CN108464300B - A kind of method and device system preparing glass freezing liquid - Google Patents
A kind of method and device system preparing glass freezing liquid Download PDFInfo
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- CN108464300B CN108464300B CN201810345241.8A CN201810345241A CN108464300B CN 108464300 B CN108464300 B CN 108464300B CN 201810345241 A CN201810345241 A CN 201810345241A CN 108464300 B CN108464300 B CN 108464300B
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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
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
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Abstract
The present invention relates to a kind of method and device systems for preparing glass freezing liquid.In the method for the invention, the glass freezing liquid includes dimethyl sulfoxide, ethylene glycol, carbohydrate, proline and seralbumin；Described method includes following steps: using buffer solution carbohydrate to get sugar juice；Dimethyl sulfoxide and ethylene glycol are added in the sugar juice, is uniformly mixed to get dimethyl sulfoxide-ethylene glycol-sugar juice；After dimethyl sulfoxide-the ethylene glycol-sugar juice is cooling, proline solution and seralbumin is added, is uniformly mixed to get proline-seralbumin-dimethyl sulfoxide-ethylene glycol-sugar juice；Constant volume and bacteria removing are carried out to get glass freezing liquid to the proline-seralbumin-dimethyl sulfoxide-ethylene glycol-sugar juice.The method of the invention can effectively avoid proline and sero-abluminous denaturation, the stability for enhancing the glass freezing liquid, extend its shelf-life.
Description
Technical field
The present invention relates to reproductions to freeze field, in particular to a kind of method and device for preparing glass freezing liquid
System.
Background technique
Reproduction freezen protective refers to through special safeguard measure and cooling process, makes reproduction cell and embryo etc. -196
Stop metabolism under the conditions of DEG C, and replys the technology of metabolic capability after heating up.With vitro fertilization-embryo implanting and its derivative skill
The continuous development of art, the reproduction freezen protective technology for being related to reproduction cell and embryo have become human ancillary reproductive field or dynamic
Essential component part in object breeding field.
Currently, conventional reproduction Refrigeration Technique includes slow freezing method and vitrifying freeze process.Wherein, slow freezing method is adopted
Reproduction cell is saved with the method for slow cooling and quick-thawing, this method accurately controls cooling, and refrigerating effect according to cooling instrument
It is bad.And vitrifying freeze process be by glass freezing liquid in refrigerating process rapid curing, form structureless hyaloid
Substance, to reduce the formation of intraor extracellular ice crystal.
It is short the time required to vitrifying freeze process compared with slow freezing method, it can be completed within 1 minute, do not depend on precision instrument,
Meanwhile sample quickly passes through danger area warm area in refrigerating process, reduces damage of the ice crystal to cytoskeleton, film integrality etc..
Therefore, vitrification has become reproduction Refrigeration Technique new direction.
In view of important function of the glass freezing liquid in refrigerating process, to glass freezing formula of liquid or its preparation work
The improvement of skill is of great significance.
It is permeability protective agent dimethyl sulfoxide and ethylene glycol etc. in reduction glass freezing liquid to reproduction cell or embryo
The toxic action of tire, the prior art propose a kind of novel glass freezing liquid.Compared with Conventional vitrification freezing liquid, novel glass
Proline is added in glass freezing liquid, so as to reduce the dosage of dimethyl sulfoxide and ethylene glycol etc., vitrifying is effectively reduced
The cytotoxicity of freezing liquid obtains ideal embryo development rate, Clinical Pregnancy Rate in and implantation rate (referring to Publication No.
The Chinese invention patent application of CN104839144A).
However, applicant actually prepare and use process in find, aforementioned new-type of vetrifying freezing liquid there are proline,
The defects of seralbumin is easy inactivation, and the stability of solution is poor, service life is short.In addition, due to dimethyl sulfoxide and second two
The viscosity of alcohol is big, and mixing difficulty is big, and preparation process is time-consuming and laborious.
In view of this, the present invention is specifically proposed.
Summary of the invention
The first object of the present invention is to provide a kind of method for preparing glass freezing liquid, and the method for the invention first will
Sugar juice is uniformly mixed with dimethyl sulfoxide, ethylene glycol, then is uniformly mixed with proline solution and serum albumin solution, can
Proline and sero-abluminous denaturation, the stability for enhancing the glass freezing liquid are effectively avoided, its shelf-life is extended.
The second object of the present invention is to provide a kind of apparatus system for preceding method, and the system passes through pump, spray
Cooperation between head and blower realizes that automatic cycle of the viscous liquid between the first container and second container mixes, reduces people
Power operation, reduces the working strength of operating personnel, meanwhile, after dimethyl sulfoxide or ethylene glycol release amount of heat, liquid exists
Flowing in first pipe is conducive to heat being transmitted to the external world, the reduction of accelerating liquid temperature, after avoiding by first pipe
Proline heat is denaturalized during continuous, shortens preparation time, maintains proline activity；
Further, apparatus system of the present invention is by adjusting the distance between spray head and the first container bottom surface, first
The draft capacity of the pumpability of pump and the first blower can remain that nozzle is located at first and holds in solution mixed process
Under the liquid level of device, the liquid level of the jet stream and the first container that avoid nozzle generates fierce collision, to avoid proline, serum
Albumin etc. inactivates in mixed process because generating a large amount of bubbles；
Further, the release to promote heat, it is preferable that also set up in the first pipe of apparatus system of the present invention
Have a thermometer and cooling device, the thermometer is used to monitor the temperature of liquid in first pipe, the cooling device for pair
The liquid overheated in first pipe carries out fast cooling；It is further preferred that the nozzle setting of apparatus system of the present invention exists
The bottom surface and side of spray head form penetrating for different directions alternatively, the nozzle being arranged on the same face is in an angle radially
Stream, promotes the mixing of liquid；
It is further preferred that being coated with adherent layer, the adherent layer on the inner and outer wall of second container of the present invention
Viscous liquid in mixed process can be prevented to be attached on the inside and outside wall of second container.
The third object of the present invention is to provide a kind of method for preparing glass freezing liquid using aforementioned device system, institute
The method of stating can be realized quickly and automatically circulation mixing, reduces operating personnel's working strength, further avoids dried meat in mixed process
The deactivations such as propylhomoserin and seralbumin.
The fourth object of the present invention is to provide the glass freezing liquid according to made of preceding method, the glass freezing
Liquid has the advantages that easy in inactivation, stability is not good, service life is long.
The fifth object of the present invention is to provide a kind of glass freezing kit, and the kit includes foregoing glass
Freezing liquid and other auxiliary reagents, have the advantages that easy in inactivation, stability is not good, service life is long.
In order to realize above-mentioned purpose of the invention, the following technical scheme is adopted:
A method of preparing glass freezing liquid, wherein the glass freezing liquid includes dimethyl sulfoxide, second two
Alcohol, carbohydrate, proline and seralbumin；Described method includes following steps:
(1) using buffer solution carbohydrate to get sugar juice；
(2) dimethyl sulfoxide and ethylene glycol are added in the sugar juice, is uniformly mixed to get dimethyl sulfoxide-second two
Alcohol-sugar juice；
(3) after the dimethyl sulfoxide-ethylene glycol-sugar juice is cooling, proline solution and seralbumin is added, mixes
It closes uniformly to get proline-seralbumin-dimethyl sulfoxide-ethylene glycol-sugar juice；
(4) proline-seralbumin-dimethyl sulfoxide-ethylene glycol-sugar juice is carried out at constant volume and degerming
Reason is to get glass freezing liquid.
The invention further relates to a kind of hybrid system for preceding method, the hybrid system includes the first container, second
Container, first pipe, second pipe, the first pump and the first blower, in which:
The first container includes the first main body, for accommodating liquid and the second container；
The second container includes the second main body and spray head, and second main body is connected to the spray head, on the spray head
It is provided with nozzle；
The second container is placed in the first container, and the first container and the second container pass through first pipe
Connection；
The first pump is provided in the first pipe, first pump through described first for managing liquid from the first container
Road is pumped in the second container；
The second container is connected to first blower by second pipe, and first blower is managed by described second
Road provides lasting wind pressure for the second container, the wind pressure by the liquid in second main body through the spray head force feed extremely
In first main body of the first container.
The invention further relates to a kind of methods for preparing glass freezing liquid, and the method is in the mistake for preparing glass freezing liquid
Liquid mixing is carried out using aforementioned device system in journey.
The invention further relates to the glass freezing liquid according to made of preceding method.
The invention further relates to a kind of glass freezing kit, the kit includes foregoing glass freezing liquid and other
Auxiliary reagent.
Specific embodiment
The present invention relates to a kind of methods for preparing glass freezing liquid, wherein the glass freezing liquid includes dimethyl
Sulfoxide, ethylene glycol, carbohydrate, proline and seralbumin；
Described method includes following steps:
(1) using buffer solution carbohydrate to get sugar juice；
(2) dimethyl sulfoxide and ethylene glycol are added in the sugar juice, is uniformly mixed to get dimethyl sulfoxide-second two
Alcohol-sugar juice；
(3) after the dimethyl sulfoxide-ethylene glycol-sugar juice is cooling, proline solution and seralbumin is added, mixes
It closes uniformly to get proline-seralbumin-dimethyl sulfoxide-ethylene glycol-sugar juice；
(4) proline-seralbumin-dimethyl sulfoxide-ethylene glycol-sugar juice is carried out at constant volume and degerming
Reason is to get glass freezing liquid.
In conventional glass freezing liquid in process for preparation, the proline solution of low viscosity is often first prepared, then is pressed
Viscosity sequentially adds serum albumin solution, sugar juice, dimethyl sulphoxide solution and ethylene glycol solution from low to high, to lower system
The difficulty of solution mixing during standby.Inventors have found that although this method can reduce the mixing difficulty of viscous liquid, but diformazan
Base sulfoxide and ethylene glycol solution discharge big calorimetric in dilution, the proline and serum being easy to cause in glass freezing liquid
Albumin denaturation, so as to cause the reduction of its stability, shortened shelf life.In order to solve the above technical problems, the present invention is to vitrifying
The preparation method of freezing liquid makes inventive improvements.Specifically, the present invention is first by sugar juice and dimethyl sulfoxide and ethylene glycol
It is uniformly mixed, then adds proline solution and serum albumin solution, therefore, proline and blood is being added in the method for the invention
Before pure albumen, the heat of dimethyl sulfoxide and ethylene glycol has discharged, and will not make proline and seralbumin that heat occur
Denaturation.Therefore, compared with conventional method, preparation method of the present invention is capable of the stability of reinforcing glass freezing liquid, extends
Its shelf-life.
In some specific embodiments, the glass freezing liquid includes that 7.5v/v%~15v/v% dimethyl is sub-
Sulfone, 7.5v/v%~15v/v% ethylene glycol, 0.5~2M carbohydrate and 0.5~2M proline.
In some specific embodiments, in the glass freezing liquid, the concentration of the dimethyl sulfoxide is 7.5v/
V%, 8v/v%, 8.5v/v%, 9v/v%, 9.5v/v%, 10v/v%, 10.5v/v%, 11v/v%, 11.5v/v%, 12v/
V%, 12.5v/v%, 13v/v%, 13.5v/v%, 14.5v/v% or 15v/v%.
In some specific embodiments, the concentration of the ethylene glycol is 7.5v/v%, 8v/v%, 8.5v/v%, 9v/
V%, 9.5v/v%, 10v/v%, 10.5v/v%, 11v/v%, 11.5v/v%, 12v/v%, 12.5v/v%, 13v/v%,
13.5v/v%, 14.5v/v% or 15v/v%.
In some specific embodiments, the concentration of the carbohydrate is 0.5M, 1M, 1.5M, 2M, 2M.
In some specific embodiments, the concentration of the proline is 0.5M, 1M, 1.5M, 2M, 2M.
In some specific embodiments, the sero-abluminous concentration be 15w/v%, 16w/v%, 17w/v%,
18w/v%, 19w/v% or 20w/v%.
In some specific embodiments, the carbohydrate is selected from monosaccharide, disaccharides or trisaccharide.
In some specific embodiments, it is preferable that the monosaccharide, disaccharides or trisaccharide are selected from glucose, galactolipin, cream
Sugar, sucrose, trehalose, gossypose, more preferably sucrose.
The invention further relates to a kind of hybrid system for preceding method, the hybrid system includes the first container, second
Container, first pipe, second pipe, the first pump and the first blower, in which:
The first container includes the first main body, for accommodating liquid and the second container；
The second container includes the second main body and spray head, and second main body is connected to the spray head, on the spray head
It is provided with nozzle；
The second container is placed in the first container, and the first container and the second container pass through first pipe
Connection；
The first pump is provided in the first pipe, first pump through described first for managing liquid from the first container
Road is pumped in the second container；
The second container is connected to first blower by second pipe, and first blower is managed by described second
Road provides lasting wind pressure for the second container, the wind pressure by the liquid in second main body through the spray head force feed extremely
In first main body of the first container.
It needs that the highly viscous solution such as dimethyl sulfoxide, ethylene glycol and carbohydrate are added during preparing glass freezing liquid,
In mix stages there are larger difficulty, need for a long time stirring repeatedly that can obtain uniformly mixed solution, it is bothersome laborious.Especially
It is aforementioned preparation process of the present invention, first mixes highly viscous solution, add the solution of low viscosity, wherein high viscosity solution
Mixing more increases the difficulty of mixing.In addition, the heat dissipation performance of solution is poor since viscosity is big, dimethyl sulfoxide and ethylene glycol are dilute
The heat discharged during releasing is not easy to spread, and the heat dissipation time is long.
To solve the above problems, applicant devises the above-mentioned apparatus system for being exclusively used in preparation method of the present invention, institute
System is stated by the cooperation between pump, spray head and blower, is penetrated liquid with certain speed from the spray head of second container using blower
Stream enters the first container, mixes with the liquid in the first container, and the liquid of the first container further passes through the first pump quilt
It is pumped in the second container, is moved in circles with this, form the automatic cycle mixing between liquid, manpower operation can be reduced,
The working strength of operating personnel is reduced, meanwhile, solution can be with accelerated heat by first pipe in automatic cycle mixed process
Scatter and disappear, shorten heat dissipation the time.
In some specific embodiments, the second container and the first container are with the spray head of the second container
The adjustable mode of height distance between the bottom surface of the first container is oppositely arranged, and the pumpability of first pump is big
It is small adjustable, the adjustable section of the draft capacity of first blower.
The method of the invention needs to add the proline and seralbumin of higher proportion during the preparation process, mixed
During conjunction, it is also easy to produce bubble, leads to albuminous degeneration.Therefore, if there are difference in height, sprays for the liquid level in nozzle and the first container
Fierce collision can occur for the jet stream and liquid level formed at mouth, be easy to cause the generation of a large amount of bubbles.To solve the above problems, this hair
Bright described device system by second container and the first container with the bottom surface of the spray head of the second container and the first container it
Between the adjustable mode of height distance be oppositely arranged, will so as to the height by adjusting spray head and the first container bottom
Nozzle submerges in a liquid, meanwhile, pumping, the pneumatic velocity of the first pump and the first blower are adjusted, nozzle can be made mixed in circulation
Submergence is remained during closing in a liquid, and then can effectively avoid liquid and a large amount of bubbles of liquid level direct collision generation.
In some specific embodiments, for the release for promoting heat, it is preferable that the of apparatus system of the present invention
It is additionally provided with thermometer and cooling device on one pipeline, wherein the thermometer is used to monitor the temperature of liquid in first pipe,
The cooling device is used to carry out fast cooling to the liquid overheated in first pipe, by between thermometer and cooling device
Cooperation can fast implement the reduction of fluid temperature in first pipe；
In some specific embodiments, the spray head is rectangular, cylindrical or arc, it is preferable that the spray head is
It is rectangular.
In some specific embodiments, the cross section of the nozzle is round or polygonal, it is preferable that the nozzle
Cross section be circle.
In some specific embodiments, the bottom surface and/or side of the spray head is arranged in the nozzle.
Preferably, it is the jet stream for forming different directions, promotes the mixing of liquid, in some specific embodiments, institute
State the spray head is arranged in nozzle bottom surface and side.
In some specific embodiments, the nozzle being arranged on the same face is parallel to each other radially or in a folder
Angle.
For the jet stream for forming different directions, promote the mixing of liquid, in some specific embodiments, is arranged same
Face top nozzle is in an angle radially.
In some specific embodiments, the angle is 5~45 °；More preferably 10~30 °.
In some specific embodiments, the nozzle is multiple, preferably 20~100, more preferably 50~100
It is a.
In some specific embodiments, the diameter of the nozzle is 10~500 μm, preferably 50~500 μm, more excellent
It is selected as 100~300 μm.
In some specific embodiments, second main body is integrally formed with the spray head or second main body
It is fixedly connected with the spray head by screw thread-screw hole.
To avoid viscous liquid from being adhered on second container surface, it is preferable that in some specific embodiments, described
Adherent layer is coated on the inner and outer wall of two containers.
Automatically to add solution to be diluted or mixed, in some specific embodiments, the system is also wrapped
Include the third container for accommodating liquid to be mixed, the third container and the first container or the second container pass through the
The connection of three pipelines, is provided with the second pump on the third pipeline, and liquid is pumped to described the from third container by second pump
In one container or the second container.
In some specific embodiments, described device system further includes bracket, and the bracket includes pedestal, is located at institute
It states the support rod on pedestal and the lateral hold assembly of the support bar top is set, the transverse direction hold assembly is used
In the clamping second container.
In some specific embodiments, the pedestal is sucker.
In some specific embodiments, the support rod it is height-adjustable.
In some specific embodiments, the support rod includes the first loop bar and the second loop bar, first loop bar
It is connected with the pedestal, second loop bar is socketed in first loop bar, and second loop bar can be along first loop bar
It slides up and down, is fixed between second loop bar and first loop bar by screw-bolt.
In some specific embodiments, it is described transverse direction hold assembly include transverse bar and be located at the transverse bar end
Clamping piece.
In some specific embodiments, the adjustable in length of the transverse bar.
In some specific embodiments, the gripping width of the clamping piece is adjustable.
In some specific embodiments, the clamping piece is made of elastic material.
The invention further relates to preparation method above-mentioned, the step (2) of the method, (3) use device system above-mentioned
System carries out the mixing of solution.
In some specific embodiments, in mixed process, the bottom surface of the spray head Yu the first container is adjusted
Height distance, control it is described first pump and first blower conveying capacity, thus keep the first main body in liquid always
The nozzle is not crossed.
The invention further relates to the glass freezing liquid according to made of preceding method.Glass freezing liquid of the present invention has
The advantage that easy in inactivation, stability is not good, service life is long.
The invention further relates to a kind of glass freezing kit, the kit packet foregoing glass freezing liquid is auxiliary with other
Help reagent.Kit of the present invention has the advantages that easy in inactivation, stability is not good, service life is long
Compared with prior art, the invention has the benefit that
(1) the method for the invention takes special Loading sequence to prepare glass freezing liquid, avoids dimethyl sulfoxide and second
The heat that glycol discharges in dilution causes proline and seralbumin to be denaturalized, and increases the stabilization of glass freezing liquid
Property, extend its shelf-life.
(2) the present invention also provides a kind of mixing device system for being exclusively used in preceding method, the hybrid system passes through pump, spray
Cooperation between head and blower realizes that automatic cycle mixes viscous liquid between the first vessel and second vessel, reduces manpower
Operation, reduces the working strength of operating personnel, while first pipe, and the thermometer and cooling device that are disposed thereon can be accelerated
Radiating rate shortens preparation time.
(3) apparatus system of the present invention by be arranged the second container spray head and the first container bottom surface it
Between height distance, and adjust the first pump and the pumping of the first blower, pneumatic velocity, can make nozzle in circulation mixed process
Remain submergence in a liquid, and then effectively liquid and liquid level direct collision avoided to generate a large amount of bubbles, avoid proline and
Seralbumin inactivation.
(4) bottom surface and side of spray head is arranged in the nozzle of apparatus system of the present invention, alternatively, being arranged on the same face
Nozzle be radially in an angle, form the jet stream of different directions, promote the mixing of liquid.
Detailed description of the invention
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art
Embodiment or attached drawing needed to be used in the description of the prior art be briefly described, it should be apparent that, it is described below
Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor
It puts, is also possible to obtain other drawings based on these drawings.
Fig. 1 is the schematic diagram of 2 described device system of embodiment；
Fig. 2 is the support schematic diagram of 2 described device system of embodiment；
Fig. 3 is the longitudinally cutting figure of preferred sprinkler described in embodiment 2；
Appended drawing reference:
1- the first container；
The first main body of 11-；
2- second container；
The second main body of 21-；
22- spray head；
221- nozzle；
3- third container；
The first blower of 4-；
5- first is pumped；
6- second is pumped；
7- bracket；
71- support rod；
The first loop bar of 711-；
The second loop bar of 712-；
72- pedestal；
73- transverse direction hold assembly；
731- transverse bar；
732- clamping piece；
8- first pipe；
9- second pipe；
10- third pipeline；
12- cooling device；
13- thermometer.
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
The conventional products obtained can be bought by city.
Embodiment 1
A kind of preparation method of glass freezing liquid, wherein the formula of the glass freezing liquid is as follows: contain 10v/
V% ethylene glycol, 10v/v% dimethyl sulfoxide, 1.0M proline, 0.5M sucrose and 20w/v% human serum albumins DPBS.
The preparation method is (500ml) as follows:
(1) proline, sucrose and human serum albumins are weighed according to formula, for use；
(2) DPBS buffer 100mL is measured, proline is added in DPBS buffer, concussion shakes up 10 minutes, room temperature
It is lower to stand 30 minutes to get proline-DPBS buffer；
(3) human serum albumins being added in step (2) proline-DPBS buffer, concussion mixes 10 minutes,
30 minutes are stood at room temperature to get human serum albumins-proline-DPBS buffer；
(4) DPBS buffer 260mL is measured, sucrose is added in the DPBS buffer measured, concussion shakes up 10 minutes, room
Temperature is lower to stand 30 minutes to get sucrose-DPBS buffer；
(5) ethylene glycol 50mL is measured, step (4) sucrose-DPBS buffer is slowly dropped to, is stirred when being added dropwise
(about 20 minutes) after completion of dropwise addition, persistently stir 10 minutes, stand 1 hour to get ethylene glycol-sucrose-DPBS buffer；
(6) dimethyl sulfoxide 50mL is measured, step (5) ethylene glycol-sucrose-DPBS buffer, side are slowly dropped to
Side stirring (about 20 minutes) is added, after completion of dropwise addition, persistently stirs 10 minutes, stands 1 hour to get dimethyl sulfoxide-second two
Alcohol-sucrose-DPBS buffer；
(7) human serum albumins in step (2)-proline-DPBS buffer is added to step (6) described dimethyl
Sulfoxide-ethylene glycol-sucrose-DPBS buffer, concussion mixing 20 minutes, stands 10 minutes to get human serum albumins-dried meat ammonia
Acid-dimethyl sulfoxide-ethylene glycol-sucrose-DPBS buffer；
(8) by step (7) human serum albumins-proline-dimethyl sulfoxide-ethylene glycol-sucrose-DPBS buffer
It is transferred in volumetric flask, is supplied with DPBS buffer to get glass freezing liquid；
(9) it is filtered using 0.22 μm of aperture filter, sealing, 4 DEG C of constant temperature are protected from light holding.
Embodiment 2
A kind of apparatus system being used to prepare glass freezing liquid, described device service system are held by the first container 1, second
Device 2, third container 3, the first blower 4, first pump 5, second pump 6, bracket 7, first pipe 8, second pipe 9, third pipeline 10,
Cooling device 12 and thermometer 13 form, in which:
The first container includes the first main body 11, for accommodating liquid and the second container 2；
The second container includes the second main body 21 and spray head 22, and second main body 21 passes through spiral shell with the spray head 22
Line-screw hole is fixedly connected；Be provided with nozzle 221 on the spray head 22, the spray head 22 be it is rectangular, the setting of nozzle 221 exists
The bottom surface and side of the spray head 22；The nozzle 221 is 100, and the diameter of the nozzle 221 is 200 μm, is arranged same
Face top nozzle 221 is in 30 ° of angles radially；
The second container 2 is placed in the first container 1, and the first container 1 passes through first with the second container 2
Pipeline 8 is connected to；The second container 2 is with the first container 1 with the spray head 21 of the second container 2 and the first container 1
Bottom surface between the adjustable mode of height distance be oppositely arranged, on the inside and outside wall of the second container 2 be coated with it is anti-sticking
Layer；
Be provided with the first pump 5 in the first pipe 8, first pump 5 for by liquid from the first container 1 through described the
One pipeline 8 is pumped in the second container 2, the adjustable section of pumpability of first pump 5, in the first pipe 8
It is provided with thermometer 13 and cooling device 12；
The second container 2 and first blower 4 are connected to by second pipe 9, and first blower 4 passes through described the
Two pipelines 9 are that the second container 2 provides lasting wind pressure, and the wind pressure is by second main body 21 liquid through the spray
First 22 force feed is into the first main body 11 of the first container 1, the adjustable section of the draft capacity of first blower 4；
The third container 3 is connected to the second container 2 by third pipeline 10, is provided on the third pipeline 10
Liquid is pumped in the second container 2 by the second pump 6, second pump 6 from third container 3；
The bracket 7 includes pedestal 72, the support rod 71 on the pedestal 72 and is arranged in the support rod
The lateral hold assembly 73 at 71 tops, the transverse direction hold assembly 73 is for clamping the second container 2；The pedestal 72 is to inhale
Disk；The support rod it is height-adjustable, the support rod 71 include the first loop bar 711 and the second loop bar 712, the first set
Bar 711 is connected with the pedestal 72, and second set of rod set 712 connects in first loop bar 711, second loop bar 712
It can be slided up and down along first loop bar 711, pass through screw-bolt between second loop bar 712 and first loop bar 711
It is fixed；The transverse direction hold assembly 73 includes transverse bar 731 and the clamping piece 732 positioned at 731 end of transverse bar, the cross
To the adjustable in length of bar 731, the gripping width of the clamping piece 732 is adjustable, and the clamping piece 732 is by elastic material system
At.
Embodiment 3
A kind of preparation method preparing glass freezing liquid using 2 described device of embodiment, wherein the glass freezing
The formula of liquid is as follows: ethylene glycol containing 10v/v%, 10v/v% dimethyl sulfoxide, 1.0M proline, 0.5M sucrose and 20w/
The DPBS (same with embodiment 1) of v% human serum albumins.
The preparation method is (500ml) as follows:
(1) proline, sucrose and human serum albumins are weighed according to formula, for use；
(2) DPBS buffer 100mL is measured, proline is added in DPBS buffer, concussion shakes up 10 minutes, room temperature
It is lower to stand 30 minutes to get proline-DPBS buffer；
(3) human serum albumins is added in step (2) proline-DPBS buffer, concussion mixes, at room temperature
30 minutes are stood to get human serum albumins-proline-DPBS buffer；
(4) DPBS buffer 260mL is measured, sucrose is added in the DPBS buffer measured, concussion shakes up, quiet at room temperature
30 minutes are set to get sucrose-DPBS buffer；
(5) sucrose-DPBS is transferred in the third container 3 of 2 described device system of embodiment, the second pump 5 passes through
Sucrose-DPBS the buffer is transferred in the second container 2 by third pipeline 10, and the first blower 4 will by second pipe 9
Sucrose-DPBS buffer force feed into the first container 1, by support rod 71 adjust the spray head 22 of the second container 2 with
Difference in height between 1 bottom surface of the first container, makes liquid not have the nozzle 221 on the spray head 22；
(6) 50mL ethylene glycol is measured, is transferred in third container 3, is transferred to the second of second container 2 using the second pump 6
In main body 21, after transfer, the second pump 6 is closed；First blower 4 is using wind pressure by the ethylene glycol solution from the spray
It is spouting with mode of jet at first 22 nozzle 221；The first pump 5 is opened, by the liquid pumping in the first container 1 to institute
It states in second container 2；The conveying capacity of first blower 4 and first pump 5 is adjusted, thus guarantee in cyclic process, the
Liquid in one container 1 did not had the nozzle 221 on the spray head 21 always, it is ensured that jet stream is below liquid level and in the first container
Liquid mixing, opens simultaneously cooling device 12, cools down to the liquid in first pipe, and is managed by thermometer 13 to first
Temperature in road is monitored, and loops back and forth like this mixing 3 minutes, later, stands 10min to get ethylene glycol-sucrose-DPBS
Buffer；
(7) dimethyl sulfoxide is mixed with the ethylene glycol-sucrose-DPBS buffer referring to step (6) mode, and
15 minutes are stood to get dimethyl sulfoxide-ethylene glycol-sucrose-DPBS buffer；
(8) referring to step (6) mode by human serum albumins-proline-DPBS buffer and dimethyl sulfoxide-second
Glycol-sucrose-DPBS buffer is uniformly mixed, and stands 10 minutes to get human serum albumins-proline-dimethyl sulfoxide-second
Glycol-sucrose-DPBS buffer；
(9) by step (8) human serum albumins-proline-dimethyl sulfoxide-ethylene glycol-sucrose-DPBS buffer
It is transferred in volumetric flask, is supplied with DPBS buffer to get glass freezing liquid；
(10) it is filtered using 0.22 μm of aperture filter, sealing, 4 DEG C of constant temperature are protected from light holding.
Embodiment 4
Glass freezing liquid is prepared referring to 1 the method for embodiment, difference is only that the formula of the vetrifying solution is such as
Shown in lower: dimethyl sulfoxide containing 10v/v%, 7.5v/v% ethylene glycol, 2.0mol/L proline, 0.5mol/L sucrose and 20v/
The DPBS of v% human serum albumins.
Embodiment 5
Glass freezing liquid is prepared referring to 3 the method for embodiment, difference is only that the formula of the vetrifying solution is such as
Shown in lower: dimethyl sulfoxide containing 10v/v%, 7.5v/v% ethylene glycol, 2.0mol/L proline, 0.5mol/L sucrose and 20v/
The DPBS of v% human serum albumins.
Embodiment 6
Glass freezing liquid is prepared referring to 1 the method for embodiment, difference is only that the formula of the vetrifying solution is such as
Shown in lower: dimethyl sulfoxide containing 7.5v/v%, 7.5v/v% ethylene glycol, 2.0mol/L proline, 0.5mol/L sucrose and 20v/
The DPBS of v% human serum albumins.
Embodiment 7
Glass freezing liquid is prepared referring to 3 the method for embodiment, difference is only that the formula of the vetrifying solution is such as
Shown in lower: dimethyl sulfoxide containing 7.5v/v%, 7.5v/v% ethylene glycol, 2.0mol/L proline, 0.5mol/L sucrose and 20v/
The DPBS of v% human serum albumins.
Comparative example 1
A kind of preparation method of glass freezing liquid, wherein the formula of the glass freezing liquid is as follows: contain 10v/
V% ethylene glycol, 10v/v% dimethyl sulfoxide, 1.0M proline, 0.5M sucrose and 20w/v% human serum albumins DPBS.
The preparation method is (500ml) as follows:
(1) proline, sucrose and human serum albumins are weighed according to formula, for use；
(2) DPBS buffer 360mL is measured, proline is added in DPBS buffer, concussion shakes up 10 minutes, room temperature
It is lower to stand 30 minutes to get proline-DPBS buffer；
(3) human serum albumins is added in step (2) described buffer, concussion mixes 10 minutes, stands at room temperature
30 minutes to get human serum albumins-proline-DPBS buffer；
(4) sucrose is added in step (3) described buffer, concussion shakes up 10 minutes, stand 30 minutes at room temperature to get
Sucrose-human serum albumins-proline-DPBS buffer；
(5) ethylene glycol 50mL is measured, step (4) described buffer is slowly dropped to, stirs (about 20 minutes) when being added dropwise,
It after completion of dropwise addition, persistently stirs 10 minutes, it is slow to get ethylene glycol sucrose-human serum albumins-proline-DPBS to stand 1 hour
Fliud flushing；
(6) dimethyl sulfoxide 50mL is measured, step (5) described buffer is slowly dropped to, stirs while adding (about 20 points
Clock), it after completion of dropwise addition, persistently stirs 10 minutes, stands 1 hour to get dimethyl sulfoxide-ethylene glycol sucrose-human seralbumin egg
White-proline-DPBS buffer；
(7) by step (6) human serum albumins-proline-dimethyl sulfoxide-ethylene glycol-sucrose-DPBS buffer
It is transferred in volumetric flask, is supplied with DPBS buffer to get glass freezing liquid；
(8) it is filtered using 0.22 μm of aperture filter, sealing, 4 DEG C of constant temperature are protected from light holding.
Comparative example 2
Glass freezing liquid is configured referring to 1 the method for comparative example, difference is only that the formula of the vetrifying solution is such as
Shown in lower: dimethyl sulfoxide containing 10v/v%, 7.5v/v% ethylene glycol, 2.0mol/L proline, 0.5mol/L sucrose and 20v/
The DPBS of v% human serum albumins.
Comparative example 3
Glass freezing liquid is prepared referring to 1 the method for embodiment, difference is only that the formula of the vetrifying solution is such as
Shown in lower: dimethyl sulfoxide containing 7.5v/v%, 7.5v/v% ethylene glycol, 2.0mol/L proline, 0.5mol/L sucrose and 20v/
The DPBS of v% human serum albumins.
Experimental example 1
During embodiment 1,3~7 and comparative example 1~3 prepare glass freezing liquid, gas in process for preparation is observed
Steep production, mixing time and heat dissipation time (refer to mixing time when ethylene glycol and dimethyl sulfoxide is added, radiate the time), tool
Body testing result is referring to table 1.
According to experimental data described in table 1 it is found that compared with embodiment 1,4,6 and comparative example 1~3, using of the invention real
Mixing time and heat dissipation time can be shortened to contract by applying 2 described device of example preparation glass freezing liquid (that is, embodiment 3,5,7)
The short total time for preparing glass freezing liquid, while being also avoided that in mixed process and generating bubble.
1 glass freezing liquid process for preparation of table is with specific reference to index
| Bubble production | Mixing time | It radiates the time | |
| Embodiment 1 | A large amount of bubbles are generated in whipping process, it is prolonged not dissipate | 1 hour | 2 hours |
| Embodiment 3 | There are few bubble generations in whipping process | 6 minutes | 30 minutes |
| Embodiment 4 | A large amount of bubbles are generated in whipping process, it is prolonged not dissipate | 1 hour | 2 hours |
| Embodiment 5 | There are few bubble generations in whipping process | 6 minutes | 30 minutes |
| Embodiment 6 | A large amount of bubbles are generated in whipping process, it is prolonged not dissipate | 1 hour | 2 hours |
| Embodiment 7 | There are few bubble generations in whipping process | 6 minutes | 30 minutes |
| Comparative example 1 | A large amount of bubbles are generated in whipping process, it is prolonged not dissipate | 1 hour | 2 hours |
| Comparative example 2 | A large amount of bubbles are generated in whipping process, it is prolonged not dissipate | 1 hour | 2 hours |
| Comparative example 3 | A large amount of bubbles are generated in whipping process, it is prolonged not dissipate | 1 hour | 2 hours |
Experimental example 2
Glass freezing liquid is prepared according to embodiment 1,3~7 and 1~3 the method for comparative example, it is detected and matches fresh
Set, 4 DEG C the refrigerating effect being kept in dark place for 1 month, 4 DEG C 2 months and 4 DEG C after being kept in dark place 3 months is kept in dark place.Specific inspection
Survey method is as follows:
One, glass freezing and defrosting
Mouse GV phase egg mother cell is obtained, first places it in equilibrium liquid and balances 4~5 minutes, it is cold to be subsequently placed in vitrifying
Freeze in liquid 1~2 minute, is put into liquid nitrogen and saves 5 hours or more later, be finally sequentially placed into thawing solution 1~4 each 5 minutes.
Wherein, equilibrium liquid is slow for the DPBS containing 7.5% ethylene glycol, 7.5% dimethyl sulfoxide and 20% human serum albumins
Fliud flushing；Thawing solution 1 is the DPBS buffer of sucrose containing 1M and 20%HSA；Thawing solution 2 is sucrose containing 0.5M and 20% human seralbumin
DPBS buffer, the thawing solution 3 of albumen are the DPBS buffer of sucrose containing 0.25M and 20%HSA, thawing solution 4 is containing 20%HSA
DPBS buffer.
Two, cell survival rate judges after thawing
The cell to thaw is observed, the cell that good, the complete cell of cellular morphology is judged to surviving by ovarian cumulus covering is on the contrary
Then it is determined as that dead cell, the ratio between survivaling cell number and total number of cells are survival rate.Specific testing result is referring to table 2.
Comparing embodiment 1,4,6 and the glass freezing liquid of embodiment 3,5,7 are to mouse GV phase egg mother cell survival rate
Influence, the results showed that, for the glass freezing liquid of Fresh,
Embodiment 1,4~7 all has the excellent effect that freezes, the survival rate of mouse GV phase egg mother cell 90% with
On；But with the extension of holding time, the glass freezing liquid prepared using 2 described device system of the embodiment of the present invention is (that is, implement
Example 3,5,7) still keep good and freeze effect, mouse GV phase cell thaw after survival rate 86% or more, the shelf-life is extremely
Less at 3 months or more；And it is of the invention
The vetrifying solution of embodiment 1,4,6 still maintains in preservation 1~2 month and preferably freezes effect, mouse GV
For the survival rate of phase cell 76% or more, but after holding 3 months, survival rate freezes significant effect decline down to 62~66%.
Experimental data shown in comparing embodiment 1,3,5 and comparative example 1~3 is it is found that either Fresh, or long-term guarantor
The effect that freezes of the glass freezing liquid deposited, comparative example 1~3 has obviously been inferior to example of the embodiment of the present invention 1,3,5.
Survival rate after the freezing of 2 different glass freezing liquid of table
Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Present invention has been described in detail with reference to the aforementioned embodiments for pipe, but those skilled in the art should understand that: its
It is still possible to modify the technical solutions described in the foregoing embodiments, or to some or all of the technical features
It is equivalently replaced；And these are modified or replaceed, various embodiments of the present invention skill that it does not separate the essence of the corresponding technical solution
The range of art scheme.
Claims (37)
1. a kind of method for preparing glass freezing liquid, which is characterized in that wherein, the glass freezing liquid includes dimethyl Asia
Sulfone, ethylene glycol, carbohydrate, proline and seralbumin；Described method includes following steps:
(1) using buffer solution carbohydrate to get sugar juice；(2) dimethyl sulfoxide and ethylene glycol are added in the sugar juice,
It is uniformly mixed to get dimethyl sulfoxide-ethylene glycol-sugar juice；(3) when the dimethyl sulfoxide-ethylene glycol-sugar juice is cooling
Afterwards, proline solution and seralbumin is added, is uniformly mixed to get proline-seralbumin-dimethyl sulfoxide-second two
Alcohol-sugar juice；(4) proline-seralbumin-dimethyl sulfoxide-ethylene glycol-sugar juice is carried out at constant volume and degerming
Reason is to get glass freezing liquid.
2. the method according to claim 1, wherein the glass freezing liquid includes the v/v% of 7.5 v/v% ~ 15
Dimethyl sulfoxide, 7.5 v/v% ~ 15 v/v% ethylene glycol, 0.5 ~ 2 M carbohydrate, proline 0.5 ~ 2 M and 15 w/v ~ 20 w/v blood
Pure albumen.
3. according to the method described in claim 2, it is characterized in that, the carbohydrate is selected from monosaccharide, disaccharides or trisaccharide.
4. according to the method described in claim 3, it is characterized in that, the monosaccharide, disaccharides or trisaccharide are selected from glucose, gala
Sugar, lactose, sucrose, trehalose, gossypose.
5. according to the method described in claim 3, it is characterized in that, the carbohydrate is sucrose.
6. according to the method described in claim 2, it is characterized in that, the seralbumin is human serum albumins.
7. apparatus system of the one kind for any one of claim 1 ~ 6 the method, which is characterized in that the hybrid system includes
The first container, second container, first pipe, second pipe, the first pump and the first blower, in which:
The first container includes the first main body, for accommodating liquid and the second container；
The second container includes the second main body and spray head, and second main body is connected to the spray head, is arranged on the spray head
There is nozzle；
The second container is placed in the first container, and the first container and the second container are connected by first pipe
It is logical；
The first pump is provided in the first pipe, first pump through the first pipe for pumping liquid from the first container
It send into the second container；
The second container is connected to first blower by second pipe, and first blower is by the second pipe
The second container provides lasting wind pressure, and the wind pressure is by the liquid in second main body through the spray head force feed to described
In first main body of the first container.
8. apparatus system according to claim 7, which is characterized in that the second container is with the first container with described
The adjustable mode of height distance between the spray head of second container and the bottom surface of the first container is oppositely arranged, and described first
The adjustable section of the pumpability of pump, the adjustable section of the draft capacity of first blower.
9. apparatus system according to claim 7, which is characterized in that be additionally provided in the first pipe cooling device and
Thermometer, for cooling down to the liquid in first pipe, the thermometer is used for in first pipe the cooling device
Fluid temperature be monitored.
10. apparatus system according to claim 7, which is characterized in that the spray head is rectangular, cylindrical or arc.
11. apparatus system according to claim 7, which is characterized in that the nozzle be arranged in the spray head bottom surface and/
Or side.
12. device according to claim 11 system, which is characterized in that the nozzle be arranged in the spray head bottom surface and
Side.
13. apparatus system according to claim 7, which is characterized in that the nozzle is multiple.
14. apparatus system according to claim 13, which is characterized in that the nozzle is 20 ~ 100.
15. apparatus system according to claim 14, which is characterized in that the nozzle is 50 ~ 100.
16. apparatus system according to claim 7, which is characterized in that the diameter of the nozzle is 10 ~ 500 μm.
17. apparatus system according to claim 16, which is characterized in that the diameter of the nozzle is 50 ~ 500 μm.
18. apparatus system according to claim 17, which is characterized in that the diameter of the nozzle is 100 ~ 300 μm.
19. device according to claim 11 system, which is characterized in that nozzle on the same face phase radially is arranged in
Mutually in parallel or in an angle.
20. apparatus system according to claim 19, which is characterized in that be arranged in the same face top nozzle is in one radially
Angle.
21. apparatus system according to claim 20, which is characterized in that the angle is 5 ~ 45 °.
22. apparatus system according to claim 21, which is characterized in that the angle is 10 ~ 30 °.
23. apparatus system according to claim 7, which is characterized in that second main body and the spray head are integrally formed,
Or second main body is fixedly connected with the spray head by screw thread-screw hole.
24. apparatus system according to claim 7, which is characterized in that coated on the inner and outer wall of the second container
There is adherent layer.
25. apparatus system according to claim 7, which is characterized in that the system also includes for accommodating solution to be mixed
Third container, the third container is connected to the first container or the second container by third pipeline, the third
The second pump is provided on pipeline, liquid is pumped to the first container or the second container from third container by second pump
In.
26. apparatus system according to claim 7, which is characterized in that described device system further includes bracket, the bracket
It is described including pedestal, the support rod on the pedestal and the lateral hold assembly that the support bar top is arranged in
Lateral hold assembly is for clamping the second container.
27. apparatus system according to claim 26, which is characterized in that the pedestal is sucker.
28. apparatus system according to claim 26, which is characterized in that the support rod it is height-adjustable.
29. apparatus system according to claim 28, which is characterized in that the support rod includes the first loop bar and second set
Bar, first loop bar are connected with the pedestal, and second loop bar is socketed in first loop bar, and second loop bar can
It slides up and down along first loop bar, is fixed between second loop bar and first loop bar by screw-bolt.
30. apparatus system according to claim 28, which is characterized in that the transverse direction hold assembly includes transverse bar and position
Clamping piece in the transverse bar end.
31. apparatus system according to claim 30, which is characterized in that the adjustable in length of the transverse bar；The folder
The gripping width of gripping member is adjustable；The clamping piece is made of elastic material.
32. the method according to claim 1, wherein the step (2), (3) use claim 7 ~ 31 any
Apparatus system described in carries out the mixing of solution.
33. according to the method for claim 32, which is characterized in that in mixed process, adjust the spray head and described the
The height distance of the bottom surface of one container controls the conveying capacity of first pump and first blower, to keep the first main body
In liquid do not had the nozzle always.
34. glass freezing liquid made of any one of according to claim 1 ~ 6 or 32 ~ 33 the methods.
35. a kind of glass freezing kit, which is characterized in that the kit includes glass freezing described in claim 34
Liquid and other auxiliary reagents.
36. glass freezing kit according to claim 35, which is characterized in that the glass freezing liquid includes
The v/v% of 7.5 v/v% ~ 15 dimethyl sulfoxide, 7.5 v/v% ~ 15 v/v% ethylene glycol, 0.5 ~ 2 M carbohydrate, 0.5 ~ 2 M of proline
With the w/v seralbumin of 15 w/v ~ 20.
37. glass freezing kit according to claim 35, which is characterized in that other described auxiliary reagents are by balancing
Liquid, thawing solution 1, thawing solution 2, thawing solution 3 and thawing solution 4 form, and the equilibrium liquid is sub- containing 7.5% ethylene glycol, 7.5% dimethyl
The DPBS buffer of sulfone and 20% human serum albumins；The thawing solution 1 is the DPBS containing 1 M sucrose and 20% human serum albumins
Buffer；The thawing solution 2 be the DPBS buffer containing 0.5 M sucrose and 20% human serum albumins, the thawing solution 3 be containing
The DPBS buffer of 0.25 M sucrose and 20% human serum albumins, the thawing solution 4 are the DPBS containing 20% human serum albumins
Buffer.
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| CN109221081B (en) * | 2018-09-07 | 2021-02-12 | 银丰低温医学科技有限公司 | Multi-tissue vitrification in-situ storage device |
| AU2020256873B2 (en) * | 2019-04-09 | 2023-08-17 | Institute Of Chemistry, Chinese Academy Of Sciences | Thawing fluid, preparation method therefor and use thereof |
| CN110432259B (en) * | 2019-08-26 | 2021-07-23 | 中国科学院理化技术研究所 | Freezing protection solution, cell cryopreservation solution containing freezing protection solution and application of freezing protection solution in cell cryopreservation |
| CN117136833B (en) * | 2023-08-18 | 2024-05-24 | 生态环境部华南环境科学研究所(生态环境部生态环境应急研究所) | Sargassum periwinkle germplasm preservation method |
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| CN102771473A (en) * | 2012-04-25 | 2012-11-14 | 余文莉 | Freezing liquid for preserving embryo, preparation method and application thereof |
| CN104145943A (en) * | 2014-07-21 | 2014-11-19 | 重庆市红汇脐血干细胞中心有限公司 | Cryopreservation protection liquid for Wharton jelly tissues of human umbilical cord and preparation and application of cryopreservation protection liquid |
| CN104839144A (en) * | 2015-04-30 | 2015-08-19 | 北京大学第三医院 | Vitrification freezing fluid of oocytes |
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|---|---|---|---|---|
| CN102771473A (en) * | 2012-04-25 | 2012-11-14 | 余文莉 | Freezing liquid for preserving embryo, preparation method and application thereof |
| CN104145943A (en) * | 2014-07-21 | 2014-11-19 | 重庆市红汇脐血干细胞中心有限公司 | Cryopreservation protection liquid for Wharton jelly tissues of human umbilical cord and preparation and application of cryopreservation protection liquid |
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Effective date of registration: 20190621 Address after: 213100 E4 Building, No. 9 Changyang Road, Changzhou West Taihu Science and Technology Industrial Park, Wujin District, Changzhou City, Jiangsu Province Patentee after: Jiangsu Kanglin Medical Technology Co., Ltd. Address before: No. 49 Huayuan North Road, Haidian District, Beijing 100000 Patentee before: The Third Affiliated Hospital of Peking University |