Method and system for storing human cell sample

Abstract

The embodiment of the invention belongs to the technical field of sample storage, and relates to a method for human cell sample storage, which comprises the following steps: S1, sample pretreatment: cleaning and centrifuging a to-be-stored cell sample by using different pretreatment liquids according to the cell type to obtain a cell suspension; s2, preparing and adding a composite cryoprotectant, namely preparing the composite cryoprotectant of a corresponding formula according to the cell type, adding the composite cryoprotectant into the cell suspension in proportion, and uniformly mixing; s3, gradient freezing: putting the cell suspension added with the composite cryoprotectant into a gradient freezing instrument, cooling by stages according to a preset rate, and meanwhile, applying a constant magnetic field with the strength of 0.1 T to assist in freezing; s4, long-term storage: transferring the cells subjected to gradient freezing into intelligent storage equipment, refrigerating and storing the cells through gas-phase liquid nitrogen under the condition of-196 DEG C, monitoring storage environment parameters in real time, and performing exception handling. Ice crystal damage can be effectively reduced, and the stability of cell storage is guaranteed.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of sample storage, in particular to a method and system for human cell sample storage. BACKGROUND

[0002] In the field of human cell sample storage, the existing technology has many deficiencies, which seriously affects the long-term activity and function of cells and restricts their wide application in clinical and scientific research.

[0003] In terms of cryoprotectants, traditional dimethyl sulfoxide (DMSO) is toxic, which can cause mitochondrial dysfunction, DNA methylation abnormalities, and even induce kidney injury or allergic reactions in patients, and can also damage the lipid structure of the cell membrane, reducing the killing ability of immune cells (such as NK cells) after thawing. Although new natural antifreeze proteins (AFPs) or biomimetic materials (such as graphene oxide) can reduce ice crystal damage, they are difficult to produce on a large scale, have high costs, and have not completely solved the biocompatibility problem, for example, the extraction and purification of AFPs is extremely costly, which limits its popularization in clinical practice.

[0004] Ice crystal damage and cell structure destruction are prominent problems. Ice crystals formed during the freezing process can directly pierce the cell membrane, causing the contents of the cell to leak out. For cells with high water content (such as brain tissue and red blood cells), ice crystal damage is particularly severe. For example, during embryo freezing, ice crystals can cause osmotic imbalance, leading to cell dehydration or rupture. Although vitrification technology reduces ice crystal formation through ultra-fast cooling, it requires the use of high concentrations of cryoprotectants (such as 60% DMSO), which further exacerbates the risk of toxicity, and has extremely high requirements for operation precision, making it difficult to apply on a large scale.

[0005] Long-term storage has stability and metabolic abnormalities. Long-term cryopreservation (such as more than 20 years) can cause a decrease in mitochondrial membrane potential, a decrease in ATP synthesis, and accumulation of reactive oxygen species (ROS), leading to oxidative damage. For example, umbilical cord blood hematopoietic stem cells (HSPCs) have a significantly decreased megakaryocyte differentiation capacity after cryopreservation, and metabolic interventions such as sulforaphane are needed to partially restore their function. At the same time, freezing can cause DNA methylation abnormalities or changes in histone modification, affecting the "identity memory" of cells. For example, egg freezing can cause imprinting gene abnormalities, increasing the risk of offspring development.

[0006] Different cell types face different challenges. Immune cells (such as NK cells) are more sensitive to freezing, and their survival rate and cytotoxicity decrease significantly after thawing. Synaptic connections of neuronal cells are easily damaged after cryopreservation, affecting their electrophysiological function. The specific functions of certain cells are difficult to preserve after cryopreservation. For example, mesenchymal stem cells (MSCs) still have differentiation ability after 20 years of cryopreservation, but their paracrine function (such as the secretion of anti-inflammatory factors) may decrease.

[0007] In view of this, the present application is proposed. SUMMARY

[0008] The purpose of the embodiments of the present application is to propose a method and system for storing human cell samples to solve the technical problem that human cell samples are prone to ice crystal damage and cell structure destruction during long-term storage.

[0009] To solve the above technical problems, the embodiments of the present application provide a method for storing human cell samples, which adopts the technical solutions as follows: A method for storing human cell samples, comprising the following steps: S1: sample pretreatment, different pretreatment liquids are used to clean and centrifuge the cell samples to be stored according to the cell type, and a cell suspension is obtained; S2: preparation and addition of a composite cryoprotectant, a composite cryoprotectant with a corresponding formula is prepared according to the cell type, and is added to the cell suspension in proportion and mixed uniformly; S3: gradient freezing, the cell suspension with the added composite cryoprotectant is placed in a gradient freezing instrument, and is cooled at a preset rate in stages, while a constant magnetic field with a strength of 0.1T is applied to assist freezing; S4: long-term storage, the cell after gradient freezing is transferred to an intelligent storage device, and is stored at -196℃ by gas-phase liquid nitrogen refrigeration, and the storage environment parameters are monitored in real time and abnormal treatment is performed.

[0010] Further, in S1, the specific parameters of the sample pretreatment are: For immune cells, the pretreatment liquid is phosphate buffer solution containing 2% fetal bovine serum, the centrifugal speed is 800r / min, and the centrifugal time is 5min; For stem cells, the pretreatment liquid is phosphate buffer solution containing 5% fetal bovine serum, the centrifugal speed is 1000r / min, and the centrifugal time is 8min; For neuron cells, the pretreatment liquid is phosphate buffer solution containing 3% fetal bovine serum and 0.1% nerve growth factor, the centrifugal speed is 600r / min, and the centrifugal time is 6min.

[0011] Further, in S2, the volume ratio of the composite cryoprotectant to the cell suspension is 1:3, and the formula of the composite cryoprotectant is: The composite cryoprotectant for immune cells: 7% trehalose, 3% pullulan, and 0.3% tea polyphenol; The composite cryoprotectant for stem cells: 9% trehalose, 4% pullulan, and 0.4% tea polyphenol; Neuronal cells with complex cryoprotectant: 6% trehalose, 2.5% pullulan and 0.2% tea polyphenols.

[0012] Further, in S3, the parameters of the gradient freezing are: From room temperature to -10℃ at a rate of 1℃ / min, holding for 10min; From -10℃ to -40℃ at a rate of 5℃ / min, holding for 15min; From -40℃ to -80℃ at a rate of 10℃ / min, holding for 20min.

[0013] Further, in S4, the intelligent storage device adopts a modular design, has an automatic nitrogen supplement function, a remote monitoring platform and a standby refrigeration system.

[0014] To solve the above technical problems, the embodiment of the present application also provides a device for storing human cell samples, which adopts the technical scheme as follows: A system for storing human cell samples, comprising: A pretreatment module for cleaning and centrifuging the sample cells to be stored with different pretreatment liquids according to the cell type, comprising a pretreatment liquid storage unit and an adjustable parameter centrifuge; A complex cryoprotectant preparation and adding module for preparing a complex cryoprotectant of a corresponding formula according to the cell type and adding it to the cell suspension in proportion; A gradient freezing module composed of a gradient freezing instrument and a magnetic field generating device, which can control the cooling rate, holding time and provide a constant magnetic field; An intelligent storage module, including a modular storage device, a sensor group, a remote monitoring platform, an automatic nitrogen supplement device and a standby refrigeration system; A quality control module for detecting the functional state of the cells before and after storage and generating a quality report.

[0015] Further, the centrifuge of the pretreatment module has an adjustable speed range of 600-1000r / min and an adjustable centrifugation time range of 5-8min.

[0016] Further, the complex cryoprotectant preparation and adding module includes a raw material storage unit, a proportioning and mixing unit and an adding device, and the proportioning and mixing unit can automatically adjust the proportion of each raw material according to the preset formula.

[0017] Further, the sensor group of the intelligent storage module includes a temperature sensor, a humidity sensor and an oxygen concentration sensor, and the remote monitoring platform can receive sensor data and send an alarm signal when the parameters are abnormal.

[0018] Further, the quality monitoring module combines flow cytometry and RT-PCR technology to detect the survival rate, differentiation potential and killing activity of cells, and comprises a sample processing unit, a detection instrument and a data analysis unit.

[0019] Compared with the prior art, the embodiments of the application have the following beneficial effects: The method for storing human cell samples disclosed in the application comprises: S1: sample pretreatment, different pretreatment liquids are used to clean and centrifugally process the cell samples to be stored according to the cell types, and cell suspensions are obtained; S2: preparation and addition of a composite cryoprotectant, a composite cryoprotectant with a corresponding formula is prepared according to the cell types, and is added into the cell suspensions in proportion and uniformly mixed; S3: gradient freezing, the cell suspensions added with the composite cryoprotectant are placed in a gradient freezing instrument, and are cooled at a preset rate in stages, while a constant magnetic field with a strength of 0.1 T is applied to assist freezing; S4: long-term storage, the cells after gradient freezing are transferred into an intelligent storage device, and are stored by gas-phase liquid nitrogen refrigeration at-196 DEG C, the storage environment parameters are monitored in real time and abnormal treatment is performed. The application can effectively reduce the toxicity of the cryoprotectant, reduce ice crystal damage, ensure the stability of long-term storage of cells, improve the adaptability to different cell types, and is convenient to operate and quality monitor. BRIEF DESCRIPTION OF DRAWINGS

[0020] In order to more clearly illustrate the scheme in the application, the drawings needed in the description of the embodiments of the application will be briefly introduced as follows. Obviously, the drawings in the following description are some embodiments of the application, and other drawings can be obtained by those skilled in the art without creating laborious work on the basis of these drawings.

[0021] Figure 1 is a flow chart of an embodiment of the method for storing human cell samples according to the application; Figure 2 is a structural schematic diagram of an embodiment of the system for storing human cell samples according to the application. DETAILED DESCRIPTION

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the application belongs; the terminology used in the specification of the application is only for the purpose of describing specific embodiments and is not intended to limit the application; the specification of the application and the claims and the above drawing description of the application use the terms "include" and "have" and any variations thereof, which are intended to cover the non-exclusive inclusion; the specification of the application and the claims or the above drawing use the terms "first", "second" and the like, which are used to distinguish different objects, not to describe a specific sequence.

[0023] Reference to“an embodiment” herein means that a particular feature, structure, or characteristic described in connection with the embodiment can be included in at least one embodiment of the application. The appearances of the phrase“in one embodiment” in various places in the specification are not necessarily all referring to the same embodiment, nor are they necessarily all referring to a common set of embodiments, of the application, that are alternatives to all other embodiments. It is explicitly contemplated that embodiments described herein can be combined to include claims directed to combinations of the embodiments.

[0024] In order to better understand the technical solutions of the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings.

[0025] With reference to Figure 1 , a flow chart of one embodiment of a method for human cell sample storage according to the present application is shown. The method for human cell sample storage includes the following steps: S1: sample pretreatment, different pretreatment solutions are used to clean and centrifuge the cell sample to be stored according to the cell type, and a cell suspension is obtained.

[0026] In this embodiment, “sample pretreatment” refers to the cleaning and centrifugation operation of the cells before the human cell sample enters the freezing storage process, which aims to remove the culture medium, metabolic waste and other impurities remaining in the cell culture process, so that the cells are in a suitable state for subsequent processing. In the specific operation, the human cells to be stored are first taken out from the culture environment, and the corresponding type of pretreatment solution is added. Then, the pretreatment solution containing the cells is transferred to a centrifuge tube and placed in a centrifuge for centrifugation. The centrifugal force is used to separate the cells from the liquid, and the cells are precipitated by centrifugation, which facilitates the subsequent removal of the supernatant and the collection of the cells.

[0027] It should be noted that the pretreatment solution refers to a solution used to clean the cells, and its composition is adjusted according to the cell type to maintain the activity of the cells during the pretreatment process.

[0028] S2: preparation and addition of complex cryoprotectant, prepare the complex cryoprotectant of the corresponding formula according to the cell type, add it to the cell suspension in proportion and mix uniformly.

[0029] In this embodiment, the composite cryoprotectant refers to an agent composed of multiple components for protecting cells from damage during freezing. The preparation process is as follows: according to the formula corresponding to the cell type, mix the components in proportion and dissolve them in a sterile solvent to form a uniform solution. When adding, slowly add the prepared composite cryoprotectant to the pretreated cell suspension. During the addition process, gently stir with low force and speed to avoid mechanical damage to the cells caused by vigorous stirring, and to ensure that the composite cryoprotectant and the cell suspension are fully mixed, ensuring that each cell can contact the protective agent. The volume ratio of the composite cryoprotectant to the cell suspension is determined according to the cell type to achieve the best protection effect.

[0030] S3: Gradient freezing, place the cell suspension with added composite cryoprotectant in a gradient freezer, and gradually cool it in stages at a preset rate, while applying a constant magnetic field with a strength of 0.1 T to assist freezing.

[0031] In this embodiment, "gradient freezing" refers to a freezing method that gradually cools the cell suspension in stages at different preset cooling rates, which allows the cells to gradually adapt to the low temperature environment and reduces cell damage. In specific implementation, the cell suspension with added composite cryoprotectant is placed in a cryopreservation tube, and then the cryopreservation tube is placed in a gradient freezer. The gradient freezer is a device that can accurately control the cooling rate. According to the preset program, it first cools from room temperature to the first temperature node at a certain rate and maintains for a period of time, allowing the cells to adapt to the temperature; then it cools to the next temperature node at another rate, and so on, until the set low temperature is reached. At the same time, during the entire freezing process, a constant magnetic field with a strength of 0.1 T is applied through a magnetic field generating device. This value can ensure the auxiliary effect while avoiding adverse effects on the cells. The constant magnetic field refers to a magnetic field with a magnetic field strength that does not change with time. Its role is to assist in inhibiting ice crystal growth and reducing ice crystal damage to cells.

[0032] S4: Long-term storage, transfer the gradient-frozen cells to an intelligent storage device, store them at -196°C through gas-phase liquid nitrogen refrigeration, and monitor the storage environment parameters in real time and handle exceptions.

[0033] In this embodiment, the gradient-frozen cells are quickly transferred from the gradient freezer to an intelligent storage device. The intelligent storage device refers to a cell storage device with automatic monitoring, automatic adjustment, and remote control functions. It uses gas-phase liquid nitrogen for refrigeration, and the storage temperature can be set to -196°C, which is the boiling point of liquid nitrogen. At this temperature, cell metabolism is almost completely stopped, allowing for long-term storage.

[0034] Optionally, the sensor group in the intelligent storage device includes various sensors that can monitor parameters such as temperature, humidity, and oxygen concentration of the storage environment in real time and transmit the monitored data to the remote monitoring platform in real time. The remote monitoring platform is connected to the storage device through a network, and relevant personnel can check the storage environment parameters at any time through the platform. When an abnormal parameter is detected, the remote monitoring platform immediately sends an alarm signal and automatically starts the standby refrigeration system and the automatic nitrogen supplement device to maintain the stability of the storage environment and ensure the safety of the cells.

[0035] It should be noted that the gaseous liquid nitrogen refers to liquid nitrogen in a gaseous state, which can reduce the pollution risk caused by direct contact of cells with liquid nitrogen compared with liquid liquid nitrogen.

[0036] The application can effectively reduce the toxicity of cryoprotective agents, reduce ice crystal damage, ensure the stability of long-term cell storage, improve the adaptability to different cell types, and is convenient to operate and quality control.

[0037] In some optional implementations of the embodiment, in S1, the specific parameters of the sample pretreatment are: For immune cells, the pretreatment solution is phosphate buffered saline containing 2% fetal bovine serum, the centrifugal speed is 800 r / min, and the centrifugal time is 5 min; For stem cells, the pretreatment solution is phosphate buffered saline containing 5% fetal bovine serum, the centrifugal speed is 1000 r / min, and the centrifugal time is 8 min; For neuron cells, the pretreatment solution is phosphate buffered saline containing 3% fetal bovine serum and 0.1% nerve growth factor, the centrifugal speed is 600 r / min, and the centrifugal time is 6 min.

[0038] In the embodiment, for immune cells such as NK cells, the pretreatment solution is phosphate buffered saline (PBS) containing 2% fetal bovine serum. Fetal bovine serum refers to serum extracted from fetal bovine blood, which contains rich nutrients and can maintain the activity of immune cells; PBS is a commonly used phosphate buffered saline, which has a similar osmotic pressure to intracellular fluid and can avoid damage to cells due to osmotic pressure imbalance. The centrifugal speed is set to 800 r / min, which can effectively precipitate the cells without causing cell rupture due to excessive centrifugal force; the centrifugal time is 5 min to ensure sufficient precipitation of the cells. For stem cells such as mesenchymal stem cells, the pretreatment solution is PBS containing 5% fetal bovine serum, which can provide more nutritional support for stem cells. The centrifugal speed is 1000 r / min and the centrifugal time is 8 min, because stem cells are relatively larger in size than immune cells, and require slightly higher centrifugal force and longer time for sufficient precipitation. For neuron cells, the pre-treatment solution is PBS containing 3% fetal bovine serum and 0.1% nerve growth factor. Nerve growth factor is a protein that can promote the growth, development and survival of neurons, and the addition of this component can reduce the damage of neuron cells during the pre-treatment process. The centrifugal speed is 600 r / min, and the centrifugal time is 6 min. Since neuron cells are relatively fragile, a lower centrifugal speed and appropriate time can avoid damage to them.

[0039] In some optional implementations of the embodiment, in S2, the volume ratio of the composite cryoprotectant to the cell suspension is 1:3, and the formula of the composite cryoprotectant is: The composite cryoprotectant for immune cells: 7% trehalose, 3% pullulan, and 0.3% tea polyphenol; The composite cryoprotectant for stem cells: 9% trehalose, 4% pullulan, and 0.4% tea polyphenol; The composite cryoprotectant for neuron cells: 6% trehalose, 2.5% pullulan, and 0.2% tea polyphenol.

[0040] It should be noted that the volume ratio of the composite cryoprotectant to the cell suspension is 1:3, which can form an effective protective environment around the cells, and at the same time, the excessive protective agent will not affect the cell activity.

[0041] In this embodiment, the composite cryoprotectant for immune cells is composed of 7% trehalose, 3% pullulan, and 0.3% tea polyphenol. Among them, trehalose is a natural sugar that can form a protective film on the cell surface and stabilize the cell membrane structure; pullulan is a natural high molecular polymer that can increase the viscosity of the protective agent and reduce the formation of ice crystals; tea polyphenol has antioxidant effect and can scavenge active oxygen generated during the freezing process of cells, reducing oxidative damage.

[0042] Alternatively, the proportion of each component in the composite cryoprotectant for stem cells is adjusted. 9% trehalose and 4% pullulan can provide stronger protection for stem cells, and 0.4% tea polyphenol can enhance the antioxidant effect to meet the needs of long-term storage of stem cells. Alternatively, in the composite cryoprotectant for neuron cells, 6% trehalose, 2.5% pullulan, and 0.2% tea polyphenol are considered in the formula according to the characteristics of neuron cells, which can ensure the protection effect while avoiding the burden on the cells caused by excessive components. In some optional implementations of the embodiment, in S3, the parameters of the gradient freezing are: From room temperature to -10℃ at a rate of 1℃ / min, and keep for 10 min; From -10℃ to -40℃ at a rate of 5℃ / min, and keep for 15 min; From -40℃ to -80℃ at a rate of 10℃ / min, hold for 20min.

[0043] In this embodiment, the gradient freezing is carried out in three stages. In the first stage, the temperature is decreased from room temperature to -10℃ at a rate of 1℃ / min. The room temperature is generally about 25℃. The slower rate of temperature decrease to -10℃ allows the cells to gradually adapt to the low temperature, reducing the damage caused by the rapid crystallization of water in the cells. In the second stage, the temperature is decreased from -10℃ to -40℃ at a rate of 5℃ / min, and held for 15min. At this stage, the water in the cells begins to crystallize more, and appropriately increasing the rate of temperature decrease can reduce the formation of large ice crystals. Holding for 15min allows the cells to reach a relatively stable state in this temperature range.

[0044] In the third stage, the temperature is decreased from -40℃ to -80℃ at a rate of 10℃ / min, and held for 20min. At lower temperatures, rapid cooling can further inhibit the growth of ice crystals, and holding for 20min prepares for the subsequent transfer to a lower-temperature storage environment.

[0045] In some optional implementations of this embodiment, in S4, the intelligent storage device adopts a modular design, has an automatic nitrogen supplement function, a remote monitoring platform, and a backup refrigeration system.

[0046] In this embodiment, the intelligent storage device adopts a modular design and is composed of multiple independent functional modules, such as a storage module, a refrigeration module, a monitoring module, etc. Each module can be maintained and replaced individually, facilitating the maintenance and upgrading of the device. Specifically, the automatic nitrogen supplement function monitors the liquid level of liquid nitrogen through a liquid level sensor. When the liquid level is below a set value, the device automatically starts the nitrogen supplement pump to transport liquid nitrogen from the storage tank to the storage device, ensuring that the liquid nitrogen level is maintained within an appropriate range. The remote monitoring platform is connected to the storage device through a wireless network and receives data transmitted by sensors in real time. Relevant personnel can access the platform through terminals such as computers and mobile phones to view the data. When the temperature exceeds the fluctuation range of ±2℃, the humidity is abnormal, or the oxygen concentration exceeds the standard, the platform immediately sends an alarm signal through methods such as SMS and APP push, and simultaneously automatically starts the backup refrigeration system. The backup refrigeration system uses independent liquid nitrogen storage and transportation devices to ensure that the storage temperature can still be maintained at -196℃ when the main system fails.

[0047] With reference to Figure 2 , a flowchart of one embodiment of a system for human cell sample storage according to the present application is shown. The system for human cell sample storage includes: a pretreatment module for washing and centrifuging the sample cells to be stored with different pretreatment liquids according to the cell type, including a pretreatment liquid storage unit and an adjustable parameter centrifuge. A complex cryoprotectant preparation and adding module is used to prepare a complex cryoprotectant corresponding to a cell type and add it to a cell suspension in proportion; A gradient freezing module is composed of a gradient freezing instrument and a magnetic field generating device, which can control the cooling rate, holding time and provide a constant magnetic field; An intelligent storage module includes a modular storage device, a sensor group, a remote monitoring platform, an automatic nitrogen supplementing device and a backup refrigeration system; A quality monitoring module is used to detect the functional state of the cells before and after storage and generate a quality report. The system for storing human cell samples provided by the application can effectively reduce the toxicity of the cryoprotectant, reduce ice crystal damage, ensure the stability of long-term cell storage, improve the adaptability to different cell types, and facilitate operation and quality monitoring.

[0048] In an embodiment, the pre-treatment liquid storage unit of the pre-treatment module is a plurality of independent sealed containers, each storing a pre-treatment liquid for different types of cells such as immune cells, stem cells and neuron cells. The containers are marked with clear labels to avoid confusion. Optionally, the centrifugal device is a table centrifuge, the speed of which can be adjusted in the range of 600-1000r / min, and the time can be set in the range of 5-8min. During operation, according to the cell type, the corresponding speed and time parameters are selected, the centrifugal tube containing the cells and the pre-treatment liquid is placed in the rotor of the centrifuge, the cover is closed, and the centrifuge is started to complete the centrifugal operation.

[0049] In an embodiment, the raw material storage unit is a sealed storage tank with stirring function, which respectively stores trehalose, pullulan, tea polyphenol and other raw materials. A material level sensor is arranged on the storage tank to monitor the remaining amount of raw materials and send a reminder when the remaining amount is insufficient. Optionally, the proportioning and mixing unit is an automatic proportioning device. According to the pre-set cryoprotectant formula corresponding to the cell type, each raw material is accurately extracted by a metering pump and delivered to a mixing tank. The stirring device in the mixing tank stirs at a speed of 300r / min for 10min to fully dissolve and mix the raw materials to form a complex cryoprotectant. Optionally, the adding device is a peristaltic pump, which slowly pumps the prepared cryoprotectant into the container containing the cell suspension at a volume ratio of 1:3. The flow rate of the pump is set to 5ml / min, and a magnetic stirrer below the container stirs at a speed of 100r / min to ensure uniform mixing of the cryoprotectant and the cell suspension.

[0050] In an embodiment, the gradient freezing instrument is a program-controlled freezing instrument, which is internally provided with a temperature sensor and a heating / cooling device, and can accurately control the cooling rate with an error range of ±0.5℃ / min. Optionally, the magnetic field generating device is an electromagnetic coil wrapped around the outside of the cryogenic instrument cavity. By adjusting the current size, the coil generates a constant magnetic field of 0.1T. The magnetic field strength can be calibrated by a gauss meter.

[0051] In an embodiment, each storage unit of the modular storage device is an independent stainless steel cavity with a volume of 50L, which can accommodate multiple cryopreservation boxes. The temperature sensor in the sensor group has an accuracy of ±0.5℃, the humidity sensor has an accuracy of ±2%RH, and the oxygen concentration sensor has an accuracy of ±0.1%. Optionally, the sensor transmits the monitoring data to the control mainboard of the device through a wired manner, and the control mainboard sends the data to the remote monitoring platform through a wireless network. The remote monitoring platform adopts a cloud server architecture and has functions such as data storage, curve display, and alarm setting, and can monitor multiple storage devices at the same time. Optionally, the automatic nitrogen supplementing device is composed of a liquid nitrogen storage tank, a solenoid valve, and a pipeline. When the liquid level sensor detects that the liquid level of the liquid nitrogen is lower than 30% of the total capacity, the solenoid valve automatically opens, and the liquid nitrogen flows into the storage device through the pipeline until the liquid level reaches 80% of the total capacity, and the solenoid valve is closed. Optionally, the standby refrigeration system and the main refrigeration system use independent pipelines and control circuits. When the temperature sensor of the main system detects a temperature higher than -190℃ for 5 minutes continuously, the standby system automatically starts.

[0052] In an embodiment, the sample processing unit includes a cell counting plate, a centrifuge tube, a pipette, and other tools for processing the removed storage cell sample, such as dilution, staining, etc.

[0053] Optionally, the detection instrument includes a flow cytometer and an RT-PCR instrument. The flow cytometer is used to detect the cell survival rate. Through fluorescence staining method, live cells and dead cells will present different fluorescence signals, and the instrument can automatically count and calculate the survival rate. The RT-PCR instrument is used to detect the expression amount of specific genes in cells to evaluate the differentiation potential and functional state of cells, such as detecting the expression of pluripotency genes such as Oct4 and Sox2 in stem cells to evaluate their differentiation potential. Optionally, the data analysis unit is a computer installed with special software. The software can automatically receive the data of the detection instrument, generate a quality detection report containing parameters such as survival rate and gene expression amount, and store the report in a database for easy tracing and querying.

[0054] It should be understood that although the steps in the flowcharts of the drawings are shown in a sequential order following the arrows, the steps are not necessarily executed in the order indicated by the arrows. Unless otherwise specifically noted, the execution of the steps is not strictly limited in order, and the steps can be executed in other orders. Moreover, at least some of the steps in the flowcharts of the drawings can include multiple sub-steps or multiple stages, which are not necessarily executed at the same time, but can be executed at different times, and the execution order is not necessarily sequential, but can be round-robin or alternately executed with at least some of the steps or sub-steps or stages of other steps.

[0055] Obviously, the above-described embodiments are only some of the embodiments of the present application, but not all the embodiments. The preferred embodiments of the present application are shown in the drawings, but do not limit the patent scope of the present application. The present application can be implemented in many different forms, and conversely, the purpose of providing these embodiments is to make the disclosure of the present application more thorough and comprehensive. Although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments, or make equivalent replacements to some of the technical features. Any equivalent structure made by using the content of the specification and drawings, directly or indirectly applied to other related technical fields, is also within the scope of the patent protection of the present application.

Claims

1. A method for human cell sample storage, characterized by, The method comprises the following steps: S1: sample pretreatment, different pretreatment solutions are used to clean and centrifuge the cell samples to be stored according to the cell type, and a cell suspension is obtained; S2: preparation and addition of a composite cryoprotectant, a composite cryoprotectant of a corresponding formula is prepared according to the cell type, and is added to the cell suspension in a proportion and mixed uniformly; S3: gradient freezing, the cell suspension added with the composite cryoprotectant is placed in a gradient freezing instrument, and is cooled at a preset rate in stages, while a constant magnetic field with a strength of 0.1 T is applied to assist freezing; S4: long-term storage, the cell after gradient freezing is transferred to an intelligent storage device, and is stored at-196 DEG C through gas-phase liquid nitrogen refrigeration, the storage environment parameters are monitored in real time and abnormal treatment is performed.

2. The method for human cell sample storage according to claim 1, characterized in that, In S1, the specific parameters of the sample pretreatment are as follows: For immune cells, the pretreatment solution is phosphate buffer solution containing 2% fetal bovine serum, the centrifugal speed is 800 r / min, and the centrifugal time is 5 min; For stem cells, the pretreatment solution is phosphate buffer solution containing 5% fetal bovine serum, the centrifugal speed is 1000 r / min, and the centrifugal time is 8 min; For neuron cells, the pretreatment solution is phosphate buffer solution containing 3% fetal bovine serum and 0.1% nerve growth factor, the centrifugal speed is 600 r / min, and the centrifugal time is 6 min.

3. The method for human cell sample storage according to claim 1, wherein, In S2, the volume ratio of the composite cryoprotectant to the cell suspension is 1:3, and the formula of the composite cryoprotectant is as follows: The composite cryoprotectant for immune cells: 7% trehalose, 3% pullulan and 0.3% tea polyphenol; The composite cryoprotectant for stem cells: 9% trehalose, 4% pullulan and 0.4% tea polyphenol; The composite cryoprotectant for neuron cells: 6% trehalose, 2.5% pullulan and 0.2% tea polyphenol.

4. The method for human cell sample storage according to claim 1, wherein, In S3, the parameters of the gradient freezing are as follows: From room temperature to-10 DEG C at a rate of 1 DEG C / min, keep for 10 min; From-10 DEG C to-40 DEG C at a rate of 5 DEG C / min, keep for 15 min; From-40 DEG C to-80 DEG C at a rate of 10 DEG C / min, keep for 20 min.

5. The method for human cell sample storage according to claim 1, wherein, In S4, the intelligent storage device adopts a modular design, has an automatic nitrogen supplement function, a remote monitoring platform and a standby refrigeration system.

6. A system for human cell sample storage, characterized by, It comprises: A pretreatment module for cleaning and centrifuging the sample cells to be stored according to the cell type, comprising a pretreatment solution storage unit and an adjustable parameter centrifugal device; A composite cryoprotectant preparation and addition module for preparing a composite cryoprotectant of a corresponding formula according to the cell type and adding it to the cell suspension in a proportion; A gradient freezing module composed of a gradient freezing instrument and a magnetic field generating device, which can control the cooling rate, the holding time and provide a constant magnetic field; An intelligent storage module comprising a modular storage device, a sensor group, a remote monitoring platform, an automatic nitrogen supplement device and a standby refrigeration system; A quality monitoring module for detecting the functional state of the cells before and after storage and generating a quality report.

7. The system for storage of human cell samples according to claim 6, characterized in that, The centrifugal device of the pretreatment module has an adjustable rotating speed range of 600-1000 r / min and an adjustable centrifugal time range of 5-8 min.

8. The system for storage of human cell samples according to claim 6, wherein, The composite cryoprotectant preparation and adding module comprises a raw material storage unit, a proportioning and mixing unit and an adding device, and the proportioning and mixing unit can automatically adjust the proportion of each raw material according to a preset formula.

9. The system for storage of human cell samples according to claim 6, wherein, The sensor group of the intelligent storage module comprises a temperature sensor, a humidity sensor and an oxygen concentration sensor, and the remote monitoring platform can receive sensor data and send an alarm signal when parameters are abnormal.

10. The system for storage of human cell samples according to claim 6, wherein, The quality monitoring module combines flow cytometry and RT-PCR technology to detect the survival rate, differentiation potential and killing activity of cells, and comprises a sample processing unit, a detection instrument and a data analysis unit.

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