Device and method for freezing biological solutions

Abstract

To provide systems and methods for use in freezing liquid mixtures or suspensions containing sensitive substances, such as biopharmaceutical materials, under sterile conditions and in small-volume containers.SOLUTION: The disclosed device enables control of ice nucleation of a solution while monitoring the layer of volume that freezes, and also enables control of an ice growth rate in a bottom up geometry, and comprises a heat transfer surface 101 with means to control temperature, a holder 102 for multiple containers 109, and pressing means 103 to press the holder against the heat transfer surface. The disclosed method comprises the steps for pre-cooling the device to a temperature substantially below the solution ice nucleation temperature, placing a container into the holder, contacting the container with the heat transfer surface until a fraction of 10% of the total sample volume is frozen, interrupting the contact, and contacting the container with the heat transfer surface at a predefined freezing rate, such that the freezing of the biological solution is homogeneous until all the volume of the solution is frozen.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present disclosure generally relates to the storage and handling of sensitive materials, such as biological materials, in multiple small volume containers. ) Devices and methods for freezing liquid mixtures or suspensions containing substances, in particular The biological material is, inter alia, a biological cell, a blood cell, a virus, a protein, an antibody. In particular, the present disclosure provides a method for controlling ice nucleation and crystal growth during freezing of small amounts of aqueous mixtures in multiple containers. Improve reproducibility. [Background technology]

[0002] Cryopreservation of delicate materials is important for many applications related to the advancement of cell biology. The resulting derivatives are generally used for biotransformation, including preservation banking of the genetic material. They are kept frozen for production and distribution control.

[0003] One major limitation of existing systems stems from the complexities associated with the freezing and thawing phenomena. This can be severely compromised by inefficient cryopreservation. This is particularly important for cell therapy due to certain therapeutic and safety constraints.

[0004] Cryopreservation involves different methods such as adding cryoprotectants, cooling (freezing), heating (thawing), and mixing. These processes determine the physicochemical stability of biological products. Because cryopreservation involves a process sequence, inconsistencies can occur at the earliest stages (cooling and freezing). This tends to spread and amplify from the final stage (thawing and mixing) to the final stage (thawing and mixing). Maximizing freezing consistency is crucial to maximizing the overall cryopreservation of biological products. It's important.

[0005] Many variables contribute to the inconsistency in freezing, such as natural convection, ice nucleation temperature, and ice crystal Growth rate, supercooling, etc. are involved. The two major issues are freezing consistency, which is difficult to control. It was related to sex, specifically to natural convection and ice nucleation.

[0006] Natural convection is a mechanism that can be used to prevent inhomogeneity in solute distribution (cold concentration or freezing) that occurs in frozen solutes of biological products. Concentration gradient-driven convection is known to be important. This means that convection displaces the solute towards the bottom and center of the cylindrical container. This is because a non-convective freezing geometry is used, i.e., one-way freezing from bottom to top. The formation of ice dendrites due to directional freezing dampens natural convection, thus It has been reported that this prevents low temperature concentration (see Non-Patent Documents 1 and 2).

[0007] Another critical aspect in cryopreservation is the control of ice nucleation temperature and ice nucleation site. In the freezing process, the aqueous solution must be cooled to a temperature below the melting point before ice nucleation occurs. This tends to cool the cells to a condition known as supercooling, which can lead to increased cell viability after thawing. It was stated that supercooling impairs the preservation capacity and therefore the effectiveness of the entire cryopreservation process. To reduce this, several techniques for controlling ice nucleation have been proposed. By manually forming the pots, by electrofreezing, by mechanical methods (shaking, ultra small ice crystals or heterogeneous ice nucleating agents by sonication, shock cooling, or pressure shift There are techniques such as introducing the above into a sample (see Non-Patent Document 3). [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Rodrigues MA, Balzan G, Rosa M, Gomes D, de Azevedo EG, Singh SK, et al. The importance of heat flow direction for reproducible and homogeneous freezing of bulk protein solutions. Biotechnol Prog 2013;29:1212-21. doi:10.1002 / btpr.1771. [Non-patent document 2] Rosa M, Tiago JM, Singh SK, Geraldes V, Rodrigues MA. Improving Heat Transfer at the Bottom of Vials for Consistent Freeze Drying with Unidirectional Structured Ice. AAPS PharmSciTech 2016;17:1049-59. doi:10.1208 / s12249-015-0437-3. [Non-patent document 3] John Morris G, Acton E. Controlled ice nucleation in cryopreservation- A review. Cryobiology 2013;66:85-92. doi:10.1016 / j.cryobiol.2012.11 Summary of the Invention [Problem to be solved by the invention]

[0009] Although many methods have been proposed, most have not been developed with a high level of reproducibility under sterile conditions. Standardize or integrate multiple small volume containers with a cryopreservation system that maintains cell viability throughout the cryopreservation process. It is difficult to ensure the strength and reproducibility of the freeze-thaw cycle. Most methods were applied to volumes larger than 10 ml. Large volumes can increase the heat transfer rate of solutions. Due to the larger area and thermal inertia, heat flux can be more easily controlled. Conversely, for small volumes such as 100 μl, the amount of The required conditions are to enable local ice nucleation and not impair unidirectional bottom-up freezing. Controlling this faces several technical challenges.

[0010] One of the problems associated with freezing systems is the nucleation temperature and The reproducibility of the first ice crystal position is difficult to control. This variation is due to non-uniformity between vials. This can lead to variations in cell characteristics and ultimately to variations in the quality of cell preservation. , which involves promoting ice nucleation by rapidly cooling the container base. To allow rapid cooling, the vessel thermal resistance at the bottom must be minimized. This improves the contact between the heat transfer plate and the container bottom, i.e., the internal A liquid, polymer, paste, or adhesive with a lower freezing point than water that minimizes air This can be achieved by using a conductive material such as followed this approach for freeze-drying applications, where a sticky material was attached to the bottom of the container. This reduces the voids formed by the concave surface of a typical glass vial, thereby increasing the thermal conductivity coefficient. Although it is effective, Rosa et al. This approach requires significant changes to the vial design, adding a lot of structural complexity and Significant changes to the vial design required regulatory validation. For example: Adhesive materials can be a source of contamination in clean rooms and are also used for cell cryopreservation. It may also react with typical materials used in container construction. Another significant technical difficulty is The purpose of this method is to enable control over ice nucleation and ice growth rates in small volumes. The rapid cooling of the base required to promote ice nucleation at the bottom is not due to ice nucleation alone. Instead, it may result in completely uncontrolled freezing of the sample. , e.g., when the mass is less than the container mass, thermal inertia influences the dynamics of the freezing process. . [Means for solving the problem]

[0011] Ideally, freezing occurs during ice nucleation to decouple ice nucleation from rate-controlled freezing. Ensure that the liquid content is not more than 20%, preferably less than 10%. This requires freezing the majority of the sample at a controlled rate in a bottom-up direction. Therefore, the cooling at the bottom is strong and short enough to allow ice nucleation of the solution. It is necessary to monitor the freezing of the body layer under supercooling conditions. This can be achieved by, for example, placing the container on a cold surface. By performing a vessel-based quench to a low temperature (typically below -40°C), However, consistent ice nucleation in a group of multiple vessels can be achieved. To do this, a) all the containers are placed simultaneously on a pre-cooled heat transfer surface, and b ) The contact between the container bottom and the heat transfer surface is equal regardless of surface roughness variations. Two criteria are critical: surface roughness variation becomes inconsistent as the container base area decreases; Decoupling ice nucleation from ice growth rate in unidirectional geometries To achieve this, the height of the liquid that can supercool by thermal diffusion must be minimized before an ice nucleation event occurs. height / width asymmetrical to monitor the amount of sample freezing that would otherwise occur uncontrollably. It is desirable that the aspect ratio is not less than 1. Therefore, the container for unidirectional freezing is The bottom has a small area, e.g., a small diameter, which can complicate heat transfer consistency due to surface roughness variations. For example, it is convenient to have a volume of less than 2 ml. Only a small amount (up to 20%) is supercooled, while the remaining liquid remains at freezing temperature (near 0 °C) to avoid excessive ice growth during ice nucleation. Otherwise, a large portion of the sample (greater than 20%) will freeze uncontrollably.

[0012] Ice nucleation implies the formation of an interface at the boundary of a new phase. A liquid cooled above the freezing point but below the maximum heterogeneous ice nucleation temperature (melting point) It is said to be supercooled. The present disclosure aims to solve the above-mentioned problems.

[0013] These and other objects, features and advantages of the present disclosure are further illustrated by the following detailed description, taken in conjunction with the accompanying drawings. It will be clear from the description.

[0014] For a better understanding of the present disclosure, reference is made to the accompanying drawings, which illustrate preferred embodiments of the present disclosure. Although exemplary embodiments are shown, these embodiments are not meant to limit the scope of this application. [Brief explanation of the drawings]

[0015] [Figure 1]1 shows a schematic perspective view of an embodiment of a freezing device with locked down press, where reference numerals indicate the following: 100 freezing head, 101 heat transfer surface, 102 holder, 103 pressing means, 104 pressing means frame, 105 operator, 106 hinge, 107 pivot, 108 compression device. [Figure 2] 1 shows a schematic perspective overall view of an embodiment of the freezing device with the press unlocked, where reference numerals indicate: 100 freezing head, 101 heat transfer surface, 102 holder, 103 pressing means, 104 pressing means frame, 105 operator, 106 hinge, 107 pivot, 108 presser, 109 container. [Figure 3] 1 shows a schematic overall perspective view of an embodiment of a freezing device with a cooling system, where reference numerals indicate the following: 102 holder, 110 system, 111 insulating frame, 112 fin support, 113 coolant reservoir. [Figure 4] 1 shows a schematic general perspective view of an embodiment of a freezing device, where the reference numerals indicate the following: 102 holder, 111 insulating frame, 112 fin support, 114 system, 115 fins. [Figure 5] Two embodiments of the heat transfer surface are shown schematically, where reference numbers indicate: 101a flat configuration, 101b recessed configuration, 116 channels, 117 recesses in the heat transfer surface. [Figure 6] An embodiment of the holder arrangement is shown diagrammatically, where the reference numerals indicate the following: 102a compressible means with spring arrangement, 118 spring, 119 contact layer. [Figure 7] An example of a holder configuration is shown diagrammatically, where the reference numerals indicate the following: 102b compressible means with tab configuration, 119 contact layer, 120 tab. [Figure 8]An example of a holder arrangement is shown diagrammatically, where the reference numerals indicate the following: 102c compressible means with pin arrangement, 109 container, 119 contact layer, 121 pin. [Figure 9] An example of a vessel configuration is shown diagrammatically, where reference numerals indicate the following: 109 vessel, 122 vessel wall, 123 vessel bottom. [Figure 10] 6 is a graph showing the effect of using (black line) or not using (dashed line) a contact-promoting material on the heat transfer surface (see FIG. 5 ) on the ice nucleation times of an aqueous solution containing 10% trehalose obtained with a device according to an embodiment described above. [Figure 11] 6 is a graph showing the effect of using (black line) or not using (dashed line) a contact promotion material on the heat transfer surface (see FIG. 5 ) on the ice nucleation times of an aqueous solution containing 10% DMSO obtained with a device according to an embodiment described above. DETAILED DESCRIPTION OF THE INVENTION

[0016] In this disclosure, unidirectional geometry refers to the development and progression of an ice front along a selected axis. This means the creation of a unidirectional temperature gradient along the axis. The geometry is the orientation along a vertical axis that causes the ice front to develop and advance from base to top. This means creating a directional temperature gradient.

[0017] In the present disclosure, controlled ice nucleation occurs within a short time after the container contacts the heat transfer surface. This refers to the formation of the first ice crystals in the turbulent liquid, preferably in less than 1 minute. In particular, it refers to the formation of the first ice crystals in the liquid at the bottom of the container. The amount of liquid that forms on the surface and freezes instantly during ice nucleation (local supercooling) is 20%. There shouldn't be more.

[0018] In some embodiments, the freezing is unidirectional from the bottom to the top of the container. Due to the insulation provided by the conductive material, freezing is achieved in a controlled manner, which is comparable to other similar technologies. When doing so, one advantage of the present disclosure is that the freezing is not radial. This is because freezing of the total contents is controlled, uniform, and more effective, regardless of the total sample volume. be.

[0019] One surprising advantage of the method of the present invention is that freezing occurs in the same freeze cycle. This means that the contents of each vial freeze uniformly and are uniformly frozen. This means that the vial will freeze just like the other vials in the freezing cycle. The freezing effectiveness is high whether it contains 1 liter or 100 ml of solution.

[0020] In another embodiment, the present disclosure relates to a device for freezing a biological solution, the device comprising: S, a heat transfer surface (101); a holder (102) having a recess for receiving a container (109) for said biological solution; a pressing means (103) for pressing the holder (102) against the heat transfer surface (101); wherein the container (109) is placed in contact with the heat transfer surface (101), The heat from the heat transfer surface (101) is transferred to the container (109) and allows for controlled ice nucleation. and separating the container (109) from the heat transfer surface (101) and 109) into contact with the heat transfer surface (101), means (103); Equipped with The freezing of the biological solution has a bottom-up geometry.

[0021] In one embodiment, the pressing means (103) faces the holder (102). By applying a uniformly distributed pressure to the holder (102), preferably by manual or mechanical uniform distribution. A compressing device (108) may be included to apply cloth pressure to the holder (102).

[0022] In one embodiment, the holder (102) may be made of a low thermal conductivity material. Preferably, and for better results, the low thermal conductivity material is a plastic, It may be made of ceramic or composite material.

[0023] In some embodiments, the device disclosed herein comprises at least one container ( 109).

[0024] In one embodiment, the pressing means presses a portion (123) of the container against the heat conducting and placing the heat transfer surface (101) in contact with the vessel. a portion (123) of the container (109) and separate the container (109) from the heat transfer surface (101). and repositioning a portion of the vessel (123) in contact with the heat transfer surface (101). Preferably, the portion of the container is the bottom of the container.

[0025] In one embodiment, the holder (102) is compressible between a first position and a second position. A pressure selectable from a spring (118), tab (120), or pin (212) is provided to allow the The first position may include a front collapsible means (102a, 102b, 102c). The bottom (123) of the container is placed in contact with the heat transfer surface (101), and Position 2 places the bottom (123) of the container away from the heat transfer surface (101). do.

[0026] In one embodiment, the holder (102) is compressible between a first position and a second position. The first position may be a compressible holder, and the second position may be a position where the container bottom (123) is and the second position is in contact with the heat transfer surface (101), and the second position is in contact with the bottom (12) of the container. 3) is positioned away from the heat transfer surface (101).

[0027] In one embodiment, the holder (102) is configured to accommodate the flat shape of the heat transfer surface (101). It can be pressed against the state (101a).

[0028] In one embodiment, the holder (102) is a recessed portion of the heat transfer surface (101). The heat transfer surface (101b) can be pressed against the recess (11) of the heat transfer surface (101b). 7) is a depression with a depth between 0.5 and 3 mm.

[0029] In one embodiment, the heat transfer surface (101) comprises: Preferably, there may be a channel (116) for removing excess contact promoting material. do.

[0030] In one embodiment, the channel (116) has a depth between 0.5 and 3 mm. The thickness may be between 1 and 5 mm.

[0031] In some embodiments, the device disclosed herein further comprises a press means frame. (104), and the pressing means frame (104) includes a hinge (106) and an operator. (105).

[0032] In one embodiment, the heat transfer surface (101) is made of stainless steel, copper, aluminum, The conductive material may be selected from the group consisting of aluminum, silicon dioxide, tungsten, tungsten carbide ...

[0033] In one embodiment, the device further comprises a coolant reservoir for storing a coolant. A server (113) may be provided.

[0034] In some embodiments, the device further comprises insulating the heat transfer surface from room temperature. The device may be provided with an insulating frame (111) for protection of the user and for preventing the device from being damaged.

[0035] In one embodiment, the device further comprises fin supports for uniform heat exchange. A support (112) may be provided.

[0036] In some embodiments, the device further provides a large contact area with the coolant. The device may be provided with fins (115) for the purpose.

[0037] In one embodiment, the device further comprises a heat transfer surface (101) and a chamber (102). The contact layer (119) may be provided to remove residual air between the container (109) and the The contact layer (119) can be a liquid, a paste, a paper, or an adhesive, and preferably The contact layer (119) has a height of 0.1 mm to 3 mm.

[0038] In one embodiment, the container (109) has a height of 5 mm to 50 mm and a width of 4 mm to 50 mm. It may have a diameter of 10 mm.

[0039] In one embodiment, the container has a height / width aspect ratio greater than 1. can be done.

[0040] In some embodiments, the container wall (122) may have a thickness of between 0.4 mm and 2 mm. The container bottom (123) can have a thickness of 0.2 mm to 2 mm.

[0041] In one embodiment, the vessel wall (122) and the vessel bottom (123) are made of different materials. Created at no charge.

[0042] In some embodiments, the vessel wall (122) is made of a polymer, ceramic, glass, or is made of other low thermal conductivity materials.

[0043] In one embodiment, the container bottom (123) is made of stainless steel, aluminum or or other highly conductive materials, or polymers, glass, or other It is made using such low conductivity materials.

[0044] In one embodiment, the device is a freezing device.

[0045] The present disclosure relates to a method of operating the device disclosed herein, the method comprising the steps of: Tep, i.e. pre-cooling the device; placing a vessel (109) containing a biological solution in the holder (102); The bottom (123) of the container is pressed against the heat transfer surface (101) using a pressing means (103). contacting the removing the container (109) from contact with the heat transfer surface (101); The container bottom (123) is pressed against the heat transfer surface (101) by the pressing means (103). ) rearranging the A controlled freezing rate is established so that the freezing of the biological solution has a bottom-up geometry. determining the Equipped with.

[0046] In some embodiments, the controlled freezing rate of the biological solution in the container However, the temperature is preferably 0.1°C / min to 100°C / min, more preferably 0.5°C / min to 10°C / min, and even more preferably 1 °C / min to 5°C / min.

[0047] In one embodiment, the step of pre-cooling the device comprises pre-cooling the device to a temperature below -20°C, preferably below -20°C. Preferably, the reaction is carried out at a temperature below -30°C, more preferably below -40°C.

[0048] In one embodiment, the container (109) is separated from the heat transfer surface (101). The step comprises: maintaining the biological solution at a temperature of about 0° C. while transferring the vessel (109) to the heat transfer vessel (109). The conductive surface (101) is preferably separated from the conductive surface (101) so as to leave a gap of 0.1 mm to 15 mm. This can be implemented by placing

[0049] In one embodiment, the container (109) is brought back into contact with the heat transfer surface (101). The step of pressing the holder (102) against the heat transfer surface (101) to fix the container (109) in contact with said heat transfer surface (101). This can be done.

[0050] In some embodiments, the biological solution comprises a microorganism, a tissue, a living cell, a stem cell, a primary cell, or a (Primary) cells, cell lines, live or attenuated viruses, nucleic acids, monoclonal antibodies monoclonal antibodies, polyclonal antibodies, biomolecules, non-peptide analogs, peptides, Protein, RNA, DNA, oligonucleotide, virus particle, or combination thereof can have:

[0051] The present disclosure further relates to biological solutions frozen according to the methods disclosed below.

[0052] In some embodiments, the present disclosure provides a method for bottom-up geometry freezing and freezing of a sample solution. and / or ice nucleation, said method comprising the following steps: pre-cooling the device to a temperature significantly below the ice nucleation temperature of said solution; The device has a heat-conducting surface at the bottom and a pressing means. Top and placing a vessel containing a biological solution in a holder, said holder comprising: the step of forming a substrate from a low thermal conductivity material; The container is placed on the heat transfer surface until 10% of the total volume of the sample is frozen. and interrupting contact between the vessel and the heat transfer surface; The container is then placed on the heat transfer surface at a predetermined freezing rate to ensure uniform freezing of the biological solution. contacting; and finally freezing the solution until the entire amount is frozen. Equipped with.

[0053] In other embodiments, the present disclosure relates to methods having multiple samples.

[0054] In another embodiment, the present disclosure provides a method for controlling the flow of the biological solution in the container. The freezing rate is preferably 0.1°C / min to 100°C / min, more preferably 0.5°C / min to 10°C / min, and even more preferably 0.5°C / min to 10°C / min. The preferred method is 1°C / min to 5°C / min.

[0055] In another embodiment, the present disclosure provides a method for treating a cancer cell comprising pre-cooling the device to a temperature below -20°C. Preferably, the process is carried out at a temperature lower than -30°C, more preferably lower than -40°C. Regarding.

[0056] In another embodiment, the present disclosure provides a method for treating a heat transfer surface by eliminating contact between the container and the heat transfer surface. The isolating step may include: placing the vessel on the heat transfer surface while maintaining the biological solution at a temperature of about 0°C. Preferably, the device is arranged so as to separate it from the surface, leaving a gap of 0.1 mm to 15 mm. This relates to a method for implementing the above.

[0057] In another embodiment, the present disclosure provides a method for freezing a container by contacting the container with the heat transfer surface at a predetermined freezing rate. The step of pressing the holder against the heat transfer surface to contact the container with the heat transfer surface. The method is carried out by placing the electrodes in contact with each other.

[0058] In another embodiment, the present disclosure provides a method for treating a disease comprising administering to a subject a biological solution ... Cells, primary cells, cell lines, live or attenuated viruses, nucleic acids, Monoclonal (monoclonal) antibodies, polyclonal antibodies, biomolecules, non-peptide analogs, Peptides, proteins, RNA molecules, DNA molecules, oligonucleotides, virus particles, or a combination thereof.

[0059] In another embodiment, the present disclosure provides a bottom-up geometry freezing and / or sample dissolving method. 1. A device for ice nucleation of a liquid, said device comprising: a heat conducting surface at the bottom; a holder having at least one cavity for the container and made of a low thermal conductivity material; The container is placed in contact with the heat transfer surface, and heat from the heat transfer surface is transferred to the container. and moving the container away from the heat transfer surface and back onto the heat transfer surface. a pressing means configured to re-contact the Equipped with The heat transfer is performed from the bottom of the container to allow for controlled ice nucleation. Regarding chairs.

[0060] In another embodiment, the present disclosure provides a method for pressing a pressing member, the method comprising: pressing a pressing member against the holder; Apply cloth pressure to the holder, preferably applying manual or mechanical evenly distributed pressure to the holder. The present invention relates to a device having a compression device for applying pressure.

[0061] In another embodiment, the present disclosure provides a method for manufacturing a ceramic material having low thermal conductivity, comprising the steps of: or a composite material.

[0062] In another embodiment, the present disclosure relates to a device comprising at least one container.

[0063] In another embodiment, the present disclosure provides a method for pressing a portion of the container into the heat conductive a heat transfer surface disposed in contact with a portion of the vessel to transfer heat from the heat transfer surface to a portion of the vessel; Pulling the container away from the heat transfer surface and placing a portion of the container in contact with the heat transfer surface. The container is configured to be repositioned in a desired position, and preferably the portion of the container is the bottom of the container. Regarding the vise.

[0064] In another embodiment, the present disclosure provides a method for manufacturing a hologram display device, the method comprising: a compressible means selected from a spring, tab, or pin to enable the first position positions the bottom of the container in contact with the heat transfer surface, and the second position positions the bottom of the container the bottom of the device being positioned away from the heat transfer surface.

[0065] In another embodiment, the present disclosure provides a method for manufacturing a hologram display device, the method comprising: a compressible holder, the first position being such that the container bottom is in contact with the heat transfer surface; and the second position positions the container bottom away from the heat transfer surface. , relating to the device.

[0066] In another embodiment, the present disclosure provides a method for manufacturing a heat transfer device, the method comprising: Related to devices.

[0067] In another embodiment, the present disclosure provides a method for manufacturing a heat transfer device, comprising: Preferably, the recessed flat shape of the heat transfer surface is between 0.5 and 3 mm. The present invention relates to a device in which the recess has a depth in the cavity.

[0068] In another embodiment, the present disclosure provides a method for removing excess liquid, comprising: Preferably, the device has a channel for removing excess contact promoting material. .

[0069] In another embodiment, the present disclosure provides a press means frame having a hinge and an operator. Related to devices.

[0070] In another embodiment, the present disclosure provides a heat transfer surface comprising a material selected from the group consisting of stainless steel, copper, aluminum, The present invention relates to devices made of conductive materials selected from the group consisting of silicon, silicon dioxide, silicon dioxide, silicon dioxide nanotubes ...

[0071] In another embodiment, the present disclosure includes a coolant reservoir for storing a coolant, Regarding the device.

[0072] In another embodiment, the present disclosure provides a method for insulating the heat transfer surface from room temperature and for use in a The device includes an insulating frame to protect the user.

[0073] In another embodiment, the present disclosure provides a fin support for uniform heat exchange over exposed area. The present invention relates to a device having a holding body.

[0074] In another embodiment, the present disclosure provides a cooling system including fins to increase the contact area with the coolant. Related to devices.

[0075] In another embodiment, the present disclosure provides a method for treating a heat transfer surface comprising the steps of: The present invention relates to a device comprising a contact layer for removing

[0076] In another embodiment, the present disclosure provides a method for manufacturing a device comprising: Preferably, the contact layer has a height of 0.1 mm to 3 mm. do.

[0077] In another embodiment, the present disclosure provides a container having a height / width aspect ratio greater than 1. , relating to the device.

[0078] In another embodiment, the present disclosure provides a method for manufacturing a container in which the container wall and the container bottom are made of different materials. Related to the device.

[0079] In another embodiment, the present disclosure provides a method for manufacturing a container wall comprising the steps of: relates to devices made of other low thermal conductivity materials.

[0080] In another embodiment, the present disclosure provides a container wherein the bottom is made of stainless steel, aluminum or or other highly conductive materials, or made of polymers, glass, or other This invention relates to devices constructed using such low conductivity materials.

[0081] In another embodiment, the present disclosure relates to a device, wherein said device is a freezing device. do.

[0082] In another embodiment, the present disclosure relates to a biological solution frozen according to the present disclosure.

[0083] <Detailed explanation> As mentioned above, one major issue in cryopreservation of delicate substances such as biological materials and solutions is the A major limitation is the inconsistency associated with the freezing and thawing phenomenon. Many variables affect the freezing process. The two major issues involved in consistency were related to natural convection and ice nucleation.

[0084] In this specification, it is preferable to provide a group of samples with high reproducibility for small volumes of less than 2 ml. A novel device and method is disclosed that allows for unidirectional bottom-up freezing within a container.

[0085] Further disclosed is a method for enabling unidirectional bottom-up freezing in a group of small volume containers, The method includes the following steps: a step of pre-cooling the container to a temperature of 1000°C, and a step of setting the multi-container in a holder and placing the holder in the heat transfer chamber. pressing the containers against a cold surface simultaneously to bring all of the containers into contact with the cold surface; Use an intermediate contact promoting material (e.g., liquid or polymer) to allow ice nucleation. After the ice nucleation step and a small volume layer, the holder and the container are removed from the cold surface. and finally, the ice growth rate in a unidirectional bottom-up geometry. and pressing the holder against the cold surface while controlling the pressure.

[0086] Thus, the liquid biological solution or The device for freezing biological solutions, which are suspensions, uses a downward force to force the container to conduct heat. The ice nucleation process includes a driver that aligns the surface in a locked-down position. Intermediate contact promotion to ensure that the amount of liquid frozen at once is no more than 20% of the total volume The start occurs when the container is pressed simultaneously against a heat-conducting surface using the adhesive. To avoid uncontrollable freezing of liquid after an ice nucleation event, drivers should The container is set in the lock-up position, and a gap of 0.1mm to 15mm is created between the container and the heat. The ice crystals then assume a standby position away from the conducting surface. Finally, the ice crystals grow in one direction under controlled ice growth rates. To promote bottom-up freezing, press down the driver again to bring it into contact with the heat-conducting surface at the bottom of the container. Promotes contact and is typically 0.1°C / min to 100°C / min based on the thermal sensitivity of the biological material. Apply the selected freezing rate by cooling the heat transfer surface at 1000 kJ / min.

[0087] The device for freezing biological solutions described herein for carrying out the above-mentioned method is A freezing head (100) having a heat transfer surface (101) comprising: a holder (102) having a receiving cavity into which the container (109) is fitted; The freezing table may also have a pressing means (103). and can use several refrigerants and / or other known cooling systems. The freezing head and cooling system are connected to the container and / or the system (114). Or, the insulation may take on a related form, such as in the embodiment of FIG.

[0088] In certain embodiments shown in Figures 1-9, which are not intended to limit the scope of this application, data preserved by the process of freezing and / or thawing, storing and transporting biological materials; Indicates a vice.

[0089] In some embodiments, the container (109) can be used for freezing and / or thawing, storing, and transporting the These vessels are especially designed to hold biological samples during all processes. The container (109) is constructed using biologically safe materials to keep the container safe. The cavity of the mold (102) can accommodate different sample volumes and different desired thermal processes. The specification of the vessel (109) takes into account the entire specific heat exchange process. The container (109) has an aspect ratio (height / width) greater than 1 and a height of 5 mm to 50 mm. and a diameter of 4 mm to 10 mm. The thickness of the container bottom (123) can be varied from 0.2 mm to 2 mm. It can be made thick.

[0090] In some embodiments, to maintain the unidirectional freezing process proposed by the present application, the container wall The container wall (122) and the container bottom (123) can be made of different materials. The container (109) is made of polymer, ceramic, glass, or other low thermal conductivity material. The bottom of the container (123) is made of stainless steel. Constructed of high conductivity materials such as stainless steel, aluminum, or other materials, or made of polymers, glass, The container (109) can be constructed using low-conductivity materials such as stainless steel or other materials. The bottom may be either low or high heat transfer, based on the thermodynamics assumed for the particular heat exchanger employed. The differences between materials can be significant, especially when frozen in small samples. Vertically driven heat flux gradient without significant influence from the vessel wall (122) This makes it easier to obtain a container (10 9) because the mass ratio is important.

[0091] In one embodiment, the cooling system (114) is a heat transfer system for the freezing head (100). The conductive surface (101) and other elements that allow the temperature in the container (109) to be controlled. The integral part is represented by a heat flow controlled by a cooling system (114). Any electronic, mechanical or machined device used to create the bundle In Figure 4, an example of a cooling system setup is shown. The fins (115) Heat is transferred between the coolant, which may be dry ice, liquid nitrogen, or other. The coolant is stored in a coolant reservoir (113). The fin support (112) supports the fins (11). 5), and the container (109) and holder (102) are attached to the heat transfer surface (101). This helps to avoid temperature fluctuations when the container (109) is placed in contact with the heat transfer surface ( When the heat transfer surface (101) contacts the container (109), the inertia from the heat transfer surface (101) A small layer of the sample (less than 20% of the volume) was frozen and the heat transfer surface (10 1) increases the temperature of the heat transfer surface (101) during the formation of the first ice layer in the container (109). As the temperature from the surface increases, ice growth becomes slower and the temperature of the fin support (112) Therefore, it is controlled.

[0092] In one embodiment, a horizontal plate, so-called heat transfer surface (101), is always The freeze head (100) is connected to a vessel (109) or vessels (109) for containing a biological solution. The freezing head (100) is specially constructed to accommodate the holder (102) into which the freezing head (100) is introduced. ) and corresponding included parts and embodiments are used to transfer heat from the cooling system (114). Controlling the heat conduction flux that passes through the conducting surface (101) and ultimately reaches the vessel bottom (123) The heat transfer surface (101) is designed to have special properties to transfer heat from the container ( One possible form of enhancing heat transfer to the vessel (109) is to provide the vessel (109) with a heat conducting surface. Press to forcefully seal the (101) and close all voids between them vertically. This is due to the existence of means (103).

[0093] In some embodiments, the heat transfer surface (101) is made of stainless steel, copper, aluminum, or other conductive material, and its dimensions may have different sizes. The container (109) can be modified to accommodate several containers or holders (102). The size of the heat transfer surface (101) depends on the type and size of the item that the upper surface will accommodate. 1cm based on the number 2 ~150cm 2 The heat conduction table The thickness of the surface (101) can be from 1 mm to 4 mm. Surface (101) is shown in a rectangular shaped embodiment, but may be any other shape depending on the device design. However, the dimensions of the heat transfer surface (101) in a particular form may be: Considering the properties from the entire device, it can have a known global thermal conductivity and inertia. The heat transfer surface (101) shall be secured by screws, any type of adhesive or paste. The cooling system (114) can be attached by other locking means. can also be compressed.

[0094] In some embodiments, the heat transfer surface (101) and the container (109) or holder (102) To remove residual air between the Other means of eliminating voids can be used. This feature improves heat transfer and In the case of adhesive or paste, the container (109) or It can be added to the holder (102). The height from the contact layer is 0.1mm to 3mm. Furthermore, for non-solid contact layers, the height can be fixed and can be The vessel (109) or holder (102) is in contact with the heat conducting surface (101) while immersed. It can be moved closer or further away to change its range from the heat flux during the process.

[0095] Here, two examples of the shape of the heat transfer surface (101) are, but are not limited to, a flat shape. The recessed version (101a) or recessed version (101b) described herein may be used. 1b) maintains a specific fluid height between the heat transfer surface (101) and the container (109). The heat transfer surface has channels (116) and depressions (117) for the purpose of The depressions (117) in the heat transfer surface are optimal for holding a layer of a specific height of the fluid used. If the user overfills the heat transfer surface recess (117), excess flow The fluid is vented through channels (116) to maintain the desired fluid height. The recess (117) is a recess having a depth between 0.5 and 3 mm. (116) unloads excess fluid from the depressions (117) on the heat transfer surface and also transfers the fluid to the heat transfer surface. The excess fluid is directed to the other sink holes in the device. The channel (116) can be between 0.5 and 3 mm deep. The thickness may be between 1 and 5 mm.

[0096] In one embodiment, the holder (102) is designed to receive the container (109); It also has different cavity designs and sizes based on the type of container (109) that is housed. Furthermore, this configuration allows for a reduction in the number of containers (109) used and the number of containers (109) used. The shape of the heat transfer surface (101) can be different. The material used for insulating the sides and top of the container (109) has low thermal conductivity. The holder (102) may be made of plastic, ceramic, composite or other material. ) can have embodiments made of any other material with some functionality.

[0097] Three embodiments of the holder (102) are shown in Figures 6, 7 and 8. These embodiments are: It is envisaged that it will be possible to control the precise moment at which the container (109) is pressed against the heat transfer surface (101). These are used to create a pressing means for pressing the container (109) onto the heat transfer surface (101). It is designed for use with, but not limited to, air contact layers (119 In these examples, the embodiment of ) is removed, in other cases it can be removed by e.g. , which may be a liquid or other that is applied to the holder (102) or the heat transfer surface (101). The contact layer (119) can be replaced with a contact-promoting material that can When added directly to the adhesive, it can be made with any adhesive, including Teflon, polymers, or other materials. The thickness of this layer is 0.1 to 2 mm. However, the additional thermal resistance this adds can range from The thermal flux device must be considered.

[0098] In one embodiment, the holder (102a) has a spring configuration, and the spring (118) The mechanical resistance of the container ( The pressing means (103) presses the container (109) against the spring elastic force. This locks the heat transfer surface (101) when a sufficient downward force is applied to overcome the DOWN (Fig. 1) position and start the thermal process again. Lock-up (Fig. 2) When in the compressible holder (102), the compressible holder (102) has a distance from the heat transfer surface (101). This distance can range from 0.1 to 10 mm, and the lockdown position When the holder (10) is in the position shown in Fig. 1, this distance is practically 0 mm. 2) itself, and in additional embodiments, The spring (118) may be made of metal, polymer or other material. The diameter of the spring (118) may range from 3 mm to 10 mm. They can be of various shapes and numbers, ranging from The extension of the spring (118) that moves from the bottom surface of the holder (102) is 0.1 mm. The distance from the holder (102) to the heat transfer surface ( 101).

[0099] In one embodiment, the holder (102b) having a tab configuration has a tab (120) and a The mechanical resistance of the container ( The pressing means (103) presses the tab against the container (109) with elastic force. When a downward force sufficient to break the tab is applied, this method The surface (101) is aligned in the lockdown position (FIG. 1) and the thermal process is started again. When in the lock-up (Fig. 2) position, the holder (102) is positioned at a distance from the heat transfer surface (101). The distance may range from 0.1 to 10 mm, and the locking When the cam is in the down position (Fig. 1), this distance is essentially 0 mm. The handle (102) has a tab (120) which may be an extension from the handle (102) itself, allowing for additional implementations. The tab (120) may be made of metal, polymer, or other material. The width of the tab (120) may range from 2 mm to 1 The dimensions vary from 0mm to 15mm, and the length varies from 4mm to 15mm. The tabs (120) can be arranged in several configurations and numbers. The extension of the tab (120) moving from the bottom surface of the holder (102) can be from 0.1 mm to 10 mm. mm and the distance from the holder (102) to the heat transfer surface (101) Controls the initial distance to

[0100] In one embodiment, the holder (102c) with a pin configuration provides lock-up (see FIG. 2) When in position, it overcomes the mechanical resistance from the pins (121) and A downward force is applied to the container (109) which breaks or deflects the pin. The pin is attached to the holder (102). They can be arranged in any number or shape in the cavity in the holder, and can be part of the holder. or in additional embodiments may be made of any polymer, metal, or otherwise. It can be kept in standby position for any other process such as gluing, coating or The pin (121) can be disposable if it is destructible, or If the container (109) is flexible, it may be possible to use it several times. 19) or the distance to the heat transfer surface (101) is 0.1 mm to 10 mm depending on the configuration. The distance may range from

[0101] In one embodiment, the pressing means (103) presses the container (109) based on the configuration. The driver is mechanically actuated to press against the heat transfer surface (101) or the contact layer (119). The lock-down and lock-up positions are shown in Figures 1 and 2, respectively. When the pressing means (103) is set to the lock-up position (Fig. 2), the container ( 109) is in a standby position separated from the heat transfer surface (101) by an air gap, and this air gap The gap can vary from 0.1mm to 10mm. (Fig. 1) position, the existing voids are removed vertically. 9) and the heat transfer surface (101) there is an interface layer (119) or any fluid, paste 1 and 2, the pressing means (1) The pressing means (103) can also be electrically controlled. It can also be operated by a mechanical actuator controlled by the actuator. In the present invention, the pressurizer (108) is a heavy pressurizer that defines the area that contacts the container (109). It is a component that allows all the vessels (109) to reach a well-distributed pressure zone. In this embodiment, the pressing device (108) is locked down. While moving the device to the on-position (Fig. 1), align the compression device (108) with all the containers (109). The press means frame (104) is supported by an adjustable pivot (107). Together, the position of this pivot (107) determines the position of the compression device (108) during the application of downward force from this mechanism. ) is forced to remain in a horizontal position. This configuration allows the hole to be filled even if not all cavities are filled with containers (109). The mechanism has a mechanical stop that allows for normal use of the handle (102). The hinge (106) and the operator (105) are also shown.

[0102] Another aspect of the present disclosure relates to a method for freezing a biological solution using the device described above. In this regard, the method comprises the following steps: Pre-cooling the heat transfer surface (101) to a low temperature (e.g., below -40°C) and At least one container (109) is placed in the holder (102), and a pressing means (1 03) by pressing the holder (102) into a lockdown position, The bottoms of all the containers are simultaneously contacted in (101) to allow controlled ice nucleation. and After ice nucleation of a small amount of ice (≦20% of the total amount), the pressing means (103) is locked up. position, and after ice nucleation, the liquid content should be kept in a suitable position to avoid uncontrolled freezing. The chemical solution is maintained at a temperature of approximately 0°C, and the heat transfer surface is only separated by an air gap of 0.1 mm to 15 mm. a step of causing the holder (102) to assume a standby position spaced apart from the holder (101); , The pressing means (103) is set in the lockdown position and the holder (102) is placed on the heat transfer surface. (101) and bring the bottom (123) of the container into contact with the heat transfer surface (101). facilitating the The device was set to a controlled freezing rate to promote a unidirectional bottom-up freezing geometry. and Equipped with.

[0103] An important aspect of the present disclosure is the use of a condenser to promote controlled mechanical ice nucleation within all containers. To obtain good contact between the container bottom (123) and the heat transfer surface (101). Therefore, the flat shape (101a) and the recessed shape (101b) are used as the cooling source to induce ice nucleation. A heat transfer surface (101) having a recessed form (101b) can be used. ) can be filled with a contact promoting material (e.g., a liquid or polymer) to a specific height, as described above. This improves the heat transfer between the heat conducting surface (101) and the container (109). The configuration (101b) allows all containers to be at the same height regardless of the number of containers used. It allows for advanced materials.

[0104] The following describes the ice nucleation of different aqueous solutions during the freezing process with and without the use of contact promoting materials. Another embodiment will be detailed in due course.

[0105] In one embodiment, several containers filled to a liquid height of 6 mm were placed in the holder ( 102) and pressed against the heat transfer surface (101) by a pressing means (103). For this experimental test, two different aqueous solutions were used: a 10% trehalose solution ( 10) and 10% DMSO solution (Figure 11) were used.

[0106] Figures 10 and 11 show the ice nucleation of two aqueous solutions in a container with a liquid height of 6 mm. As can be seen in Figure 10, the ice nucleation process is When using the ion exchanger, all samples nucleated within 1-20 seconds, while contact-promoted ice formation was observed. When no material is used, this range increases to 26-67 seconds. Looking at nucleation (Figure 10), 66% of the samples using contact-promoting materials were formed within 5 seconds (5–10 sec), whereas the ice nucleation time distribution without the contact promoter is 25 Similar results were obtained for ice nucleation in 10% DMSO solution (Figure 11). This is also observed in ice nucleation, where ice nucleation is faster (5-20°C) when using contact-promoting materials. seconds), it can be seen that this occurs within a shorter time range.

[0107] In some embodiments, the methods and devices of the present invention involve not only freezing of biological samples but also It is also used for decompression.

[0108] Therefore, the use of a flat morphology and a heat transfer surface without a contact promoting material results in extensive ice nucleation. Compared with the time spectrum, the depression morphology with a specific height of contact-promoting material shows a higher degree of ice formation. This results in controlled nucleation, which may affect other performance aspects and properties of the frozen solution. Yes.

[0109] Another important aspect of the present disclosure is the decoupling of ice nucleation from controlled rate freezing. For this purpose, the amount of liquid frozen in the ice nucleation step is set to be more than 20%. It is necessary to ensure that the difference is not more than 10%, preferably not more than 10%. The remaining liquid volume freezes at a controlled rate. After ice nucleation in a small volume of solution, The sample must be removed from the heat transfer surface to prevent the solution from continuing to freeze uncontrollably. Therefore, after the time required for ice nucleation has elapsed, the sample is slightly removed from the heat transfer surface. This allows the pre-nucleation solution portion to remain frozen and the liquid portion to be kept at approximately 0°C. Finally, the amount of solution in which all samples were frozen and the remaining liquid at the same temperature (0°C) were Freezing in a unidirectional bottom-up manner with controlled freezing rates while holding the body It is possible.

[0110] As used herein, the term "comprising" means including the described features, integers, steps, etc. A group is intended to indicate the presence of a component, but not one or more other features, integers, does not preclude the presence or addition of any step, component, or group thereof. Figure.

[0111] Those skilled in the art will appreciate that unless otherwise indicated, the particular sequence of steps described are merely illustrative and may be varied without departing from the disclosure. Unless otherwise stated, the steps listed are not sequential and should be used where possible. , the steps may be performed in any convenient or desirable order.

[0112] The present disclosure should not be viewed as limited to the described embodiments, and those skilled in the art will appreciate that One would expect there to be many possible changes to

[0113] The above-described embodiments may be combined. The claims further describe specific embodiments of the present disclosure. The embodiment will be clarified.

Claims

1. A device for freezing a sample solution held in a container and forming ice nuclei, comprising: a thermally conductive surface disposed on the bottom of the device; a holder made of a low thermal conductivity material and having at least one cavity configured to receive the container; a pressing means configured to press a bottom of the container housed in the cavity of the holder against the heat-conducting surface, transfer heat from the heat-conducting surface to the container, and release the container from the heat-conducting surface when 10% of the total volume of the sample has frozen, and then bring the container back into contact with the heat-conducting surface; Equipped with heat transfer is effected from the bottom of the vessel to allow for controlled ice nucleation; the holder has compressible means selected from a spring, a tab, or a pin and disposed between the cavity of the holder and the bottom of the container so as to be movable between a first position in which the bottom of the container contacts the heat transfer surface and a second position in which the bottom of the container is spaced from the heat transfer surface; device.

2. 2. The device of claim 1, wherein the pressing means comprises a constrictor configured to apply an evenly distributed pressure to the holder.

3. 3. The device according to claim 1, wherein the low thermal conductivity material of the holder is plastic or ceramic.

4. 4. A device according to any one of claims 1 to 3, wherein the pressing means is configured to place a portion of the container in contact with the heat transfer surface, transfer heat from the heat transfer surface to the container, move the container away from the heat transfer surface, and then return the portion of the container to contact with the heat transfer surface.

5. A device according to any one of claims 1 to 4, wherein the holder is configured to be pressed against a flat or recessed surface of the heat conducting surface.

6. A device according to any one of claims 1 to 5, wherein the recessed surface of the heat transfer surface comprises a recess having a depth in the range of 0.5 to 3 mm.

7. The device of claim 6 , wherein the heat-conducting surface includes a channel communicating with the recess.

8. The device according to any one of claims 1 to 7, wherein the heat conducting surface is made of a heat conducting material selected from stainless steel, copper, aluminum, or combinations thereof.

9. The device of any one of claims 1 to 8, further comprising at least one of a coolant reservoir for storing coolant, cooling fins, or fin supports.

10. A device according to any one of claims 1 to 9, comprising an insulating frame arranged to insulate the heat conducting surface from room temperature and for protecting a user.

11. A device according to any one of the preceding claims, comprising a contact layer for removing residual air between the heat transfer surface and the container.

12. 12. The device of claim 11, wherein the contact layer is a liquid, a paste, a paper, or an adhesive.

13. A device according to any one of the preceding claims, wherein the container wall and the container bottom are made of different materials.

14. The device according to any one of claims 1 to 13, wherein the container wall is made of polymer, ceramic, glass or other low thermal conductivity material.

15. The device according to any one of the preceding claims, wherein the bottom of the container is made of a high thermal conductivity material or is made of a low thermal conductivity material.

16. A device according to any one of claims 1 to 15, comprising a plurality of containers.

17. A method for freezing a sample solution and forming ice nuclei using a device according to any one of claims 1 to 16, comprising: pre-cooling a heat transfer surface of a device to a temperature below the ice nucleation temperature of the solution, said device having said heat transfer surface at a bottom and a pressing means; placing a container containing a biological solution in a holder, the holder comprising a low thermal conductivity material; bringing the container into contact with the heat transfer surface by pressing the holder against the heat transfer surface until 10% of the total volume of the sample is frozen; After 10% of the total volume of the sample has frozen, interrupting contact between the container and the heat transfer surface by pulling the container away from the heat transfer surface; bringing the container into contact with the heat transfer surface by pressing the bottom of the container against the heat transfer surface until the entire volume of the biological solution is frozen at a predetermined freezing rate to ensure homogeneous freezing of the solution; A method comprising:

18. 18. The method of claim 17, wherein the controlled freezing rate of the biological solution in the container is between 0.1°C / min and 100°C / min.

19. 19. The method of claim 17 or 18, wherein the step of pre-cooling the device is carried out at a temperature below -20°C.

20. 20. The method of any one of claims 17 to 19, wherein the step of interrupting contact between the container and the heat transfer surface is performed by positioning the container away from the heat transfer surface while maintaining the biological solution at a temperature of about 0°C.

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