CN109997841B - Human gamete rapid freezing and thawing visualization environment cabin maintaining atmospheric air atmosphere - Google Patents
Human gamete rapid freezing and thawing visualization environment cabin maintaining atmospheric air atmosphere Download PDFInfo
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- CN109997841B CN109997841B CN201910278850.0A CN201910278850A CN109997841B CN 109997841 B CN109997841 B CN 109997841B CN 201910278850 A CN201910278850 A CN 201910278850A CN 109997841 B CN109997841 B CN 109997841B
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- 238000010257 thawing Methods 0.000 title claims abstract description 22
- 239000012298 atmosphere Substances 0.000 title claims abstract description 21
- 238000012800 visualization Methods 0.000 title claims abstract description 12
- 238000007710 freezing Methods 0.000 title abstract description 35
- 230000008014 freezing Effects 0.000 title abstract description 35
- 238000005485 electric heating Methods 0.000 claims abstract description 26
- 230000007613 environmental effect Effects 0.000 claims abstract description 17
- 230000000149 penetrating effect Effects 0.000 claims abstract description 12
- 230000000007 visual effect Effects 0.000 claims abstract description 5
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 4
- 229910052594 sapphire Inorganic materials 0.000 claims description 12
- 239000010980 sapphire Substances 0.000 claims description 12
- 230000001954 sterilising effect Effects 0.000 claims description 6
- 238000004659 sterilization and disinfection Methods 0.000 claims description 6
- 238000010438 heat treatment Methods 0.000 claims description 5
- 238000007789 sealing Methods 0.000 claims description 4
- 239000010963 304 stainless steel Substances 0.000 claims description 3
- 229910000589 SAE 304 stainless steel Inorganic materials 0.000 claims description 3
- 239000003292 glue Substances 0.000 claims description 3
- 238000000034 method Methods 0.000 abstract description 14
- 238000001816 cooling Methods 0.000 abstract description 9
- 238000004017 vitrification Methods 0.000 abstract description 8
- 238000009529 body temperature measurement Methods 0.000 abstract description 2
- 238000002425 crystallisation Methods 0.000 abstract description 2
- 230000008025 crystallization Effects 0.000 abstract description 2
- 230000001052 transient effect Effects 0.000 abstract description 2
- 239000000523 sample Substances 0.000 description 20
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 16
- 239000007788 liquid Substances 0.000 description 8
- 229910052757 nitrogen Inorganic materials 0.000 description 8
- 210000004027 cell Anatomy 0.000 description 7
- 239000003223 protective agent Substances 0.000 description 5
- 241000894006 Bacteria Species 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 239000012472 biological sample Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000003958 fumigation Methods 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 241000700605 Viruses Species 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 108010066057 cabin-1 Proteins 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 210000000973 gametocyte Anatomy 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
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Classifications
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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
- A01N1/10—Preservation of living parts
- A01N1/14—Mechanical aspects of preservation; Apparatus or containers therefor
- A01N1/142—Apparatus
- A01N1/144—Apparatus for temperature control, e.g. refrigerators or freeze-drying apparatus
- A01N1/145—Stationary or portable vessels generating cryogenic temperatures, e.g. liquid nitrogen baths
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Dentistry (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Sampling And Sample Adjustment (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
一种维持常压空气氛围的人类配子快速冻融可视化环境舱，主要由环境舱主体、高导热底板、上法兰盖板、O型垫圈、预紧螺丝、毛细管、除菌干燥器、热电偶及电加热膜组成，所述的环境舱主体为扁平状空心圆柱结构，与所述的高导热底板、上法兰盖板共同组成封闭腔体。所述的上法兰盖板的中央嵌有高透的石英玻璃，扣于环境舱主体的顶面，环境舱主体的侧面开设毛细管接口、热电偶穿舱接口、电加热膜穿舱接口以及连接杆。本发明可实现人类配子样本在恒定常压条件下的快速冻融，并且具备全程可视化条件和实时测温能力，适用于观测低温降温或复温过程中配子的瞬态玻璃化或者结晶化冻融过程。
A visual environment chamber for rapid freezing and thawing of human gametes that maintains an atmosphere of atmospheric pressure and an electric heating film, the main body of the environmental chamber is a flat hollow cylindrical structure, and the high thermal conductivity bottom plate and the upper flange cover plate together form a closed cavity. The center of the upper flange cover plate is embedded with high-transparency quartz glass, which is buckled on the top surface of the main body of the environmental chamber. The side of the main body of the environmental chamber is provided with a capillary interface, a thermocouple penetrating interface, an electric heating film penetrating interface and a connection. rod. The invention can realize rapid freezing and thawing of human gamete samples under constant and normal pressure conditions, and has the ability of whole-process visualization and real-time temperature measurement, and is suitable for observing transient vitrification or crystallization freezing and thawing of gametes in the process of low temperature cooling or rewarming. process.
Description
Technical Field
The invention relates to a visual environment cabin for rapidly freezing and thawing human gametes in a normal-pressure air atmosphere, in particular to a device which can repeatedly load gamete samples, can make the samples contact with a low-temperature cold surface at intervals but can be rapidly cooled, and can always ensure an aerobic normal-pressure environment and ensure high-transparency and visible inside.
Background
The principle of the vitrification freezing technology is that a biological sample is directly thrown into liquid nitrogen or low-temperature steam together with a freezing protective agent to obtain enough cooling rate, so that ice crystals are not formed inside and outside cells, and physical damage to the cells is avoided. The vitrification freezing preservation method has the advantages of short and simple operation time, low requirement on equipment, good freezing preservation effect and the like. In order to load the gamete sample repeatedly, make the sample contact with low temperature cold surface interval but rapid cooling to and the device that guarantees aerobic ordinary pressure environment and inside high see-through all the time during and, design and development a human gamete that maintains ordinary pressure air atmosphere visual environment cabin of freezing and thawing fast has important value.
The published papers and patents on the vitrification freezing technology are basically focused on the research of vitrification freezing carriers and the steps of the freezing and thawing process, for example, chinese patent CN106153622A discloses a vitrification freezing observation device, which can be applied to screening of optimal freezing protective agents and optimization of tissue equilibrium time, and can conveniently observe the vitrification states of gametes in different freezing protective agents and the surface area changes of tissues in the equilibrium process of the tissues in the freezing agents during the processes of rapid freezing and thawing, so as to complete the preliminary vitreous state screening work and the optimization and determination of tissue equilibrium time, but the whole device is exposed in the atmosphere of liquid nitrogen and nitrogen, and gametocytes have no isolation function, are easily polluted by bacteria and uncontrollable rewarming. For another example, chinese patent CN206238188U discloses a liquid nitrogen fumigation box, which is equipped with a gamete carrier rack convenient for height adjustment and a corresponding buckle device, so that the carrier rack can be freely adjusted according to the liquid level height of liquid nitrogen, thereby keeping gametes in a freezing storage environment satisfying the conditions all the time. In this patent gamete carrier is arranged in liquid nitrogen fumigation case, has not opened the observation window, still does not possess the condition of visual observation gamete freeze thawing phenomenon.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides the rapid freezing and thawing visualization environment cabin for the human gamete for maintaining the atmospheric air atmosphere, the rapid freezing and thawing visualization environment cabin for the gamete is simple and reliable, can realize rapid freezing to deep low temperature and controlled rewarming, can repeatedly load gamete samples, can realize rapid freezing and thawing of the human gamete samples under the constant and atmospheric conditions, has the whole-course visualization condition and the real-time temperature measurement capability, and is suitable for observing the transient vitrification or crystallization freezing and thawing process of the gamete in the low-temperature cooling or rewarming process.
The technical solution of the invention is as follows:
a human gamete rapid freeze-thawing visualization environment cabin maintaining an atmospheric air atmosphere is characterized by comprising an environment cabin main body, a high heat conduction bottom plate, an upper flange cover plate, an O-shaped gasket, a pre-tightening screw, a capillary tube, a sterilization dryer, a thermocouple and an electric heating film, wherein the environment cabin main body is of a flat hollow cylindrical structure and forms a closed cavity together with the high heat conduction bottom plate and the upper flange cover plate;
the outer side surface of the environment cabin main body is provided with a capillary tube interface, a thermocouple cabin penetrating interface, an electric heating film cabin penetrating interface and a connecting rod; the high heat conduction bottom plate is embedded in the bottom surface of the opening of the environment cabin main body, the high heat conduction bottom plate and the opening are bonded and sealed by low-temperature glue, and a groove and a counter bore are etched in the high heat conduction bottom plate and used for positioning and placing a gamete sample; the center of the upper flange cover plate is embedded with high-transparency quartz glass, the upper flange cover plate is buckled on the top surface of the environmental chamber main body and forms detachable sealing connection through an O-shaped gasket and a pre-tightening screw, and the thermocouple is embedded and installed on the high-heat-conductivity bottom plate; the electric heating film is an annular heating film and is tightly adhered to the outer ring of the high-heat-conductivity bottom plate, the capillary tube interface is communicated with the outlet of the degerming dryer through the capillary tube, the inlet of the degerming dryer is communicated with the atmosphere, and the thermocouple cabin-penetrating interface is used for the airtight cabin-penetrating access of the thermocouple; the electric heating film cabin-penetrating interface is used for the airtight cabin-penetrating access of a lead of the electric heating film, and the connecting rod is used for being connected with an external movement executing mechanism.
The environmental chamber body is made of thin-walled 304 stainless steel.
The high heat conduction bottom plate is made of high-transparent sapphire.
When the rapid freezing and thawing environmental chamber is used, the working process is as follows:
opening an upper flange cover plate of the environment chamber, loading a gamete sample (which can contain a freezing protective agent), and dripping or fixing the gamete sample into a groove or a counter bore preset on a high-thermal-conductivity sapphire base plate; and ensuring that the thermocouple cabin penetrating interface and the electric heating film cabin penetrating interface are respectively connected with the fixed thermocouple and the electric heating film in the environment cabin. And covering the upper flange cover plate, and sealing by an O-shaped gasket and a pre-tightening screw.
And the connecting rod is connected to an external motion actuating mechanism and fixed, the environmental chamber is moved to the position right above the low-temperature cold surface, the capillary tube interface is communicated with the capillary tube, the other end of the capillary tube is communicated with the outlet of the degerming dryer, and the inlet of the degerming dryer is communicated with the atmosphere.
During the process of quick freezing, will high heat conduction sapphire bottom plate in close contact with the cold surface of low temperature, the high sapphire that passes through of high heat conduction bottom plate can transmit the cold volume of liquid nitrogen for the gamete sample of its upper surface fast, make gamete sample and the cold surface interval contact of low temperature and rapid cooling.
And during the cell recovery process, the high-heat-conduction sapphire base plate is far away from the low-temperature cold surface, the electric heating film is started to heat, the heat is rapidly transferred to the gamete sample on the high-heat-conduction base plate, and the frozen cells are controlled to be rewarming.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) the environment cabin body of the human gamete rapid freezing and thawing visualization environment cabin maintaining the atmosphere of normal pressure air is communicated with the normal pressure air through the capillary all the time, so that the damage to a biological sample caused by negative pressure generated in the cooling process of the closed cavity is avoided; this ordinary pressure air passes through the desicator drying, the purification of bacteria removing device, not only can avoid inside fogging influence to observe, avoids bacterial virus or impurity infection moreover, has improved biological freezing and has deposited the security.
(2) According to the connecting rod of the environment cabin main body, the whole position of the environment cabin is moved to be right above the low-temperature cold surface through the external movement executing mechanism, and then the environment cabin is moved downwards until the high-heat-conduction bottom plate at the bottom of the environment cabin main body is in close contact with the low-temperature cold surface, so that the rapid cooling is performed. The movement can prevent the sample from being influenced by low temperature in a standby state, and once the sample needs to be cooled, the sample can be quickly cooled.
(3) The freezing carrier medium adopts transparent sapphire, is transparent, is beneficial to backlight penetration and imaging, has extremely high thermal conductivity, and can realize rapid cooling. And a thermocouple and an electric heating film are also arranged on the high-heat-conduction bottom plate, so that rapid controlled rewarming of the gamete sample can be realized.
(4) The invention not only ensures the basic physical condition of the frozen gamete sample on the whole, but also provides possibility for observing the freezing and thawing process of the gamete cells in real time in the cold state process from room temperature to 196 ℃ below zero.
Drawings
FIG. 1 is a schematic perspective view of a human gamete rapid freezing and thawing visualization environment cabin maintaining an atmospheric air atmosphere according to the present invention;
FIG. 2 is a schematic structural view of the environmental chamber body of FIG. 1;
FIG. 3 is a schematic structural diagram of the high thermal conductivity bottom plate of the environmental chamber in FIG. 1;
in the figure: 1 is an environment chamber main body, 2 is a high heat conduction bottom plate, 3 is an upper flange cover plate, 4 is an O-shaped gasket, 5 is a pre-tightening screw, 6 is a capillary tube, 7 is a sterilization dryer, 8 is a thermocouple, 9 is an electric heating film, 10 is a capillary tube interface, 11 is a thermocouple cross-chamber interface, 12 is an electric heating film cross-chamber interface, 13 is a connecting rod, and 14 is a low-temperature cold surface.
Detailed Description
The invention is described in detail below with reference to the figures and specific embodiments.
Referring to fig. 1 to 3, it can be seen that the human gamete rapid freeze thawing visualization environment chamber for maintaining the atmospheric air atmosphere of the present invention includes an environment chamber main body 1, a high thermal conductivity bottom plate 2, an upper flange cover plate 3, an O-ring gasket 4, a pre-tightening screw 5, a capillary tube 6, a sterilization dryer 7, a thermocouple 8 and an electrical heating film 9, wherein the environment chamber main body 1 is a flat hollow cylindrical structure, and forms a closed cavity together with the high thermal conductivity bottom plate 2 and the upper flange cover plate 3; the outer side surface of the environment cabin body 1 is provided with a capillary tube interface 10, a thermocouple cabin penetrating interface 11, an electric heating film cabin penetrating interface 12 and a connecting rod 13; the high heat conduction bottom plate 2 is embedded in the bottom surface of the opening of the environment chamber main body 1, the high heat conduction bottom plate and the opening are bonded and sealed by low-temperature glue, and a groove and a counter bore are etched in the high heat conduction bottom plate 2 and used for positioning and placing a gamete sample; the center of the upper flange cover plate 3 is embedded with high-permeability quartz glass, the upper flange cover plate 3 is buckled on the top surface of the environmental chamber main body 1 and is in detachable sealing connection with a pretightening screw 5 through an O-shaped gasket 4, and the thermocouple 8 is embedded and installed on the high-heat-conductivity bottom plate 2; the electric heating film 9 is an annular heating film and is tightly adhered to the outer ring of the high heat conduction bottom plate 2, the capillary tube interface 10 is communicated with the outlet of the sterilization dryer 7 through the capillary tube 6, the inlet of the sterilization dryer 7 is communicated with the atmosphere, and the thermocouple cabin penetrating interface 11 is used for the airtight cabin penetrating access of the thermocouple 8; the electric heating film cabin-penetrating interface 12 is used for the airtight cabin-penetrating access of the lead of the electric heating film 9, and the connecting rod 13 is used for being connected with an external movement executing mechanism.
The environmental chamber body 1 is made of thin-walled 304 stainless steel.
The high heat conduction bottom plate 2 is made of high-transparent sapphire.
The O-shaped gasket 4 is resistant to low temperature.
The capillary tube interface 10 is communicated with the capillary tube 6, the other end of the capillary tube 6 is communicated with the outlet of the degerming dryer 7, and the inlet of the degerming dryer 7 is communicated with the atmosphere. The pressure in the cabin is equal to the atmosphere, and the cabin is dry and free from fogging and external pollution.
The connecting rod 13 is used for being connected with an external motion executing mechanism, so that the whole position of the environment cabin can be moved right above the low-temperature cold surface 14, and then the environment cabin is moved downwards until the high-heat-conduction bottom plate 2 at the bottom of the environment cabin body 1 is in close contact with the low-temperature cold surface 14 to cool.
The thermocouple 8 is embedded on the high heat conduction bottom plate 2 and is used for measuring the temperature of a measured sample; the electric heating film 9 is an annular heating film, is tightly adhered to the outer ring of the high heat conduction bottom plate 2 and is used for rewarming the sample.
The pre-tightening screw 5 is screwed into a threaded hole on the top side of the upper flange cover plate 3 of the environmental chamber, and is provided with 8 threaded holes with the aperture of 3mm, so that the environmental chamber main body 1 and the upper flange cover plate 3 are sealed after being installed.
When the gamete rapid freeze-thawing environmental chamber is used, the working process is as follows:
opening an upper flange cover plate 3 of the environment chamber main body 1, loading a gamete sample (which can contain a freezing protective agent), and dripping or fixing the gamete sample into a groove or a counter bore preset on a high-heat-conductivity sapphire bottom plate 2; and ensuring that the thermocouple cabin-penetrating interface 11 and the electric heating film cabin-penetrating interface 12 are respectively connected with the fixed thermocouple 8 and the electric heating film 9 in the environment cabin 1. The upper flange cover plate 3 is covered and sealed by an O-shaped gasket 4 and a pretightening screw 5.
The connecting rod 13 is connected to an external motion actuating mechanism and fixed, the environmental chamber 1 is moved to the position right above the low-temperature cold surface 14, the capillary tube interface 10 is communicated with the capillary tube 6, the other end of the capillary tube 6 is communicated with the outlet of the degerming dryer 7, and the inlet of the degerming dryer 7 is communicated with the atmosphere.
During the process of quick freezing, will high heat conduction sapphire bottom plate 2 closely contact low temperature cold surface 14, high heat conduction sapphire of bottom plate 2 can transmit the cold volume of liquid nitrogen for the gamete sample of its upper surface fast, can make sample and low temperature cold surface 14 interval contact and rapid cooling.
During the process of cell recovery, the high-heat-conductivity sapphire baseplate 2 is far away from the low-temperature cold surface 14, the electric heating film 9 is started to heat, heat is rapidly transferred to gametes on the high-heat-conductivity baseplate 2, and the frozen cells are controlled to rewarm.
Claims (3)
1. A visual environment cabin for maintaining the rapid freeze thawing of human gametes in an atmospheric air atmosphere is characterized by comprising an environment cabin main body (1), a high heat conduction bottom plate (2), an upper flange cover plate (3), an O-shaped gasket (4), a pre-tightening screw (5), a capillary tube (6), a sterilization dryer (7), a thermocouple (8) and an electric heating film (9), wherein the environment cabin main body (1) is of a flat hollow cylindrical structure and forms a closed cavity together with the high heat conduction bottom plate (2) and the upper flange cover plate (3);
the outer side surface of the environment cabin main body (1) is provided with a capillary tube interface (10), a thermocouple cabin penetrating interface (11), an electric heating film cabin penetrating interface (12) and a connecting rod (13); the high heat conduction bottom plate (2) is embedded in the bottom surface of the opening of the environmental chamber main body (1), the high heat conduction bottom plate and the opening are bonded and sealed by low-temperature glue, and a groove and a counter bore are etched in the high heat conduction bottom plate (2) and used for positioning and placing a gamete sample; the high-transparency quartz glass is embedded in the center of the upper flange cover plate (3), the upper flange cover plate (3) is buckled on the top surface of the environmental chamber main body (1) and is in detachable sealing connection with a pretightening screw (5) through an O-shaped gasket (4), and the thermocouple (8) is embedded on the high-heat-conduction bottom plate (2); the electric heating film (9) is an annular heating film and is tightly adhered to the outer ring of the high heat conduction bottom plate (2), the capillary tube interface (10) is communicated with the outlet of the degerming dryer (7) through the capillary tube (6), the inlet of the degerming dryer (7) is communicated with the atmosphere, and the thermocouple cabin-penetrating interface (11) is used for the airtight cabin-penetrating access of the thermocouple (8); the electric heating film cabin-penetrating interface (12) is used for the airtight cabin-penetrating access of a lead of the electric heating film (9), and the connecting rod (13) is used for being connected with an external movement executing mechanism.
2. The human gamete rapid freeze-thaw visualization environment chamber maintaining atmospheric air atmosphere of claim 1, characterized in that the environment chamber body (1) is made of thin wall 304 stainless steel.
3. The human gamete rapid freeze-thaw visualization environment chamber maintaining atmospheric air atmosphere of claim 1 or 2, characterized in that the high thermal conductivity bottom plate (2) is made of high transparent sapphire.
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| CN109292448A (en) * | 2018-10-23 | 2019-02-01 | 上海原能细胞生物低温设备有限公司 | Biological sample intelligent storage mechanism |
| CN109520196A (en) * | 2018-12-29 | 2019-03-26 | 北京大学第三医院 | Stifling household freezer and stifling freezing method |
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