CN106818705B - Embryo vitrification freezer and using method - Google Patents
Embryo vitrification freezer and using method Download PDFInfo
- Publication number
- CN106818705B CN106818705B CN201710010574.0A CN201710010574A CN106818705B CN 106818705 B CN106818705 B CN 106818705B CN 201710010574 A CN201710010574 A CN 201710010574A CN 106818705 B CN106818705 B CN 106818705B
- Authority
- CN
- China
- Prior art keywords
- embryo
- cavity
- needle
- compression
- freezing
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- 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
Landscapes
- Physics & Mathematics (AREA)
- Thermal Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Mechanical Engineering (AREA)
- Health & Medical Sciences (AREA)
- Dentistry (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention discloses a blank vitrification freezer which comprises a shell, wherein the upper half part of an inner cavity of the shell is divided into a compression cavity I and a compression cavity II which are mutually independent, and the lower half part of the inner cavity of the shell is a waste liquid cavity; a pull rod piston positioned in the compression cavity I is hermetically and slidably connected with the inner side wall of the compression cavity I, and the pull rod is fixedly connected with the pull rod piston; a compression rod piston positioned in the compression cavity II is hermetically and slidably connected with the inner side wall of the compression cavity II, and the compression rod is fixedly connected with the compression rod piston; a refrigerating fluid suction pipe communicated with the top of the waste liquid cavity is arranged on the side wall of the shell; the lower end of the freezing liquid suction pipe is provided with a freezing cavity, and the end part of the embryo needle is positioned in the freezing cavity. The invention also discloses a using method of the embryo vitrification freezer.
Description
Technical Field
The invention relates to the technical field of reproductive medicine medical treatment and health, in particular to a embryo vitrification freezer and a using method thereof.
Background
When a patient is treated by IVF (ICSI) -in vitro fertilization (intracytoplasmic sperm injection technology), a plurality of good-quality embryos are generally obtained in one superovulation treatment period, but all or the rest of good-quality embryos can not be transplanted or are not suitable to be transplanted at one time in the period. The embryo freezing preservation can lead the patient to obtain the opportunity of embryo transplantation for a plurality of times in one treatment period, lead the utilization rate of the embryo to reach the maximum limit and greatly improve the success rate of IVF (ICSI) treatment.
The embryo cryopreservation technology is to adopt a slow or fast cooling method to freeze the embryo at low temperature and then store the embryo at ultralow temperature in liquid nitrogen at-196 ℃. The prior embryo cryopreservation technology comprises a slow programmed freezing technology and a vitrification freezing technology, and the vitrification freezing technology has the advantages of simplicity, rapidness, high clinical pregnancy rate and high embryo implantation rate compared with the slow programmed freezing technology, so that the prior reproductive centers basically adopt vitrification freezing embryos for preservation. In the process of vitrification freezing, after the embryo is placed in the freezing liquid in the last step, the embryo is placed on the carrying rod in 60 seconds and is put into liquid nitrogen, but the embryo floats on the upper layer of the freezing liquid and is difficult to absorb in 60 seconds, and especially for laboratory workers who are not skilled in working operation, how to simplify the vitrification freezing operation of the embryo and avoid operation errors is needed, and the problem that the improvement of the survival rate (the current survival rate is 94%) of the vitrification frozen embryo after recovery is urgently needed to be solved by the industry.
Disclosure of Invention
The invention aims to provide the embryo vitrification freezer which has the advantages of simple structure, convenient use and high survival rate of the vitrified frozen embryos after recovery and the use method.
In order to solve the technical problem, the invention provides a blank vitrification freezer, which comprises a shell, a pull rod, a pressure rod, a pull rod piston, a pressure rod piston, a spring, a freezing liquid suction pipe and a blank needle, wherein the pull rod is connected with the pull rod piston; the upper half part of the inner cavity of the shell is divided into a compression cavity I and a compression cavity II which are independent from each other, the lower half part of the inner cavity of the shell is a waste liquid cavity, and the compression cavity I and the compression cavity II are both communicated with the waste liquid cavity;
the top end of the shell is provided with two openings which are respectively communicated with the compression cavity I and the compression cavity II;
the pull rod piston positioned in the compression cavity I is connected with the inner side wall of the compression cavity I in a sealing and sliding manner, and the bottom of the pull rod penetrates through an opening communicated with the compression cavity I and then is fixedly connected with the pull rod piston; the upper end and the lower end of the compression cavity I are respectively provided with a pull rod piston upper limit limiting ring and a pull rod piston lower limit limiting ring which are used for limiting the sliding position of the pull rod piston;
a compression rod piston positioned in the compression cavity II is connected with the inner side wall of the compression cavity II in a sealing and sliding manner, and the bottom of the compression rod penetrates through an opening communicated with the compression cavity II and then is fixedly connected with the compression rod piston; a compression rod piston upper limit limiting ring and a spring fixing ring which are used for limiting the sliding position of the compression rod piston are respectively arranged at the upper end and the lower end of the compression cavity II, a spring is arranged between the compression rod piston and the spring fixing ring, and two ends of the spring are respectively abutted against the compression rod piston and the spring fixing ring;
a refrigerating fluid suction pipe communicated with the top of the waste liquid cavity is arranged on the side wall of the shell; the lower end of the freezing liquid suction pipe is provided with a freezing cavity, and the end part of the embryo needle is positioned in the freezing cavity.
As an improvement of the embryo vitrification freezer of the present invention: the embryo needle comprises an embryo needle upper section and an embryo needle lower section which are hollow and communicated up and down, and the top of the embryo needle upper section extends into the freezing cavity;
the top of the upper section of the embryo needle is in a net shape and is hemispherical, and the aperture of a mesh is smaller than the diameter of an embryo;
the lower end of the lower section of the embryo needle is an opening end.
Remarks explanation: the top of the upper section of the embryo needle is in a net-shaped hemisphere shape; the structure can intercept the embryo, so that the embryo is always positioned at the top of the upper section of the embryo needle and cannot enter the freezing cavity and the freezing liquid suction pipe; but also ensure the passage of liquid.
The cross section of the inner cavity of the upper section of the embryo needle and the lower section of the embryo needle is larger than the diameter of the embryo, so that the embryo moves in the embryo needle.
As a further improvement of the embryo vitrification freezer of the present invention: the included angle between the upper section of the embryo needle and the lower section of the embryo needle is 145-155 degrees (preferably 150 degrees).
As a further improvement of the embryo vitrification freezer of the present invention:
the top end of the pull rod is provided with a pull plate, the size of the pull plate is larger than that of an opening communicated with the compression cavity I, and the pull plate is positioned above the compression cavity I;
the top end of the pressing rod is provided with a pressing plate, the size of the pressing plate is larger than that of an opening communicated with the compression cavity II, and the pressing plate is located above the compression cavity II.
As a further improvement of the embryo vitrification freezer of the present invention: the volume of compression chamber I is greater than the volume of compression chamber II.
As a further improvement of the embryo vitrification freezer of the present invention: the cross section of the compression cavity I is larger than that of the compression cavity II.
As a further improvement of the embryo vitrification freezer of the present invention: and the outer surface of the shell is provided with balance liquid scale marks.
As a further improvement of the embryo vitrification freezer of the present invention: the freezing cavity (15) is a spherical cavity.
The invention also provides a using method of the embryo vitrification freezer, which comprises the following steps:
1) firstly, pressing the pull plate downwards to enable the pull rod to move downwards in the compression cavity I until the pull rod piston is contacted with the lower limit limiting ring of the pull rod piston; then the pressing plate is pressed downwards, the pressing rod moves downwards in the compression cavity II, and the pressing rod piston compresses the spring until the spring is compressed to the limit; the air in the shell is compressed and then is discharged out of the shell through a refrigerating fluid suction pipe and an embryo needle;
firstly, placing the lower section opening end of the embryo needle into culture solution containing embryos, then loosening the pressing plate, restoring the pressing rod under the action of the elastic force of the spring, enabling the pressure in the shell to be smaller than the environmental pressure, and generating suction force to suck the culture solution containing the embryos; the culture solution containing the embryo sequentially passes through the lower section of the embryo needle and the upper section of the embryo needle and enters the top of the upper section of the embryo needle in the freezing cavity; the embryo is trapped on the top of the upper section of the embryo needle; the culture solution firstly enters the freezing cavity through the meshes at the top of the upper section of the embryo needle and then flows into the waste liquid cavity along the freezing liquid suction pipe;
2) placing the open end of the lower section of the embryo needle into the balancing liquid, pulling the pull plate upwards to enable the pull rod to move upwards in the compression chamber I, sucking the balancing liquid from the open end of the lower section of the embryo needle under the action of air pressure difference inside and outside the shell, and enabling the balancing liquid to enter the freezing chamber through meshes at the tops of the lower section of the embryo needle, the upper section of the embryo needle and the upper section of the embryo needle in sequence so as to realize replacement of the culture solution; when the rod piston is pulled up to the bottom end to be parallel to the scale mark of the equilibrium liquid under the action of the pull rod, keeping for 6-10 minutes (for example, 8 minutes) and then performing the following step 3);
remarks explanation: the step 2) is used for replacing all culture solution in the embryo needle, the freezing cavity and the freezing liquid suction pipe into balance liquid; the replaced culture solution and the redundant balancing solution flow into the waste liquid cavity from the freezing cavity through the freezing liquid suction pipe;
3) placing the opening end of the lower section of the embryo needle into the refrigerating fluid, continuously pulling the pulling plate upwards until the pull rod piston is contacted with the upper limit limiting ring of the pull rod piston, and sucking the refrigerating fluid from the opening end of the lower section of the embryo needle under the action of the air pressure difference inside and outside the shell; the freezing liquid enters the freezing cavity through meshes at the tops of the lower section of the embryo needle, the upper section of the embryo needle and the upper section of the embryo needle in sequence so as to realize the replacement of the balance liquid; holding for 30. + -. 3 seconds and then performing the following step 4)
Remarks explanation: the step 3) is used for quickly and completely replacing the refrigerating fluid by the balance fluid in the embryo needle, the freezing cavity and the refrigerating fluid suction pipe; the replaced balance liquid and the redundant refrigerating liquid flow into the waste liquid cavity from the refrigerating cavity through the refrigerating liquid suction pipe;
4) and the pressing plate is pressed downwards, the pressing rod moves downwards in the compression cavity II, the embryo positioned in the top of the upper section of the embryo needle is discharged to the embryo slide of the carrying rod from the embryo needle by the gas in the shell under the action of the air pressure difference between the inside and the outside of the shell, and then the carrying rod is rapidly put into liquid nitrogen (rapidly put into the liquid nitrogen, which is the conventional technology).
As an improvement of the method of use of the embryo vitrification freezer of the present invention:
when the piston of the pull rod is pulled up to the bottom end to be parallel to the scale line of the balance liquid under the action of the pull rod, the balance liquid sucked from the open end of the lower section of the embryo needle is 200 mu L;
when the pull rod piston is continuously pulled up to be in contact with the upper limit limiting ring of the pull rod piston, 200 microliter of refrigerating fluid is sucked from the opening end of the lower section of the embryo needle.
The invention has the following technical advantages:
1. in the embryo freezing process, the embryos are sucked and released for 1 time respectively under the microscope, and the embryos do not need to be sucked and released for 3 times under the microscope like the prior art, so that the operation is simplified;
2. the device is characterized in that a large-volume compression cavity I and a small-volume compression cavity II are respectively arranged, and the large-volume compression cavity I is used for absorbing balance liquid and refrigerating liquid with larger volumes and is realized by drawing a pull rod; the compression cavity II with small volume can provide sensitive suction and discharge force for sucking and discharging embryos with small volume (namely, culture solution containing the embryos), and is realized by a compression spring;
3. the lower end of the freezing liquid suction pipe is provided with a spherical freezing cavity, the freezing cavity in the shape can contain enough balance liquid or freezing liquid, and water in cytoplasm of the embryo cells can be replaced by a low-temperature protective agent in the balance liquid; the end of the embryo needle extending into the freezing cavity is in a reticular hemisphere shape, which allows liquid to pass through and can intercept the embryo at the end of the embryo needle in the reticular hemisphere shape, so that the embryo is always in the embryo needle and cannot flow into the freezing liquid suction pipe.
4. The embryo needle is divided into an embryo needle lower section and an embryo needle upper section, the embryo needle lower section and the embryo needle upper section form a certain angle (namely, the included angle is 145-155 degrees, preferably 150 degrees), when an embryo is placed on an embryo slide on the carrying rod, the embryo needle lower section can be parallel to the microscope carrying platform, and the position of the embryo in the embryo needle can be clearly observed.
In conclusion, the invention greatly simplifies the process of embryo vitrification freezing, when in use, embryos (namely culture solution containing embryos) needing vitrification freezing and storage are sucked into an embryo needle, then balance solution and freezing solution are sequentially sucked, and finally the embryos are placed on a carrying rod and are rapidly put into liquid nitrogen. The survival rate can reach 98 percent after the method is adopted.
Drawings
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1 is a schematic view of the overall structure of the embryo vitrification freezer of the present invention;
fig. 2 is a schematic cross-sectional view of fig. 1.
Detailed Description
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto.
The compression chamber I91 and the compression chamber II 92 are isolated from each other and are not communicated with each other. The lower half part of the inner cavity of the shell 1 is a waste liquid cavity 93, and the compression cavity I91 and the compression cavity II 92 are both communicated with the waste liquid cavity 93; waste chamber 93 is used to absorb waste/excess embryo culture fluid, equilibration fluid or freezing fluid.
The top end of the shell 1 is provided with two openings which are respectively communicated with a compression cavity I91 and a compression cavity II 92.
The pull rod piston 4 positioned in the compression cavity I91 is connected with the inner side wall of the compression cavity I91 in a sealing and sliding manner, and the bottom of the pull rod 2 penetrates through an opening communicated with the compression cavity I91 and then is fixedly connected with the pull rod piston 4; the upper end and the lower end of the compression chamber I91 are respectively provided with a pull rod piston upper limit limiting ring 7 and a pull rod piston lower limit limiting ring 11 which are used for limiting the sliding position of the pull rod piston 4. The pull rod piston upper limit limiting ring 7 and the pull rod piston lower limit limiting ring 11 limit the position of the pull rod piston 4 between the two, and have the function of preventing the pull rod piston 4 from falling out of the compression chamber I91. The top of pull rod 2 is provided with arm-tie 21, and the size of arm-tie 21 is greater than the open-ended size of intercommunication compression chamber I91, and arm-tie 21 is located the top of compression chamber I91.
The compression rod piston 5 positioned in the compression chamber II 92 is connected with the inner side wall of the compression chamber II 92 in a sealing and sliding manner, and the bottom of the compression rod 3 is fixedly connected with the compression rod piston 5 after penetrating through an opening communicated with the compression chamber II 92; and a compression rod piston upper limit limiting ring 8 and a spring fixing ring 12 for limiting the sliding position of the compression rod piston 5 are respectively arranged at the upper end and the lower end of the compression cavity II 92, a spring 6 is arranged between the compression rod piston 5 and the spring fixing ring 12, and two ends of the spring 6 are respectively abutted against the compression rod piston 5 and the spring fixing ring 12. When the plunger piston 5 is in contact with the plunger piston upper limit ring 8, the plunger piston 5 is just in contact with the spring 6, but does not exert pressure on the spring 6. The diameter of the concentric circle of the spring 6 is slightly smaller than that of the pressure rod piston 5, so that the spring 6 can be prevented from contacting with the inner wall of the compression cavity II 92. The upper limit limiting ring 8 of the pressure rod piston and the spring fixing ring 12 limit the position of the pressure rod piston 5 between the upper limit limiting ring and the spring fixing ring, and have the function of preventing the pressure rod piston 5 from falling out of the compression cavity II 92. The top end of the compression rod 3 is provided with a pressure plate 31, the size of the pressure plate 31 is larger than that of an opening communicated with the compression cavity II 92, and the pressure plate 31 is positioned above the compression cavity II 92.
The pulling plate 21 and the pressing plate 31 are both arranged to facilitate the use and operation.
A refrigerating fluid suction pipe 14 communicated with the top of the waste fluid cavity 93 is arranged on the side wall of the shell 1; the lower end of the refrigerating fluid suction pipe 14 is provided with a refrigerating cavity 15, and the refrigerating cavity 15 is a spherical cavity.
The embryo needle 10 comprises an embryo needle upper section 101 and an embryo needle lower section 102 which are both hollow and connected up and down, and the embryo needle upper section 101 is communicated with the embryo needle lower section 102; the top of the upper section 101 of the embryo needle extends into the freezing chamber 15, i.e. the top of the upper section 101 of the embryo needle is located in the freezing chamber 15. The inside diameter of the embryo needle 10 (i.e., the inside diameter of the upper section 101 of the embryo needle and the inside diameter of the lower section 102 of the embryo needle) is slightly larger than the diameter of the embryo at the early stage, so that the embryo can move smoothly in the embryo needle 10.
The top of the upper section 101 of the embryo needle is in a net-shaped semispherical shape, and the aperture of a mesh is smaller than the diameter of an embryo; therefore, the embryo can be intercepted, so that the embryo is always positioned at the top of the upper section 101 of the embryo needle and cannot enter the freezing cavity 15 and the freezing liquid suction pipe 14; but also ensures that liquid (including culture solution, balancing solution and freezing solution) enters the freezing cavity 15 through the meshes. The lower end of the lower section 102 of the embryo needle is an open end which is a round opening for facilitating the suction of embryos.
The included angle between the embryo needle upper section 101 and the embryo needle lower section 102 is 145-155 degrees (preferably 150 degrees). At the angle, under a stereo microscope, the embryo vitrification freezer is held by a hand, the lower section 102 of the embryo needle can be parallel to a microscope stage, when an embryo is placed on the carrying rod, the position of the embryo in the lower section 102 of the embryo needle can be observed, and when the embryo just reaches the opening end of the lower section 102 of the embryo needle, the opening end of the lower section 102 of the embryo needle is placed on the embryo carrying piece of the carrying rod, so that the embryo is placed on the embryo carrying piece, and the quantity of refrigerating fluid on the embryo carrying piece is minimum.
The outer surface of the housing 1 is provided with balance liquid graduation marks 16.
The invention discloses an embryo vitrification freezer, belonging to a disposable device.
The practical use process of the invention is as follows:
1) firstly, the pulling plate 21 is pressed downwards, so that the pulling rod 2 moves downwards in the compression cavity I91 until the pulling rod piston 4 is contacted with the pulling rod piston lower limit limiting ring 11; then the pressing plate 31 is pressed downwards, the pressing rod 3 moves downwards in the compression cavity II 92, and the pressing rod piston 5 compresses the spring 6 until the spring 6 is compressed to the limit; the air in the shell 1 is compressed and then discharged out of the shell 1 through the freezing liquid suction pipe 14 and the embryo needle 10;
the open end of the lower segment 102 of the embryo needle is firstly placed into a culture solution containing embryos; then the pressure plate 31 is released, the pressure rod 3 is restored under the action of the elastic force of the spring 6, so that the pressure in the shell 1 is lower than the ambient pressure; the culture solution containing the embryo is sucked by the generated suction force, and the culture solution containing the embryo sequentially passes through the lower section 102 and the upper section 101 of the embryo needle and enters the top of the upper section 101 of the embryo needle in the freezing cavity 15; the embryo is trapped on top of the upper section 101 of the embryo needle; the culture solution firstly enters the freezing cavity 15 through the mesh at the top of the upper section 101 of the embryo needle and then flows into the waste liquid cavity 93 along the freezing liquid suction pipe 14. This process requires observation using a microscope stage, since the embryos are barely visible to the naked eye.
2) Placing the open end of the lower section 102 of the embryo needle into the balancing liquid, pulling the pull plate 21 upwards to enable the pull rod 2 to move upwards in the compression chamber I91, sucking the balancing liquid from the open end of the lower section 102 of the embryo needle under the action of the difference of air pressure inside and outside the shell 1, and enabling the balancing liquid to enter the freezing chamber 15 through meshes at the tops of the lower section 102, the upper section 101 and the upper section 101 of the embryo needle in sequence so as to realize replacement of the culture liquid; when the rod piston 4 is pulled up to the bottom end parallel to the scale line 16 of the equilibrium liquid by the action of the rod 2, it is held for 8 minutes and then the following step 3) is performed.
When the pull rod piston 4 is pulled up to the bottom end to be parallel to the scale line 16 of the equilibrium liquid, the volume of the equilibrium liquid sucked from the opening end of the lower segment 102 of the embryo needle is 200 muL. The balance liquid is wasted due to the overlarge volume of the sucked balance liquid, and the subsequent refrigerating liquid to be sucked is less than 200 mu L; too low will result in less than 200 μ L of balancing fluid being drawn, thereby affecting the pre-balancing of the embryo.
Remarks explanation: the step 2) has the function of completely replacing the culture solution in the embryo needle 10, the freezing cavity 15 and the freezing liquid suction pipe 14 with balance liquid; the displaced culture fluid and excess equilibration fluid flow from freezing chamber 15 through the cryostraw 14 into waste chamber 93.
3) Placing the opening end of the lower section 102 of the embryo needle into the refrigerating fluid, continuously pulling the pulling plate 21 upwards until the pull rod piston 4 is contacted with the upper limit limiting ring 7 of the pull rod piston, and sucking the refrigerating fluid from the opening end of the lower section 102 of the embryo needle under the action of the difference of the air pressure inside and outside the shell 1; the freezing liquid sequentially passes through the meshes at the tops of the lower section 102, the upper section 101 and the upper section 101 of the embryo needle and enters the freezing cavity 15, so that the balance liquid is replaced; held for 30 seconds and then subjected to step 4) described below.
In this step, the volume of the freezing fluid sucked from the open end of the lower section 102 of the embryo needle is 200. mu.L.
Remarks explanation: the step 3) has the function of quickly replacing the refrigerating fluid by the balance fluid in the embryo needle 10, the freezing cavity 15 and the refrigerating fluid suction pipe 14; the displaced balancing fluid and excess cooling fluid will flow from the cooling chamber 15 through the cooling fluid suction pipe 14 into the waste fluid chamber 93;
4) then, the pressing plate 31 is pressed downwards, the pressing rod 3 moves downwards in the compression cavity II 92, and under the action of the difference between the air pressure inside and outside the shell 1, the air in the shell 1 discharges the embryo positioned at the top of the upper section 101 of the embryo needle (the top of the upper section 101 of the embryo needle is positioned in the freezing cavity 15) from the embryo needle 10 to the embryo slide of the carrying rod, and then the carrying rod is quickly put into liquid nitrogen. This process requires observation using a microscope stage, since the embryos are barely visible to the naked eye.
The carrier rod can be produced by KITAZATO Biopharma
And (4) vitrifying the freezing carrying rod. The carrying rod is used as a carrier for freezing embryos, embryo information can be marked on the carrying rod and is placed in liquid nitrogen for storage, and when the embryos need to be revived, the needed frozen embryos can be taken out from the liquid nitrogen according to the marked information for unfreezing.
The invention firstly puts the embryo into the vitrification solution (equilibrium solution) with lower concentration, and balances for a certain time, so that the low-temperature protective agent in the equilibrium solution permeates into cytoplasm; the embryos are then transferred to a higher concentration of vitrification solution (freezing fluid) which both shortens the equilibration time and prevents the chemical toxicity of the cryoprotectant and limits its excessive penetration.
By adopting the invention, the survival rate of the embryo can reach 98%.
Comparative example 1, the use of only the spring 6 in example 1 in the compression chamber ii 92 was eliminated, and the rest was the same as example 1.
When the device is used for embryo vitrification freezing experiments, the embryo suction and release force generated by the push-pull compression bar 3 is difficult to control and the embryo cannot be well sucked and released; but also causes the unfavorable condition that the freezing liquid containing the embryo is too big when being placed on the embryo slide of the carrying rod; thus, the final embryo survival rate using this device was only 78%.
Comparative example 2, eliminating the entire compression chamber ii 92 of example 1, includes the corresponding elimination of all of the following: the opening of the compression cavity II 92, the compression rod 3, the compression rod piston 5, the spring 6, the compression rod piston upper limit limiting ring 8 and the spring fixing ring 12 are communicated. The rest is equivalent to embodiment 1.
When the device is used for embryo vitrification freezing experiments, the suction and discharge force generated by the push-pull rod 2 is too large, and the embryo is possibly damaged or lost; thus, the final embryo survival rate using this device was only 61%.
Comparative example 3-1, the position of the scale line 16 of the balancing liquid on the housing 1 is moved upward relative to example 1, that is, when the pull rod piston 4 is pulled up until the bottom end is parallel to the scale line 16 of the balancing liquid, the volume of the balancing liquid sucked from the opening end of the lower section 102 of the embryo needle is changed from 200 μ L to 300 μ L. The rest is equivalent to embodiment 1.
When the device is used for embryo vitrification freezing experiments, the unfavorable condition that the equilibrium liquid cannot be discharged well in the step 3) can be caused, so that the freezing liquid is diluted; thus, the final embryo survival rate using this device was only 85%.
Comparative example 3-2, the position of the scale line 16 of the equilibration fluid on the housing 1 was shifted downwards relative to example 1, i.e. when the plunger 4 was pulled up until the bottom end was parallel to the scale line 16 of the equilibration fluid, the volume of equilibration fluid sucked in from the open end of the lower segment 102 of the embryo needle was changed from 200 μ L to 100 μ L. The rest is equivalent to embodiment 1.
When the device is used for embryo vitrification freezing experiments, equilibrium liquid is not filled in the freezing cavity 15, the cryoprotectant cannot completely permeate into embryo cell cytoplasm, and ice crystals can be formed in cells when the cryoprotectant is subsequently placed into liquid nitrogen, so that the adverse condition of cell damage is caused; thus, the final embryo survival rate using this device was only 80%.
Comparative example 4, the cross sections of the compression chamber I91 and the compression chamber II 92 are changed into the same size, and of course, the height of the compression chamber I91 is still larger than that of the compression chamber II 92, namely, the volume of the compression chamber I91 is still larger than that of the compression chamber II 92. The rest is equivalent to embodiment 1.
When the device is used for performing an embryo vitrification freezing experiment, the sensitivity is reduced when a culture solution containing an embryo is sucked and discharged, and the unfavorable condition that the size of liquid drops is difficult to master can occur when the embryo is placed on an embryo slide of a carrying rod; thus, the final embryo survival rate using this device was only 76%.
Comparative example 5, the "connection of the refrigerant fluid suction pipe 14 to the top of the waste fluid chamber 93" in example 1 was changed to "connection of the refrigerant fluid suction pipe 14 to the middle of the waste fluid chamber 93"; the rest is equivalent to embodiment 1.
When the device is used for embryo vitrification freezing experiments, the disadvantage that when the liquid level of the waste liquid exceeds the position where the freezing liquid suction pipe 14 is communicated with the waste liquid cavity 93, the waste liquid can dilute the freezing liquid in the freezing liquid suction pipe 14 and even in the freezing cavity 15 can occur; thus, the final embryo survival rate using this device was only 82%.
Comparative example 6, only the freezing chamber 15 connected with the freezing liquid suction pipe 14 in example 1 is eliminated, and the rest is identical to example 1.
When the device is used for embryo vitrification freezing experiments, the phenomenon that the cryoprotectant cannot well replace water in the embryos and ice crystals can be formed in cells when the embryos are placed in liquid nitrogen subsequently can be caused, so that the adverse condition of cell damage is caused; thus, the final embryo survival rate using this device was only 66%.
Finally, it should be noted that: the above embodiments are only used for illustrating the technical solution of the present invention, and not for limiting the same; while the invention has been described in detail with reference to exemplary embodiments thereof, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.
Claims (9)
1. Embryo vitrification freezer which characterized in that: comprises a shell (1), a pull rod (2), a pressure rod (3), a pull rod piston (4), a pressure rod piston (5), a spring (6), a freezing liquid suction pipe (14) and an embryo needle (10); the upper half part of the inner cavity of the shell (1) is divided into a compression cavity I (91) and a compression cavity II (92) which are independent of each other, the lower half part of the inner cavity of the shell (1) is a waste liquid cavity (93), and the compression cavity I (91) and the compression cavity II (92) are both communicated with the waste liquid cavity (93);
the top end of the shell (1) is provided with two openings which are respectively communicated with a compression cavity I (91) and a compression cavity II (92);
the pull rod piston (4) positioned in the compression cavity I (91) is connected with the inner side wall of the compression cavity I (91) in a sealing and sliding manner, and the bottom of the pull rod (2) penetrates through an opening communicated with the compression cavity I (91) and then is fixedly connected with the pull rod piston (4); the upper end and the lower end of the compression cavity I (91) are respectively provided with a pull rod piston upper limit limiting ring (7) and a pull rod piston lower limit limiting ring (11) which are used for limiting the sliding position of the pull rod piston (4);
a compression rod piston (5) positioned in the compression chamber II (92) is connected with the inner side wall of the compression chamber II (92) in a sealing and sliding manner, and the bottom of a compression rod (3) is fixedly connected with the compression rod piston (5) after passing through an opening communicated with the compression chamber II (92); the upper end and the lower end of the compression cavity II (92) are respectively provided with a compression bar piston upper limit limiting ring (8) and a spring fixing ring (12) which are used for limiting the sliding position of the compression bar piston (5), a spring (6) is arranged between the compression bar piston (5) and the spring fixing ring (12),
a refrigerating fluid suction pipe (14) communicated with the top of the waste fluid cavity (93) is arranged on the side wall of the shell (1); the lower end of the freezing liquid suction pipe (14) is provided with a freezing cavity (15), and the end part of the embryo needle (10) is positioned in the freezing cavity (15);
the embryo needle (10) comprises an embryo needle upper section (101) and an embryo needle lower section (102) which are hollow and communicated up and down, and the top of the embryo needle upper section (101) extends into the freezing cavity (15);
the top of the upper section (101) of the embryo needle is in a net-shaped hemispherical shape, and the aperture of a mesh is smaller than the diameter of an embryo;
the lower end of the lower section (102) of the embryo needle is an opening end.
2. The embryo vitrification freezer of claim 1, wherein:
the included angle between the embryo needle upper section (101) and the embryo needle lower section (102) is 145-155 degrees.
3. The embryo vitrification freezer according to claim 1 or 2, characterized in that: the top end of the pull rod (2) is provided with a pull plate (21), the size of the pull plate (21) is larger than that of an opening communicated with the compression cavity I (91), and the pull plate (21) is positioned above the compression cavity I (91);
the top end of the pressure lever (3) is provided with a pressure plate (31), the size of the pressure plate (31) is larger than the size of an opening communicated with the compression cavity II (92), and the pressure plate (31) is positioned above the compression cavity II (92).
4. The embryo vitrification freezer according to claim 1 or 2, characterized in that: the volume of the compression cavity I (91) is larger than that of the compression cavity II (92).
5. The embryo vitrification freezer according to claim 4, characterized in that: the cross section of the compression cavity I (91) is larger than that of the compression cavity II (92).
6. The embryo vitrification freezer according to claim 1 or 2, characterized in that: the outer surface of the shell (1) is provided with balance liquid scale marks (16).
7. The embryo vitrification freezer according to claim 1 or 2, characterized in that: the freezing cavity (15) is a spherical cavity.
8. The use method of the embryo vitrification freezer is characterized by comprising the following steps:
1) firstly, the pulling plate (21) is pressed downwards to enable the pulling rod (2) to move downwards in the compression cavity I (91) until the pulling rod piston (4) is contacted with the pulling rod piston lower limit limiting ring (11); then the pressing plate (31) is pressed downwards, the pressing rod (3) moves downwards in the compression cavity II (92), and the pressing rod piston (5) compresses the spring (6) until the spring (6) is compressed to the limit; the air in the shell (1) is compressed and then is discharged out of the shell (1) through the freezing liquid suction pipe (14) and the embryo needle (10);
firstly, the opening end of the lower section (102) of the embryo needle is placed into culture solution containing embryos, then the pressing plate (31) is loosened, the pressing rod (3) is restored under the action of the elastic force of the spring (6), so that the pressure in the shell (1) is smaller than the environmental pressure, and the produced suction force sucks the culture solution containing the embryos; the culture solution containing the embryo sequentially passes through the lower section (102) of the embryo needle and the upper section (101) of the embryo needle and enters the top of the upper section (101) of the embryo needle in the freezing cavity (15); the embryo is trapped on top of the upper section (101) of the embryo needle; the culture solution firstly enters the freezing cavity (15) through the meshes at the top of the upper section (101) of the embryo needle and then flows into the waste liquid cavity (93) along the freezing liquid suction pipe (14);
2) placing the open end of the lower section (102) of the embryo needle into a balance liquid, pulling the pull plate (21) upwards to enable the pull rod (2) to move upwards in the compression chamber I (91), sucking the balance liquid from the open end of the lower section (102) of the embryo needle under the action of the difference of air pressure inside and outside the shell (1), and enabling the balance liquid to enter the freezing chamber (15) through meshes at the tops of the lower section (102) of the embryo needle, the upper section (101) of the embryo needle and the upper section (101) of the embryo needle in sequence so as to realize replacement of the culture solution; when the pull rod piston (4) is pulled up to the bottom end to be parallel to the balance liquid scale mark (16) under the action of the pull rod (2), keeping for 6-10 minutes and then performing the following step 3);
3) placing the opening end of the lower section (102) of the embryo needle into the refrigerating fluid, continuously pulling the pull plate (21) upwards until the pull rod piston (4) is contacted with the upper limit limiting ring (7) of the pull rod piston, and sucking the refrigerating fluid from the opening end of the lower section (102) of the embryo needle under the action of the difference of the air pressure inside and outside the shell (1); the freezing liquid enters the freezing cavity (15) through meshes at the tops of the lower section (102) of the embryo needle, the upper section (101) of the embryo needle and the upper section (101) of the embryo needle in sequence so as to realize the replacement of the balance liquid; holding for 30. + -. 3 seconds and then performing the following step 4)
4) And the pressing plate (31) is pressed downwards, the pressing rod (3) moves downwards in the compression cavity II (92), and the embryo positioned in the top of the upper section (101) of the embryo needle is discharged to an embryo slide of the carrying rod from the embryo needle (10) by the air in the shell (1) under the action of the air pressure difference between the inside and the outside of the shell (1).
9. The method of using an embryo vitrification freezer according to claim 8, characterized in that:
when the pull rod piston (4) is pulled up to the bottom end to be parallel to the balance liquid scale mark (16) under the action of the pull rod (2), the balance liquid sucked from the opening end of the lower section (102) of the embryo needle is 200 mu L;
when the pull rod piston (4) is continuously pulled up to be in contact with the upper limit limiting ring (7) of the pull rod piston, the amount of the freezing liquid sucked from the opening end of the lower section (102) of the embryo needle is 200 muL.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710010574.0A CN106818705B (en) | 2017-01-06 | 2017-01-06 | Embryo vitrification freezer and using method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710010574.0A CN106818705B (en) | 2017-01-06 | 2017-01-06 | Embryo vitrification freezer and using method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106818705A CN106818705A (en) | 2017-06-13 |
| CN106818705B true CN106818705B (en) | 2022-02-11 |
Family
ID=59117928
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710010574.0A Active CN106818705B (en) | 2017-01-06 | 2017-01-06 | Embryo vitrification freezer and using method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106818705B (en) |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1308441C (en) * | 2005-01-28 | 2007-04-04 | 中国农业大学 | Vitrification freezing, simple defreezing and direct implanting method for embryo |
| CN101671627B (en) * | 2008-09-12 | 2012-05-23 | 陈子江 | Vitrified refrigeration carrier and use method thereof |
| CN101664704A (en) * | 2009-09-28 | 2010-03-10 | 赖大邃 | Cell minim suction storer |
| CN103650702B (en) * | 2013-12-09 | 2015-04-08 | 东北农业大学 | Seeding strip dot-matrix type glue dispensing apparatus |
| CN206403019U (en) * | 2017-01-06 | 2017-08-15 | 金华市人民医院 | Embryo vitrifying freeze device |
-
2017
- 2017-01-06 CN CN201710010574.0A patent/CN106818705B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN106818705A (en) | 2017-06-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP5278978B2 (en) | Tubular for vitrification preservation of animal embryos or eggs | |
| CN101779623B (en) | Cryopreservation method for oocyte / embryo and frozen carrier thereof | |
| US20120251999A1 (en) | Vitrification systems and methods | |
| CN101003791A (en) | Refrigeration method for preserving embryo and oocyte by vitrification of glasscapillary | |
| WO2006012613A1 (en) | Delivery of high cell mass in a syringe and related methods of cryopreserving cells | |
| JP5051716B2 (en) | Germ cell device and germ cell cryopreservation method | |
| CN112292033B (en) | Improved ultrafast cooling system and method of use | |
| EP2613733A1 (en) | A device for manipulating biological materials in a process of cryopreservation and a use of such a device | |
| Tomita et al. | Cryopreservation of a small number of human sperm using enzymatically fabricated, hollow hyaluronan microcapsules handled by conventional ICSI procedures | |
| CN111700061A (en) | Closed vitrification carrier and application thereof | |
| CN201222947Y (en) | Needling type carrier for vitrification of tissue piece | |
| CN106818705B (en) | Embryo vitrification freezer and using method | |
| Araki et al. | Single human sperm cryopreservation method using hollow-core agarose capsules | |
| JP2007505100A (en) | Improving post-thaw viability of cryopreserved biological material by applying hydrostatic pressure | |
| JP2009148221A (en) | Freeze preservation vessel for reproductive cell and method for freeze preservation of reproductive cell | |
| CN104012521A (en) | Non-contact type liquid drop method refrigerating device and refrigerating method | |
| CN206403019U (en) | Embryo vitrifying freeze device | |
| US20190133111A1 (en) | Method and device for monitoring the temperature of a cryopreserved biological sample | |
| JPWO2016111217A1 (en) | Hollow fiber cryopreservation tool and cell cryopreservation method | |
| Nagy et al. | The human embryo: vitrification | |
| CN214229620U (en) | Low-temperature semen sample preservation and checking device for reproductive center | |
| WO2018199106A1 (en) | Frozen biological cell pellet preparation device | |
| CN104378979A (en) | Automatic operation method and system for rapid vitrification freezing of living cells | |
| CN206078795U (en) | Cell glass thaws and deposits device | |
| CN205569837U (en) | A static separating bracket for fat transplantation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |