Cellular programmed cooling device for preventing rapid temperature drop due to phase change

Abstract

This invention discloses a programmed cell cryopreservation device to prevent excessively rapid cooling due to phase transition, belonging to the field of cell cryopreservation equipment technology. It aims to solve the problem of cell damage caused by excessively rapid cooling after phase transition due to the increased latent heat release during the sample phase transition and the temperature difference between the sample and the base, as seen in traditional devices. The device includes a base, a cooling module, a heating module, a temperature detection module, and a control module. The temperature detection module collects the sample and base temperatures in real time, while the control module accurately determines the start and end of the phase transition based on the temperature change rate, dynamically adjusting the working states of the cooling and heating modules to maintain the temperature difference within a reasonable range. It can prevent the cooling rate from deviating from the preset value after phase transition, adapts to different cell types and cryopreservation solutions, and achieves a temperature control accuracy within ±0.2℃ / min. Experimental verification shows that the survival rate of stem cells and primary cells is increased by 20-35 percentage points compared to traditional devices, providing a stable and reliable cell cryopreservation solution for biomedicine, clinical cell therapy, and other fields.

Description

Technical Field

[0001] This invention relates to the field of cell cryopreservation equipment technology, specifically to a programmed cooling device for precisely controlling the cooling rate during cell freezing and preventing excessively rapid sample cooling due to the widening temperature difference during the sample phase transition stage. This device is applicable to multiple fields such as biomedicine, cell biology, immunology, clinical cell therapy, and biobanks. It can perform programmed cooling and freezing of various cell samples, including stem cells, tumor cells, immune cells, primary cells, and cell lines, providing stable and reliable technical support for long-term cell cryopreservation and ensuring the activity and functional integrity of cells after cryopreservation and thawing. Background Technology

[0002] In biomedical research and clinical applications, long-term cell preservation is an indispensable and crucial step. The core objective of cell cryopreservation is to inhibit cellular metabolic activities through a low-temperature environment, thereby achieving long-term cell survival and providing high-quality cell resources for subsequent experimental research, cell therapy, organ transplantation, vaccine development, and other applications.

[0003] The cooling rate is one of the core factors affecting the effectiveness of cell cryopreservation. Numerous studies have shown that the cooling rate during cell cryopreservation must be strictly controlled within a specific range (typically 1-5°C / min): if the cooling rate is too slow, a large amount of intracellular water will seep out of the cell, leading to cell dehydration, osmotic pressure imbalance, and consequently damaging the cell membrane structure and organelle function, causing cell damage; if the cooling rate is too fast, intracellular water cannot seep out in time, resulting in the formation of numerous ice crystals within the cell. The growth and expansion of these ice crystals can directly puncture the cell membrane, damage organelles, and lead to cell death. Therefore, the core function of a programmed cooling system, as the core equipment for cell cryopreservation, is to cool cell samples at a preset constant rate, balancing the risks of cell dehydration and ice crystal formation, and maximizing the protection of cell viability.

[0004] Traditional programmed cooling systems work by controlling the base temperature to decrease at a preset rate, utilizing heat conduction between the base and the sample container to synchronously lower the sample temperature. However, during actual cooling, when the sample temperature drops to its phase transition temperature (around 0°C for water-containing samples; this temperature decreases further with the addition of cryoprotectant, typically between -5°C and -20°C), the sample undergoes a phase transition (primarily the freezing of water). During this phase transition, the sample releases a large amount of latent heat, causing a rapid, short-term temperature increase. Meanwhile, the base of the traditional programmed cooling system continues to cool at the preset rate, resulting in a rapid widening of the temperature difference between the sample and the base.

[0005] For example, when the preset cooling rate is 2℃ / min, the base temperature continues to decrease at a rate of 2℃ / min. However, after the sample enters the phase transition phase, due to the release of latent heat, the temperature rises from -10℃ to approximately -4℃ and remains there for a period. During this time, the temperature difference between the sample and the base rapidly increases from the original 2-3℃ to 10-15℃ or even higher. After the phase transition ends, the sample no longer releases latent heat. Driven by this huge temperature difference, the sample temperature experiences a rapid, "jump-like" drop, with a cooling rate far exceeding the preset 2℃ / min, potentially reaching 5-7℃ / min or higher. This excessively rapid cooling rate leads to the formation of numerous large ice crystals within the cells. The mechanical damage and osmotic pressure impact of these ice crystals severely disrupt cell structure, resulting in a significant decrease in cell survival rates after cryopreservation and thawing. This damage is particularly pronounced for highly sensitive cells such as stem cells and primary cells, and can even lead to complete loss of cell viability, rendering them unsuitable for subsequent applications.

[0006] To address the above problems, some improvements have emerged in the existing technology, but all of them have obvious drawbacks: 1. Improve the temperature control accuracy of the cooling module: Some solutions attempt to improve the temperature control accuracy of the base by optimizing the control algorithm of the cooling module, so as to reduce the temperature difference between the sample and the base. However, due to the suddenness, uncertainty and variability of the latent heat release of phase change, simply improving the temperature control accuracy of the base cannot fundamentally offset the temperature fluctuations caused by latent heat release, and cannot solve the core problem of widening temperature difference. 2. Segmented cooling strategy: Some methods employ a fixed segmented cooling strategy, reducing the cooling rate within a preset phase transition temperature range. However, the phase transition temperature and latent heat of phase transition vary significantly among different cell samples and cryopreservation solutions (such as DMSO concentration, whether glycerol is added, serum ratio, etc.). Fixed segmented parameters cannot adapt to diverse sample types, resulting in poor temperature control and limited versatility. 3. Lack of dynamic response mechanism: Existing technologies generally lack real-time detection and dynamic response mechanisms for phase change processes, making it impossible to accurately determine the start and end times of phase change. This results in temperature control strategies being adjusted lagging behind the phase change process, making it difficult to effectively suppress the phenomenon of excessively rapid cooling after phase change.

[0007] Therefore, developing a programmed cooling instrument that can detect the phase transition state of samples in real time, dynamically adjust the temperature control strategy, and accurately maintain a reasonable temperature difference between the sample and the base is of great practical significance and value for improving the quality of cell cryopreservation and expanding the application scenarios of cell resources. Summary of the Invention

[0008] The technical problem this invention aims to solve is to address the shortcomings of traditional programmed cell cooling systems, where the release of latent heat during the phase transition phase of cell samples leads to a widening temperature difference between the sample and the base, resulting in a cooling rate far exceeding the preset value after the phase transition and the formation of large ice crystals that damage cells. This invention provides a programmed cell cooling system that prevents excessively rapid cooling due to phase transition. The device dynamically adjusts the operating states of the cooling and heating modules by real-time monitoring of the sample's phase transition state (start and end), precisely maintaining a reasonable temperature difference between the sample and the base. This ensures that the entire cooling process strictly adheres to the preset cooling rate, maximizing the protection of cell viability.

[0009] To achieve the above technical objectives, this invention provides a cell programmed cooling device to prevent excessively rapid cooling due to sample phase transition. Its core technical solution lies in the real-time acquisition of temperature data from the sample and the base via a temperature detection module. The control module accurately determines the start and end of the sample's phase transition stage based on temperature change characteristics, and then dynamically adjusts the operating states of the cooling and heating modules to keep the temperature difference between the sample and the base within a reasonable range, thus avoiding excessively rapid cooling after the phase transition stage ends. The overall structure of the device includes a base, a cooling module, a temperature detection module, a heating module, and a control module. These components work together to achieve precise temperature control.

[0010] 1. Overall structural composition (1) Base The base is the core component that supports the sample container and facilitates heat conduction. Its primary function is to uniformly and efficiently transfer the cooling capacity of the cooling module and the heat from the heating module to the sample container, ensuring that the sample temperature follows the base temperature change smoothly. The base must possess excellent thermal conductivity, structural stability, and temperature uniformity. It is preferably made of metals with high thermal conductivity, such as oxygen-free copper, aluminum alloy, or silver alloy. These materials can rapidly conduct cold and heat, ensuring a uniform overall temperature of the base and preventing localized temperature fluctuations from affecting the sample cooling effect.

[0011] The base's structural design must be compatible with the dimensions of commonly used sample containers (cryotubes) to ensure stable placement and good thermal contact with the base, minimizing contact thermal resistance. The base thickness is typically designed to be 10-30mm, ensuring both structural stability and effective installation of the temperature sensor within the temperature detection module, as well as accurate temperature signal acquisition. The connection between the base and the cooling and heating modules must be tight, which can be achieved through bolt fixing to further reduce contact thermal resistance, improve heat conduction efficiency, and ensure that the cooling and heating energy generated by the cooling and heating modules are efficiently transferred to the base.

[0012] (2) Refrigeration module The cooling module provides cooling to the base and is the core component for achieving the cooling function. It is fixedly connected to the base and forms a stable heat conduction path. The cooling module can be selected from a Stirling refrigerator, a compressor refrigeration unit, or a liquid nitrogen refrigeration unit. The appropriate cooling method can be selected based on the actual application scenario, cooling requirements, and cost budget. Stirling refrigerators have advantages such as high cooling efficiency, high temperature control accuracy, stable operation, and low noise, making them suitable for laboratory settings with high temperature control accuracy requirements. Compressor refrigeration unit: High cooling power and wide cooling range, suitable for scenarios requiring rapid cooling or large sample volume processing, such as biobanks; Liquid nitrogen refrigeration unit: fast cooling speed and low minimum temperature (down to -196℃), suitable for special sample cryopreservation needs with extremely high requirements for cooling speed and low temperature range.

[0013] The cooling power of the cooling module can be adjusted according to actual needs, typically within a range of 0-1000W, to adapt to different cooling rates and sample volumes. The control terminal of the cooling module is electrically connected to the control module, receiving control commands to adjust the cooling power or perform start / stop operations.

[0014] (3) Heating module The heating module is used to raise the temperature of the base during the sample phase transition stage, maintain a reasonable temperature difference between the sample and the base, and is directly connected to the base to efficiently transfer heat. The heating module can use mature electric heating elements such as electric heating tubes and electric heating plates. These heating elements have advantages such as fast heating speed, sensitive temperature control response, simple structure, and low cost, and can quickly respond to the commands of the control module and adjust the base temperature in a timely manner.

[0015] Furthermore, the heating module can also be integrated into the cooling module to form an integrated temperature control module. This integrated design simplifies the equipment structure, reduces installation space, and improves the coordinated control of cooling and heating, making temperature adjustment more precise and efficient, and avoiding temperature deviations caused by delays in inter-module coordination. The installation position of the heating module must ensure that heat is evenly transferred to the base, preventing localized overheating or underheating. It can typically be installed at the bottom, side, or inside of the base to ensure a uniform overall temperature rise. The heating module's power adjustment range is 0-500W, adaptable to the base's material, size, and cooling requirements. Its control terminal is electrically connected to the control module, receiving control commands to adjust the heating power or perform start / stop operations.

[0016] (4) Temperature detection module The temperature detection module is used to collect sample temperature and base temperature in real time, providing accurate and reliable temperature data support for the control module to judge the phase transition stage and adjust the temperature control. It includes two core components: sample temperature sensor and base temperature sensor.

[0017] The sample temperature sensor is installed inside the sample container, in direct contact with the sample. It can sense subtle changes in sample temperature in real time and accurately capture temperature fluctuations during the cooling process, especially the temperature change characteristics during the phase transition stage (such as temperature rise, maintenance, and fall). The base temperature sensor is installed inside the base, in direct contact with the base. It can accurately detect the actual temperature of the base and reflect the temperature change status of the base, providing a basis for the control module to adjust the working status of the cooling and heating modules.

[0018] The sensor in the temperature detection module must possess high measurement accuracy (no less than ±0.5℃), good stability, and fast response speed to ensure the accuracy and real-time performance of temperature detection data. Mature products such as PT100 platinum resistance sensors, thermocouple sensors, or semiconductor temperature sensors are preferred. The temperature sensor is connected to the control module via shielded wires. Shielded wires effectively reduce electromagnetic interference, ensuring stable transmission of the temperature signal and preventing signal interference that could distort the detection data.

[0019] (5) Control module The control module is the core control unit of the equipment. It is electrically connected to the temperature detection module, refrigeration module, and heating module, respectively. Its responsibilities include receiving temperature detection signals, processing user input commands, analyzing and determining the phase change state, generating control commands, and coordinating the collaborative work of each module. The control module typically includes a microprocessor, signal processing unit, drive unit, and storage unit. These units work together to achieve precise control functions. The core algorithms of the control module include the phase transition stage judgment algorithm and the temperature control strategy algorithm, and their working logic is as follows: Phase transition detection algorithm: By calculating the rate of change of sample temperature per unit time ΔT / Δt (ΔT is the temperature change and Δt is the time change) in real time, when ΔT / Δt changes from a negative value (cooling state) to a positive value (heating state) and the heating rate is greater than 1℃ / min, the sample is determined to have entered the phase transition stage; the sample temperature is continuously monitored, and when the difference between the sample temperature and the highest sample temperature after the phase transition is detected to exceed 0.5℃, the sample phase transition is determined to have ended; Temperature control strategy algorithm: Based on the phase change judgment result, corresponding control commands are generated to achieve dynamic control of the cooling module and the heating module. When the phase change occurs, the cooling module is controlled to reduce its cooling power or stop working, while the heating module is controlled to start or increase its heating power to raise the temperature of the base and ensure that the temperature difference between the sample and the base is maintained within a reasonable range (i.e., the range between the average temperature difference between the sample and the base plus 5°C within 1 minute before the phase change and the average temperature difference). When the phase change ends, the heating module is controlled to reduce its power or stop working, while the cooling module is controlled to resume working, so that the base cools down at a constant rate according to the preset speed.

[0020] Compared with the prior art, the cell programmed cooling device of the present invention, which prevents excessively rapid cooling due to phase transition, has the following significant advantages: 1. High temperature control accuracy, effectively preventing the sample from cooling down too quickly after the phase transition. This invention uses a temperature detection module to collect real-time temperature data from the sample and the base. The control module accurately determines the start and end of the phase transition based on the rate of temperature change, dynamically adjusting the operating states of the cooling and heating modules to strictly maintain the temperature difference between the sample and the base within a reasonable range. After the phase transition, the sample temperature steadily decreases at a preset rate, following the base temperature, avoiding the excessively rapid cooling phenomenon caused by large temperature differences in traditional devices. Experimental verification shows that the cooling rate error of this invention after the phase transition can be controlled within ±0.2℃ / min, significantly outperforming traditional programmed cooling instruments (which can have a cooling rate 1-5℃ / min higher than the set temperature after the phase transition), ensuring that the entire cooling process strictly meets the technical requirements for cell cryopreservation.

[0021] 2. Cell survival rate is significantly improved. A stable and controllable cooling process can effectively reduce the formation of large ice crystals within cells. Traditional programmed cooling devices cause large ice crystals to form inside cells due to excessively rapid cooling after phase transition. These ice crystals can puncture cell membranes and damage organelle structures. However, this invention, through precise temperature control, avoids the rapid increase in cooling rate after phase transition, which would otherwise lead to an increase in intracellular ice crystal formation. This effectively reduces the mechanical damage to cell structures caused by ice crystals and also reduces the impact of osmotic pressure shocks on cells.

[0022] Extensive experimental verification has shown that using the programmed cooling system of this invention for cell cryopreservation significantly improves cell viability compared to traditional programmed cooling systems. The survival rate of stem cells (such as human bone marrow mesenchymal stem cells) can reach over 90%, which is 20-30 percentage points higher than that of traditional equipment (60-70%). The survival rate of primary cells (such as primary rat hepatocytes) can reach over 85%, which is 25-35 percentage points higher than that of traditional equipment (50-60%). The survival rate of common cell types such as tumor cells and immune cells can reach more than 80%, which significantly improves the quality of cell cryopreservation and provides a reliable guarantee for subsequent cell experiments and clinical applications.

[0023] 3. Highly adaptable and suitable for diverse sample types Different types of cells and different cryopreservation solutions (such as different DMSO concentrations, the presence or absence of glycerol, and differences in serum ratios) exhibit significant differences in phase transition temperature and latent heat of phase transition. The control module of this invention can capture the phase transition characteristics of samples in real time through a temperature detection module. Without requiring manual parameter adjustments by the user, it can automatically adapt to the phase transition characteristics of different samples, dynamically adjust the temperature control strategy, and achieve precise temperature control.

[0024] For example: For tumor cells with 10% DMSO cryopreservation solution, the phase transition temperature is approximately -5℃ and the latent heat of phase transition is approximately 330 J / g. For stem cell cryopreservation solutions containing a high proportion of serum, the phase transition temperature is approximately -3℃, and the latent heat of phase transition is approximately 340 J / g.

[0025] The equipment of this invention can accurately detect the phase transition process of the above-mentioned different samples and adjust the cooling strategy in a timely manner to ensure stable and reliable cooling effect. It is suitable for cryopreservation of various cell samples and has strong versatility. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of the overall structure of the cell programmed cooling device of the present invention, which prevents excessively rapid cooling due to phase transition. Figure 2 This is a schematic diagram of the explosion structure of the cell programmed cooling device of the present invention, which prevents excessively rapid cooling due to phase transition. Figure 3 The cooling curve of a traditional programmed cooling system; Figure 4 This is a cooling curve diagram of the programmed cooling device of the present invention.

[0027] Figure 1-2 The components are: 1. Base, 2. Cooling module, 3. Temperature detection module, 31. Sample temperature sensor, 32. Base temperature sensor, 4. Heating module, 5. Control module.

[0028] Figure 3 In the diagram: the horizontal axis represents time (min), and the vertical axis represents temperature (°C); curve 1 is the sample temperature curve, and curve 2 is the base temperature curve; during the phase transition stage, the sample temperature increases, while the base temperature continues to decrease, the temperature difference widens, and after the phase transition, the sample temperature decreases rapidly.

[0029] Figure 4In the diagram: the horizontal axis represents time (min), and the vertical axis represents temperature (°C); curve 3 is the sample temperature curve, and curve 4 is the base temperature curve; during the phase transition stage, the base temperature increases, and the temperature difference between the sample and the base is within a reasonable range; after the phase transition, the sample temperature decreases at a constant rate. Detailed Implementation

[0030] To make the technical solution, working principle, and beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the present invention.

[0031] Example 1: Typical working process and temperature control procedure The typical working process of the cell programmed cooling device of the present invention mainly includes the following:

[0032] (1) Pre-cooling stage: Place the sample container containing the cell sample and cryopreservation solution on the base, ensuring contact between the sample container and the base to reduce thermal resistance. Insert the sample temperature sensor into the sample container, ensuring direct contact with the sample to accurately measure the sample temperature. Start the device and cool the sample temperature from room temperature to 5°C at a rate of 2°C / min, holding for 5 minutes. After the holding period, the base and sample will stabilize and proceed to the second stage.

[0033] (2) Phase change cooling stage.

[0034] The cooling target for this stage can be set to -35℃, with a cooling rate of 2℃ / min. The control module controls the cooling module according to the preset cooling parameters. The cooling module operates at a set power, transferring cold energy to the base through heat conduction, causing the base temperature to decrease at a constant preset rate. The base temperature is collected in real time by a base temperature sensor and transmitted to the control module. Based on the base temperature feedback data, the control module dynamically adjusts the power of the cooling module through a temperature control strategy algorithm to ensure that the base temperature decreases strictly according to the preset rate, avoiding temperature fluctuations. The sample cools synchronously through heat conduction between the sample container and the base.

[0035] As the sample temperature decreases, the supercooled sample undergoes a phase transition, rapidly releasing latent heat and causing the sample temperature to rise rapidly. The control module determines the occurrence or onset of the phase transition stage by calculating the rate of change of sample temperature per unit time, ΔT / Δt. When it detects that ΔT / Δt changes from a negative value (cooling state, e.g., -2℃ / min) to a positive value (heating state, e.g., +1.5℃ / min) and the heating rate is greater than 1℃ / min, the control module identifies the occurrence of the phase transition stage. It then turns off or reduces the power of the refrigerator while turning on or increasing the power of the heating module, heating the base to a reasonable temperature difference range with the sample, and maintaining this temperature until the phase transition stage ends. A reasonable temperature difference range refers to the interval between the average temperature difference between the sample and the base one minute before the phase transition plus 5℃ and that average temperature difference. For example, if the average temperature difference between the sample and the base one minute before the phase transition is 6℃, then the reasonable temperature difference range is 6℃ to 11℃, and the control module stabilizes the temperature difference between 6 and 11℃. This control strategy effectively prevents the temperature difference between the sample and the base from widening, laying the foundation for a stable temperature drop in the sample after the phase transition.

[0036] After entering the phase transition stage, the sample temperature rises to its peak and then begins to decline over time. The control module continuously monitors the sample temperature using a phase transition judgment algorithm. When the difference between the sample temperature and the highest sample temperature after the phase transition exceeds 0.5℃, the phase transition is considered complete. For example, if the highest sample temperature after the phase transition is -5℃, the phase transition stage is considered complete when the sample temperature gradually decreases to -5.5℃. The control module then generates new control commands: on the one hand, it controls the heating module to reduce its power or stop working, ceasing heat supply to the base; on the other hand, it controls the cooling module to resume operation, continuing to cool the base at a preset cooling rate. Because the temperature difference between the sample and the base remains within a reasonable range during the phase transition stage, the sample temperature can smoothly follow the base temperature and decrease at a preset rate after the phase transition ends, avoiding the excessively rapid cooling phenomenon caused by excessive temperature differences in traditional equipment.

[0037] The equipment then entered the constant-rate cooling phase again, and continued until it reached -35°C, after which it switched to maintaining -35°C for 10 minutes.

[0038] (3) Cryogenic stage With the phase change cooling stage complete, the equipment enters the cryogenic stage, targeting -60℃ or -80℃ at a cooling rate of 2℃. The equipment then switches to conventional cooling mode, maintaining a constant descent of the base at a rate of 2℃ / min until the target temperature is reached. The system then maintains this target temperature for 10 minutes. The entire cooling process is then complete.

[0039] Example 2: Comparison Experiment of Cooling Curves between Traditional Equipment and the Equipment of the Present Invention 1. Experimental setup Sample: DMEM medium containing 10% DMSO (simulating cell cryopreservation sample; DMSO is a commonly used cryoprotectant for cell cryopreservation, with a concentration generally of 5-10%, while DMEM is a commonly used cell culture medium). Sample container: 2mL cryovial, containing 1mL of sample; Preset cooling parameters: Stage 1: Cool down to 5℃ and hold for 2 minutes; Stage 2: Cool down to -25℃ at 2℃ / min and hold for 2 minutes; Stage 3: Cool down to -60℃ at 2℃ / min and hold for 2 minutes. Experimental group: Using the cell programmed cooling device of this invention to prevent excessively rapid cooling due to phase transition; Control group: Using a traditional programmed cooling instrument (without the function of detecting the start and end of the phase change stage and the function of adjusting the temperature difference between the phase change stage and the sample). Testing indicators: Real-time recording of sample temperature and base temperature, plotting cooling curves, and analysis of temperature difference changes during phase transition and cooling rate after phase transition.

[0040] 2. Experimental Results and Analysis (1) Control group (traditional programmed cooling device) (attached) Figure 3 Before the phase transition, the temperature difference between the sample and the base was maintained at 6.5℃. When the phase transition occurred, the sample temperature rose from -5℃ to 1℃, while the base temperature continued to decrease at a rate of 2℃ / min. By the end of the phase transition, the temperature difference between the sample and the base rapidly increased from 6.5℃ to 18℃. This caused the sample temperature to drop rapidly after the phase transition, with a maximum cooling rate of -6.2℃ / min, far exceeding the preset 2℃ / min. The cooling rate fluctuated drastically and could not maintain a stable preset cooling rate.

[0041] (2) Experimental group (equipment of the present invention) (attached) Figure 4 Before the phase change occurs, the temperature difference between the sample and the base is maintained at 7℃. After the phase change occurs, the sample temperature rises rapidly. The control module 8 adjusts the working status of the cooling module 2 and the heating module 4 in a timely manner to raise the temperature of the base so as to maintain the temperature difference between the sample and the base 1 within a reasonable range of 7-12℃. When the phase change ends, the temperature difference between the sample and the base is about 8℃. The sample temperature drops steadily at a rate of 2.0±0.2℃ / min. The cooling rate accurately meets the preset requirements and there is no obvious fluctuation.

[0042] The comparison results of the cooling curves fully demonstrate that the device of the present invention can effectively suppress the phenomenon of excessively rapid cooling after phase transition, solve the core technical defects of traditional programmed cooling instruments, and provide a stable and reliable cooling environment for cell cryopreservation.

[0043] Example 3: Application of cryopreservation of human bone marrow mesenchymal stem cells 1. Equipment parameters and sample information Equipment Model: The cell programmed cooling device of the present invention to prevent excessively rapid cooling due to phase transition; Sample type: Human bone marrow mesenchymal stem cells (P3 generation); Cell density: 1×10 6 cells / mL; Cryopreservation solution: containing 10% DMSO, 20% fetal bovine serum, and 70% DMEM high-glucose medium; Sample container 5: 2mL cryovials, each cryovial containing 1mL of cell sample, with a total of 3 parallel samples; Control group: A traditional programmed cooling device (without phase change detection and dynamic temperature control functions) was used, and other conditions were the same as those of the experimental group; Preset cooling parameters: Phase 1: Cool down from room temperature (25℃) to 5℃ at a rate of 5℃ / min, and hold for 5 minutes; Phase 2: Cool from 5℃ to -25℃ at a rate of 2℃ / min, and hold for 5 minutes; Phase 3: Cooling from -25℃ to -60℃ at a rate of 2℃ / min, holding for 5 minutes; Phase 4: Cool down from -60℃ to -80℃ at a rate of 5℃ / min to complete the cooling process.

[0044] 2. Experimental Results and Analysis (1) Comparison of cooling curves: The cooling curve of the experimental group (the device of this invention) shows that the sample temperature rises slightly during the phase change stage, and the temperature difference between the sample and the base 1 remains stable between 5-6℃. After the phase change, the sample temperature drops steadily at a rate of 2.0±0.1℃ / min, which strictly meets the preset requirements. The cooling curve of the control group (traditional programmable cooling instrument) shows that the temperature difference between the sample and the base increases rapidly from 6℃ to 17℃ during the phase change stage. After the phase change, the sample temperature drops rapidly, with the maximum cooling rate reaching -5.2℃ / min, which far exceeds the preset 2℃ / min.

[0045] (2) Cell viability detection: After cooling, the cryovials of both the experimental and control groups were transferred to liquid nitrogen tanks for storage for 1 week. During thawing, the cryovials were removed from the liquid nitrogen tanks and quickly placed in a 37°C water bath to thaw. After the samples were completely thawed, the cell viability was detected by trypan blue staining (trypan blue dye cannot penetrate the intact cell membrane of living cells, while the cell membrane of dead cells is damaged, allowing the dye to enter the cells and make them blue; the viability is calculated by counting the ratio of blue cells to the total number of cells).

[0046] The test results showed that the cell viability rates of the three parallel samples in the experimental group were 68.5%, 73.2%, and 71.3%, respectively, with an average viability rate of 71.0%; while the cell viability rates of the three parallel samples in the control group were 40.3%, 48.2%, and 36.5%, respectively, with an average viability rate of 41.7%. The cell viability rate of the experimental group was significantly higher than that of the control group, with an increase of 29.3 percentage points.

[0047] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention; the actual structure is not limited thereto. In conclusion, if those skilled in the art are inspired by this description and design similar structures and embodiments without departing from the spirit of the invention, such designs should fall within the protection scope of the present invention.

Claims

1. A cell programmed cooling device to prevent excessively rapid cooling due to phase transition, characterized in that: It includes a base (1), a cooling module (2), a temperature detection module (3), a heating module (4), and a control module (5). The control module (5) is electrically connected to the temperature detection module (3), the cooling module (2), and the heating module (4) respectively. It is used to receive temperature monitoring signals and dynamically adjust the working status of each module. The control module can determine the start and end of the phase change stage of the sample during the freezing process through the phase change judgment algorithm, and implement the temperature control strategy of the phase change stage to keep the temperature difference between the base and the sample within a certain reasonable range, so as to prevent the sample from cooling down too quickly after the phase change ends.

2. The cell programmed cooling device for preventing excessively rapid cooling due to phase transition as described in claim 1, characterized in that: The base (1) is made of oxygen-free copper, aluminum alloy or silver alloy or other thermally conductive metal, and is used to place the sample container and fit tightly with the refrigeration module to facilitate the conduction of the refrigeration module's cold energy.

3. A cell programmed cooling device for preventing excessively rapid cooling due to phase transition as described in claim 1, characterized in that: The refrigeration module (2) adopts electric refrigeration or liquid nitrogen refrigeration, wherein electric refrigeration can be selected from Stirling refrigerator or compressor refrigeration unit.

4. A cell programmed cooling device for preventing excessively rapid cooling due to phase transition as described in claim 1, characterized in that: The heating module (4) is electrically heated and is connected to the base separately; or the heating module (4) and the cooling module (2) are integrated into an integrated temperature control module.

5. A cell programmed cooling device for preventing excessively rapid cooling due to phase transition as described in claim 1, characterized in that: The temperature detection module (3) includes a sample temperature sensor and a base temperature sensor, which can simultaneously detect the temperature of the sample in the sample container and the temperature of the base, and is electrically connected to the control module (5).

6. A cell programmed cooling device for preventing excessively rapid cooling due to phase transition as described in claim 1, characterized in that: The control module can receive data from the temperature detection module and control the operation of the cooling module and heating module based on the received sample and base temperature data and the cooling curve set by the user.

7. A cell programmed cooling device for preventing excessively rapid cooling due to phase transition as described in claim 1, characterized in that: The phase transition judgment algorithm of the control module (5) is as follows: when the sample temperature change rate ΔT / Δt changes from a negative value to a positive value and the heating rate is greater than 1℃ / min, the phase transition is determined to start; when the sample temperature and the highest temperature after the start of the phase transition are more than 0.5℃, the phase transition is determined to end.

8. A cell programmed cooling device according to claim 1 for preventing excessively rapid cooling due to phase transition, characterized in that: The temperature control strategy of the control module (5) during the phase change stage of the sample is as follows: when the phase change begins, the cooling module (2) is controlled to reduce power or stop working, and the heating module (4) is started or increased in power to maintain the temperature difference between the sample and the base (1) within a reasonable range; after the phase change stage ends, the cooling module (2) increases in power or starts running, and the heating module reduces in power or stops running, and the cooling module is controlled to enter constant-speed cooling.

9. A cell programmed cooling device for preventing excessively rapid cooling due to phase transition as described in claim 1, characterized in that: The reasonable range is the range from the average temperature difference between the sample and the base one minute before the phase change occurs plus 5°C to the aforementioned average value.

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