CN113403191B - Modularized low-temperature preservation and rapid rewarming device for biological samples - Google Patents
Modularized low-temperature preservation and rapid rewarming device for biological samples Download PDFInfo
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- CN113403191B CN113403191B CN202110644749.XA CN202110644749A CN113403191B CN 113403191 B CN113403191 B CN 113403191B CN 202110644749 A CN202110644749 A CN 202110644749A CN 113403191 B CN113403191 B CN 113403191B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/12—Means for regulation, monitoring, measurement or control, e.g. flow regulation of temperature
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0278—Physical preservation processes
- A01N1/0284—Temperature processes, i.e. using a designated change in temperature over time
Abstract
The invention discloses a modularized low-temperature preservation and rapid rewarming device for a biological sample, which comprises a biological sample packaging system, a slow-speed program cooling system, a liquid nitrogen cooling system, a radio frequency rewarming system and a jet rewarming system, wherein the biological sample packaging system is connected with the slow-speed program cooling system; the radio frequency rewarming chip with high specific surface area and ultrathin wall surface is designed, so that large-volume vitrification storage of biological samples can be realized, and the volume limit of the traditional vitrification container is broken through; in addition, the two modes of jet flow rewarming and radio frequency rewarming are coupled in the rewarming stage to achieve the effect of quick rewarming, so that the damage of recrystallization and devitrification to the biological sample in the rewarming process of slow low-temperature storage or vitrification storage can be effectively reduced; on one hand, the invention can realize the slow low-temperature preservation of the biological sample of the low-concentration protective agent, inhibit the regrowth of ice crystals in the rewarming process and improve the quality of the sample after the low-temperature preservation; on the other hand, the large-volume vitrification preservation of the biological sample can be realized, and a novel method is provided for realizing the high-quality preservation of the biological sample.
Description
Technical Field
The invention relates to the technical field of low-temperature storage, in particular to a modular low-temperature storage and rapid rewarming device for a biological sample.
Background
Cryopreservation refers to long-term storage of cells or tissues under low temperature conditions, and is widely applied in modern medicine, particularly reproductive medicine, organ storage and transportation, cell therapy, sample bank construction and the like. The vitrification freezing method is considered as the best cryopreservation mode of biological materials such as biological tissues, cells and the like, and has very important application value in the aspect of the construction of biological sample banks. The vitrification method is that a biological sample is cooled to a temperature far lower than the crystallization point of the biological sample through a very fast cooling rate, so that the formation and the growth of ice crystals in a solution are greatly limited, and finally, the transformation process to an amorphous solid state is realized. The way of realizing vitrification preservation is not limited to two categories: firstly, the concentration of the low-temperature protective agent is increased, and vitrification preservation can be realized only by low cooling rate; and secondly, the concentration of the protective agent is reduced, the volume of the sample is reduced, the specific surface area is increased, and ultra-fast freezing is realized to realize vitrification. For the former, because the protective agent has high concentration and strong toxicity, a complicated elution procedure is required, and the subsequent clinical application standard is difficult to achieve. Therefore, the vitrification method of ultra-fast freezing by increasing the specific surface area is currently the focus of research.
In addition, in the vitrification preservation method, the most common temperature recovery mode is adopted by 37 ℃ water bath temperature recovery in the temperature recovery process, but the temperature rise rate is slow, and the temperature rise rate is not fast enough, so that the devitrification phenomenon can be caused, and fatal ice crystal damage can be generated to a biological sample. Radio frequencies generate induced eddy currents in the conductor with resistive losses that generate heat. The metal layer in the rf re-warming chip 110 may generate induced eddy current in the rf field, which may provide a temperature rise rate of over 1000 ℃/min. But the single radio frequency rewarming is not enough for vitrification rewarming with low protective agent concentration, the rewarming speed is further improved by coupling jet rewarming, the jet rewarming means that warm water forms jet flow to impact the surface of a rewarmed object at high speed for rewarming, because the jet flow boundary layer is extremely thin, the jet flow rewarming has very high heat exchange efficiency, the rewarming speed can be further improved, the overheating phenomenon possibly generated by the radio frequency rewarming can be inhibited, and the method is a key technology for realizing high-speed cooling or rewarming of a sample.
Another key point of the current research is that technologies such as intracellular delivery of a natural non-permeable protective agent are combined to realize DMSO-free preservation of a biological sample, but the preservation method also faces cell damage caused by recrystallization during rewarming, and the quality of the biological sample after rewarming can be further improved by combining the use of radio frequency rewarming and jet flow rewarming, and other substances do not need to be introduced into a biological sample system like magnetic nanoparticle rewarming and gold nanoparticle laser rewarming, so that potential toxicity to the biological sample and subsequent tedious elution steps are avoided.
Disclosure of Invention
The present invention provides a modular device for low-temperature preservation and rapid rewarming of a biological sample, which solves the above problems in the prior art.
In order to achieve the purpose, the invention provides the following technical scheme:
a modularized low-temperature preservation and rapid rewarming device for a biological sample comprises a biological sample packaging system, a slow-speed program cooling system, a liquid nitrogen cooling system, a radio-frequency rewarming system and a jet rewarming system; the system also comprises a control system;
the biological sample packaging system comprises a radio frequency rewarming chip and a heat sealing device; the radio frequency rewarming chip is a rectangular bagged structure formed by a metal composite film, wherein the innermost layer of the radio frequency rewarming chip is made of a biocompatible material, so that a biological sample can be isolated from a metal layer, and the potential toxicity of the metal material to the biological sample is avoided; the middle layer of the radio frequency rewarming chip is made of metal materials, generates a heat effect in an alternating magnetic field, can improve the rewarming rate of the biological sample, reduces the damage of recrystallization and devitrification phenomena to the biological sample, and can provide good light shielding and oxygen isolating effects to better protect the biological sample; the outermost layer of the radio frequency rewarming chip is polyethylene glycol terephthalate, so that good mechanical performance can be provided, the chip has good mechanical strength, the sample leakage is prevented, and the polyethylene glycol terephthalate has good printing performance and can well display sample information;
The slow-speed program cooling system comprises a program cooling box, isopropanol, a deep low-temperature refrigerator and a liquid nitrogen tank; the program cooling box comprises a shell, a box support and a box cover, wherein the shell and the box cover are buckled with each other, the box support is positioned in the shell, a plurality of chip placing cavities are arranged in the box support at intervals, and intervals are arranged between outer walls corresponding to the chip placing cavities;
the isopropanol is a solution in the cooling box, is positioned between the shell and the box support, plays a role in slowing down the cooling rate, and the box support isolates the isopropanol solution from the sample to avoid the isopropanol from directly contacting the sample.
The liquid nitrogen cooling system comprises liquid nitrogen, a Dewar flask and a storage rack;
the radio frequency rewarming system comprises a radio frequency generator and a cooling water circulating device, and the cooling water circulating device is used for cooling the radio frequency generator;
the jet flow rewarming system comprises a jet flow generator, a high-pressure pump, a circulating pump, a water tank and a constant temperature tank; the jet generator comprises a shell, a chip placing groove which penetrates through the shell vertically is arranged in the shell, and limiting blocks are integrally formed on two sides of the bottom of the chip placing groove; the chip placing groove is provided with a plurality of through holes which are obliquely arranged downwards, the chip placing groove is communicated with the water distributing groove through the through holes, and the top of the water distributing groove is communicated with a connecting pipe which is communicated with a water inlet pipe; the water tank and the thermostatic bath form circulation through a water inlet pipe and a water return pipe, the water inlet pipe and the water return pipe are both provided with a manual valve, wherein the water inlet pipe is provided with a high-pressure pump for water inlet of water flow, and the water return pipe is provided with a circulating pump for water return of water flow;
A coil of the radio frequency generator is positioned in the water tank, and the jet flow generator is positioned in the coil of the radio frequency generator;
further, the control system comprises a temperature sensing device, a pressure sensor, a water level detector and a controller.
Further, the through holes on the partition plate can be horizontally arranged.
Furthermore, the volume of the radio frequency rewarming chip is 0ml-50 ml.
Furthermore, the radio frequency rewarming chip is used for storing biological samples, such as viruses, bacteria, cells, cell clusters and tissues.
Furthermore, the radio frequency rewarming module is coupled with the jet flow rewarming module, so that the rewarming speed is further improved, and the overheating phenomenon of the radio frequency rewarming chip in a radio frequency field can be prevented.
Further, a method for using the modular biological sample low-temperature storage and rapid rewarming device is as follows:
firstly, transferring a biological sample into a radio frequency rewarming chip, and then packaging by using a heat sealing machine;
when slow low-temperature storage is carried out, the radio frequency rewarming chip is transferred to a program cooling box, then transferred to a deep low-temperature refrigerator for slow cooling, taken out from the program cooling box after staying overnight, and transferred to liquid nitrogen for low-temperature storage;
When the biological sample is subjected to vitrification preservation, the radio frequency rewarming chip packaged with the biological sample is directly transferred into liquid nitrogen for cooling, and then is preserved in a liquid nitrogen tank;
when the temperature recovery operation is carried out, firstly, the jet flow generator is replaced by a required model, then the temperature of the jet flow for temperature recovery is set in the intelligent constant temperature circulator, the working parameters of the high-pressure pump and the circulating pump are set, after the setting is finished, after the constant temperature tank of the intelligent constant temperature circulator is stable at the set temperature, the manual valve is opened, the high-pressure pump and the circulating pump are opened, and the jet flow is formed in the jet flow generator;
then starting a cooling water circulation device of a radio frequency generator, setting the frequency, the intensity and the duration of a radio frequency field in the radio frequency generator when the temperature of cooling water is reduced to be below 20 ℃, starting the radio frequency generator, transferring a radio frequency rewarming chip to the side of rewarming equipment by using liquid nitrogen after the radio frequency reaches the set intensity and is stable, then quickly transferring the radio frequency rewarming chip to a radio frequency rewarming chip placement position, quickly rewarming, closing the radio frequency generator after rewarming is finished, closing the cooling water circulation device, and closing a jet flow rewarming system; during rewarming, radio frequency rewarming and jet flow rewarming are used in a matched mode, so that rewarming speed can be increased, damage to the biological sample caused by recrystallization and devitrification is restrained, and quality of the biological sample after rewarming is improved.
Furthermore, the jet flow rewarming means that warm water is formed into jet flow and impacted on the surface of a rewarmed object at high speed for rewarming, and the jet flow rewarming has high heat exchange efficiency due to the extremely thin jet flow boundary layer, so that the rewarming speed can be further improved, the overheating phenomenon possibly generated by radio frequency rewarming can be inhibited, and the quality of the rewarmed biological sample can be further improved.
Furthermore, the flow of the modularized low-temperature biological sample storage and rapid rewarming device is to realize large-volume vitrification of the biological sample, perform rewarming by matching with radio frequency and jet flow, inhibit the devitrification phenomenon in the rewarming process and reduce the damage to the biological sample.
Furthermore, the flow of the modularized low-temperature biological sample storage and rapid rewarming device is to store the biological sample by slow freezing and perform rewarming by matching with radio frequency and jet flow, so that the recrystallization phenomenon in the rewarming process is reduced, and the damage to the biological sample is reduced.
Compared with the prior art, the invention has the beneficial effects that:
(1) the radio frequency rewarming chip adopting the composite membrane structure can provide good biocompatibility, excellent mechanical strength and printing performance at the same time, and can be matched with a radio frequency field for rapid rewarming; by adopting the design of high specific surface area and ultrathin wall surface, the rapid cooling and rewarming can be realized, simultaneously the sample suspension with upgraded amount can be contained, and the rapid vitrification preservation of the biological sample can be realized;
(2) The jet flow rewarming system adopts a replaceable jet flow generator, different jet flow generator cavities adopt different apertures, angles and distribution modes, various jet flows can be formed, and in addition, the flow and the flow rate of the pump can be adjusted, so that abundant jet flow parameter adjustment choices are provided, and rewarming requirements of different biological samples are met;
(3) the improved program cooling box can be adapted to a square radio frequency rewarming chip;
(4) the method for heating the metal composite film by the radio frequency field is adopted to rapidly rewarm the biological sample, other heating media such as magnetic nanoparticles and the like are not required to be introduced into a biological sample system, and the subsequent complicated elution process and residual risk can be avoided;
(5) by adopting radio frequency rewarming coupled jet rewarming, the biological sample stored at low temperature can be rewarmed quickly and uniformly, and the recrystallization and devitrification phenomena in the biological sample system are effectively inhibited;
(6) the invention can inhibit the recrystallization phenomenon in the rewarming process of the slow low-temperature preservation and improve the slow low-temperature preservation quality of the biological sample;
(7) the invention designs the radio frequency rewarming chip with high specific surface area and ultrathin wall surface, can realize the large-volume vitrification preservation of the biological sample, breaks through the volume limitation of the traditional vitrification container, and provides a novel method for realizing the high-quality preservation of the biological sample;
(8) The invention designs a combined radio frequency rewarming, jet rewarming and low-temperature preservation process.
Drawings
Fig. 1 is a partial schematic view of a modular biological sample cryopreservation and rapid rewarming device.
Fig. 2 is a block diagram of a modular biological sample cryopreservation and rapid rewarming device.
Fig. 3 is a flow chart of the use of the modular biological sample cryopreservation and rapid rewarming device.
Fig. 4 is a schematic structural diagram of a radio frequency rewarming chip in a modular biological sample cryopreservation and rapid rewarming device.
Fig. 5 is a schematic structural diagram of a jet generator in a modular biological sample cryopreservation and rapid rewarming device.
Fig. 6 is a cross-sectional view of a jet generator in a modular biological specimen cryopreservation and rapid rewarming device.
Fig. 7 is another angled cross-sectional view of a jet generator in a modular biological specimen cryopreservation and rapid rewarming device.
Fig. 8 is a schematic view of the structure of the rf generator in the modular cryopreservation and rapid rewarming device for biological samples.
Fig. 9 is a schematic structural diagram of a cassette holder in the modular biological sample cryopreservation and rapid rewarming device.
Detailed Description
The technical solution of the present patent will be described in further detail with reference to the following embodiments.
Referring to fig. 1-9, a modular biological sample cryopreservation and rapid rewarming device includes a biological sample packaging system 100, a slow-speed programmed cooling system 200, a liquid nitrogen cooling system 300, a radio frequency rewarming system 400, and a jet rewarming system 500; also included is a control system 600;
the biological sample packaging system 100 comprises a radio frequency rewarming chip 110 and a heat sealing device;
the biological sample packaging system comprises a radio frequency rewarming chip 110 and a heat sealing device; the radio frequency rewarming chip is a rectangular bagged structure formed by a metal composite film, wherein the innermost layer 111 of the radio frequency rewarming chip is made of a biocompatible material, a biological sample can be isolated from a metal layer, and the potential toxicity of the metal material to the biological sample is avoided; the intermediate layer 113 of the radio frequency rewarming chip is made of metal materials, generates a heat effect in an alternating magnetic field, can improve the rewarming rate of the biological sample, reduces the damage of recrystallization and devitrification phenomena to the biological sample, and can provide good light shielding and oxygen isolating effects to better protect the biological sample; the outermost layer 113 of the radio frequency rewarming chip is polyethylene terephthalate, so that good mechanical performance can be provided, the chip has good mechanical strength, the sample is prevented from leaking, and the polyethylene terephthalate has good printing performance and can well display sample information;
In this embodiment, the metal material of the middle layer 113 of the rf rewarming chip is aluminum foil;
in addition, the radio frequency rewarming chip adopts the design of high specific surface area and ultrathin wall surface, and can contain upgraded sample suspension while realizing rapid cooling and rewarming. The metal layer in the radio frequency re-heating chip can generate induced eddy current in a radio frequency field, and the temperature rise rate of over 1000 ℃/min can be provided in the radio frequency field;
specifically, the heat sealing device is an existing air heater;
the slow programmed cooling system 200 comprises a programmed cooling box 210, isopropanol, a deep low temperature refrigerator and a liquid nitrogen tank;
the program cooling box 210 comprises a shell 211, a box support 212 and a box cover 213, wherein the shell 211 and the box cover 213 are buckled with each other, the box support 212 is positioned in the shell 211, a plurality of chip placing cavities 214 are arranged in the box support 212 at intervals, and intervals are arranged between outer walls corresponding to the chip placing cavities 214;
the liquid nitrogen cooling system 300 comprises liquid nitrogen, a Dewar flask and a storage rack;
the radio frequency rewarming system 400 comprises a radio frequency generator 401 and a cooling water circulating device 402, wherein the cooling water circulating device 402 is used for cooling the radio frequency generator 401;
specifically, the radio frequency generator 401 is a D5 alternating radio frequency generator of NanoScale Biomagnetics;
Specifically, the cooling water circulation device 402 is a constantly-popular YT1 cooling water circulation machine;
the jet flow rewarming system 500 comprises a jet flow generator 510, a high-pressure pump 520, a circulating pump 530, a water tank 540 and a thermostatic bath 550;
the jet generator 510 comprises a shell, wherein a vertically penetrating chip placement groove 511 is vertically arranged in the shell, and two sides of the bottom of the chip placement groove 511 are integrally formed with a limiting block 519, and the limiting block 519 is used for bearing the radio frequency rewarming chip 110 so that the radio frequency rewarming chip 110 cannot be separated from the bottom of the chip placement groove 511, and water flow is not prevented from being discharged from the bottom of the chip placement groove 511;
the chip placing groove 511 is provided with water distributing grooves 512 at two sides, a partition 513 is arranged between the water distributing grooves 512 and the chip placing groove 511, the partition 513 is provided with a plurality of through holes which are obliquely arranged downwards, the water distributing grooves 512 and the chip placing groove 511 are communicated through the through holes, wherein the top of the water distributing grooves 512 is communicated with a connecting pipe 514, and the connecting pipe 514 is communicated with a water inlet pipe 521;
the thermostatic bath 550 is a ZX-20B intelligent thermostatic circulator of Shanghai informed experience instrument technology priority company;
the water tank 540 and the thermostatic bath 550 form a circulation through a water inlet pipe 521 and a water return pipe 531, manual valves are mounted on the water inlet pipe 521 and the water return pipe 531, a high-pressure pump 520 is mounted on the water inlet pipe 521 and used for water inflow, a circulating pump 530 is mounted on the water return pipe 531 and used for water return, and thermostatic water enters the jet flow generator from the thermostatic bath 550 through the water inlet pipe 521;
The inlets of the water inlet pipe 521 and the water return pipe 531 are both provided with filters, the filters are DF core drilling filters, and PPF polypropylene fiber filter elements are adopted;
wherein the coils of the radio frequency generator 401 are located within the water bath 540 and the jet generator 510 is located within the coils of the radio frequency generator 401;
the control system 600 comprises a temperature sensing device, a pressure sensor, a water level detector and a controller;
the use method of the modular biological sample low-temperature storage and rapid rewarming device is specifically as follows:
firstly, transferring a biological sample into a radio frequency rewarming chip 110, and then packaging by using a heat sealing machine;
when slow low-temperature storage is carried out, the radio frequency rewarming chip 110 is transferred to the program cooling box 210, then transferred to a deep low-temperature refrigerator for slow cooling, and after the night, the radio frequency rewarming chip 110 is taken out of the program cooling box 210 and transferred to liquid nitrogen for low-temperature storage;
when the biological sample is subjected to vitrification preservation, the radio frequency rewarming chip 110 packaged with the biological sample is directly transferred to liquid nitrogen for cooling, and then is preserved in a liquid nitrogen tank;
when the rewarming operation is performed, firstly, the jet flow generator 510 is changed into a required model, then the temperature of the jet flow rewarming is set in the intelligent constant temperature circulator 500, the working parameters of the high-pressure pump and the circulating pump are set, after the setting is completed, after the constant temperature tank of the intelligent constant temperature circulator 500 is stable at the set temperature, the manual valve is opened, the high-pressure pump 501 and the circulating pump 502 are opened, and the jet flow is formed in the jet flow generator 510;
Then, starting a cooling water circulating device 402 of a radio frequency generator 401, setting the frequency, the intensity and the duration of a radio frequency field in the radio frequency generator 401 when the temperature of cooling water is reduced to below 20 ℃, starting the radio frequency generator 401, transferring the radio frequency rewarming chip 110 to a rewarming device by using liquid nitrogen after the radio frequency reaches the set intensity and is stable, then quickly transferring the radio frequency rewarming chip 110 to a placing position 22 of the radio frequency rewarming chip 110 for quick rewarming, after the rewarming is finished, closing the radio frequency generator 401, closing the cooling water circulating device 402, and closing the jet flow rewarming system 500; during rewarming, radio frequency rewarming and jet flow rewarming are used in a matched mode, so that rewarming speed can be increased, damage to the biological sample caused by recrystallization and devitrification is restrained, and quality of the biological sample after rewarming is improved.
The jet flow rewarming means that warm water forms jet flow to impact on the surface of a rewarmed object at high speed for rewarming, and because the jet flow boundary layer is extremely thin, the jet flow rewarming has high heat exchange efficiency, the rewarming speed can be further improved, the overheating phenomenon possibly generated by radio frequency rewarming can be inhibited, and the quality of the rewarmed biological sample can be further improved.
The flow of the modularized low-temperature biological sample storage and rapid rewarming device is to store the biological sample by slow freezing and perform rewarming by matching with radio frequency and jet flow, so that the recrystallization phenomenon in the rewarming process is reduced, and the damage to the biological sample is reduced.
On one hand, the invention can realize the slow low-temperature preservation of the biological sample of the low-concentration protective agent, inhibit the regrowth of ice crystals in the rewarming process and improve the quality of the sample after the low-temperature preservation.
On the other hand, the biological sample can be stored, the volume limit of the traditional vitrification container is broken through, and a novel method is provided for realizing the high-quality storage of the biological sample.
Example 1, a freezing experiment of a549 cell mass, in this example, the biocompatible material is polyethylene (PE for short);
a549 cell pellet was equilibrated in 2.5% DMSO, 2.5% EG, 0.05M trehalose in DMEM for 10min at 4 deg.C, and then 5min in 5% DMSO, 5% EG, 0.1M trehalose in DMEM. Then dividing the cell mass suspension into two parts, respectively adding 1mL of the cell mass suspension into a radio frequency rewarming chip (15mm wide, 70mm long and 1.5mm thick) and a 1.5mL freezing tube, respectively transferring the cell mass suspension into a matched program cooling box and a commercial program cooling box, putting the cell mass suspension into a refrigerator at minus 80 ℃ for overnight, and then transferring the cell mass suspension into liquid nitrogen for storage for 24 hours; and the radio frequency rewarming chip of the frozen sample adopts radio frequency-jet flow rapid rewarming, and the freezing tube adopts water bath rewarming. After rewarming, the structural integrity of the cell mass is quantitatively analyzed by combining a microscopic bright field picture with an image processing means, and the activity of the cell mass after cryopreservation is analyzed by using a CCK-8 kit. The results are shown in Table 1.
TABLE 1 comparison of structural integrity and relative viability after rewarming of A549 cell pellets stored at low temperature
| Group of | Structural integrity | Relative vigor |
| Fresh group | 85％ | 100％ |
| Freezing tube group | 73％ | 30％ |
| Radio frequency rewarming chip group | 82％ | 78％ |
The time required from-196 ℃ to 20 ℃ during rewarming was recorded by fiber optic thermometry and the results are shown in Table 2.
TABLE 2 comparison of RF-jet rewarming time of RF rewarming chip with the rewarming time of water bath for cryopreserved tube
| Group of | Time required for rewarming |
| Freezing tube water bath rewarming | 140s |
| RF-jet current rewarming of RF rewarming chip | 8s |
The results show that the cell mass of the radio frequency rewarming chip set is significantly superior to the cryopreserved tube set in both structural integrity and relative viability.
Example 2, a freezing experiment of a549 cell mass, in this example, the biocompatible material is polyethylene (PE for short);
a549 cells were equilibrated in 10% DMSO in DMEM for 10min at 4 ℃. Then dividing the cell suspension into two parts, respectively adding the two parts of 1mL into a radio frequency rewarming chip (15mm wide, 70mm long and 1.5mm thick) and a 1.5mL freezing tube, respectively transferring the two parts to a matched programmed cooling box and a commercial programmed cooling box, putting the boxes into a refrigerator at minus 80 ℃ for overnight, and then transferring the boxes into liquid nitrogen for storage for 24 hours. And the radio frequency rewarming chip of the frozen sample adopts radio frequency-jet flow rapid rewarming, and the freezing tube adopts water bath rewarming. And analyzing the survival rate of the cell clusters after the cryopreservation by using AO/PI fluorescent staining after rewarming. The results are shown in Table 3.
TABLE 3 comparison of survival rates after rewarming of A549 cells stored at low temperature
| Group of | Survival rate |
| Fresh group | 98％ |
| Freezing tube group | 94％ |
| Radio frequency rewarming chip group | 97％ |
The results show that the cell survival rate of the radio frequency rewarming chip set is obviously superior to that of a freezing storage tube set.
Although the preferred embodiments of the present patent have been described in detail, the present patent is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present patent within the knowledge of those skilled in the art.
Claims (9)
1. A modularized low-temperature preservation and rapid rewarming device for a biological sample comprises a biological sample packaging system, a slow-speed program cooling system, a liquid nitrogen cooling system, a radio-frequency rewarming system and a jet rewarming system; the method is characterized in that:
the biological sample packaging system comprises a radio frequency rewarming chip and a heat sealing device; the radio frequency rewarming chip is a rectangular bagged structure formed by a metal composite film, wherein the innermost layer of the radio frequency rewarming chip is made of a biocompatible material, so that a biological sample can be isolated from a metal layer, and the potential toxicity of the metal material to the biological sample is avoided; the middle layer of the radio frequency rewarming chip is made of metal materials and generates a heat effect in an alternating magnetic field; the metal layer in the radio frequency re-heating chip can generate induced eddy current in a radio frequency field, and the temperature rise rate of over 1000 ℃/min can be provided in the radio frequency field; the metal material of the middle layer of the radio frequency rewarming chip is aluminum foil;
The outermost layer of the radio frequency rewarming chip is polyethylene glycol terephthalate, so that good mechanical performance can be provided, the chip has good mechanical strength, the sample leakage is prevented, and the polyethylene glycol terephthalate has good printing performance and can well display sample information;
the slow-speed program cooling system comprises a program cooling box, a deep low-temperature refrigerator and a liquid nitrogen tank; the program cooling box comprises a shell, a box support and a box cover, wherein the shell and the box cover are buckled with each other, the box support is positioned in the shell and is immersed in an isopropanol solution, a plurality of chip placing cavities are arranged in the box support at intervals, and intervals are arranged between the outer walls corresponding to the chip placing cavities;
the liquid nitrogen cooling system comprises liquid nitrogen, a Dewar flask and a storage rack;
the radio frequency rewarming system comprises a radio frequency generator and a cooling water circulating device, and the cooling water circulating device is used for cooling the radio frequency generator; the radio frequency generator is a D5 alternating radio frequency generator of NanoScale Biomagnetics;
the jet flow rewarming system comprises a jet flow generator, a high-pressure pump, a circulating pump, a water tank and a constant temperature tank; the jet generator comprises a shell, a chip placing groove which penetrates through the shell vertically is arranged in the shell, and limiting blocks are integrally formed on two sides of the bottom of the chip placing groove; the chip placing groove is provided with a plurality of through holes which are obliquely arranged downwards, the chip placing groove is communicated with the water distributing groove through the through holes, and the top of the water distributing groove is communicated with a connecting pipe which is communicated with a water inlet pipe; the water tank and the thermostatic bath form circulation through a water inlet pipe and a water return pipe, the water inlet pipe and the water return pipe are both provided with a manual valve, wherein the water inlet pipe is provided with a high-pressure pump for water inlet of water flow, and the water return pipe is provided with a circulating pump for water return of water flow; a coil of the radio frequency generator is positioned in the water tank, and the jet flow generator is positioned in the coil of the radio frequency generator;
The radio frequency rewarming module is coupled with the jet flow rewarming module, so that the rewarming speed is further improved, and the overheating phenomenon of the radio frequency rewarming chip in a radio frequency field can be prevented.
2. The modular biological specimen cryopreservation and rapid rewarming device of claim 1 further comprising a control system comprising a temperature sensing device, a pressure sensor, a water level detector and a controller.
3. The modular biological specimen cryopreservation and rapid rewarming device of claim 1 wherein the through holes in the partition are horizontally disposed.
4. The device for cryopreservation and rapid rewarming of a modular biological sample according to claim 1, wherein the volume of the rf rewarming chip is 0ml to 50 ml.
5. The device as claimed in claim 1, wherein the RF rewarming chip is used for storing biological samples such as viruses, bacteria, cells, cell clusters, and tissues.
6. The modular biological sample cryopreservation and rapid rewarming device of claim 1, wherein the method of using the modular biological sample cryopreservation and rapid rewarming device is as follows:
Firstly, transferring a biological sample into a radio frequency rewarming chip, and then packaging by using a heat sealing machine;
when slow low-temperature storage is carried out, the radio frequency rewarming chip is transferred into a program cooling box, then the radio frequency rewarming chip is transferred into a deep low-temperature refrigerator to carry out slow cooling, the radio frequency rewarming chip is taken out of the program cooling box after staying overnight, and the radio frequency rewarming chip is transferred into liquid nitrogen to carry out low-temperature storage;
when the biological sample is subjected to vitrification preservation, directly transferring the radio frequency rewarming chip encapsulated with the biological sample into liquid nitrogen for cooling, and then preserving in a liquid nitrogen tank;
when the temperature recovery operation is carried out, firstly, the jet flow generator is replaced by a required model, then the temperature of the jet flow for temperature recovery is set in the intelligent constant temperature circulator, the working parameters of the high-pressure pump and the circulating pump are set, after the setting is finished, after the constant temperature tank of the intelligent constant temperature circulator is stable at the set temperature, the manual valve is opened, the high-pressure pump and the circulating pump are opened, and the jet flow is formed in the jet flow generator;
then starting a cooling water circulation device of a radio frequency generator, setting the frequency, the intensity and the duration of a radio frequency field in the radio frequency generator when the temperature of cooling water is reduced to be below 20 ℃, starting the radio frequency generator, transferring a radio frequency rewarming chip to the side of rewarming equipment by using liquid nitrogen after the radio frequency reaches the set intensity and is stable, then quickly transferring the radio frequency rewarming chip to a radio frequency rewarming chip placement position, quickly rewarming, closing the radio frequency generator after rewarming is finished, closing the cooling water circulation device, and closing a jet flow rewarming system; during rewarming, radio frequency rewarming and jet flow rewarming are used in a matched mode, so that rewarming speed can be increased, damage to the biological sample caused by recrystallization and devitrification is restrained, and quality of the biological sample after rewarming is improved.
7. The device for modular low-temperature preservation and rapid rewarming of biological samples according to claim 1, wherein the jet rewarming means that warm water is formed into jet flow and impacted on the surface of a rewarmed object at a high speed for rewarming, and the jet flow rewarming has high heat exchange efficiency due to the extremely thin jet flow boundary layer, so that the rewarming speed can be further improved, the overheating phenomenon possibly generated by radio frequency rewarming can be inhibited, and the quality of the biological samples after rewarming can be further improved.
8. The modular biological sample cryopreservation and rapid rewarming device of claim 1 wherein the flow of the modular biological sample cryopreservation and rapid rewarming device is to vitrify the biological sample in large volume, and rewarming with radio frequency and jet to suppress the devitrification during rewarming and reduce the damage to the biological sample.
9. The modular biological sample cryopreservation and rapid rewarming device of claim 1, wherein the flow of the modular biological sample cryopreservation and rapid rewarming device is to freeze and preserve the biological sample slowly, and rewarm with radio frequency and jet flow to inhibit recrystallization during rewarming and reduce damage to the biological sample.
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