CN114608932A

CN114608932A - Automatic thawing system for cryopreservation of biological samples

Info

Publication number: CN114608932A
Application number: CN202210387928.4A
Authority: CN
Inventors: 姚树龙; 钱晓玲
Original assignee: Shanghai Guansai Biotechnology Co ltd
Current assignee: Shanghai Guansai Biotechnology Co ltd
Priority date: 2022-04-13
Filing date: 2022-04-13
Publication date: 2022-06-10

Abstract

The invention discloses an automatic thawing system for cryopreservation of biological samples, which comprises an electric module, a support frame, a circuit main board, a control assembly and a metal block for placing samples, wherein a vertical groove is formed in the support frame, the electric module is installed in the vertical groove, the metal block is installed at the upper end of the electric module, the circuit main board is installed on one side of the support frame, a sample groove is formed in the upper end of the metal block, an IR temperature sensor is installed on the side surface of the metal block, and a heating rod and a thermistor are arranged inside the metal block. In the implementation process of the invention, the accurate control and real-time detection of the heating process of the biological sample are realized through the optimized control strategy, the defects caused by a water bath method are completely avoided, the unfreezing standardization of the biological sample is realized, the control strategy can be further put into GMP and clinical application through SOP, the problems of pain points and difficulties in the prior art are practically solved, and the method has important significance for the development of the biological industry.

Description

Automatic thawing system for cryopreservation of biological samples

Technical Field

The invention belongs to the technical field of unfreezing of a frozen and preserved biological sample, and particularly relates to an automatic unfreezing system for the frozen and preserved biological sample.

Background

Cryopreservation is a technique for preserving biological agents at low temperatures (at or below-80 ℃ C., for long periods of time) and has become a valuable technique for the routine manipulation of cells and tissues in the field of bioscience. Normal cells, tumor cells and cell lines, human and animal germ cells, transgenic yeast and bacteria are common cell types that researchers need regular cryopreservation.

With the development of freezing technology, it is now generally recognized that both cooling and thawing during cryopreservation must occur at extremely rapid rates, and that thawing is more difficult than freezing. Optimal thawing of biological samples is critical to ensure success of downstream studies, but little attention remains in sample thawing. Many areas, such as biological specimen banks, cell therapy, monoclonal antibodies, cell-based drug discovery, assisted reproduction, and other cell-based assays, can benefit from standardized cell thawing to increase their productivity and help them fulfill the mission of improving human health.

During thawing of frozen samples, recrystallization of ice may begin at temperatures as low as-100 ℃, and as the temperature rise progresses, the temperature difference between the thawed sample and the thawing environment becomes smaller, and the rate of heat transfer to the thawed sample will decrease proportionally. As the melting point is approached, the temperature difference between the thawed sample and the thawing environment is typically less than 50 ℃, and intracellular ice crystals, which may not be harmful after freezing, may grow to a destructive size.

Previous studies have shown that the longer the thawing time, the greater the damage to the cells. Compared with slow thawing, fast thawing of the sample can improve the cell viability after thawing. The rapid temperature increase allows the sample to thaw while minimizing the time for ice recrystallization and exposure of the cells to high osmotic pressure and Cryoprotectant (CPAs) concentrations. The most common method of rapid thawing is to remove the sample from the ultra-low temperature storage vessel and immediately place it in a warm (37 ℃) water bath. The time from the removal at low temperature to the introduction into the warm water bath ("air time") is of critical importance and should be as short as possible (a few seconds). Prolonged times at temperatures above-80 ℃ (nominally >30 seconds) can result in slow sample warming, which can impair cell viability and function. For this reason, the thawing process typically requires the transfer and transport of frozen samples in dry ice (-79 ℃) or liquid nitrogen (LN 2) (LN 2 bath) to keep the samples at a super-low temperature before thawing. The most common thawing procedure is to place the sample in a warm water (37 ℃) bath, gently mixing or stirring to reduce the formation of sudden thermal gradients within the sample throughout the thawing process, which prevents the formation of localized high temperature (about >10 ℃) microenvironments in the sample, and avoids exposure of some of the sample to high temperature environments, and thus toxicity associated with CPAs. During thawing, the thawed sample is kept in a warm water bath until the last visible ice dissipates, then the sample is removed and placed on a cold shelf or ice, and finally diluted with culture medium for use.

It has been found that one of the major thermodynamic obstacles to the rapid thawing process is the latent heat of fusion. Latent heat of fusion refers to the heat removed from water at freezing point to convert water into ice or added to ice to convert it into water. The latent heat of water is large, corresponding to the heat required to raise the temperature of an equivalent volume of water to 79.9 ℃. Latent heat is the largest obstacle during thawing. The water recrystallizes already before reaching the thawing temperature and as the "thawing plateau" is approached, the rate of recrystallization will increase rapidly and a large number of crystals may grow.

The overall sample quality is severely affected by many factors of the thawing process, including air time, thawing temperature, thawing rate, final sample temperature, thawing time, homogeneity and dilution process. Other concerns include sterility, repeatability, controllability, documentation and cleanliness. In general, the thawing process should be performed as quickly as possible to minimize air time, heat the sample quickly until the ice is frozen out (typically 0 ℃) and mixing continues, then dilute immediately to reduce the cytotoxic damage of the cryoprotectant to the sample.

The traditional water bath unfreezing method has the following defects: (1) water wicks into the vial cap threads and is sealed in an improperly maintained water bath, making the contents of the vial highly likely to be contaminated, (2) the inability to use the water bath as part of the aseptic process, (3) the large subjective differences in determining thaw time, final temperature, and endpoint for different users, and (4) the limitations of using water baths in GMP or clinical settings.

In order to avoid the above disadvantages, the current better operation of thawing with a water bath requires the following points:

1. the time between the removal of the sample from storage and the placement in the thawing apparatus for passive heating, i.e., the "air time" is minimized.

2. Rather than completely submerging the vial, the lid and O-ring area are kept above the water line to reduce the risk of contamination.

3. The sample is mixed uniformly during the heating process to minimize thermal gradients within the sample.

4. Once all the ice had melted, the sample was removed without allowing the sample to warm to the bath temperature.

5. Once the sample was thawed prior to dilution, the sample was kept at the cool temperature (. about.4 ℃).

6. The samples are diluted with medium in a single or multi-step process (the method depends on cryoprotectant concentration and cell type).

Although the disadvantages caused by water bath can be avoided to a certain extent by strictly following the above operations, the water bath thawing and the current specification and document intensive research and clinical environment are even less able to meet the GMP requirements. To meet this demand, a number of devices are being developed to provide rapid, controlled, repeatable and recorded sample thawing. These devices fall into the category of "dry defrosters" in which the sample is heated in a dry oven. While there is concern that dry thawing will have a reduced heat transfer efficiency compared to a wet water bath, the reported thawing rates are comparable between the two methods. In addition, dry defrosters have many advantages over water baths, including improved processing and reduced risk of contamination and user error, among others.

Therefore, the development of a 'dry-type unfreezer' which has similar or better unfreezing effect with the traditional water bath method, can avoid the defects of the water bath and can meet the requirements of current industrial specifications or GMP (good manufacturing practice), realizes the requirements on controllability, recordability, repeatability and the like in the unfreezing process of low-temperature frozen samples, thereby benefiting the biological samples from standardized unfreezing, improving the productivity of the biological samples and helping the biological samples to finish the mission of improving the human health

Disclosure of Invention

In view of the above situation, in order to overcome the defects of the prior art, the present invention provides an automatic thawing system for cryopreservation of biological samples, which effectively solves the problems in the background art.

In order to achieve the purpose, the invention provides the following technical scheme: an automatic thawing system for freezing and preserving biological samples comprises an electric module, a supporting frame, a circuit main board, a control assembly and a metal block for placing samples,

the supporting frame is provided with a vertical groove, the electric module is arranged in the vertical groove, the metal block is arranged at the upper end of the electric module, the circuit main board is arranged at one side of the supporting frame,

the sample groove is formed in the upper end of the metal block, the IR temperature sensor is installed on the side face of the metal block, and the heating rod and the thermistor are arranged inside the metal block.

Preferably, the control assembly comprises a power main switch, a start button switch and a pause button switch, and the power main switch, the start button switch and the pause button switch are all mounted on the circuit main board.

Preferably, an RS485 communication interface and an RGB indicator lamp panel are further arranged on the circuit main board.

Preferably, the metal block is fixedly connected with the electric module through a connecting piece.

Preferably, the lower end of the electric module is fixedly connected to the fixed base, and the fixed base is provided with a screw hole.

Compared with the prior art, the invention has the beneficial effects that:

through the control strategy of optimizing, realized accurate control and real-time detection to biological sample heating process, avoided the drawback that the water bath method brought completely, realized the standardization that the biological sample unfreezes moreover, can get into GMP and clinical application with this control strategy through the SOP more, solve the pain point and difficult problem of prior art conscientiously, have important meaning to the development of biological industry.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:

FIG. 1 is a schematic view of the internal structure of the present invention;

FIG. 2 is a schematic view of the present invention;

FIG. 3 is a schematic view of the support frame and the metal block of the present invention;

FIG. 4 is an enlarged schematic view of the stand of the present invention;

fig. 5 is a schematic view of the combination of the metal block and the electromotive module according to the present invention.

In the figure: 1. a metal block; 2. a heating rod; 3. a circuit main board; 4. an RS485 communication interface; 5. a power supply main switch; 6. an electromotive module; 7. a fixed base; 8. a support frame; 9. an RGB indicator panel; 10. a pause button switch; 11. a thermistor; 12. starting a button switch; 13. an IR temperature sensor; 14. a sample tank; 15. a housing.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments; all other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

As shown in fig. 1-5, the invention discloses an automatic thawing system for cryopreservation of biological samples, comprising an electric module, a support frame, a circuit main board, a control assembly and a metal block for placing samples,

a vertical groove is arranged on the supporting frame, the electric module is arranged in the vertical groove, the metal block is arranged at the upper end of the electric module, the circuit main board is arranged at one side of the supporting frame,

the upper end of the metal block is provided with a sample groove, the side surface of the metal block is provided with an IR temperature sensor, and a heating rod and a thermistor are arranged inside the metal block.

The control assembly comprises a power supply main switch, a start button switch and a pause button switch, and the power supply main switch, the start button switch and the pause button switch are all installed on the circuit main board.

And an RS485 communication interface and an RGB indicator lamp panel are also arranged on the circuit main board.

The metal block is fixedly connected with the electric module through a connecting piece.

The unfreezing control system comprises operation/control software, a circuit mainboard, a thermistor, an electric module, an IR temperature sensor, a communication module and a heating rod, wherein the operation/control software is connected with the circuit mainboard through RS485 and sends an operation instruction to a microprocessor (CPU) on the mainboard; the circuit main board is connected with the thermistor, the IR temperature sensor, the electric module and the heating rod, the CPU on the circuit main board collects data of the thermistor and the IR temperature sensor and working states of the electric module and the heating rod and transmits the data to the operation software, the operation software carries out comprehensive processing according to the data of the thermistor and the IR temperature sensor and the working states of the electric module and the heating rod, sends an instruction to the CPU, and controls the heating rod and the electric module through the CPU.

Meanwhile, the system can operate independently without depending on operation/control software, and in this state, the system is operated by a control program built in the CPU and control buttons connected to the circuit board.

During thawing, the contact surface of the sample well that is in physical contact with the outer surface of the vial is heated to a constant temperature, which may be set by software or a built-in program, in the range of 37-50 ℃. A microprocessor (CPU) located on the circuit board may detect the temperature of the thawed sample in real time via an IR temperature sensor, and the CPU may use a built-in function to identify the end time of the sample thawing process.

The control process of the software or the built-in program comprises the following steps: switching on a power supply, enabling the system to enter a starting stage, initializing parameters, acquiring thermistor parameters by a CPU (central processing unit) and converting the thermistor parameters into real-time temperature, determining a value according to a difference value between the process set temperature and the real-time temperature, and starting a heating rod to heat a metal block (a sample groove) if the temperature is lower than the set temperature; and meanwhile, the state of the electric module is collected, and the electric module is adjusted to be in an opening and closing state. After the initialization phase is finished, the program enters the next control process, and the process comprises the following steps: the method comprises the steps of placing a biological sample in a sample groove in a metal block, adjusting an electric module to be in a clamping state, collecting the temperature of the sample in real time by an IR temperature sensor, transmitting the data to a CPU, controlling a heating rod by the CPU to heat the metal block, detecting the temperature of the sample in the heating process in real time by the IR temperature sensor, controlling the electric module by a program to enable the metal block (the sample groove) to be separated from the biological sample when the temperature reaches a thawing end point temperature set by the program, finishing thawing, and entering the next period.

The metal block (sample tank) is formed by combining two independent metal blocks (two half tanks), wherein one stretches out a protruding tray in the sample tank bottom, and another piece is opened at the sample tank bottom has a recess to receive another tray part when two metal blocks are closed, thereby form a closed sample tank, prevent simultaneously that the card from breaking down when placing the sample, avoid influencing normal thawing process. And the two metal blocks are respectively provided with a heating rod for heating the metal blocks to a set temperature and providing heat in the unfreezing process. The bottom parts of the two heating blocks are respectively provided with a thermistor for detecting the real-time temperature of the heating blocks, and the temperature of the heating blocks is controlled by a microprocessor which receives the temperature signal feedback from the thermistor. Meanwhile, an IR temperature sensor is installed at the sample tank and used for collecting the temperature of the outer surface of the unfrozen sample in real time, the temperature data is transmitted to the CPU in real time, when the temperature is detected to reach the unfreezing end point temperature set by a program, the sample tank is driven by the electric module, and the electric module is adjusted to be in an opening and closing state, so that the sample tank is separated from the unfrozen sample, the unfreezing process is finished, and the next period is entered.

Between the metal block and the sample tank, there is a flexible heat conducting material attached to the sample tank, which is used to eliminate or reduce the air gap between the thawed sample and the metal block (sample tank) and provide a uniform path through which heat can be transferred from the metal block (sample tank) to the thawed sample. Preferably, the flexible material is selected from the group consisting of a Gap Pad TGP 800VOS thermally conductive flexible material manufactured by Henkel corporation, and the thickness of the flexible material is selected to be 1.02mm, which is sufficient to ensure sufficient thermal contact, and the flexible material will have a thermal conductivity of greater than 0.5 Watts/meter Kelvin, which ensures good thermal conductivity. The flexible material and the sample cell may be removably coupled together.

The system is simultaneously developed with independent software, the software is provided with a user interface, and the name of a thawed sample can be input through the user interface before thawing; after the sample name is input, placing the sample in a sample tank, clicking 'start', starting the system to record the temperature of the unfrozen sample, and displaying the temperature data of the unfrozen sample in a software interface in real time in the form of data and a curve; if the sample is placed wrongly, the electric module drives the metal block to be separated from the sample by clicking 'pause', so that unfreezing is stopped. After each thawing cycle is completed, the software automatically stores the recorded sample name, time, temperature data and profile and selects whether to print or not according to the user's requirements. The data is stored in the format of xls, the temperature curve is stored in the format of pdf, and the data is not modifiable.

The system can be operated independently of the control software. The method comprises the following specific steps: turning on a power switch, enabling the system to enter a self-checking stage of each component, entering a preheating stage after the self-checking is finished, and heating a metal block (a sample groove) to a temperature preset by a program; after the preheating stage is finished, the system prompts to place a sample to be thawed, after the sample is placed, a 'start' button is pressed, the sample tank clamps the sample through the electric module, and the thawing stage is started until the thawing end point temperature preset by a program is reached; after the thawing end temperature is reached, the system automatically sends an instruction to the driving module, the driving module drives the metal block (sample groove) to be separated from the sample, thawing is completed, and the system enters the next period. Specifically, if the sample is placed by mistake, the "pause" button is pressed, and the metal block (sample tank) is separated from the sample, so that thawing is stopped, and overheating of the sample is prevented.

The end temperature of the preheating stage was set to 50 ℃ and the end temperature of the thawing stage was set to 0 ℃.

The present invention may provide audio or visual feedback to allow the user to be informed of the device status and the status of the sample thawing process. Different stages are accompanied by different combined sound and light signals to indicate the stage and the operation steps to be taken. Meanwhile, when the system fails, corresponding sound and light signal combinations are provided so as to find and eliminate the failure.

In further embodiments, another method of thawing a sample within a vessel may be provided. The method may include equilibrating the sample and vessel to an intermediate temperature. The intermediate temperature may be between-78 ℃ and-70 ℃. The sample may be equilibrated to an intermediate temperature range by placing the sample vessel in a container containing solid carbon dioxide.

The thermistor is an NTC type resistor.

The electric module is driven by a direct current brushless motor.

The IR temperature sensor is an ADC type infrared sensor.

The heating rod is a resistance type heating rod.

In the initialization stage, a heating program of the heating rod is determined by a built-in program of a CPU (central processing unit), and a metal block connected with the heating rod is heated to a preset temperature; the electric module is in an opening and closing state.

The time for the initialization phase is set to 180-300s, and the single sample thawing period is set to about 150-180s according to the program.

The output force of the driving module is 5-30N.

The lamp strip is an RGB lamp strip specially designed, and the sound signal is generated by a buzzer.

The circuit main board is a 4-layer PCB main board.

The power main switch is a ship-type switch.

The start button switch and the pause button switch are self-resetting.

The fixed base is made of 304 stainless steel.

The lower end of the electric module is fixedly connected to the fixed base, and the fixed base is provided with a screw hole so as to be fixedly connected with the shell 15.

It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims

1. An automatic thawing system for the cryopreservation of biological samples, characterized in that: comprises an electric module, a supporting frame, a circuit main board, a control component and a metal block for placing a sample,

2. The automated thawing system for cryopreservation of biological samples according to claim 1, wherein: the control assembly comprises a power supply main switch, a starting button switch and a pause button switch, and the power supply main switch, the starting button switch and the pause button switch are all installed on the circuit main board.

3. The automated thawing system for cryopreservation of biological samples according to claim 1, wherein: still be provided with RS485 communication interface and RGB pilot lamp plate on the circuit mainboard.

4. The automated thawing system for cryopreservation of biological samples according to claim 1, wherein: the metal block is fixedly connected with the electric module through a connecting piece.

5. The automated thawing system for cryopreservation of biological samples according to claim 1, wherein: the lower end of the electric module is fixedly connected to the fixed base, and a screw hole is formed in the fixed base.