Air suction-type process cooling method, thermally insulated box, and cooling instrument

Abstract

The present invention relates to an air suction-type process cooling method, a thermally insulated box, and a cooling instrument, and relates to the technical field of cryopreservation equipment for tissues and organs, cells, and subcellular components. The method comprises the following steps: placing a cavity structure I, in which a sample to be cryopreserved is placed, in a cavity structure II containing ultralow-temperature cold air; and by means of process control, causing the ultralow-temperature cold air in the cavity structure II to enter the cavity structure I, so that the cooling capacity of the ultralow-temperature cold air entering the cavity structure I consumes heat released in the freezing process of the sample to be cryopreserved. The present invention implements an active cooling process, quickly crosses the freezing point, and prevents repeated freezing and thawing, thereby improving the yield and cell viability of tissues and organs, cells, and subcellular components. Additionally, liquid nitrogen does not need to be used in the cooling process, the overall structure is simple, maintenance costs are low, and the process is safe.

Description

A method for air-inhalation programmed cooling, a heat preservation box and a cooling device Technical Field

[0001] The present invention relates to the technical field related to freezing equipment for tissue organs, cells and their subcellular components, and in particular to an aspiration-type programmed cooling method, a heat preservation box and a cooling device. Background Art

[0002] During the preservation of tissues, organs, cells, and their subcellular components, the freezing solution, tissues, organs, cells, and their subcellular components release a large amount of heat when freezing, and the freezing points of various components are inconsistent. This leads to repeated freezing and thawing of tissues, organs, cells, and their subcellular components, which in turn causes the cells or subcellular components to rupture, ultimately resulting in a decrease in the number of viable cells and decreased cell viability. After crossing the freezing point (generally a temperature between -30°C and -40°C), the sample is kept slowly cooled at a rate of 1-2°C / min to promote the growth of ice crystals, which ultimately improves cell viability and activity.

[0003] Currently, there are three common cooling methods in the industry: (1) Programmable cooling instruments that use liquid nitrogen as a cooling medium. This instrument is large, expensive, and consumes liquid nitrogen during the cooling process, which poses a significant safety hazard to pressure vessels. (2) Programmable cooling boxes that use isopropyl alcohol as an intermediate medium. This cooling box uses a passive cooling mode and cannot control the sample cooling rate. The cell yield and survival rate of cells recovered after freezing are low. In addition, the flammable and explosive chemical agent, isopropyl alcohol, is used during the freezing process and is highly toxic to the human body and poses a safety hazard. (3) Programmable cooling boxes made of thermal insulation materials. Although toxic isopropyl alcohol is not required, this cooling box uses a passive cooling mode and cannot control the sample cooling rate. The cell yield and survival rate of cells recovered after freezing are low. For example, the patent name is "A New Type of Test Tube Programmable Cooling Box"; the patent authorization announcement number is CN 203916695U. The existing programmable cooling boxes use a passive cooling principle and cannot change the cooling speed according to the actual needs of the user.

[0004] Since cells and cryopreservation fluid release heat when they freeze, the released heat will cause some cells in the test tube to freeze and thaw repeatedly. The repeated freezing and thawing of cells during the freezing process will greatly affect the activity and survival rate of the cells. As shown in Figure 1, the cell freezing curve using an ordinary program cooling box shows that the temperature in the test tube has a significant upward trend at around -12°C. The increased temperature will cause cells and cryopreservation fluid to freeze and thaw repeatedly, thereby affecting the activity and survival rate of the cells. In order to achieve programmed cooling and avoid the use of dangerous substances such as liquid nitrogen and isopropyl alcohol, the present invention provides an aspiration-type programmed cooling method, a heat preservation box, and a cooling device.

[0005] Summary of the Invention

[0006] The present invention aims to solve the problems that existing programmable cooling devices are large in size and expensive and that ordinary cooling devices cannot control the cooling speed, resulting in low cell viability after recovery. An aspiration-type programmed cooling method, an insulation box and a cooling device are provided.

[0007] The technical solution of the present invention to solve the above technical problems is as follows:

[0008] In the first aspect, a method for aspiration-type programmed cooling comprises the following steps: placing a cavity structure I containing a sample to be frozen in a cavity structure II containing ultra-low temperature cold air, and allowing the ultra-low temperature cold air to enter the cavity structure I through program control, so that the cooling capacity of the ultra-low temperature cold air entering the cavity structure I consumes the heat released during the freezing process of the sample to be frozen.

[0009] The ultra-low temperature cold air is generally the cold air in an ultra-low temperature refrigerator, and the temperature is generally below -60°C; the sample to be frozen is at least one of a cell suspension, exosomes, and organoids.

[0010] Compared with the prior art, the present invention has the following beneficial effects:

[0011] (1) The present invention uses program control to allow ultra-low temperature cold air to enter the cavity structure I where the sample to be frozen is placed, so that the coldness of the ultra-low temperature cold air entering the cavity structure I consumes the heat released during the freezing process of the sample to be frozen, ensuring that the cooling rate is controllable within the freezing temperature range, overcoming the problem of repeated freezing and thawing of cells caused by heat release of cells and freezing solution near the freezing point, thereby improving the yield and cell viability of tissue organs, cells and their subcellular components;

[0012] (2) Compared with the programmed cooling method using isopropyl alcohol as the intermediate medium, the present invention does not require flammable, explosive and toxic isopropyl alcohol, is less expensive, more environmentally friendly, and has a better cell freezing effect.

[0013] Furthermore, the method further includes the following step: when the sample to be frozen is frozen and cooled to between -30°C and -40°C (i.e., after the sample to be frozen crosses the latent hot spot), room temperature air is allowed to enter the cavity structure I through program control to slow down the cooling rate of the cavity structure I or to recover the temperature, thereby continuously decreasing the temperature of the sample to be frozen. The room temperature air temperature is generally above 12°C.

[0014] Compared with the prior art, the above-mentioned further beneficial effect of the present invention is: when the sample to be frozen crosses the freezing point and is cooled to between -30°C and -40°C, the cavity temperature is restored to a slow cooling state by pumping in room temperature air, so that the sample continues to maintain a slow cooling rate of 1 to 2°C / min, which is conducive to the reasonable growth of ice crystals and ultimately improves cell viability and cell activity; the present invention can also freely set the target heating point and target heating rate of the cavity according to the needs of the user, meeting the research and exploration of the optimal freezing curve of different cells in the field of biomedicine.

[0015] Further, the method includes the following specific steps:

[0016] (1) placing the sample to be frozen into the cavity structure I of the thermal insulation box, placing the thermal insulation box into the cavity structure II containing ultra-low temperature cold air, and collecting the temperature in the cavity structure I of the thermal insulation box in real time through a temperature sensor;

[0017] (2) The target temperature and target cooling rate are set by the program. After the temperature collected by the temperature sensor reaches the target temperature, the cooling rate is calculated based on the collected temperature. The cooling rate is compared with the target cooling rate. Based on the comparison result, one of the operations (A) to (C) is performed:

[0018] (A) When the cooling rate is slower than the target cooling rate of 1°C / min, the ultra-low temperature cold air is allowed to enter the cavity structure I of the thermal insulation box, so that the cooling capacity of the ultra-low temperature cold air entering the cavity structure I of the thermal insulation box consumes the heat released during the freezing process of the sample to be frozen, so that the temperature in the cavity structure I of the thermal insulation box continues to decrease;

[0019] (B) when the collected cooling rate is faster than the program target cooling rate by 1°C / min, the room temperature air is allowed to enter the cavity structure I to slow down the cooling rate or increase the temperature of the cavity structure I;

[0020] (C) When the difference between the cooling rate and the target cooling rate is within 1°C / min, the ultra-low temperature cold air or the room temperature air is stopped from entering the cavity structure I of the insulation box.

[0021] The above-mentioned cooling rate is generally calculated by calculating the temperature change value collected in the last 3 seconds to obtain the cooling rate (°C / min).

[0022] Furthermore, the target temperature is a specific temperature point between 8°C and -60°C. The program can set multiple target temperature points and target cooling / heating rates, and the target cooling rate is between +2°C / min and -45°C / min.

[0023] It should be noted that the program can freely select the sample temperature or cavity temperature as the target temperature point and set the corresponding cooling rate according to different frozen samples. The program can set multiple groups of target temperature points and corresponding cooling rates.

[0024] In the second aspect, the above-mentioned method of air-intake programmed cooling adopts an insulation box for air-intake programmed cooling, and the insulation box includes an insulation box cover and an insulation box base, the inner side wall of the insulation box cover and the inner side wall of the insulation box base form a cavity structure I, and a temperature sensor is arranged in the cavity structure I; an upper cover through hole communicating with the cavity structure I is arranged on the wall of the insulation box cover, and a base through hole communicating with the cavity structure I is arranged on the wall of the insulation box base, a spoiler is arranged at the base through hole and / or the upper cover through hole and located in the cavity structure I, and an air pipe communicating with the cavity structure I is arranged on the wall of the insulation box cover and / or the wall of the insulation box base.

[0025] Compared with the prior art, the beneficial effect of the above-mentioned insulation box for aspiration-type programmed cooling used in the present invention is: compared with the programmed cooling box of insulation material, the present invention realizes temperature-controlled programmed cooling, can quickly cross the freezing point, and has a better cell freezing effect.

[0026] Furthermore, the cavity structure I includes an upper cavity and a lower cavity, the upper cover of the insulation box is provided with the upper cavity, and the base of the insulation box is provided with the lower cavity; a sample tube holder is provided in the lower cavity, and the sample tube holder is provided with a sample tube, and at least one temperature sensor is inserted into the insulation box cavity or the sample tube.

[0027] Furthermore, the spoiler includes an upper cover spoiler and a base spoiler; the top wall of the upper cover of the thermal insulation box is provided with the upper cover through hole, and the upper cover spoiler is provided at the upper cover through hole and located in the upper cavity; the base through hole is provided on the bottom wall of the base of the thermal insulation box, and the base spoiler is provided at the base through hole and located in the lower cavity.

[0028] Furthermore, a side through hole on the upper cover of the heat preservation box is also provided on the side wall thereof and is communicated with the upper cavity. The through hole on the upper cover may also be a hollow copper tube.

[0029] In the third aspect, the above-mentioned method of aspirated programmed cooling uses an aspirated programmed cooling instrument, which includes a programmed cooling instrument host and an insulation box. The insulation box is arranged in an ultra-low temperature device, and the temperature sensor used in the insulation box is electrically connected to the programmed cooling instrument host, and the air pipe used in the insulation box is connected to the programmed cooling instrument host.

[0030] Wherein, the trachea is preferably a flexible trachea.

[0031] Compared with the prior art, the above-mentioned aspiration type programmed cooling device used in the present invention has the following beneficial effects:

[0032] (1) The aspiration-type programmed cooling device of the present invention can achieve the same cell freezing effect as a large-scale programmed cooling device, and has the advantages of being simple, efficient, small in size, low in cost, and having few safety hazards;

[0033] (2) The aspirated programmed cooling apparatus of the present invention realizes an active cooling program, quickly crosses the freezing point, avoids repeated freezing and thawing, and thus improves the yield and cell viability of tissues, organs, cells and their subcellular components.

[0034] Furthermore, the program cooling instrument host includes a touch screen computer all-in-one, a temperature sensor plug, an tracheal plug, a solenoid valve group, a micro diaphragm air pump, an IO module, a temperature acquisition module and a switching power supply; the program cooling instrument is provided with the switching power supply, the touch screen computer all-in-one is connected to the micro diaphragm micro air pump through the motor speed control module, the temperature sensor is electrically connected to the temperature sensor plug, and the tracheal plug is connected to the trachea; the temperature sensor plug is electrically connected to the touch screen computer all-in-one through the temperature acquisition module, and the temperature in the cavity structure of the insulation box is collected and monitored in real time; the touch screen computer all-in-one is connected to the solenoid valve group through the IO module, and the connection between the trachea and the blowing port or the suction port of the micro diaphragm micro diaphragm pump is switched.

[0035] Furthermore, the program cooling device host also includes a pressure sensor; the pressure sensor is arranged in the program cooling device host; the pressure sensor circuit is electrically connected to the touch screen computer all-in-one through the IO module, and the pressure sensor air circuit is connected to the trachea to monitor the air pressure status of the trachea.

[0036] In addition, the program cooling device main unit includes a frame shell. The ultra-low temperature device can be an ultra-low temperature refrigerator. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] FIG1 is a temperature curve diagram of cells frozen using a conventional program cooling box according to the present invention;

[0038] FIG2 is a schematic structural diagram of the assembly of the temperature reducing device and the ultra-low temperature device of the present invention;

[0039] FIG3 is a schematic structural diagram of a temperature reducing device according to the present invention;

[0040] FIG4 is a schematic structural diagram of a program cooling device host of the present invention;

[0041] FIG5 is a schematic structural diagram of the heat preservation box of the present invention;

[0042] FIG6 is a top view of the upper cover spoiler of the present invention;

[0043] FIG7 is a perspective view of the upper cover spoiler of the present invention;

[0044] FIG8 is a front view of the upper cover spoiler of the present invention;

[0045] FIG9 is a perspective view I of the base spoiler of the present invention;

[0046] FIG10 is a top view of the base spoiler of the present invention;

[0047] FIG11 is a perspective view II of the base spoiler of the present invention;

[0048] FIG12 is a diagram showing the air flow state in the heat preservation box when air is extracted without the upper and lower spoiler devices of the present invention, using SolidWorks software to simulate the air flow state, wherein the left figure is a top view and the right figure is a side view;

[0049] FIG13 is a diagram showing the air flow state in the heat preservation box when the upper and lower spoilers are added according to the present invention, simulated using SolidWorks software during air extraction, wherein the upper figure is a top view and the lower figure is a side view;

[0050] FIG14 is a graph showing the effect of the presence or absence of a turbulence device on the sample temperature when measuring the actual sample temperature using the same exhaust device according to the present invention;

[0051] FIG15 is a diagram showing the effects of the cooling methods of the FC group, CH group, and SCM group on the temperature curve crossing the freezing point;

[0052] FIG16 is a diagram showing the effect of the BCM group cooling method on the temperature curve crossing the freezing point according to the present invention;

[0053] FIG17 is a diagram showing cell adhesion in the FC group of the present invention;

[0054] FIG18 is a diagram showing the cell adhesion of the CH group of the present invention;

[0055] FIG19 is a diagram showing cell attachment in the SCM group of the present invention;

[0056] FIG20 is a diagram showing cell adhesion in the BCM group of the present invention.

[0057] In the accompanying drawings, the list of components represented by each reference number is as follows: 1-Programmed cooling instrument, 2-Ultra-low temperature device, 21-Programmed cooling instrument main unit, 22-Trachea, 23-Insulation box, 24-Temperature sensor, 210-Frame housing, 211-Touch screen computer all-in-one, 212-Temperature sensor plug, 213-Trachea plug, 214-Pressure sensor, 215-Switching power supply, 216-Solenoid valve group, 217-Micro diaphragm air pump, 218-IO module, 219-Motor speed control module, 2120-Temperature acquisition module, 230-Insulation box upper cover, 231-Upper cover side through hole, 232-Sample tube bracket, 233-Insulation box base, 234-Trachea plug, 235-Base through hole, 236-Base spoiler, 237-Sample tube, 238-Upper cover spoiler, 239-Upper cover through hole. DETAILED DESCRIPTION

[0058] The principles and features of the present invention are described below. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0059] The following examples describe the manufacturers and performance parameters of the components used (or how they were implemented):

[0060] The insulation box cover 230 is made of high-density polyethylene foam, a heat-insulating material; the upper cover spoiler 238, the base spoiler 236, the upper cover through-hole 239, and the base through-hole 235 are made of aluminum alloy; the sample tube holder 232 is made of stainless steel; the hollow copper tube is a hollow copper tube with a diameter of 6 mm; the joint of the air pipe 22 is made of copper; the air pipe 22 is a polymer material polyurethane (PU), and the air pipe 22 is a PU material with an outer diameter of 8 mm. The air pipe 22 can achieve the goal of not hindering the closing of the refrigerator door and keeping the middle of the air pipe 22 from being completely flattened by the refrigerator door, and air can pass normally after the refrigerator door is closed; the wire is 0.75 square millimeter copper wire; the sample tube 237 is a 2ml or 5ml cell freezing tube; the temperature sensor 24 is a PT100 platinum resistance temperature sensor 24, brand: Ai Ruidi, model: ARD-CLPT100; the rack shell 210 is made of stainless steel Made of sheet metal; touch screen all-in-one computer 211, 15-inch industrial tablet computer brand: Yanjiang Technology; temperature sensor plug 212, model: KPS-SJYCT, PT100 platinum thermal resistor connector brand: Capeson; tracheal plug 213, model PM10, brand: AirTac; pressure sensor 214, model ZSE30A-C6H-N-MGD(0), brand: SMC; switching power supply 215, model MS-150-24, brand: Mean Well; solenoid valve group 216, model 3V206NCB, brand: AirTac; micro diaphragm air pump 217, model 6010D, brand: Ibis; IO module 218, model G5NB-1A-E, brand: Zhongsheng Technology; motor speed control module 219, model PWM-02, PWM pulse output module brand: Zhongsheng Technology; temperature acquisition module 2120, model ZS-PT100-8 brand: Zhongsheng Technology.

[0061] Example 1

[0062] This embodiment relates to an aspiration-type programmed cooling method, comprising the following steps: placing a cavity structure I containing a sample to be frozen in a cavity structure II containing ultra-low temperature cold air, and allowing the ultra-low temperature cold air to enter the cavity structure I through program control, so that the cooling capacity of the ultra-low temperature cold air entering the cavity structure I consumes the heat released during the freezing process of the sample to be frozen.

[0063] The ultra-low temperature cold air is generally the cold air in an ultra-low temperature refrigerator, and the temperature is generally below -60°C; the sample to be frozen is at least one of a cell suspension, exosomes, and organoids.

[0064] The present invention uses program control to allow ultra-low temperature cold air to enter the cavity structure I where the sample to be frozen is placed, so that the coldness of the ultra-low temperature cold air entering the cavity structure I consumes the heat released during the freezing process of the sample to be frozen, ensuring that the cooling rate is controllable within the freezing temperature range, overcoming the problem of repeated freezing and thawing of cells caused by heat release of cells and freezing solution near the freezing point, thereby improving the yield and cell viability of tissue organs, cells and their subcellular components; compared with the programmed cooling method using isopropyl alcohol as an intermediate medium, the present invention does not require flammable, explosive and toxic isopropyl alcohol, has lower cost, is more environmentally friendly, and has better cell freezing effect.

[0065] Preferably, in this embodiment, the following steps are also included: when the sample to be frozen is frozen and cooled to between -30°C and -40°C, room temperature air is allowed to enter the cavity structure I through program control to slow down the cooling rate of the cavity structure I or raise the temperature, and the temperature of the sample to be frozen continues to drop.

[0066] Wherein, the room temperature air temperature is generally above 12°C.

[0067] Preferably, in this embodiment, the following specific steps are included:

[0068] (1) placing the sample to be frozen into the cavity structure I of the heat preservation box 23, placing the heat preservation box 23 into the cavity structure II containing ultra-low temperature cold air, and collecting the temperature in the cavity structure I of the heat preservation box 23 in real time through the temperature sensor 24;

[0069] (2) The target temperature and target cooling rate are set by the program. After the temperature collected by the temperature sensor 24 reaches the target temperature, the cooling rate is calculated based on the collected temperature. The cooling rate is compared with the target cooling rate (°C / min). Based on the comparison result, one of the operation methods (A) to (C) is performed:

[0070] (A) When the cooling rate is lower than the target cooling rate of 1°C / min, the ultra-low temperature cold air is allowed to enter the cavity structure I of the thermal insulation box 23, so that the cooling capacity of the ultra-low temperature cold air entering the cavity structure I of the thermal insulation box 23 consumes the heat released during the freezing process of the sample to be frozen, so that the temperature in the cavity structure I of the thermal insulation box 23 continues to decrease;

[0071] (B) when the collected cooling rate is higher than the program target cooling rate by 1°C / min, the room temperature air is allowed to enter the cavity structure I to slow down the cooling rate or raise the temperature of the sample to be frozen;

[0072] (C) When the difference between the cooling rate and the target cooling rate is within 1°C / min, the ultra-low temperature cold air or the room temperature air is stopped from entering the cavity structure I of the heat preservation box 23.

[0073] The cooling rate is generally calculated by taking the temperature change value collected in the last 3 seconds to obtain the cooling rate (°C / min).

[0074] Preferably, in this embodiment, the target temperature is a specific temperature point between 8°C and -60°C, and the program can set multiple target temperature points and target cooling rates, and the target cooling rate is between +2°C / min and -45°C / min.

[0075] This embodiment also provides an insulation box 23 for air-intake programmed cooling adopted in the method for air-intake programmed cooling, the insulation box 23 comprising an insulation box upper cover 230 and an insulation box base 233, the inner side wall of the insulation box upper cover 230 and the inner side wall of the insulation box base 233 forming a cavity structure I, a temperature sensor 24 is arranged in the cavity structure I; an upper cover through hole 239 communicating with the cavity structure I is arranged on the wall of the insulation box upper cover 230, a base through hole 235 communicating with the cavity structure I is arranged on the wall of the insulation box base 233, a spoiler is arranged at the base through hole 235 or / and the upper cover through hole 239 and located in the cavity structure I, an air pipe 22 communicating with the cavity structure I is arranged on the wall of the insulation box upper cover 230 or / and the wall of the insulation box base 233.

[0076] Compared with the programmed cooling box made of heat-insulating materials, the present invention realizes temperature-controlled programmed cooling, can quickly cross the freezing point, and has a better cell freezing effect.

[0077] Preferably, in this embodiment, the cavity structure I includes an upper cavity and a lower cavity, the insulation box upper cover 230 is provided with the upper cavity, and the insulation box base 233 is provided with the lower cavity; a sample tube holder 232 is provided in the lower cavity, and the sample tube holder 232 is provided with a sample tube 237, and at least one temperature sensor 24 is inserted into the sample tube 237.

[0078] Preferably, in this embodiment, the spoiler includes an upper cover spoiler 238 and a base spoiler 236; the top wall of the insulation box upper cover 230 is provided with the upper cover through hole 239, and the upper cover spoiler 238 is provided at the upper cover through hole 239 and located in the upper cavity; the bottom wall of the insulation box base 233 is provided with the base through hole 235, and the base spoiler 236 is provided at the base through hole 235 and located in the lower cavity.

[0079] Preferably, in this embodiment, a side through hole 231 communicating with the upper cavity is also provided on the side wall of the upper cover 230 of the heat preservation box, and the upper cover through hole 239 may also be a hollow copper tube.

[0080] This embodiment also provides an air-inspired programmed cooling device 1 used in an air-inspired programmed cooling method, comprising a programmable cooling device main unit 21 and a heat preservation box 23. The heat preservation box 23 is disposed within a cryogenic device 2. A temperature sensor 24 used in the heat preservation box 23 is electrically connected to the programmable cooling device main unit 21. An air pipe 22 used in the heat preservation box 23 is connected to the programmable cooling device main unit 21. The air pipe 22 is preferably a flexible air pipe 22.

[0081] The aspiration-type programmed cooling device 1 of the present invention can achieve the same cell freezing effect as a large-scale programmed cooling device, and has the advantages of simplicity, high efficiency, small size, low cost, and small safety hazards; the aspiration-type programmed cooling device 1 of the present invention realizes an active cooling program, quickly crosses the freezing point, avoids repeated freezing and thawing, thereby improving the yield and cell viability of tissue organs, cells and their subcellular components.

[0082] Preferably, in this embodiment, the program cooling instrument host 21 includes a touch screen computer all-in-one 211, a temperature sensor plug 212, an trachea plug 234, a solenoid valve group 216, a micro diaphragm air pump 217, an IO module 218, a temperature acquisition module 2120 and a switching power supply 215; the program cooling instrument 1 is provided with the switching power supply 215, the touch screen computer all-in-one 211 is connected to the micro diaphragm air pump 217 through the motor speed control module 219, the temperature sensor 24 is electrically connected to the temperature sensor plug 212, and the trachea plug 234 is connected to the trachea 22; the temperature sensor plug 212 is electrically connected to the touch screen computer all-in-one 211 through the temperature acquisition module 2120, and the temperature in the cavity structure of the insulation box 23 is collected and monitored in real time; the touch screen computer all-in-one 211 is connected to the solenoid valve group 216 through the IO module 218, switching the connection between the trachea 22 and the blowing port or the suction port of the micro diaphragm pump.

[0083] Preferably, in this embodiment, the program cooling device host 21 also includes a pressure sensor 214; the pressure sensor 214 is arranged in the program cooling device host 21; the pressure sensor 214 circuit is electrically connected to the touch screen computer all-in-one 211 through the IO module 218, and the pressure sensor 214 air circuit is connected to the air pipe 22 to monitor the air pressure status of the air pipe 22.

[0084] In addition, the program cooling device host 21 includes a frame housing 210. The ultra-low temperature device 2 can be an ultra-low temperature refrigerator.

[0085] Experimental example

[0086] 1. Spoiler Function Verification

[0087] The present invention innovatively designs upper and lower spoiler devices (upper cover spoiler 238 and base spoiler 236) so that the cold air drawn into the insulation box 23 can flow quickly and evenly in the cavity of the insulation box 23, so as to achieve the purpose of uniform and rapid cooling of the cavity within the program set temperature range.

[0088] 1. Use SolidWorks Flow similation software to simulate

[0089] 1.1 Experimental methods

[0090] The simplified model was simulated using SolidWorks Flow similation software to study the flow of cold air inside the insulation box 23 during air extraction, ensuring that the cold air drawn into the insulation box 23 can flow quickly and evenly within the cavity 1 of the insulation box 23, achieving the purpose of uniform and rapid cooling of the cavity within the program-set temperature range.

[0091] 1.2 Experimental Results

[0092] (1) There is no upper and lower turbulence device. SolidWorks software simulates the air flow state in the heat preservation box 23 during air extraction. The cold air cannot flow quickly and evenly in the cavity of the heat preservation box 23 (as shown in Figure 12).

[0093] (2) Adding upper and lower spoiler devices SolidWorks software simulates the air flow state in the heat preservation box 23 during air extraction, and the cold air can flow quickly and evenly in the heat preservation box 23 cavity (as shown in Figure 13).

[0094] 2. Verification using actual samples

[0095] Actual sample temperature measurement, using the same exhaust device to compare the effect of the disturbance device on the sample temperature.

[0096] 2.1 Experimental methods

[0097] Program cooling box group A (Comers item number / model CellHome-30): follow the instructions of the program cooling box, that is, place the cryopreserved tubes in the program cooling box and directly put it into the -80℃ freezer.

[0098] Aspiration type programmed cooling instrument group B has no turbulence device: through the setting, the suction device starts to start when the temperature of the cryotube drops to -3℃, and stops suctioning when it reaches -20℃.

[0099] The suction-type programmed cooling apparatus group C has a turbulence device (Example 1 of the present invention): through the setting, the suction device is started when the temperature of the cryotube drops to -3°C, and the suction is stopped when it reaches -20°C.

[0100] The data of Group A, Group B, and Group C were exported, and the average values ​​of 3 independent repetitions of 3 groups were calculated using Excel and plotted, as shown in Figure 14.

[0101] 2.2 Results Analysis

[0102] As shown in Figure 14, the programmed cooling chambers in Groups A and B experienced a temperature rise near freezing, with Group B experiencing a lower temperature rise slope than Group A. This indicates that direct extraction of cold air without a turbulence device can offset the heat dissipated by cells and cryopreservatives at freezing to some extent, but cannot eliminate the adverse effect of rising freezing temperatures. Group C, which included a turbulence device, did not experience a temperature rise during the cooling process, indicating that the turbulence device disrupts the air flow within the chamber, allowing cold air to flow rapidly along the outer wall of the sample tube, offsetting the heat dissipated by cells and cryopreservatives at freezing, and thus preventing repeated freezing and thawing of cells.

[0103] 2. The influence of cooling method on the temperature curve across the freezing point

[0104] 1. Isolation and culture of primary mouse hepatocytes

[0105] The method for isolating and culturing primary mouse hepatocytes includes the following steps: according to the instructions of the primary hepatocyte isolation kit (Livuo Bio, LV-PHIK001, two-step collagenase digestion method), primary mouse hepatocytes in good condition are isolated and extracted according to the steps described in the instructions.

[0106] Resuspend primary mouse hepatocytes at 1 x 10^7 / ml in commercial cryopreservation buffer (Sigma CS10, Cat. No. C2874-100ML) and aliquot into tubes at 1 ml / tube. Punch a hole in the cap of the cryotube and insert three temperature sensors into the center of the liquid in each cryotube.

[0107] 2. Test group and control group settings

[0108] -80℃ quick freezing group FC group: cryovials were placed directly in a -80℃ freezer.

[0109] Programmed Cooling Box CH Group (Comers Product Number / Model CellHome-30): Follow the instructions of the programmed cooling box, that is, place the cryopreserved tubes in the programmed cooling box and directly place it in a -80°C freezer.

[0110] Suction programmed cooling instrument SCM group (Example 1 of the present invention): The target temperature point of the cryotube is set by the program to be cooled from -3°C to -40°C, and the target cooling rate is -37°C / min.

[0111] Large programmed cooling instrument BCM group (France, Casu, model Micro-digitcool): program setting -3℃ to -40℃, achieving a cooling rate of -37℃ / min.

[0112] The above experiment was repeated 3 times.

[0113] 3. Data processing

[0114] 1) Export the data of the FC, CH, and SCM groups, calculate the average of three independent replicates in three groups using Excel, and plot them as shown in Figure 15.

[0115] 2) Export the data of the BCM group. The results are shown in Figure 16.

[0116] 4. Results Analysis

[0117] As shown in Figures 15 and 16 above, both the FC and CH groups experienced temperature increases near freezing. The FC group, even after directly placing the cryovials at -80°C, still experienced a temperature increase. This indicates that placing the cryovials at -80°C, regardless of the method, cannot effectively offset the heat released by the cells and cryopreservation solution. This also demonstrates that this approach has little effect on crossing the freezing point and is ineffective. No temperature increase was observed in the SCM and BCM groups. Cold air and liquid nitrogen convection offsets the heat released by the cells and cryopreservation solution, preventing repeated freezing and thawing of the cells.

[0118] 3. Application Examples

[0119] 1. Primary hepatocyte cryopreservation test

[0120] 1.1 Isolation and culture of primary mouse hepatocytes

[0121] The method for isolating and culturing primary mouse hepatocytes includes the following steps: according to the instructions of the primary hepatocyte isolation kit (Livuo Bio, LV-PHIK001, two-step collagenase digestion method), primary mouse hepatocytes in good condition are isolated and extracted according to the steps described in the instructions.

[0122] Resuspend the primary mouse hepatocytes in freezing solution at 1*10^7 / ml and dispense into several tubes at 1 ml / tube.

[0123] 1.2 Test group and control group settings

[0124] -80℃ quick freezing group FC group: cryovials were placed directly in a -80℃ freezer.

[0125] Programmed Cooling Box CH Group (Comers Product Number / Model CellHome-30): Follow the instructions of the programmed cooling box, that is, place the cryopreserved tubes in the programmed cooling box and directly place it in a -80°C freezer.

[0126] Suction programmed cooling instrument SCM group (Example 1 of the present invention): The target temperature point of the cryotube is set by the program to be cooled from -3°C to -40°C, and the target cooling rate is -37°C / min.

[0127] Large programmed cooling instrument BCM group (France, Casu, model Micro-digitcool): program setting -3℃ to -40℃, achieving a cooling rate of -37℃ / min.

[0128] The above experiment was repeated 3 times.

[0129] 1.3 Cell recovery

[0130] 1) In a biosafety cabinet, add 10 ml of recovery medium (Livuo Biotechnology, Cat# LV-Rec001) to a 15 ml centrifuge tube, preheat in a 37°C water bath for 20 min, and then transfer to the biosafety cabinet.

[0131] 2) Quickly transfer the cryopreserved hepatocytes from the refrigerator to a 37°C water bath. Immerse as many cells as possible in 37°C water. Shake the cryovials clockwise, ensuring the cap remains above the water level. Thaw the cryovials for approximately 90-120 seconds, until only small pieces of ice remain floating in the cryovials. Disinfect the cryovials with 75% alcohol and transfer them to a biosafety cabinet.

[0132] 3) Aspirate the cells with a wide-mouth pipette tip and transfer dropwise to a centrifuge tube containing preheated recovery medium. Gently invert the tube 2-3 times to mix.

[0133] 4) Directly centrifuge at low speed, 50×g, at room temperature for 5 minutes, remove the supernatant, resuspend in 4 ml of plating medium, and measure the viability of hepatocytes by trypan blue exclusion. If the viability is >60%, it can be directly plated; otherwise, purification is required.

[0134] 5) Cell Purification: Centrifuge the cell suspension at 50 × g for 5 minutes at room temperature, discard the supernatant, and resuspend the cells in 2 ml of purification medium. Carefully add 1 ml of plating medium along the tube wall (do not disturb the lower layer; distinct stratification will be visible after addition). Centrifuge at 800 × g at 4°C for 20 minutes (acceleration speed: 9, deceleration speed: 1). After centrifugation, aspirate the turbid band (live cell layer) between the purification medium and plating medium and transfer it to a new 15 mL centrifuge tube. Add 2 volumes of plating medium, mix thoroughly, and centrifuge at 50 × g for 5 minutes at room temperature. Discard the supernatant and resuspend the cells in 4 ml of plating medium. Determine the viability and total cell count of hepatocytes using the trypan blue exclusion method (manual counting with a hemocytometer, not a cell counter). It is recommended to perform the assay three times and calculate the average value.

[0135] 6) According to the number of living cells 1×10^5cells / cm 2 Inoculate into collagen-coated culture plates, shake well, and culture in a 37°C / 5% CO2 incubator.

[0136] 1.4 Results Analysis

[0137] 1) Cell yield and viability

[0138] The results are shown in Table 1:

[0139] Table 1 Cell yield and viability

[0140] Note: Initial cell viability was 86.7±5.7%, and the number of viable cells was 1*10^7 / tube.

[0141] As shown in Table 1, the cell viability and cell yield of the FC and CH groups were significantly lower than those of the SCM and BCM groups due to repeated freeze-thaw cycles near freezing. However, there were no significant differences in cell viability and cell yield between the SCM and BCM groups. This suggests that an aspirated programmed cooling device can achieve the same freezing performance as a large-scale programmed cooling device, and that this device does not require liquid nitrogen consumption, making it more cost-effective and safer.

[0142] 2) Cell adhesion

[0143] Since the cell viability of the FC and CH groups was significantly lower than 60%, the FC and CH groups were purified and the viability reached more than 80% after purification. 5 cells / cm 2 Seed the cells onto a collagen-coated culture plate, shake well, and culture in a 37°C / 5% CO2 incubator. The cell adhesion is shown in Figures 17-20.

[0144] As shown in Figures 17-20 above, cells in the FC and CH groups were basically not attached to the wall, while cells in the SCM and BCM groups showed excellent attachment, healthy cells, and good growth.

[0145] 2. Exosome cryopreservation test

[0146] 2.1 Exosome Preparation

[0147] 1) Commercial human mesenchymal stem cells (hUC-MSCs, purchased from ATCC) were used to obtain seed cells through subculture.

[0148] 2) Detection of CD19 and CD105 surface markers of seed cells by flow cytometry to confirm that the seed cells are CD19 negative (positive rate is not higher than 1%) and CD105 positive (positive rate is not lower than 99%).

[0149] 3) Using qualified seed cells, divide them into 2D and 3D culture for subculture. 2D culture is cultured in T225 cell culture flasks, and 10 T225 cell culture flasks are inoculated in a single experiment; 3D culture is cultured in 3D miniSPIN bioreactor and 3D TableTrix microcarriers.

[0150] 4) hUC-MSCs culture medium uses 3D Three-dimensional stem cell culture medium.

[0151] 5) After the cells grew to 80% density, the supernatant was collected at 24 hours, 48 ​​hours, 72 hours, and 96 hours, and fresh culture medium was added each time.

[0152] 6) Terminate the culture experiment if significant changes in cell morphology occur. For 3D culture, determine cell proliferation and apoptosis in the bioreactor using FDA / PI staining. For 2D culture, seed 12-well plates in parallel and determine cell proliferation and apoptosis using FDA / PI staining at different time points.

[0153] 7) Centrifuge the supernatants of the 2D culture and 3D culture separately at 4000 g and 4° C. for 10 minutes; transfer the supernatants to new containers and filter them using a 0.45 μm filter membrane.

[0154] 8) Place the filtered supernatant into a 100 kD semipermeable membrane dialysis bag. Tie the bag tightly and place it in 4°C sterile saline. Use gravity or a crimping machine to remove contaminants and inorganic salts. Change the dialysis bag every 24 hours for a total of three times.

[0155] 9) The concentrated and purified exosomes are transferred to a cryopreservation tube using a nano-coulter apparatus for cryopreservation.

[0156] 2.2 Test group and control group settings

[0157] -80℃ quick freezing group FC group: cryovials were placed directly in a -80℃ freezer.

[0158] Programmed Cooling Box CH Group (Comers Product Number / Model CellHome-30): Follow the instructions of the programmed cooling box, that is, place the cryopreserved tubes in the programmed cooling box and directly place it in a -80°C freezer.

[0159] Suction programmed cooling instrument SCM group (Example 1 of the present invention): The target temperature point of the cryotube is set by the program to be cooled from -3°C to -40°C, and the target cooling rate is -37°C / min.

[0160] Large programmed cooling instrument BCM group (France, Casu, model Micro-digitcool): program setting -3℃ to -40℃, achieving a cooling rate of -37℃ / min.

[0161] The above experiments were repeated 3 times.

[0162] 2.3. Exosome Thawing and Counting

[0163] 1) Quickly transfer the cryopreserved exosomes from the refrigerator to a 37°C water bath. Immerse as many exosomes as possible in 37°C water and shake horizontally in a clockwise motion, ensuring the cap remains above the water level. Thaw the cryotube for approximately 90-120 seconds, until only small pieces of ice remain floating in the cryotube. Sterilize the cryotube with 75% alcohol and transfer it to a biosafety cabinet.

[0164] 2) Sampling and counting exosomes using a nano-Coulter instrument.

[0165] 2.4 Results Analysis

[0166] The results are shown in Table 2:

[0167] Table 2 Exosome number and exosome yield

[0168] Note: The starting number of exosomes is 1*10^12 / tube.

[0169] As shown in Table 2 above, the average exosome yields of the SCM group and the BCM group were significantly better than those of the FC group and the CH group, and there was no significant difference between the SCM group and the BCM group.

[0170] 3. Organoid cryopreservation

[0171] Kidney tubules are structurally similar to organoids and were used for testing in this study.

[0172] 3.1. Isolation of renal tubules

[0173] 1) SD rats were dissected (Guangdong Medical Animal Center), and the kidneys were freed and placed in PBS on ice. The kidneys were minced into 1 mm x 1 mm x 1 mm cubes using surgical scissors.

[0174] 2) Wash off the blood with PBS, repeat 3 times.

[0175] 3) Add collagenase IV digestion solution containing calcium ions and digest for 30 minutes. Use a Pasteur pipette to repeatedly pipette until the tissue blocks are basically digested.

[0176] 4) The digestion was terminated with a cell wash solution containing 10% fetal bovine serum and the cells were filtered through a 100 μm cell sieve to remove large undigested tissue pieces.

[0177] 5) Centrifuge at 50×g for 5 minutes and wash the cells three times with cell wash buffer.

[0178] 6) Add freezing solution and dispense into several tubes at 1 ml / tube.

[0179] 3.2. Program cooling

[0180] -80℃ quick freezing group FC group: cryovials were placed directly in a -80℃ freezer.

[0181] Programmed Cooling Box CH Group (Comers Product Number / Model CellHome-30): Follow the instructions of the programmed cooling box, that is, place the cryopreserved tubes in the programmed cooling box and directly place it in a -80°C freezer.

[0182] Suction programmed cooling instrument SCM group (Example 1 of the present invention): The target temperature point of the cryotube is set by the program to be cooled from -3°C to -40°C, and the target cooling rate is -37°C / min.

[0183] Large programmed cooling instrument BCM group (France, Casu, model Micro-digitcool): program setting -3℃ to -40℃, achieving a cooling rate of -37℃ / min.

[0184] The above experiment was repeated 3 times.

[0185] 3.3 Resuscitation and survival rate detection

[0186] 1) In a biosafety cabinet, add 10 ml of recovery medium (Livuo Biotechnology, Cat# LV-Rec001) to a 15 ml centrifuge tube, preheat in a 37°C water bath for 20 min, and then transfer to the biosafety cabinet.

[0187] 2) Quickly transfer the cryopreserved renal tubules from the refrigerator to a 37°C water bath. Immerse as many of them as possible in 37°C water. Shake the tubes clockwise, ensuring the caps remain above the water level. Thaw the tubes for approximately 90-120 seconds, until only small pieces of ice remain floating in the tubes. Disinfect the tubes with 75% alcohol and transfer them to a biosafety cabinet.

[0188] 3) Aspirate the cells with a wide-mouth pipette tip and transfer dropwise to a centrifuge tube containing preheated recovery medium. Gently invert the tube 2-3 times to mix.

[0189] 4) Directly centrifuge at low speed, 50×g, at room temperature for 5 min, remove the supernatant, resuspend in 4 ml of plating medium, and measure the viability of hepatocytes by FDA / PI staining.

[0190] 3.4 Results Analysis

[0191] The results are shown in Table 3:

[0192] Table 3 Renal tubular activity

[0193] Note: Initial tubular viability was 79.1±3.1.

[0194] As shown in Table 3 above, the tubular viability of the SCM group and the BCM group was significantly better than that of the FC group and the CH group, and there was no significant difference between the SCM group and the BCM group.

[0195] In summary, the present invention realizes an active cooling process, quickly crosses the freezing point, avoids repeated freezing and thawing, and thus improves the yield and cell viability of tissues, organs, cells and their subcellular components.

[0196] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A method for air-inspired programmed cooling, characterized in that: The method comprises the following steps: placing a cavity structure I containing a sample to be frozen in a cavity structure II containing ultra-low temperature cold air, and allowing the ultra-low temperature cold air to enter the cavity structure I through program control, so that the cooling capacity of the ultra-low temperature cold air entering the cavity structure I consumes the heat released during the freezing process of the sample to be frozen.

2. The method of air-inspired programmed cooling according to claim 1, characterized in that: The method further includes the following steps: when the sample to be frozen is frozen and cooled to between -30°C and -40°C, room temperature air is allowed to enter the cavity structure I through program control to slow down the cooling rate of the cavity structure I or to recover the temperature, and the temperature of the sample to be frozen continues to decrease.

3. The method of air-inspired programmed cooling according to claim 2, characterized in that: The specific steps include: (1) placing the sample to be frozen into the cavity structure I of the thermal insulation box, placing the thermal insulation box into the cavity structure II containing ultra-low temperature cold air, and collecting the temperature in the cavity structure I of the thermal insulation box in real time through a temperature sensor; (2) The target temperature and target cooling rate are set by the program. After the temperature collected by the temperature sensor reaches the target temperature, the cooling rate is calculated based on the collected temperature. The cooling rate is compared with the target cooling rate. Based on the comparison result, one of the operations (A) to (C) is performed: (A) When the cooling rate is slower than the target cooling rate of 1°C / min, the ultra-low temperature cold air is allowed to enter the cavity structure I of the thermal insulation box, so that the cooling capacity of the ultra-low temperature cold air entering the cavity structure I of the thermal insulation box consumes the heat released during the freezing process of the sample to be frozen, so that the temperature in the cavity structure I of the thermal insulation box continues to decrease; (B) when the collected cooling rate is faster than the program target cooling rate by 1°C / min, the room temperature air is allowed to enter the cavity structure I to slow down the cooling rate or increase the temperature of the cavity structure I; (C) When the difference between the cooling rate and the target cooling rate is within 1°C / min, the ultra-low temperature cold air or the room temperature air is stopped from entering the cavity structure I of the insulation box.

4. The method of air-inspired programmed cooling according to claim 3, characterized in that: The target temperature point is set between 8°C and -60°C; the target cooling rate is set between +2°C / min and -45°C / min.

5. An insulation box for air-intake programmed cooling used in the method for air-intake programmed cooling according to any one of claims 1 to 4, the insulation box comprising an insulation box cover and an insulation box base, characterized in that: The inner side wall of the upper cover of the thermal insulation box and the inner side wall of the base of the thermal insulation box form a cavity structure I, and a temperature sensor is arranged in the cavity structure I; an upper cover through hole communicating with the cavity structure I is arranged on the wall of the upper cover of the thermal insulation box, and a base through hole communicating with the cavity structure I is arranged on the wall of the base of the thermal insulation box, a spoiler is arranged at the base through hole and / or the upper cover through hole and located in the cavity structure I, and an air pipe communicating with the cavity structure I is arranged on the wall of the upper cover of the thermal insulation box and / or the wall of the base of the thermal insulation box.

6. The heat preservation box for air-intake programmed cooling according to claim 5, characterized in that: The cavity structure I includes an upper cavity and a lower cavity. The upper cover of the insulation box is provided with the upper cavity, and the base of the insulation box is provided with the lower cavity; a sample tube holder is provided in the lower cavity, and the sample tube holder is provided with a sample tube. At least one temperature sensor is inserted into the insulation box cavity structure I or the sample tube.

7. The heat preservation box for air-intake programmed cooling according to claim 6, characterized in that: The spoiler includes an upper cover spoiler and a base spoiler; the top wall of the upper cover of the thermal insulation box is provided with the upper cover through hole, and the upper cover spoiler is provided at the upper cover through hole and located in the upper cavity; the base through hole is provided on the bottom wall of the base of the thermal insulation box, and the base spoiler is provided at the base through hole and located in the lower cavity.

8. An air-intake programmed cooling apparatus used in the air-intake programmed cooling method according to any one of claims 1 to 4, characterized in that: It comprises a program cooling device host and the insulation box according to any one of claims 5 to 7, wherein the insulation box is arranged in an ultra-low temperature device, the temperature sensor used in the insulation box is electrically connected to the program cooling device host, and the air pipe used in the insulation box is connected to the program cooling device host.

9. The air-inhalation programmed cooling device according to claim 8, characterized in that: The program cooling instrument host includes a touch screen computer all-in-one, a temperature sensor plug, an tracheal plug, a solenoid valve group, a micro diaphragm air pump, an IO module, a temperature acquisition module and a switching power supply; the program cooling instrument is provided with the switching power supply, the touch screen computer all-in-one is connected to the micro diaphragm air pump through a motor speed control module, the temperature sensor is electrically connected to the temperature sensor plug, and the tracheal plug is connected to the trachea; the temperature sensor plug is electrically connected to the touch screen computer all-in-one through the temperature acquisition module, and the temperature in the cavity structure of the insulation box is collected and monitored in real time; the touch screen computer all-in-one is connected to the solenoid valve group through the IO module, and the connection between the trachea and the blowing port or the suction port of the micro diaphragm pump is switched.

10. The air-inhalation programmed cooling device according to claim 9, characterized in that: The program cooling device host also includes a pressure sensor; the pressure sensor is arranged in the program cooling device host; the pressure sensor circuit is electrically connected to the touch screen computer all-in-one through the IO module, and the pressure sensor air circuit is connected to the trachea to monitor the air pressure status of the trachea.

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