CN106614523B - Cooling and rewarming working medium driving device for vitrification preservation and control method thereof - Google Patents
Cooling and rewarming working medium driving device for vitrification preservation and control method thereof Download PDFInfo
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- CN106614523B CN106614523B CN201611010306.0A CN201611010306A CN106614523B CN 106614523 B CN106614523 B CN 106614523B CN 201611010306 A CN201611010306 A CN 201611010306A CN 106614523 B CN106614523 B CN 106614523B
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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
- A01N1/0236—Mechanical aspects
- A01N1/0242—Apparatuses, i.e. devices used in the process of preservation of living parts, such as pumps, refrigeration devices or any other devices featuring moving parts and/or temperature controlling components
- A01N1/0252—Temperature controlling refrigerating apparatus, i.e. devices used to actively control the temperature of a designated internal volume, e.g. refrigerators, freeze-drying apparatus or liquid nitrogen baths
- A01N1/0257—Stationary or portable vessels generating cryogenic temperatures
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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
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- F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
- F25—REFRIGERATION OR COOLING; COMBINED HEATING AND REFRIGERATION SYSTEMS; HEAT PUMP SYSTEMS; MANUFACTURE OR STORAGE OF ICE; LIQUEFACTION SOLIDIFICATION OF GASES
- F25D—REFRIGERATORS; COLD ROOMS; ICE-BOXES; COOLING OR FREEZING APPARATUS NOT OTHERWISE PROVIDED FOR
- F25D29/00—Arrangement or mounting of control or safety devices
- F25D29/005—Mounting of control devices
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- F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
- F25—REFRIGERATION OR COOLING; COMBINED HEATING AND REFRIGERATION SYSTEMS; HEAT PUMP SYSTEMS; MANUFACTURE OR STORAGE OF ICE; LIQUEFACTION SOLIDIFICATION OF GASES
- F25D—REFRIGERATORS; COLD ROOMS; ICE-BOXES; COOLING OR FREEZING APPARATUS NOT OTHERWISE PROVIDED FOR
- F25D3/00—Devices using other cold materials; Devices using cold-storage bodies
- F25D3/10—Devices using other cold materials; Devices using cold-storage bodies using liquefied gases, e.g. liquid air
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Abstract
The invention relates to the field of cooling/rewarming in vitrification preservation of biological materials, in particular to a cooling/rewarming working medium driving device for vitrification preservation and a control method thereof. The device comprises a high-pressure inert gas input end, a pressure regulating valve, a ball valve, a cooling and rewarming working medium container, a sample section and a liquid storage tank which are connected in series in sequence through a conduit. The invention uses high-pressure inert gas as drive to drive the cooling and rewarming working medium in the cooling and rewarming container to move at high speed through the sample section and act on the sample or a carrier thereof, thereby realizing the cooling and rewarming of the sample. The invention takes high-pressure inert gas as driving force to drive the cooling and rewarming working medium to realize the vitrification preservation of the sample, changes the prior passive action mode into initiative, and greatly improves the stability and consistency of low-temperature preservation; the driving pressure is adjusted through the pressure adjusting valve, and the large-range adjustment of the cooling and rewarming speed of the sample can be realized.
Description
Technical Field
The invention relates to the field of cooling/rewarming in vitrification preservation of biological materials, in particular to a cooling/rewarming working medium driving device for vitrification preservation and a control method thereof.
Background
Low-temperature preservation is the only effective long-term preservation means at present for a plurality of biological materials, and plays an irreplaceable role in a plurality of fields of the national civilians. In the field of clinical medicine, more and more diseases can be cured by transplantation of cells and tissues, and large-scale cryopreservation is an important means for solving the problem of clinical supply of these important biological materials. Cryo-preserved biological materials need to undergo a step-over from ambient to cryogenic temperatures, during which ice crystal growth is the biggest obstacle to preservation success. The vitrification preservation is a revolutionary new method, in the vitrification preservation process, the biological sample does not generate (or only generates a small amount of) ice crystals, the concentration of the solution does not change obviously, and the supercooling time is short, so the low-temperature damage caused by various factors can be effectively avoided, and the vitrification preservation method has a principle breakthrough compared with the traditional method.
In order to obtain the ultrahigh temperature reduction and rewarming rate required by glass transition, a thin tube (0.25 ml) is used to bear a trace of sample and is directly put into liquid nitrogen (-196 ℃) in the standard method, the sample is driven to rapidly cool through the huge temperature difference between the sample and the liquid nitrogen and the vaporization action of the liquid nitrogen on the surface of the thin tube, and in the rewarming link, the thin tube is directly put into a constant temperature water bath (about 30 ℃) and the sample is driven to rapidly heat through the temperature difference between the sample and the warm water. On the basis, Vajta et al improve the structure of the tubules, and propose an Open drawn Straw (OPS) method, which greatly improves the obtained cooling rate. Reubinnoff also successfully carries out stem cell vitrification cryopreservation for the first time by applying the method, and the subsequent research continuously improves the method, and methods such as a Closed drawn Straw (CPS), a micro suction head method (Micropipette), Cryotop, Cryotip and the like appear in sequence.
No matter how the structure is improved, various containers bearing samples in the method can play a certain thermal resistance role in heat exchange between the samples and working liquid, such as a refrigerant (liquid nitrogen) or a rewarming agent (warm water) in the process of temperature reduction or rewarming. In order to eliminate the influence of the container, a direct contact mode is adopted in another part of research, namely a trace sample is directly contacted with a refrigerant or a rewarming agent, and higher heat exchange efficiency is realized. Typical methods include Electron Microscopy Grids (EMG), cold ring method (Cryoloop), etc., with fine copper mesh and nylon rings carrying a small amount of cells, respectively. In recent research, a cell Micro-drop method (Micro Droplet, MD) has also appeared, which uses a Micro-fluidic chip to generate Micro-droplets for wrapping cells, and the Micro-droplets are directly dropped into liquid nitrogen for cooling. The method can obtain higher temperature reduction and rewarming speed, but the problem of pollution caused by recovery and direct contact of cells needs to be solved.
In general, although the research of vitrification preservation method has achieved a series of achievements, the existing cooling and rewarming technology is still not mature, which is shown in the following:
(1) the heat exchange efficiency is limited. The existing cooling method obtains a higher cooling rate through Pool boiling (Pool boiling) of a sample in liquid nitrogen in essence, but a large amount of liquid nitrogen steam is generated in the Pool boiling process to wrap the periphery of the sample, so that a heat insulation effect is generated on further cooling, and therefore the heat exchange coefficient is generally lower; for the rewarming process, the conventional rewarming method generally drives the temperature rise through the temperature difference between a sample and a constant-temperature water bath, so that the heat exchange efficiency is lower, and the rewarming speed is rapidly attenuated along with the temperature rise and the temperature difference reduction of the sample, so that the problems of devitrification and the like are easily caused;
(2) operational contingencies are of great concern. The sample is immersed in liquid nitrogen to realize cooling and is put into a constant temperature bath to realize rewarming, the working medium passively acts on the biological sample or a carrier, the cooling and rewarming speed is difficult to control, the stability and the consistency are lacked, and the regulation cannot be realized.
Disclosure of Invention
Aiming at the problems or the defects, the invention provides a desuperheating working medium driving device for vitrification storage and a control method thereof, aiming at solving the problems of limited heat exchange efficiency, large influence of accidental operation factors and the like in vitrification storage.
The specific technical scheme is as follows:
the cooling and rewarming working medium driving device for vitrification storage comprises a high-pressure inert gas input end, a pressure regulating valve, a ball valve, a cooling and rewarming working medium container, a sample section and a liquid storage tank, wherein the components are sequentially connected in series through a guide pipe, as shown in figure 1.
The invention uses high-pressure inert gas as drive to drive the cooling and rewarming working medium in the cooling and rewarming container to move at high speed through the sample section and act on the sample or a carrier thereof, thereby realizing the cooling and rewarming of the sample.
In addition, the pressure of the high-pressure inert gas input to the temperature reduction and rewarming working medium container is adjusted by using a pressure adjusting valve, and the ball valve is used for opening and closing to control the on-off of the input high-pressure inert gas. And the regulation of the temperature reduction and rewarming speed can be realized by adjusting the driving pressure through the pressure regulating valve.
The high-pressure inert gas introduced into the high-pressure inert gas input end is nitrogen with the pressure of 0.1Mpa or above.
The inner wall of the conduit is made of polytetrafluoroethylene and can resist the low temperature of 200 ℃ below zero, and the exterior of the conduit is wrapped with a heat insulation material which is made of foam.
The sample section is used for storing a sample to be subjected to vitrification preservation or a carrier thereof and is provided with an inlet and an outlet; the flow channel is provided with a cooling and rewarming working medium, and the cooling and rewarming working medium acts on the sample to perform high-intensity heat exchange in the high-speed movement process in the sample section.
The cooling and rewarming working medium comprises liquid nitrogen and an alcohol solution, wherein the liquid nitrogen is used for cooling, and the alcohol solution is used for rewarming.
The cooling and rewarming working medium container stores cooling and rewarming working media. The cooling and rewarming working medium container is composed of an outer high-strength shell and an inner heat insulation material and is provided with an input port and an output port, wherein the input port is communicated with the top layer of the inner space, the output port is communicated with the bottom of the inner space, and when the input port is communicated with high-pressure inert gas, the cooling and rewarming working medium in the cooling and rewarming working medium container is output at high speed through the output port under the action of high pressure. The output port of the cooling and rewarming working medium container is connected with the inlet of the sample section through a conduit. When in use, the cooling and rewarming working medium does not contact the input port of the cooling and rewarming working medium container, and submerges the output port of the cooling and rewarming working medium container
The pressure regulating valve regulates the pressure of the high-pressure inert gas input to the input port of the cooling and rewarming working medium container, and the pressure regulating range of the pressure regulating valve is 0.1Mpa to 20 Mpa.
And the ball valve is used for switching on and off to control the on and off of the high-pressure inert gas input to the cooling and rewarming working medium container.
The liquid storage tank is a container, is connected with the outlet of the sample section through a conduit and is used for collecting the cooling and rewarming working medium after passing through the sample section.
The cooling and rewarming process of the cooling and rewarming working medium driving device for vitrification preservation comprises the following steps:
the high-pressure inert gas drives the cooling and rewarming working medium in the cooling and rewarming working medium container to enable the cooling and rewarming working medium to move at a high speed and act on the sample or a carrier thereof in the sample section, so that ultrahigh-speed cooling and rewarming of the sample is realized.
The invention provides a driving device for a cooling and rewarming working medium for vitrification preservation and a control method thereof by utilizing a high-pressure inert gas driving technology. The method aims to drive the temperature reduction and recovery working medium to move at high speed through high-pressure inert gas and act on a sample or a carrier thereof, and the acting mode is changed from passive to active. Thereby achieving higher heat exchange efficiency and better process stability and consistency. In addition, the action rate of the working medium can be changed by adjusting the pressure of the high-pressure gas, so that the large-range adjustment of the cooling and rewarming rate is realized, and the requirements of different objects for storage can be met. The problems caused by the fact that the traditional vitrification preservation cooling medium passive mode acts on a sample are solved.
Compared with the prior art, the invention has the beneficial effects that:
(1) the invention uses high-pressure inert gas as driving force to drive the cooling and rewarming working medium to realize the vitrification preservation of the sample, changes the prior passive action mode into initiative, and greatly improves the stability and consistency of low-temperature preservation.
(2) The invention adjusts the driving pressure through the pressure adjusting valve, and can realize the large-range adjustment of the temperature reduction and recovery rate of the sample.
Drawings
FIG. 1 is a schematic diagram of the structure of the present invention and its control method;
FIG. 2 is a schematic view of a desuperheating working fluid vessel of the present invention;
FIG. 3 is a schematic view of an embodiment;
FIG. 4 is a graph showing the results of a cooling test according to an embodiment;
FIG. 5 is a graph showing the results of the rewarming test of the examples;
reference numerals: 1 high-pressure inert gas input end, 2 pressure regulating valve, 3 ball valve, 4 cooling and rewarming working medium container, 5 cooling and rewarming working medium, 6 sample section, 7 liquid storage tank, 8 conduit, 9 thermocouple, 10 data acquisition card, 11 computer.
Detailed Description
The present invention will be described in further detail with reference to the following examples and the accompanying drawings.
As shown in fig. 3, a schematic diagram of a driving device for a cooling and rewarming working medium for vitrification storage and a control method thereof includes 1 a high-pressure inert gas input end, 2 a pressure regulating valve, 3 a ball valve, 4 a cooling and rewarming working medium container, 5 a cooling and rewarming working medium, 6 a sample section, 7 a liquid storage tank, 8 a guide pipe, 9 a thermocouple, 10 a data acquisition card and 11 a computer.
The high pressure inert gas used in this example was nitrogen at a pressure of 0.1MPa or greater.
The inner wall of the conduit 8 is made of polytetrafluoroethylene and can resist the low temperature of 200 ℃ below zero, and the outside of the conduit is wrapped with a heat insulating material which is made of foam.
The sample section 6 stores a sample or its carrier to be vitrified, and has an inlet and an outlet.
The cooling and rewarming working medium 5 comprises liquid nitrogen and alcohol solution, wherein the liquid nitrogen is used for cooling, and the alcohol solution is used for rewarming.
E. Temp. -lowering working medium container
And storing the cooling and rewarming working medium 5 by using the cooling and rewarming working medium container 4. The input port of the cooling and rewarming working medium container 4 is communicated with the top layer of the inner space, the output port is communicated with the bottom of the inner space, and when the input port is communicated with high-pressure inert gas, the cooling and rewarming working medium 5 in the cooling and rewarming working medium container 4 is output at high speed through the output port under the action of high pressure. The output port of the cooling and rewarming working medium container is connected with the inlet of the sample section 6 through a conduit 8.
The pressure regulating valve 2 regulates the pressure of the high-pressure inert gas input to the input port of the cooling and rewarming working medium container 4, and the pressure regulating range of the pressure regulating valve is 0.1Mpa to 20 Mpa.
The ball valve 3 controls the on-off of the high-pressure inert gas input into the cooling and rewarming working medium container 4.
The reservoir 7 is connected to the outlet of the sample section 6 by a conduit 8.
I. Temperature data acquisition module
And D, the temperature data acquisition module consists of a thermocouple, a data acquisition card and a computer, and the thermocouple probe is inserted into the sample in the step D, transmits signals to the data acquisition card and is processed and output by the computer.
J. Process of lowering and recovering temperature
The high-pressure inert gas drives the cooling and rewarming working medium 5 in the cooling and rewarming working medium container 4, so that the cooling and rewarming working medium 5 moves at high speed and acts on the sample or a carrier thereof in the sample section 6, and the ultrahigh-speed cooling and rewarming of the sample is realized.
The method comprises the following steps:
a) the assembly of the device of the present invention is completed according to the above steps.
b) And adding liquid nitrogen into the cooling and rewarming working medium container.
c) The pressure regulating valve is regulated to make the pressure of the high-pressure inert gas input into the cooling and rewarming working medium container be 0.1Mpa (0.2Mpa,0.3Mpa,0.4Mpa,0.5Mpa,0.6Mpa and 0.7 Mpa).
d) And acquiring temperature data by using a temperature data acquisition module.
e) And controlling the input of the high-pressure inert gas passage by using a ball valve, and controlling the input of the high-pressure inert gas to be disconnected by using the ball valve after 10 seconds to finish the temperature reduction process of the sample.
f) And taking the sample section out of the device and placing the sample section into a liquid nitrogen tank for preservation.
g) The sample sections stored in the liquid nitrogen tank were taken out to complete the assembly of the apparatus of the present invention.
h) Adding an alcohol solution with the temperature of 37 ℃ and the concentration of 85 percent into the cooling and rewarming working medium container.
i) Adjusting the pressure regulator to make the pressure of the high-pressure inert gas be 0.1Mpa (0.2Mpa,0.3Mpa,0.4Mpa,0.5Mpa,0.6Mpa,0.7 Mpa).
j) And acquiring temperature data by using a temperature data acquisition module.
k) Controlling the input of high-pressure inert gas by using a ball valve, and after 10 seconds, controlling the input of high-pressure inert gas by using the ball valve to open circuit to finish the sample rewarming process
Fig. 4 is a graph of the cooling rate of the sample at different pressures. FIG. 5 is the rewarming rate of the sample at different pressures.
The result shows that the increase of the pressure of the high-pressure inert gas in the cooling process enables the flow speed of the liquid nitrogen flowing through the sample section to be fast, and the heat exchange effect of the device is enhanced. When the pressure of the high-pressure inert gas is 0.1Mpa, the cooling rate of the sample reaches 30000 ℃/min, and when the pressure of the high-pressure inert gas is increased to 0.6Mpa, the cooling rate is about 47698 ℃/min, which shows that the invention can realize the large-range adjustment of the vitrification preservation cooling rate through the control of experimental operating conditions. Through repeated experiments for a plurality of times (the times are more than or equal to 3) under the unified experimental condition, the deviation of the cooling rate can be controlled to be about 10 percent, which shows that the invention has stability and consistency in the vitrification preservation and cooling process.
For the rewarming process, the increase of the pressure of the high-pressure inert gas enables the flow rate of the alcohol flowing through the micro sample section to be fast, and the heat exchange effect of the device is enhanced. When the pressure of the high-pressure inert gas is 0.1Mpa, the rewarming speed reaches 16438 ℃/min; when the pressure of the high-pressure inert gas is increased to 0.7Mpa, the rewarming speed reaches about 47965 ℃/min, which shows that the rewarming speed of vitrification preservation can be adjusted in a large range by controlling the experimental operating conditions. Through multiple experiments under the unified experimental condition (the times are more than or equal to 3), the temperature recovery rate deviation can be controlled to be about 10 percent, which shows that the invention has stability and consistency in the process of vitrification preservation and temperature recovery.
Claims (4)
1. The utility model provides a fall rewarming working medium drive arrangement towards vitrification storage which characterized in that: the device comprises a high-pressure inert gas input end, a pressure regulating valve, a ball valve, a temperature reduction and rewarming working medium container, a sample section and a liquid storage tank, wherein the components are sequentially connected in series through a conduit;
the inner wall of the conduit is made of polytetrafluoroethylene, can resist the low temperature of-200 ℃, and is externally wrapped with a heat insulation material;
the sample section is used for storing a sample to be subjected to vitrification preservation or a carrier thereof and is provided with an inlet and an outlet; a flow passage for the cooling and rewarming working medium is arranged, and the cooling and rewarming working medium acts on the sample to perform high-intensity heat exchange in the high-speed movement process in the sample section;
the cooling and rewarming working medium comprises liquid nitrogen and an alcohol solution, wherein the liquid nitrogen is only adopted during cooling, and the alcohol solution is only adopted during rewarming;
the cooling and rewarming working medium container is used for storing cooling and rewarming working media, consists of an external high-strength shell and an internal heat insulation material, and is provided with an input port and an output port, wherein the input port is communicated with the top layer of the internal space, and the output port is communicated with the bottom of the internal space; the output port of the cooling and rewarming working medium container is connected with the inlet of the sample section through a conduit; when the cooling and rewarming device is used, the input port is not in contact with the cooling and rewarming working medium, and the output port is communicated with the inside of the cooling and rewarming working medium;
the pressure regulating valve regulates the pressure of the high-pressure inert gas input to the input port of the cooling and rewarming working medium container, and the pressure regulating range of the pressure regulating valve is 0.1Mpa to 20 Mpa;
the ball valve is used for switching on and off and controlling the on and off of high-pressure inert gas input to the cooling and rewarming working medium container;
the liquid storage tank is a container, is connected with the outlet of the sample section through a conduit and is used for collecting the cooling and rewarming working medium after passing through the sample section.
2. The vitrification storage oriented rewarming working medium driving device as set forth in claim 1, wherein: the high-pressure inert gas introduced into the high-pressure inert gas input end is nitrogen with the pressure of 0.1Mpa or above.
3. The vitrification storage oriented rewarming working medium driving device as set forth in claim 1, wherein: the heat insulation material is made of foam.
4. The control method of the vitrification storage oriented cooling recuperation working medium driving device of claim 1 comprises the following steps:
the pressure of the high-pressure inert gas input into the cooling and rewarming working medium container is adjusted by using a pressure adjusting valve, the cooling and rewarming working medium in the cooling and rewarming working medium container is driven by the high-pressure inert gas, so that the cooling and rewarming working medium moves at high speed and acts on a sample or a carrier thereof in the sample section, and the ultrahigh-speed cooling and rewarming of the sample is realized.
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