｎ2）を用いた極低温微細固体粒子連続生成装置１００を示したが、この形態でなくともよい。例えば、水素（Ｈ2）、酸素（Ｏ2）、アルゴン（Ａｒ）などを採用してもよい。
　例えば、水素（Ｈ2）を採用した場合、極低温の過冷却液体水素（ＧＨ2）と極低温水素ガス（ＬＨ2）の一成分混相流を高速にラバルノズル部内に導入し、ラバルノズル部の噴射部にて、一成分混相流を音速（混相流の音速）を超えた状態で断熱膨張させて、一成分極低温微細固体粒子を含む噴霧流を連続して生成する。
　酸素（Ｏ2）を採用した場合、極低温の過冷却液体酸素（ＧＯ2）と極低温酸素ガス（ＬＯ2）の一成分混相流を高速にラバルノズル部内に導入し、ラバルノズル部の噴射部にて、一成分混相流を音速（混相流の音速）を超えた状態で断熱膨張させて、一成分極低温微細固体粒子を含む噴霧流を連続して生成する。
　アルゴン（Ａｒ）を採用した場合、極低温の過冷却液体アルゴンと極低温アルゴンガスの一成分混相流を高速にラバルノズル部内に導入し、ラバルノズル部の噴射部にて、一成分混相流を音速（混相流の音速）を超えた状態で断熱膨張させて、一成分極低温微細固体粒子を含む噴霧流を連続して生成する。 In the above embodiment, although the nitrogen (N 2) cryogenic fine solid particles continuously generating apparatus 100 using the, may not in this form. For example, hydrogen (H 2 ), oxygen (O 2 ), argon (Ar), or the like may be employed.
For example, when hydrogen (H 2 ) is used, a one-component mixed phase flow of cryogenic supercooled liquid hydrogen (GH 2 ) and cryogenic hydrogen gas (LH 2 ) is introduced into the Laval nozzle at a high speed, and the Laval nozzle is injected. In the section, the one-component multiphase flow is adiabatically expanded in a state exceeding the speed of sound (the speed of sound of the multiphase flow) to continuously generate a spray flow containing one-component cryogenic fine solid particles.
When oxygen (O 2 ) is used, a one-component mixed phase flow of supercooled liquid oxygen (GO 2 ) and cryogenic oxygen gas (LO 2 ) at a low temperature is introduced into the Laval nozzle at a high speed, and injected into the injection part of the Laval nozzle. Thus, the one-component multiphase flow is adiabatically expanded in a state exceeding the speed of sound (sound speed of the multiphase flow) to continuously generate a spray flow containing one-component cryogenic fine solid particles.
When argon (Ar) is used, a one-component mixed phase flow of supercooled liquid argon and cryogenic argon gas at a low temperature is introduced into the Laval nozzle portion at high speed, and the one-component mixed phase flow is sonic ( Adiabatic expansion is performed in a state exceeding the sound velocity of the multiphase flow, and a spray flow containing one-component cryogenic fine solid particles is continuously generated.
　尚、極低温とは、一般的な低温（０℃）よりも低い温度であり、且つ、窒素、水素、ヘリウム、アルゴンなどの標準気圧での沸点程度以下の温度である。詳細には、例えば、窒素（Ｎ2）を用いた場合、標準気圧での窒素の沸点である７７．３６Ｋ（－１９５．７９℃）程度以下の温度であり、水素（Ｈ2）を用いた場合、標準気圧での水素の沸点である２０．２８Ｋ（－２５２．８７℃）程度以下の温度であり、酸素（Ｏ2）を用いた場合、標準気圧での酸素の沸点である９０．２Ｋ（－１８２．９６℃）程度以下の温度であり、アルゴン（Ａｒ）を用いた場合、８３．８０Ｋ（－１８９．３５℃）程度以下の温度である。 Note that the cryogenic temperature is a temperature lower than a general low temperature (0 ° C.) and a temperature equal to or lower than the boiling point at a standard pressure such as nitrogen, hydrogen, helium, or argon. Specifically, for example, when nitrogen (N 2 ) is used, the temperature is about 77.36 K (−195.79 ° C.), which is the boiling point of nitrogen at standard pressure, and hydrogen (H 2 ) is used. In this case, the temperature is about 20.28 K (−252.87 ° C.) or less, which is the boiling point of hydrogen at the standard pressure, and when oxygen (O 2 ) is used, the boiling point of oxygen at the standard pressure is 90.2 K. The temperature is about (−182.96 ° C.) or less, and when argon (Ar) is used, the temperature is about 83.80 K (−189.35 ° C.) or less.
　また、本実施形態では、極低温の過冷却液体を用いたが、この形態に限られるものではなく、例えば、過冷却状態でない、極低温の液体を用いてもよい。尚、極低温の過冷却液体を用いることにより、短時間に、容易に、一成分極低温微細固体粒子を生成することが可能である。 In this embodiment, a cryogenic supercooled liquid is used. However, the present invention is not limited to this mode. For example, a cryogenic liquid that is not in a supercooled state may be used. In addition, by using a cryogenic supercooled liquid, it is possible to easily produce one-component cryogenic fine solid particles in a short time.
　本発明の実施形態に係る凍結装置によれば、極低温微細固体粒子を含む高速噴霧流の活用により、超高熱流速による細胞のガラス化凍結制御を行うことができる。氷核規模縮小と凍結保存剤等不純物の混入を極力減らすことが可能となり、高生存率のｉＰＳ細胞やＥＳ細胞などの各種細胞を、高品質に氷核生成を抑え、急速に冷凍することによりガラス化、凍結保存技術の確立が可能となる。
　マイクロ・ナノレベルの極低温微細固体粒子の有する超高熱流束冷却効果により、従来のｉＰＳ細胞凍結法では不可能であった、凍結保護液不要かつ氷核生成を極力抑えた超高速ガラス凍結法を可能にし、生命化学領域、医用工学領域、各種医療産業へのこの新技術を提供することができる。また、この技術は、医学・医工学・生命科学分野のみならず、広範囲の異分野産業領域にわたって貢献度の高い技術となる。 According to the freezing apparatus which concerns on embodiment of this invention, vitrification freezing control of the cell by a super-high heat flow rate can be performed by utilization of the high-speed spray flow containing a cryogenic fine solid particle. By reducing the size of ice nuclei and mixing impurities such as cryopreservatives as much as possible, various cells such as iPS cells and ES cells with high survival rate can be frozen at high quality and rapidly frozen. Vitrification and cryopreservation technology can be established.
Ultra-high-speed glass freezing method that does not require cryoprotection solution and suppresses ice nucleation as much as possible with conventional iPS cell freezing method due to ultra-high heat flux cooling effect of micro / nano level cryogenic fine solid particles And can provide this new technology to the biochemical field, medical engineering field, and various medical industries. In addition, this technology has a high contribution not only in the medical, medical engineering, and life science fields but also in a wide range of different industrial fields.
　以上、本発明の実施形態について図面を参照して詳述してきたが、具体的な構成はこれらの実施形態に限られるものではなく、本発明の要旨を逸脱しない範囲の設計の変更等があっても本発明に含まれる。
　また、上述の各図で示した実施形態は、その目的及び構成等に特に矛盾や問題がない限り、互いの記載内容を組み合わせることが可能である。
　また、各図の記載内容はそれぞれ独立した実施形態になり得るものであり、本発明の実施形態は各図を組み合わせた一つの実施形態に限定されるものではない。 As described above, the embodiments of the present invention have been described in detail with reference to the drawings. However, the specific configuration is not limited to these embodiments, and there are design changes and the like without departing from the gist of the present invention. Is included in the present invention.
Further, the embodiments described in the above drawings can be combined with each other as long as there is no particular contradiction or problem in the purpose and configuration.
Moreover, the description content of each figure can become independent embodiment, respectively, and embodiment of this invention is not limited to one embodiment which combined each figure.
　１　ノズル
　３　液体窒素導管
　３ａ　バルブ
　４　窒素ガス導管
　４ａ　バルブ
　５　断熱部（真空断熱部）
　６　超音波振動子
　６ａ　超音波振動生成部
　６ｂ　超音波伝達部
　７　可動皿部
　１０　混合部
　１１　ラバルノズル部（ラバール・ノズル）
　１１ａ　導入部
　１１ｂ　縮径部（スロート部）
　１１ｃ　噴射部（拡開部）
　１８　スパイラルノズル
　６０　凍結保存容器
　６１　凍結対象
　７０　支持装置
　７１　支持部
　７２　噴霧位置変更部（噴霧位置変更手段）
　８１　駆動部
　８２　制御部
　１００　極低温微細固体粒子連続生成装置（噴霧部）
　２００　凍結装置 1 Nozzle 3 Liquid Nitrogen Conduit 3a Valve 4 Nitrogen Gas Conduit 4a Valve 5 Heat Insulation (Vacuum Insulation)
6 Ultrasonic vibrator 6a Ultrasonic vibration generating section 6b Ultrasonic transmission section 7 Movable dish section 10 Mixing section 11 Laval nozzle section (Laval nozzle)
11a introduction part 11b reduced diameter part (throat part)
11c injection part (expansion part)
18 Spiral nozzle 60 Cryopreservation container 61 Object to be frozen 70 Support device 71 Support unit 72 Spray position change unit (spray position change means)
81 Drive part 82 Control part 100 Cryogenic fine solid particle continuous production apparatus (spray part)
200 Freezing device

Claims (10)

  1. 　凍結対象として水分を含む弾性体膜包の凍結装置であって、
    　前記凍結対象を収容する凍結保存容器と、
    　前記凍結保存容器に極低温微細固体粒子を含む噴霧流を連続して噴霧して、前記凍結保存容器に収容された凍結対象をガラス化凍結させる噴霧部と、を有することを特徴とする
    　凍結装置。     A freezing device for elastic membrane capsules containing moisture as a freezing object,
    A cryopreservation container containing the object to be frozen;
    A spraying unit for continuously spraying a spray stream containing cryogenic fine solid particles on the cryopreservation container to vitrify and freeze the object to be frozen housed in the cryopreservation container. .
  2. 　前記凍結保存容器は、前記凍結対象として細胞を収容し、
    　前記噴霧部は、前記凍結保存容器に極低温微細固体粒子を含む噴霧流を連続して噴霧して、前記凍結保存容器に収容された細胞をガラス化凍結させることを特徴とする
    　請求項１に記載の凍結装置。     The cryopreservation container contains cells as the object to be frozen,
    The spray unit continuously sprays a spray flow containing cryogenic fine solid particles on the cryopreservation container to vitrify and freeze the cells stored in the cryopreservation container. The freezing device described.
  3. 　前記凍結保存容器を前記噴霧部から噴射される前記極低温微細固体粒子を含む噴霧流の下流側に支持する支持部と、
    　前記支持部に支持された前記凍結保存容器に対する前記噴霧流の噴霧位置を変更する噴霧位置変更手段と、を有することを特徴とする請求項１または請求項２に記載の凍結装置。     A support part for supporting the cryopreservation container on the downstream side of the spray flow containing the cryogenic fine solid particles ejected from the spray part;
    The freezing apparatus according to claim 1 or 2, further comprising spray position changing means for changing a spray position of the spray flow with respect to the cryopreservation container supported by the support portion.
  4. 　前記極低温微細固体粒子は、窒素、二酸化炭素、アルゴン、水素のいずれか１つ、又は、２つ以上の組み合わせにより構成されていることを特徴とする請求項１から請求項３のいずれかに記載の凍結装置。 4. The cryogenic fine solid particles are constituted by any one of nitrogen, carbon dioxide, argon, and hydrogen, or a combination of two or more thereof. The freezing device described.
  5. 　前記凍結対象は、ｉＰＳ細胞、ＥＳ細胞、血液、植物細胞、食品のいずれかであることを特徴とする請求項１に記載の凍結装置。 The freezing apparatus according to claim 1, wherein the object to be frozen is any one of iPS cells, ES cells, blood, plant cells, and food.
  6. 　前記噴霧部は、一成分極低温微細固体粒子連続生成装置であり、
    　極低温の過冷却液体と、該過冷却液体と同一の元素で構成される極低温気体の高速流とを混合して一成分混相流を生成する混合部と、
    　前記混合部の下流に設けられ、該混合部で生成された前記一成分混相流から極低温微細固体粒子を含む噴霧流を生成するラバルノズル部と、を有し、
    　前記ラバルノズル部は、前記混合部で生成された前記一成分混相流を導入する導入部と、
    　前記導入部の下流側に設けられ該導入部の開口断面積よりも小さい開口断面積の縮径部と、
    　前記縮径部の下流側に設けられ該縮径部より開口断面積よりも大きい開口断面積に形成され、且つ、下流側に向かって拡開した形状の噴射部と、を有し、
    　前記噴射部にて、前記一成分混相流を音速を超えた状態で断熱膨張させて、一成分極低温微細固体粒子を含む噴霧流を連続して生成することを特徴とする請求項１から請求項５のいずれかに記載の凍結装置。     The spray unit is a one-component cryogenic fine solid particle continuous production device,
    A mixing section that mixes a cryogenic supercooled liquid with a high-speed flow of a cryogenic gas composed of the same element as the supercooled liquid to generate a one-component mixed phase flow;
    A Laval nozzle unit that is provided downstream of the mixing unit and generates a spray flow containing cryogenic fine solid particles from the one-component mixed phase flow generated in the mixing unit,
    The Laval nozzle part introduces the one-component mixed phase flow generated in the mixing part, and
    A reduced diameter portion having an opening cross-sectional area smaller than the opening cross-sectional area of the introduction portion provided on the downstream side of the introduction portion;
    An injection portion provided on the downstream side of the reduced diameter portion, formed in an opening cross-sectional area larger than the opening cross-sectional area than the reduced diameter portion, and having a shape expanded toward the downstream side,
    The spray unit continuously generates a spray flow including one-component cryogenic fine solid particles by adiabatically expanding the one-component multiphase flow in a state exceeding the speed of sound in the injection unit. Item 6. The freezing apparatus according to any one of Items 5.
  7. 　前記ラバルノズル部に超音波を印加する超音波振動子を有することを特徴とする請求項６に記載の凍結装置。 The freezing apparatus according to claim 6, further comprising an ultrasonic vibrator for applying an ultrasonic wave to the Laval nozzle portion.
  8. 　前記凍結保存容器は、前記凍結対象と微量の凍結保存液を収容していることを特徴とする請求項１から請求項７のいずれかに記載の凍結装置。 The freezing apparatus according to any one of claims 1 to 7, wherein the cryopreservation container contains the object to be frozen and a small amount of a cryopreservation solution.
  9. 　前記凍結装置の噴霧部は、二成分極低温微細固体粒子連続生成装置であることを特徴とする請求項１から請求項５のいずれかに記載の凍結装置。 The freezing apparatus according to any one of claims 1 to 5, wherein the spraying section of the freezing apparatus is a two-component cryogenic fine solid particle continuous production apparatus.
  10. 　凍結対象として水分を含む弾性体膜包の凍結装置の凍結方法であって、
    　前記凍結装置は、前記凍結対象を収容する凍結保存容器と、
    　前記凍結保存容器に極低温微細固体粒子を含む噴霧流を噴霧する噴霧部と、を有し、
    　前記凍結対象を収容する凍結保存容器を支持部により噴霧流の下流側に支持し、
    　前記噴霧部が、前記凍結保存容器に極低温微細固体粒子を含む噴霧流を連続して噴霧して、前記凍結保存容器に収容された凍結対象をガラス化凍結させることを特徴とする
    　凍結方法。     A freezing method of a freezing apparatus for elastic membrane capsules containing water as a freezing object,
    The freezing device includes a cryopreservation container that houses the object to be frozen,
    Spraying a spray stream containing cryogenic fine solid particles in the cryopreservation container,
    Support the cryopreservation container containing the object to be frozen on the downstream side of the spray flow by the support,
    The said spraying part sprays the spray flow containing a cryogenic fine solid particle continuously in the said cryopreservation container, and freezes the object to be frozen accommodated in the said cryopreservation container, The freezing method characterized by the above-mentioned.
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