Cell freezing system

Abstract

To provide a cooling device and a cooling method causing little intracellular ice crystal formation in spite of rapid cooling.SOLUTION: A cell freezing system according to the present invention includes a slurry tank that stores a secondary coolant, and an ice making unit that is disposed inside the slurry tank and is immersed in the secondary coolant, wherein the ice making unit includes an ice making plate that circulates a primary coolant supplied from a cylinder of liquid nitrogen therein and has an ice making surface that generates ice of the secondary coolant on at least one side surface, a rotation unit that rotates or rotationally reciprocates with respect to the ice making surface, and a flow control unit that is configured as a wall including a curved surface along a part of an outer circumference of a rotation circle of the rotation unit. A freezing work area in which the cells are rapidly frozen by directly immersing the cells in the secondary refrigerant.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to a cell freezing system that produces an ice slurry for freezing, for example, animal or plant cells. [Background technology]

[0002] Cryopreservation of biological materials, such as cells, tissues, organs, blood products, embryos, sperm, stem cells, and fish eggs, requires freezing the biological material to a sufficiently low temperature. Cryopreservation of biological materials requires not only freezing the biological material but also preserving its viability, i.e., its ability to resume normal biological function after thawing. Generally, when biological materials are frozen, the internal fluid undergoes a phase transition, which can lead to the formation of ice crystals. The formation of ice crystals can damage the biological material, making it unlikely to be viable after thawing.

[0003] Therefore, there is a need to ensure the viability of cells that require transportation for biomedical applications such as cell therapy, regenerative medicine, tissue engineering, etc. Therefore, it is desirable to optimize cryopreservation conditions, especially when cryopreserving cells for therapeutic use.

[0004] Traditionally, freezing of living cells has been classified into slow and rapid cooling. A freezing rate of approximately 1-10°C / min or less is often called slow (slow) cooling, while a rate of approximately 100°C / min or more is called rapid cooling. In slow cooling, extracellular water freezes before ice forms inside the cells. Supercooled water inside the cells has a higher vapor pressure than the frozen water outside the cells, so it moves outside the cell membrane and freezes. Increased intracellular solute concentrations have a detrimental effect on cell survival. On the other hand, excessive water retention inside the cells can cause damage due to intracellular ice crystal formation and recrystallization during thawing, leading to cell death. The cooling rate has a significant impact on these phenomena.

[0005] Rapid cooling minimizes the effect of solute concentration because ice is formed uniformly, but because water is not moving out of the cell, more intracellular ice forms. On the other hand, slow cooling increases water loss from the cell, reducing internal ice formation but increasing the solution effect. Both ice crystal formation and solute effects contribute to cell damage, and optimal cooling rates can minimize their respective effects. Thermo Scientific's cryogenic storage guide recommends a cooling rate of 1°C / min. This slow cooling rate minimizes intracellular ice crystal formation over a long period of time, thereby avoiding cell damage. However, slow freezing has drawbacks, such as the long time required for complete freezing and low cell viability after thawing.

[0006] Cryoprotectants such as dimethyl sulfoxide (DMSO), glycerin, ethylene glycol (EG), and propylene glycol (PG) are used to inhibit the formation of ice crystals inside and outside cells. Rapid freezing methods using cryoprotectants are known. For example, rapid freezing involves immersing cells in a cryopreservation solution containing a high concentration of DMSO, then rapidly cooling them in a sealed space using liquid nitrogen or other methods to supercool the solution. Rapid freezing controls the movement of water molecules, suppressing the formation of ice crystals and thereby reducing freezing-related cell damage. Freezing methods using cryoprotectants are also called vitrification freezing because they suppress ice crystal formation and induce vitrification. Vitrification freezing transforms everything, including the extracellular fluid, into a glass-like state, eliminating damage from ice crystals. In theory, any type of cell can be cryopreserved with a high survival rate.

[0007] Patent Document 1 discloses a slow vitrification cryopreservation method. Rapid vitrification allows for faster freezing than slow freezing and is expected to maintain viability by preventing intracellular freezing. However, because an extremely fast cooling rate is required, specialized equipment and techniques are required.

[0008] For example, by directly immersing the sample in liquid nitrogen, intracellular freezing can be prevented and the viability can be maintained by ultra-rapid freezing. Patent Document 2 describes a method in which a biological sample such as a cell is sealed in a biological sample cryopreservation container, and then directly immersed in liquid nitrogen in the container filled with liquid nitrogen, or frozen in the gas phase above the liquid nitrogen. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] Japanese Patent Publication No. 2022-8269 [Patent Document 2] Japanese Patent Publication No. 2023-106615 [Non-Patent Document 1] https: / / static.thermoscientific.com / images / D21111~.pdf Summary of the Invention [Problem to be solved by the invention]

[0010] However, if samples are directly immersed in liquid nitrogen, the cryogenic temperature of -196°C is too low, and even if they are vitrified, there is a risk of cell damage.Furthermore, if samples are frozen in the vapor phase above liquid nitrogen, the cooling rate is slow and there is a risk of them not being vitrified.

[0011] An object of the present invention is to provide a cooling device and a cooling method that achieve rapid cooling while minimizing intracellular ice crystal formation. [Means for solving the problem]

[0012] (1) To solve the above problems, the present invention provides a slurry tank for storing a secondary refrigerant; The cell freezing system includes an ice-making unit disposed inside the slurry tank and immersed in the secondary refrigerant, the ice-making unit having an ice-making plate having an ice-making surface that circulates the primary refrigerant supplied from a liquid nitrogen cylinder therein and produces ice of the secondary refrigerant on at least one side thereof, a rotating unit that rotates or reciprocates in rotation relative to the ice-making surface, a flow control unit configured as a wall including a curved surface that follows part of the outer periphery of the rotation circle of the rotating unit, and a freezing work area in which cells are rapidly frozen by directly immersing them in the secondary refrigerant. (2) Another invention for solving the above problem is a cell freezing system as described in claim 1, in which the secondary refrigerant is adjusted so that the temperature of the slurry in the slurry tank is -120°C to -150°C. (3) Another invention for solving the above problem is the cell freezing system according to claim 1, wherein the secondary refrigerant is an aqueous ethanol solution. (4) Another invention for solving the above problem is a cell freezing system as described in claim 1, which includes a sensor that measures the temperature of the slurry in the slurry tank and controls the supply amount of the primary refrigerant or the rotation speed of the rotating part based on the temperature. (5) Another invention for solving the above problem is a cell freezing system described in any one of claims 2 to 4, wherein the rotating unit includes a secondary refrigerant flow generating unit that generates a flow of the secondary refrigerant, and a scraping unit that separates ice formed on the ice-making surface from the ice-making surface. (6) Another invention for solving the above problem is a cell freezing system as described in claim 5, wherein the flow control section is configured as a wall including a curved surface along an arc having a central angle of approximately 180° of the rotation circle of the scraping section. (7) Another invention for solving the above problem is the cell freezing system according to claim 1, wherein the rotating parts are arranged both above and below the ice making plate. [Effects of the Invention]

[0013] According to the present invention, it is possible to provide a cooling device and a cooling method that achieve rapid cooling while minimizing intracellular ice crystal formation. [Brief explanation of the drawings]

[0014] [Figure 1] 1 is a perspective view showing a schematic diagram of an embodiment of a freezing system of the present invention; [Figure 2] FIG. 1 is a perspective view showing the internal structure of a freezing system. [Figure 3] FIG. 10 is a side view showing an example of the arrangement of the ice slurry producing unit. [Figure 4] FIG. 10 is a side view showing an example of the arrangement of the ice slurry producing unit. [Figure 5] 1A is a plan view schematically showing a first embodiment of refrigerant piping in a disk portion, and FIG. 1B is a plan view schematically showing a second embodiment of refrigerant piping in a disk portion. [Figure 6] 10 is a side view schematically showing the principle by which a blade separates ice from a disk portion. FIG. [Figure 7] This is a schematic diagram of a case in which a casing is placed to cover the circumference of a fin rotation circle with a central angle of 180°. [Figure 8] FIG. 1 is a diagram showing a processing flow of a freezing system. [Figure 9] FIG. 1(A) is a plan view showing a cell sample frozen by the freezing system of the present invention, and FIG. 1(B) is a plan view showing a cell sample frozen with liquid nitrogen. DETAILED DESCRIPTION OF THE INVENTION

[0015] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. FIG. 1 is a diagram showing the configuration of a freezing system to which this embodiment is applied. The freezing system 1 shown in FIG. 1 is configured by combining an ice slurry ice maker 10 as an ice production unit, a control panel 11, a slurry tank 12, a housing 13, a heat insulating lid 13a, a cylinder 14, and the like.

[0016] The ice slurry ice maker 10 produces flake-shaped (also called thin, flake-shaped, small, or granular) ice (flake ice) by precipitating ice from an aqueous solution (brine) such as ethyl alcohol (ethanol). While the term "brine" originally refers to salt water, in this embodiment it refers to a secondary refrigerant used in an indirect freezing method. Brine should have a high specific heat capacity, good conductivity, low viscosity, and a low freezing temperature. Examples of brine that can be used include an aqueous ethyl alcohol solution, an aqueous 1-propyl alcohol (propan-1-ol, 1-propanol), and an aqueous aryl alcohol solution. However, as described below, a target cooling temperature of -120°C to -150°C is desirable for cell samples and the like, so it is desirable to use a secondary refrigerant with a composition and concentration that prevents freezing within this temperature range.

[0017] Control panel 11 controls ice slurry ice maker 10, cylinder 14, and other pumps (not shown). Control panel 11 produces ice slurry at an appropriate temperature and viscosity. Control panel 11 is equipped with an electronic circuit with a CPU and a display that accepts input of various setting conditions from the user. The display accepts input of setting conditions such as the amount of liquid nitrogen supplied from the liquid nitrogen cylinder, the set temperature of the ice slurry, ON / OFF of the motor that operates ice slurry ice maker 10, and the motor rotation speed.

[0018] The slurry tank 12 is a tank for storing brine. The slurry tank 12 is approximately 45 cm wide (longitudinal direction), 25 cm deep, and 25 cm high, and has a capacity of approximately 20 L. A portion of the ice slurry ice maker 10 is located inside the slurry tank 12. That is, the ice slurry production section 15 (see FIG. 2) of the ice slurry ice maker 10 is immersed in a predetermined amount of brine stored in the slurry tank 12. The flake ice produced by the ice slurry ice maker 10 is dispersed within the slurry tank 12 by rotating fins and blades (described below). As a result, the originally liquid brine mixes with the flake ice to form ice slurry. Approximately half of the tank's width is the ice production area, and the other half is the freezing area. A coarse-mesh stainless steel net is installed at the boundary between the two areas to prevent the ice slurry from impeding its flow. The slurry tank 12 may be made of resin. The outer surface and the outside of the bottom of the slurry tank 12 are covered with foam insulation material to a thickness of 7 to 10 cm, thereby providing a heat insulating structure.

[0019] The housing 13 covers the entire freezing system and has an insulated structure to prevent cold from escaping from the inside. The insulated lid 13a is an insulated lid that can be opened and closed using a hinge structure. With the insulated lid 13a open, the user can immerse a cell sample in the ice slurry in the slurry tank 12 and rapidly freeze it. While the ice slurry is being produced, the insulating effect of the closed insulated lid 13a prevents the cold from the ice slurry from diffusing to the outside. Because the material to be frozen must be immersed in the secondary medium, even if the secondary medium is a dangerous or toxic substance, it is difficult to completely seal the slurry tank 12. For this reason, it is desirable to have a structure that minimizes the area (opening) where the ice slurry is exposed inside the slurry tank 12 when the insulating lid 13a is opened.

[0020] In this embodiment, "directly immersing cells in a secondary refrigerant" also includes immersing a cell sample in a container that can accommodate cells at cryogenic temperatures in the secondary refrigerant. The container for containing the cells may be a resin box-shaped container, a three-sided sealed bag made of two sheets of film, or a two-sided sealed bag, a tube bag, or a tube container. The container must be capable of aseptically containing the internal solution containing the biological sample and have physical properties that allow it to withstand temperatures below -120°C, preferably below -150°C (temperatures in an ultra-low temperature freezer or in the vapor phase of liquid nitrogen), and more preferably -196°C (liquid nitrogen temperature). Suitable materials for such a container include low-density polyethylene, linear low-density polyethylene, medium-density polyethylene, high-density polyethylene, ultra-high molecular weight polyethylene, nylon, polyester, polystyrene, polyimide, ethylene-vinyl acetate copolymer, fluororesin, and laminates thereof.

[0021] The secondary refrigerant is preferably a composition that can be cooled to a temperature lower than the freezing point of the cryoprotectant for cells. For example, the vitrification transition temperature of the representative cryoprotectant VS55 is −123°C, so the cooling temperature is preferably about 3°C ​​lower, around −125°C. VS55 is a solution containing 24.2 w / v% (3.1 M) dimethyl sulfoxide, 16.8 w / v% (2.2 M) 1,2-propanediol, and 14.0 w / v% (3.1 M) formamide. Furthermore, for example, the vitrification transition temperature of dimethyl sulfoxide (DMSO) at a different concentration from the above is sometimes −132°C, so the cooling temperature is preferably around −135°C. Since the freezing point of many cryoprotectants is around −120°C to −140°C, the cooling temperature is preferably −120°C to −150°C. Therefore, the secondary medium is preferably a composition that remains liquid even at −120°C to −150°C.

[0022] Cylinder 14 is a supply source for liquid nitrogen, which is a primary refrigerant. The primary refrigerant is supplied to ice slurry preparation section 15 of ice slurry ice maker 10 via refrigerant inlet pipe 18a, vaporizes, and is discharged via refrigerant outlet pipe 18b. Cylinder 14 is disposed next to slurry tank 12. A flow meter 16 and a regulator 19 are disposed to deliver the liquid nitrogen stored in cylinder 14 to ice slurry preparation section 15. Flow meter 16 measures the flow rate of the liquid nitrogen. Regulator 19 controls the flow rate of the liquid nitrogen. Regulator 19 is also connected to control panel 11 for communication. To increase the cooling effect, regulator 19 can increase the flow rate of the liquid nitrogen. Refrigerant inlet pipe 18a and refrigerant outlet pipe 18b may be rubber hoses. For example, refrigerant inlet pipe 18a and refrigerant outlet pipe 18b may be rubber hoses outside housing 13 and metal hoses inside housing 13. However, it is desirable that the refrigerant introduction pipe 18a be protected with a heat insulating material so that the liquid nitrogen sent to the ice slurry preparation section 15 does not evaporate on the way.

[0023] FIG. 2 is a perspective view showing a schematic diagram of the internal structure of the freezing system 1. A secondary refrigerant is stored in the slurry tank 12 so as to form a brine liquid level Ws. An ice slurry ice maker 10 disposed in the ice making area of ​​the slurry tank 12 supplies ice slurry brine to a freezing work area Wa. The freezing work area Wa is a space where the freezing work of cell samples can be performed, and corresponds to approximately half of the entire area of ​​the slurry tank 12. For example, a user places the object to be frozen in a metal basket 45 and rapidly freezes it by immersing the basket 45 in the ice slurry in the freezing work area Wa of the slurry tank 12. If the secondary medium is a dangerous or toxic substance, the basket 45 may have the structure of an empty container instead of a mesh structure. In this case, the object to be frozen is placed in the container without directly touching the secondary medium, and is instantly frozen.

[0024] Ice slurry ice maker 10, located within slurry tank 12, comprises ice slurry production section 15, frame section 17, refrigerant inlet pipe 18a, refrigerant outlet pipe 18b (not shown), etc. Ice slurry production section 15 comprises cooling section 21 (see FIG. 5), rotation drive section 22 as a drive section, and rotation transmission shaft 23. Cooling section 21 comprises fins 24 as a brine flow generating section that generates a brine flow, blades 25 as scraping sections that separate ice, disk section 26 as an ice making plate, refrigerant piping 28, and casing 30 as a flow control section. Fins 24 and blades 25 together form a rotating section that rotates or reciprocates with respect to the ice making surface. Note that the "rotating section" recited in claim 1 may also comprise only fins 24 or only blades 25.

[0025] The frame portion 17 is formed, for example, by connecting rod-shaped parts to form a framework. Materials such as ordinary angle bars, round pipes, square pipes, or extruded materials can be used for the frame portion 17. In FIG. 2, the parts of the frame portion 17 are depicted as strips to avoid cluttering the drawing. It is desirable to select the material for the frame portion 17 taking into consideration the required strength and structure. From the viewpoints of mechanical strength and corrosion resistance, the frame portion 17 may be made of stainless steel. The components of the frame portion 17 can be joined by welding or screw fastening (including bolt fastening). The frame portion 17 can be made of metal or synthetic resin. As the metal, various common materials such as steel, stainless steel, and aluminum can be used. Furthermore, when using metal such as steel, various common surface treatments can be performed to prevent rust. Frame portion 17 fixes rotation drive unit 22, disk unit 26, and other components of ice slurry production unit 15. Frame portion 17 supports ice slurry production unit 15 so that rotation drive unit 22 is always positioned above the brine liquid level Ws.

[0026] Ice slurry producing unit 15 produces flake ice from brine on surface 26a (ice-making surface) of disk unit 26. Blade 25, which serves as a scraping unit, scrapes (sweeps) the produced flake ice and disperses it within slurry tank 12, producing ice slurry. Fins 24, which serve as a brine flow producing unit, produce a flow of brine containing ice slurry by rotating or reciprocating in a rotating manner. Disk portion 26 is formed from a metal plate having a rectangular (here, square) plate surface (ice-making surface) and a predetermined thickness, and is fixed to frame portion 17. Here, disk portion 26 is not limited to a rectangular shape, but may also be circular. Examples of materials for disk portion 26 include copper, stainless steel, steel that has been surface-treated to provide anti-rust effects, aluminum, and duralumin. From the perspective of preventing electrolytic corrosion due to the potential difference between metals, disk portion 26 is preferably attached to frame portion 17 via an insulating member such as resin.

[0027] The size (dimensions) of disk portion 26 can be, for example, about 30 cm square. In this embodiment, the upper ice-making surface (surface 26a) and the lower ice-making surface (surface 26b) of disk portion 26 are machined to be substantially flat and parallel to each other. Furthermore, a plurality of holes are formed inside disk portion 26, aligned parallel to each other at substantially equal intervals, so as to penetrate the interior of disk portion 26. Adjacent internal through-holes of the disk portion 26 are connected by U-shaped connecting pipes, forming a refrigerant pipe (refrigerant passage) 28 inside the disk portion 26. The disk portion 26, which serves as an ice-making plate, is preferably manufactured by casting copper pipes (refrigerant passages) using aluminum casting. To prevent electrolytic corrosion due to the potential difference between metals, the copper pipes (refrigerant passages) are preferably protected from contact with brine by painting, applying resin, or using a cover. Alternatively, the refrigerant pipes 28 may be formed inside the disk portion 26 without providing internal through-holes in the disk portion 26 by casting copper pipes bent into a zigzag or incense-like shape using aluminum casting. Liquid nitrogen is heated and vaporized inside the copper pipes (refrigerant passages) inside the disk portion 26 by heat transferred from the outside, and the vaporized liquid nitrogen flows through the refrigerant outlet pipe 18b (see FIG. 1).

[0028] The disk portion 26 is cooled by the primary refrigerant delivered from the cylinder 14 flowing through the refrigerant piping 28. For example, liquid nitrogen, which is the primary refrigerant, is at about -196°C immediately after being discharged from the cylinder 14. The disk portion 26 is configured by casting copper pipes (refrigerant piping) into aluminum plates. The ice slurry (high-temperature fluid: temperature (e.g., -120°C)) on the surface 26a of the disk portion warms the surface temperature on the liquid nitrogen (low-temperature fluid: temperature -196°C) side (e.g., -180°C), and the liquid nitrogen boils, cooling the surface.

[0029] The blade 25 scrapes off the ice that has grown on the surface 26a of the disk portion 26 and disperses it into the brine stored in the slurry tank 12. By repeating this process, the ice concentration gradually increases and an ice slurry is formed in the slurry tank 12.

[0030] The fins 24 have the function of generating a brine flow by rotating or reciprocating in rotation. The fins 24 are plate-like members extending perpendicular to the surface 26a of the disk portion 26, and may be one or more. Blade 25 is a member that scrapes off ice formed on disk portion 26, which is an ice making plate, by rotating. Blade 25 is attached to the end of fin 24, a vertically extending plate-like member, on the disk portion side. Therefore, the ice scraped off by blade 25 is mixed with brine by the rotation of fin 24, and an ice slurry-like brine flow is generated. Furthermore, it is not necessary to provide blades 25 on all fins 24. For example, the fins 24 may be formed from four blades that form a cross shape in a plan view, and the blades 25 may be arranged so as to connect to only two blades of these fins 24.

[0031] The fins 24 are connected to a rotation drive unit 22 via a rotation transmission shaft 23. A motor (fin drive motor) is incorporated in this rotation drive unit 22. As will be described later, the rotation drive unit 22 is capable of continuously rotating the fins 24 in the brine stored in the slurry tank 12 (the liquid surface is shown imaginarily by a dashed line in FIG. 2). Here, the rotational drive unit 22 can be a geared motor that is an integral combination of a motor and a speed reducer (gear). The rotational drive unit 22 is located above the brine level Ws and is disposed so as to extend outside the brine. The rotational drive unit 22 is not limited to a unit that rotates the fins 24 in one direction, but may also be a unit that rotates the fins 24 back and forth (a unit that rotates the fins back and forth in forward and reverse directions).

[0032] The casing 30 is composed of curved and flat surfaces that surround a portion of the circumference of the rotation of the fins 24. The casing 30 has the effect of collecting flake ice scraped by the blades 25 from the surface of the disk portion 26 so that it does not immediately disperse outside the rotation of the fins 24, and the effect of sending slurry ice to the freezing operation area Wa of the slurry tank 12. Furthermore, it also has the effect of preventing ice growing on the side of the disk portion 26 from entering the rotation area of ​​the fins 24. Without the casing 30, it is difficult to achieve a uniform ice concentration within the slurry tank 12 because the ice slurry brine is more viscous than a liquid. For this reason, in the past, a brine flow was created within the slurry tank 12 by pumping the brine with a pump and spraying it from a nozzle located elsewhere within the slurry tank 12. The casing 30 of this embodiment eliminates the need for a pump and the electrical energy required to operate the pump, allowing for energy-saving generation of a flow within the casing.

[0033] The height of the wall formed by the casing 30 is preferably about the same as the height of the fins 24, but it may be higher or lower. The casing 30 may be made of metal or resin. What shape of the casing 30 is effective for forming a brine flow will be described later.

[0034] The temperature sensor 40 measures the temperature of the ice slurry in the slurry tank 12. The temperature sensor 40 may be installed anywhere as long as it can measure the temperature of the ice slurry in the slurry tank 12. However, it is preferable that the temperature sensor 40 be installed in a location that does not obstruct the flow of the ice slurry. The temperature sensor 40 is connected to the control panel 11 via a wired or wireless connection. The measured temperature data is sent to a memory (not shown) that constitutes part of the control panel 11. The CPU of the control panel 11 controls the regulator 19, which controls the flow rate of the primary refrigerant, and the rotation of the fins 24 based on the measured temperature. If the control panel 11 determines that more cooling is required based on the temperature measured by the temperature sensor 40, it issues a command to the regulator 19 to increase the flow rate of liquid nitrogen. The regulator 19 may change the flow rate while monitoring the flow rate measured by the flow meter 16.

[0035] 3 and 4 are side views of the ice slurry production unit 15 as viewed from viewpoint III in FIG. 2. Because these are side views from the freezing area side, only the thickness of the casing 30 is shown outside the fins 24. A refrigerant inlet pipe 18a and a refrigerant outlet pipe 18b are connected to the disk unit 26 of the ice slurry production unit 15. The fins 24 function to rotate (agitate) the brine near the upper or lower surface of the disk unit 26. Blades 25 may be provided between the fins 24 and the disk unit 26. The blades 25 are metal blades that face the surface of the ice-making plate with a gap of approximately 0.3 mm between them. By rotating, they scrape off ice formed on the surface 26a of the disk unit 26. It is desirable that the blades 25 basically have two blades on one side, and the fins 24 have four blades on one side. For example, blades 25 of various materials and shapes are applicable. Several application examples of the blades 25 will be described later using FIG. 6.

[0036] While Fig. 3 shows a configuration in which fins 24 are arranged only on the upper surface 26a of the disk portion 26, as shown in Fig. 4, fins 24 may also be arranged on the lower surface 26b, so that flake ice can be produced on both the upper and lower sides. When fins 24 are arranged on both sides in this way, the refrigerant inlet pipe 18a and the refrigerant outlet pipe 18b must not interfere with the rotation of the fins 24. For example, the refrigerant inlet pipe 18a and the refrigerant outlet pipe 18b may be arranged so as not to interfere with the rotation of the fins 24, and may be connected to the refrigerant piping 28 from the side of the disk portion 26.

[0037] As shown in Fig. 4, blades 25 are attached to the fins 24. The blades 25 are arranged to face the surfaces 26a, 26b of the disk portion 26. Furthermore, in this embodiment, the blades 25 are arranged to contact the surfaces 26a, 26b of the disk portion 26 with an appropriately weak pressure (low surface pressure). The blades 25 have a function (sweeping function) of sweeping away ice exposed on the surfaces 26a, 26b of the disk portion 26 and separating it from the disk portion 26, as will be described later.

[0038] The blade 25 may be made of any of a variety of buffs commonly used for polishing. For example, the blade 25 may be made of urethane or other synthetic resins, metal, or wool. Examples of the blade 25 include sponges, foams, brushes, scrubbing brushes, resin mesh, and nonwoven fabrics made from the various materials described above. Examples of the blade 25 include materials with a certain degree of flexibility. The blade 25 may be made of metal. Even if the blade 25 is made of a metal blade, the blade 25 does not come into contact with the disk portion 26 via a gap. This configuration prevents corrosion of the disk portion 26, which is an ice-making plate (aluminum), due to the potential difference.

[0039] Each blade 25 is attached to a rod-shaped spoke provided on the fin 24. Four fins 24 are arranged at 90-degree intervals on each of surfaces 26a and 26b of the disk portion 26. The fins 24 are integrally joined to the rotation transmission shaft 23, which is a round rod.

[0040] The rotation transmission shaft 23 passes through the disk portion 26 in the thickness direction, avoiding the refrigerant pipes 28 (see FIG. 2), and is capable of rotating in forward and reverse directions around its axis. The rotation transmission shaft 23 is capable of rotational displacement together with the blades 25 relative to the stationary disk portion 26.

[0041] 3 and 4, the refrigerant inlet pipe 18a and the refrigerant outlet pipe 18b pass outside the slurry tank 12, enter at the brine liquid level Ws, and connect to the disk unit 26. It is desirable that the refrigerant inlet pipe 18a be protected with a heat insulating material before being connected to the disk unit 26 to prevent the liquid nitrogen from evaporating.

[0042] 5(A) is a plan view schematically showing a first embodiment of the refrigerant piping 28 of the disk portion 26. (B) is a plan view schematically showing a second embodiment of the refrigerant piping 28 of the disk portion 26. 5(A), refrigerant pipe 28 is formed in a serpentine shape with straight portions and curved portions alternately combined. Furthermore, one end of refrigerant pipe 28 is connected to refrigerant inlet pipe 18a, and the other end is connected to refrigerant outlet pipe 18b. Liquid nitrogen as a refrigerant supplied from cylinder 14 (see FIG. 1) flows inside refrigerant pipe 28 (pipe).

[0043] The outer circumferential surface of the refrigerant pipe 28 is in contact with the inner circumferential surface of the hole in the disk portion 26 so as to allow heat transfer. A copper pipe, which generally has high thermal conductivity, can be used as the material for the refrigerant pipe 28. As the refrigerant flows through the inside of the refrigerant pipe 28, heat is removed from the disk portion 26, thereby cooling the disk portion 26. It is also possible to use materials other than copper (for example, stainless steel, aluminum, duralumin, etc.) for the refrigerant pipes 28. It is also possible to form a coating with excellent thermal conductivity on the outer circumferential surface of the refrigerant pipes 28 (or the inner circumferential surface of the hole in the disk portion 26).

[0044] Furthermore, the refrigerant pipes 28 are not limited to being formed by inserting pipes as physical tubular components into the holes in the disc portion 26. For example, it is possible to omit the tubular components and directly use holes drilled inside the disc portion 26 as refrigerant pipes (refrigerant flow paths). In this case, the refrigerant flows while contacting the inner circumferential surface of the hole in the disc portion 26. Furthermore, when tubular components are omitted as described above, it is possible to form a serpentine-shaped refrigerant flow path by connecting a folded U-shaped pipe to the disc portion 26 and liquid-tightly connecting the internal space of the U-shaped pipe with the internal space of the hole in the disc portion 26.

[0045] Furthermore, it is also possible to provide a serpentine hole having a straight portion and a folded portion inside the disk portion 26. In this case, it is possible to use a casting core for forming the refrigerant flow path and form the disk portion 26 with the serpentine hole by casting.

[0046] 5A, in a plan view of the disk portion 26, the end 28b of the refrigerant pipe 28, which is connected to the refrigerant outlet pipe 18b, extends in a direction perpendicular to the other straight portions. The end 28b of the refrigerant pipe 28, which is connected to the refrigerant outlet pipe 18b, is positioned so as to overlap the other portions in the thickness direction of the disk portion 26.

[0047] Although not shown, the refrigerant pipes 28 may be formed in a shape that meanders more, and may be formed so as to overlap, for example, two, three, or more times in the thickness direction of the disk portion 26. In this way, the flow rate of the refrigerant flowing inside the disk portion 26 can be increased, and the disk portion 26 can be cooled more effectively.

[0048] Furthermore, without being limited to these, for example, the ends 28a, 28b of the refrigerant pipe 28, which are connected to the refrigerant inlet pipe 18a and the refrigerant outlet pipe 18b, may be formed to extend in a direction parallel to other straight portions when the disk portion 26 is viewed in a plan view as shown in Figure 5(B). This makes it easier to process the refrigerant pipe 28 and drill holes in the disk portion 26. It also makes it easier to make the disk portion 26 thinner.

[0049] Next, Fig. 6 shows a schematic diagram of blade 25 separating ice from disk portion 26. Blade 25 attached to fin 24 moves horizontally (rotationally) from left to right in the figure as indicated by arrow C. As shown in FIG. 6, the blade 25 can be made of a metal member with a claw at its tip end protruding in the direction of travel C. When such a claw-shaped rigid member scrapes off ice, the ice tends to disperse vertically away from the disk portion 26, facilitating mixing of the ice with the brine. It is desirable to maintain a gap H between the metal blade 25 and the disk portion 26 at approximately 0.2 mm to 0.3 mm. This prevents wear on the metal plate and the disk portion 26. Furthermore, it is desirable that the gap H be adjustable as needed. Adjustment of the gap H can be achieved not only by replacing the blade 25, but also by using a structure that allows the fixed position of the blade 25 to be controlled vertically. The blades 25 can be fixed to the fins 24 by various common methods, such as adhesive, screw (bolt) fastening, riveting, clamping, and the like. The blade 25 may be made of a material other than a metal plate, such as a synthetic resin plate. If the blade 25 is flexible, it may be in contact with the upper surface 26a of the disk portion 26 with a moderately weak pressure (low surface pressure). The blade 25 may be capable of generating friction and deforming as it moves while in contact with the surface 26a of the disk portion 26. The blade 25 strikes ice (not shown) formed on the surface 26a of the disk portion 26, applying an external force to the ice and sweeping it off the surface 26a of the disk portion 26. Furthermore, if a fin 24 is also arranged on the opposite surface (lower surface 26b) of the disk portion 26, the blade 25 will sweep off the ice according to the same principle. As a further modification, it is possible to transmit power to the blade 25 (not shown) from the side (side of the end) of the disk portion 26 without drilling a hole in the disk portion 26 through which the rotation transmission shaft 35 passes. In this case, for example, it is conceivable to use a parallel crank mechanism, with links (arms) of the parallel crank mechanism reciprocating with each other while sandwiching the disk portion 26. By employing such a mechanism, the disk portion 26 can be sandwiched between the fins 24, and the mechanism can operate like a car windshield wiper to sweep away ice.

[0050] In the explanation of Figure 6, the blade 25 separating ice from the disk portion 26 is sometimes described as "sweeping" and sometimes as "scraping," but both mean that the scraping portion separates ice from the ice-making surface.

[0051] Next, a description will be given of measures to prevent ice from adhering to the sides of the disk portion 26. In this embodiment, ice adhering to surfaces 26a and 26b of the disk portion 26 is separated from the disk portion 26 by the blades 25. However, with regard to ice adhering to portions that are not in contact with the blades 25, such as the sides of the disk portion 26, the brine flow hits the ice, but no further external force acts on it.

[0052] Therefore, if the ice adhering to disk portion 26 grows and becomes large and takes on an unexpected shape or size, the grown ice may press against surrounding equipment (e.g., refrigerant pipes 28, etc.) and place an excessive load on the surrounding equipment. Also, the grown ice may reach surfaces 26a and 26b of disk portion 26 and interfere with fins 24 and blades 25, hindering their operation.

[0053] Taking these points into consideration, it is possible to partially provide an ice adhesion prevention portion 46 on the disk portion 26, as shown in Figures 5(A) and (B). In the example of Figures 5(A) and (B), the ice adhesion prevention portion 46 is formed so as to cover the curved portion of the refrigerant pipe 28 that protrudes from the disk portion 26.

[0054] The ice adhesion prevention portion 46 can be formed, for example, from a synthetic resin having a lower thermal conductivity than the metal material of the disk portion 26. The surface of the ice adhesion prevention portion 46 can also be molded into a smooth shape without sharp corners, making it difficult for ice to adhere. In the examples of Figures 5(A) and (B), only the outline of the ice adhesion prevention portion 46 is shown by a two-dot chain line.

[0055] Furthermore, in this embodiment, since the casing 30 is provided, even if ice grows on the side of the disk portion 26, the ice will not reach the inside of the casing 30. Therefore, the casing 30 can prevent the grown ice from interfering with the operation of the fins 24.

[0056] Figure 7 is a diagram for explaining the calculation conditions. Figure 7 shows a structure in which the curved surface of the casing 30 is such that the center of the rotation circle of the fin 24 follows an arc of approximately 180°. The presence of the casing 30, which is a flow control device as shown in Figure 7, allows the brine flow to reach the freezing work area, allowing ice slurry of a uniform temperature to be produced.

[0057] Without the casing 30, the flow of brine from the ice-making area to the freezing work area is slow. Therefore, without the casing 30, it is difficult to achieve a uniform ice slurry. When the casing 30 is installed, the flow of brine from the ice-making area to the freezing work area is faster than when the casing 30 is not installed. In particular, with a casing with a central angle of 180° as shown in Figure 7, the flow rate of brine from the ice-making area to the freezing work area is faster. With the configuration of Figure 7, the inlet where the brine flows in and the outlet where it flows out are far apart, making it easier for a fast outflow flow to be formed.

[0058] Furthermore, the fins 24 are not limited to those that rotate in one direction, but may be those that rotate back and forth (those that rotate back and forth in forward and reverse directions). In the case where the fins 24 have a rotary drive unit that rotates back and forth, the casing 30 may be formed as a wall in which flat surfaces are connected to both sides of a curved surface that follows an arc.

[0059] FIG. 8 is a diagram showing a processing flow of the freezing system. The user sets the target cooling temperature on the display screen of the control panel 11 (S100). When the cryoprotectant is dimethyl sulfoxide (DMSO), the glass transition temperature of DMSO is −132° C., so the target cooling temperature can be set to 135° C., which is about 3° C. lower. When the cryoprotectant is VS55, the glass transition temperature is −123° C., so the target cooling temperature can be set to −125° C. Next, ice slurry production begins (S110). A primary refrigerant is supplied to the disk unit 26, which is an ice-making plate, to generate ice and produce ice slurry. A temperature sensor measures the temperature of the ice slurry (S120). If the measured temperature is higher than the cooling target temperature (YES in S130), the flow rate of the primary refrigerant and the rotation speed of the fins 24 are increased (S140). If the measured temperature is lower than the cooling target temperature (NO in S130), the system waits for the temperature of the ice slurry to rise (S150). Then, it is determined whether the measured temperature is approximately equal to the cooling target temperature (S160). If the measured temperature is approximately equal to the cooling target temperature, the user immerses the cell sample in the freezing operation area, and the secondary refrigerant rapidly freezes the cells (S170). If the measured temperature is not approximately equal to the cooling target temperature, the system returns to the process of S130.

[0060] Figure 9(A) is a plan view showing a cell sample frozen using the cell freezing system of the present invention, and (B) is a plan view showing a cell sample frozen using liquid nitrogen. Figure 9(A) shows a cell sample frozen using liquid nitrogen as the primary refrigerant, a 90% ethanol solution as the secondary refrigerant, and a target cooling temperature of -125°C. When ice crystals form, a cloudy, opaque appearance appears, but in the photograph of Figure 9(A), the entire sample is transparent, indicating that it has been vitrified and frozen. On the other hand, when immersed in liquid nitrogen, the cells freeze opaquely, indicating the formation of ice crystals, as shown in Figure 9(B). In the example of Figure 9(B), the cells are severely damaged, suggesting a low cell survival rate. [Explanation of symbols]

[0061] 1...Freezing system, 10...Ice slurry ice maker, 11...Control panel, 12...Slurry tank, 13...Housing, 13a...Insulated lid, 14...Cylinder, 15...Ice slurry production section, 16...Flow meter, 17...Frame section, 18a...Refrigerant inlet pipe, 18b...Refrigerant outlet pipe, 19...Regulator, 21...Cooling section, 22...Rotation drive section, 23...Rotation transmission shaft, 24...Fin, 25...Blade, 26...Disc section, 26a, 26b...Disc section surface (ice-making surface), 28...Refrigerant piping, 30...Casing, 40...Temperature sensor, 45...Cage, 46...Ice adhesion prevention section, Wa...Freezing work area, Ws...Brine liquid level

Claims

1. a slurry tank for storing a secondary refrigerant; an ice making unit disposed inside the slurry tank and immersed in the secondary refrigerant; Equipped with The ice making unit includes an ice making plate having an ice making surface that circulates a primary refrigerant supplied from a liquid nitrogen cylinder therein and produces ice of a secondary refrigerant on at least one side thereof; A rotating part that rotates or reciprocates with respect to the ice making surface; A flow control section configured as a wall including a curved surface along a part of the outer periphery of the rotation circle of the rotating section; a freezing area in which cells are rapidly frozen by directly immersing them in the secondary refrigerant; A cell freezing system comprising:

2. The secondary refrigerant is adjusted so that the temperature of the slurry in the slurry tank is −120° C. to −150° C. The cell freezing system of claim 1 .

3. The secondary refrigerant is an aqueous ethanol solution. The cell freezing system of claim 1 .

4. a sensor for measuring the temperature of the slurry in the slurry tank; controlling the supply amount of the primary refrigerant or the rotation speed of the rotating part based on the temperature. The cell freezing system of claim 1 .

5. The rotating part is a secondary refrigerant flow generator for generating the secondary refrigerant flow; A scraping unit that separates ice formed on the ice making surface from the ice making surface; The cell freezing system of claim 2 , comprising:

6. The cell freezing system according to claim 5 , wherein the flow control section is configured as a wall including a curved surface along an arc having a central angle of approximately 180° of a rotation circle of the scraping section.

7. The cell freezing system of claim 1 , wherein the rotating portion is disposed both above and below the ice making plate.

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