CN109497040A - A kind of human oocytes and embryo vitrifying freeze thawing device and its application method - Google Patents
A kind of human oocytes and embryo vitrifying freeze thawing device and its application method Download PDFInfo
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- CN109497040A CN109497040A CN201811365778.7A CN201811365778A CN109497040A CN 109497040 A CN109497040 A CN 109497040A CN 201811365778 A CN201811365778 A CN 201811365778A CN 109497040 A CN109497040 A CN 109497040A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0236—Mechanical aspects
- A01N1/0242—Apparatuses, i.e. devices used in the process of preservation of living parts, such as pumps, refrigeration devices or any other devices featuring moving parts and/or temperature controlling components
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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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- 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
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0608—Germ cells
- C12N5/0609—Oocytes, oogonia
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Abstract
A kind of human oocytes and embryo vitrifying freeze thawing device and its application method, the lower surface of cyclotron oscillation pallet and the output shaft of first motor are fixedly linked, and outlet tube slidably extends through through-hole and is fixed on extension folder by clamp assemblies；The bottom end of outlet tube offers liquid outlet, and liquid outlet is located at the top of the opening of open slot；Being connected in the top of outlet tube can move up and down along the outlet tube with the driving portion for aspirating or injecting the liquid.The present invention passes through the collective effect of outlet tube and cyclotron oscillation disk, so that egg mother cell/embryo carries out glass freezing in vitro and is in the environment of a freezing liquid or thawing solution concentration continuity gradual change during thawing, and eliminate the operation that experimenter aspirates repeatedly to embryo, blows and beats, shifts, stirs etc., effectively reduce vitro and manual operation to embryo stress sexual stimulus influence, it is beneficial to development and the filial generation safety at a specified future date of embryo, while also mitigates the working strength of experimenter.
Description
Technical field
The invention belongs to medical instruments fields, and in particular to a kind of human oocytes and embryo vitrifying freeze thawing device and
Its application method.
Background technique
With the relieving of national " two children " policy comprehensively, the country meets the man and wife of two child's standards about at 90,000,000 pairs,
In have 60,000,000 women at 35 years old or more, much all need to be by supplementary reproduction (test tube baby if this part advanced age women wants to be pregnant
Youngster) technology.But due to environmental pollution, job and life stress is big, the age increases and the superposition shadow of the factors such as bad life style
It rings, " the infertile Current Situation Investigation of the China is reported " display jointly issued according to Chinese population association, State Family Planning Commission is Chinese
For infertile disease incidence in 12.5%-15%, patient numbers are more than 40,000,000, this means that every 7-8 is to just there is 1 pair of trouble in Mr. and Mrs
There is infertile problem, these are all had to by supplementary reproduction means.
And when embryo experiments room carries out IVF-ET (in vitro fertilization-embryo transfer) operation, ova harvest can be encountered and worked as
The case where day, wife's side egg mother cell have taken out, but the bridegroom's or husband's side can not take out sperm because of various reasons can also encounter the wife's side because pernicious swollen
Tumor and be badly in need of the case where saving before receiving chemotherapy or radiation cure to fertility, just need to women's at this time
Egg mother cell carries out freezing operation.And work as vitro Development of Embryos to certain phase, it can encounter and be greater than transplanting using embryo's number
Situations such as embryo's number or wife's side endometrium are not in the best embryo transfer phase just must carry out freezen protective to embryo at this time
Operation.
Currently, the freezing of egg mother cell or embryo are broadly divided into sequencing freezing and glass freezing.Glass freezing phase
The sequencing frigorimeter because not needing purchasing expensive is freezed compared with sequencing, and there is easy to operate, time saving, refrigerating effect is good etc.
Advantage and used by most of embryo experiments rooms.
But existing glass freezing and operation of thawing often carry out in three orifice plates or four orifice plates, and often at interval of 1
~3 minutes, experimenter needs to carry out various concentration freezing liquid or thawing solution to egg mother cell/embryo with glass capillary
Transfer operation, and in order to which the substance for guaranteeing that egg mother cell/embryo can be sufficiently carried out with freezing liquid or thawing solution inside and outside cell membrane is handed over
It changes, needs that egg mother cell/embryo is placed in different liquid layers with glass capillary and the suction carried out repeatedly is blown and beaten, while also needing
Liquid level is stirred back and forth.And egg mother cell/embryo freeze-thaw process inherently cell sharply shrinkage-return it is swollen-shrinkage-is again again
Swollen process is returned, in a non-natural physiological more fragile state, and embryo is carried out with glass capillary again at this time
The operation of suction, piping and druming, carrying, agitation etc., certainly will exist influences egg mother cell/embryo's subsequent development risk, and different holes
Between the operation frequently shifted there is also small egg mother cell/embryo is removed the risk lost, the furthermore operation of high frequency repeatedly
Also increase the working strength of experimenter.
Currently, existing egg mother cell/embryo cryopreservation protective agent selects sucrose as impermeability protective agent in the world,
And selecting propylene glycol (PROH), ethylene glycol (EG) or dimethyl sulfoxide (DMSO) etc. is permeability protective agent.It is existing international
Cryoprotection agent prescription is as follows:
Formula 1:
Egg mother cell/embryo cryopreservation liquid:
Equilibrium liquid: 75% buffer (V/V)+10%SPS (V/V)+7.5%PROH (V/V)+7.5%EG (V/V)；
Freezing liquid: 60% buffer (V/V)+10%SPS (V/V)+15%PROH (V/V)+15%EG (V/V)+0.5mol/
L sucrose
Egg mother cell/embryo thawing liquid:
Thawing solution I:90% buffer (V/V)+10%SPS (V/V)+1mol/L sucrose；
Thawing solution II:90% buffer (V/V)+10%SPS (V/V)+0.5mol/L sucrose；
Thawing solution III:90% buffer (V/V)+10%SPS (V/V)+0.25mol/L sucrose；
Thawing solution IV:90% buffer (V/V)+10%SPS (V/V)
Formula 2:
Egg mother cell/embryo cryopreservation liquid:
Pre-equilibrate liquid: 95% buffer (V/V)+5%HSA (V/V)
Freezing liquid I:85% buffer (V/V)+5%HSA (V/V)+5%DMSO (V/V)+5%EG (V/V)
Freezing liquid II:75% buffer (V/V)+5%HSA (V/V)+10%DMSO (V/V)+10%EG (V/V)
Freezing liquid III:25% buffer (V/V)+5%HSA (V/V)+20%DMSO (V/V)+20%EG (V/V)+30%
Sucrose
Egg mother cell/embryo thawing liquid:
Thawing solution I:65% buffer (V/V)+5%HSA (V/V)+30% sucrose；
Thawing solution I:75% buffer (V/V)+5%HSA (V/V)+20% sucrose；
Thawing solution I:85% buffer (V/V)+5%HSA (V/V)+10% sucrose；
Thawing solution I:90% buffer (V/V)+5%HSA (V/V)+5% sucrose
Chinese patent CN 104663649A application trehalose instead sucrose joins simultaneously as impermeability cryoprotector
Propylene glycol (PROH) and ethylene glycol (EG) are closed as permeability cryoprotector in clinically obtaining good freeze thawing effect, is matched
Side are as follows:
Egg mother cell/embryo cryopreservation liquid:
Equilibrium liquid: 60% buffer (V/V)+20% human serum substitute (V/V)+10%PROH (V/V)+10%EG (V/
V)；
Freezing liquid: 40% buffer (V/V)+20% human serum substitute (V/V)+20%PROH (V/V)+20%EG (V/
V)+0.65mol/L trehalose
Egg mother cell/embryo thawing liquid:
Thawing solution I:80% buffer (V/V)+20% human serum substitute (V/V)+1mol/L trehalose；
Thawing solution II:80% buffer (V/V)+20% human serum substitute (V/V)+0.5mol/L trehalose；
Thawing solution III:80% buffer (V/V)+20% human serum substitute (V/V)+0.2mol/L trehalose；
+ 20% human serum substitute (V/V) of thawing solution IV:80% buffer (V/V)
Buffer described above includes human tubal fluid HTF1024 (SAGE, USA), human tubal fluid HTF1023 (SAGE,
USA), 3-propanesulfonic acid GMOPS (Vitrolife, Sweden) and Du Shi phosphate buffer DPBS (SAGE, USA) etc.,
Haemocyanin substitute SPS (SAGE, USA), human serum albumin HSA (Vitrolife, Sweden) are human serum substitution
Object, another V/V represent volume ratio.
But in the above formula, there is the concentration of experience noncontinuity (such as formula 1 from low to high in egg mother cell or embryo
15%) or from high to low the concentration of middle permeability cryoprotector PROH jumps directly to that (CN 104663649A is special by 7.5%
Impermeability cryoprotector trehalose concentration jumps directly to 0.5mol/L by 1mol/L in benefit formula).Although current glass
Changing freezing can be in clinically obtaining relatively satisfactory recovery survival rate, cleavage rates, Blastocyst formation rate and Clinical Pregnancy Rate in.But from
Smoothly deliver after first case human embryos vitrifying freeze thawing and (completed by Mukaida within 1998) only 20 years history so far, and
How on earth is the safety of generation filial generation after vitrifying freeze thawing, and the data for still lacking large sample at present are supported.
Therefore, vitro or operation are reduced as far as possible to the irritability stimulus effects of embryo, allow egg mother cell or embryo
Glass freezing and thaw during undergo cryoprotector concentration variation more tend to it is progressive and soft, to the body of embryo
The safety of outgrowth development and filial generation is all beneficial.
Summary of the invention
The purpose of the present invention is overcoming the deficiencies of existing technologies, provide it is a kind of can make egg mother cell/embryo in vitro into
Environment during row glass freezing and defrosting in a freezing liquid or thawing solution concentration continuity gradual change, can effectively subtract
Few vitro or operation can remove a series of peace of repetitive operations to egg mother cell/embryo irritability stimulus effects
Complete efficient vitrifying freeze thawing device and its application method.
Technical proposal that the invention solves the above-mentioned problems is:
It includes water that embodiments herein, which provides a kind of human oocytes and embryo vitrifying freeze thawing device, described device,
The chassis of setting is put down, first motor is fixed on the chassis, the output shaft of the first motor extends straight up and connects
It is connected to vibration section；The vibration section includes cyclotron oscillation pallet, the lower surface of the cyclotron oscillation pallet and the first motor
Output shaft be fixedly linked, to drive the cyclotron oscillation pallet to rotate, and the first motor is connected with the first power supply and control
Make the first switch of the first motor start and stop；
The limiting groove for accommodating accommodating plate, the limiting groove are offered on the upper surface of the cyclotron oscillation pallet
Central axis it is conllinear with the central axis of the output shaft of the first motor；The accommodating plate is eccentrically set on the limiting groove
Interior, the centre of the accommodating plate offers the open slot for holding liquid, and the notch of the open slot is open upwards；
The side on the chassis is equipped with the bracket extended straight up, is fixed with horizontal-extending extension on the bracket
Folder；And the fixing end for extending folder is fixed on the bracket, top water of the free end for extending folder to the chassis
It is flat to extend；
The through-hole penetrated through along the vertical direction is offered on the free end for extending folder, outlet tube slidably extends through described
Through-hole is simultaneously fixed on the extension folder by clamp assemblies；The bottom end of the outlet tube offers liquid outlet, the liquid outlet
Positioned at the top of the opening of the open slot, to inject the liquid into the open slot；And the outlet tube is positioned at described
The tube body for extending folder lower section is equipped with scale；
Being connected in the top of the outlet tube can move up and down along the outlet tube for aspirating or injecting the liquid
The driving portion of body, the driving portion includes that can slide up and down to the piston being arranged in the outlet tube along the vertical direction, described
The top of piston is connected by connecting rod with piston rod；The piston rod can be axially slidably disposed in institute along the outlet tube
It states in outlet tube, the piston rod runs through the top end opening of the outlet tube, and side of the piston rod towards the bracket
Equipped with the rack gear extended along the vertical direction, and the rack and pinion is meshed；
The gear is arranged on bracket by support plate, and the gear fixation is set on the output shaft of the second motor,
Second motor is fixed in the support plate, and the support plate is fixed on the bracket, and the support plate is located at institute
State the top for extending folder；
Second motor is connected with second source, the second switch of control the second motor start and stop and control described the
The speed control of the revolving speed of two motors.
Further, the first motor is fixed on the chassis by motor support plate.
Further, institute's described device further includes timer, and the timer is fixed in the support plate.
Further, the area of the opening of the open slot is greater than the area of slot bottom.
Further, the clamp assemblies are specially spring clip, the outlet tube by be located at extensions folder top with
The spring clip of lower section is fixed on the extension folder.
Further, the outer wall of the piston rod and the inner wall of the outlet tube fit.
Further, the cyclotron oscillation pallet the center point is labeled with red salient point.
Further, the liquid is glass freezing liquid or thawing solution, wherein the glass freezing liquid is that balance is slow
The impermeability frozen solution of fliud flushing, the permeability frozen solution of single concentration or single concentration.
Wherein: the ingredient of the equilibration buffer is people's Culture in oviduct liquid that volume fraction is 95% and volume fraction is
The mixed liquor of 5% human serum substitute.
The permeability frozen solution of the single concentration is people's Culture in oviduct liquid that volume fraction is 55%, volume point
Count the mixed of the human serum substitute for 5%, the dimethyl sulfoxide that volume fraction is 20% and the ethylene glycol that volume fraction is 20%
Close object.
The impermeability frozen solution of the single concentration is the sucrose that volume fraction is 95% and volume fraction is 5%
Human serum substitute mixture.
The thawing solution be volume fraction be 65% people's Culture in oviduct liquid, volume fraction be 30% sucrose and volume
The mixture for the human serum substitute that score is 5%.
The present embodiment also provides the application method of a kind of human oocytes and embryo vitrifying freeze thawing device, including following
Step:
Step 1: outlet tube 1 by driving portion extract 700ul the single concentration permeability frozen solution, and to
The equilibration buffer that 300ul is added in the open slot of plate is accommodated, and makes the permeability frozen solution of the single concentration
Balance 30min is stood at 25 DEG C with the equilibration buffer；
Step 2: adjusting outlet tube position, it is made to be located at the cyclotron oscillation pallet center of circle with the chassis vertical and liquid outlet of holding
At 2~3cm of surface, then accommodating plate is placed in the limiting groove of cyclotron oscillation pallet；
Step 3: starting to carry out glass freezing, the egg mother cell or embryo to be freezed are added into the open slot of accommodating plate
Tire opens the first switch of first motor, rotates the cyclotron oscillation pallet, and revolving speed is 100rpm, to drive accommodating
Plate circular motion；
After step 4:1min, the second switch of the second motor is opened, so that second motor driven gear rotates, gear
It is meshed with rack gear, piston rod pushes down on piston, by the permeability frozen solution of the single concentration with 100ul/min's
Flow velocity is released from liquid outlet and is injected in open slot, while opening timing device, timing 6min；
Step 5: when timer issues the prompt sound, closing out second switch, and added into the open slot with suction pipe
Enter the impermeability frozen solution of the single concentration of 400ul；
After step 6:1min, first switch is closed, with glass capillary by the egg mother cell or embryo transfer in open slot
It carries on bar to glass freezing, and is stored in the liquid nitrogen of -196 DEG C of investment rapidly；
Step 7: start before carrying out vitrifying defrosting, the equilibration buffer of 1ml is extracted with the piston rod in outlet tube,
And into open slot be added 300ul the thawing solution, the thawing solution, make the equilibration buffer is stood at 25 DEG C put down
Weigh 30min, and the accommodating plate for filling the thawing solution is placed in 37 DEG C of incubator and stands balance 30min；
Step 8: adjusting the position of outlet tube, keep its holding perpendicular with chassis, and liquid outlet is located at cyclotron oscillation pallet
At the 2~3cm of surface in the center of circle；
Step 9: start to carry out vitrifying defrosting, accommodating plate taken out out of incubator and is placed on station, then rapidly from-
The egg mother cell to be thawed is taken out in 196 DEG C of liquid nitrogen or embryo is placed into the open slot for filling the thawing solution, is led to
It crosses micro- sem observation and confirms that egg mother cell or embryo carry from freezing and unload and entered in open slot on bar, then plate will be accommodated
It is placed in the limiting groove of the cyclotron oscillation pallet, and opens first switch, rotate cyclotron oscillation pallet, and rotate
Speed is 100rpm；
After step 10:2min, the second switch of the second motor is opened, by the equilibration buffer with the stream of 100ul/min
Speed instills in the open slot, while opening timing device, timing 9min；
Step 11: when timer issues the prompt sound, second switch and first switch are closed, with glass capillary by ovum
Mother cell or embryo are transferred in gamete culture solution or spilting of an egg culture solution out of open slot, and are placed in 37 DEG C, CO2Concentration is
In 6% constant temperature and humidity incubator, in case subsequent operation.
Beneficial effects of the present invention are mainly manifested in:
1, device of the present invention passes through the collective effect of outlet tube and cyclotron oscillation disk, so that egg mother cell/embryo exists
The external environment for carrying out glass freezing and a freezing liquid or thawing solution concentration continuity gradual change are in during thawing, and
The operation that experimenter aspirates repeatedly to embryo, blows and beats, shifts, stirs etc. is eliminated, vitro and artificial is effectively reduced
Operate to embryo stress sexual stimulus influence, be beneficial at a specified future date development and the filial generation safety of embryo.Experiment people is also mitigated simultaneously
The working strength of member, other work in the off time expansion laboratory during enabling personnel to efficiently use freeze-thaw, mentions
High working efficiency.
2, the accommodating plate is eccentrically set in the limiting groove, i.e., the central axis of the described accommodating plate and the convolution are shaken
The central axis for swinging pallet deviates, so that the accommodating plate, which has, preferably rocks effect, to promote in the open slot
Liquid uniformly mixes.
3, the scale that outlet tube is equipped with facilitates experimenter accurately to draw quantitative liquid before freezing or operation of thawing start
Body (freezing liquid or equilibration buffer) avoids the waste of liquid while guaranteeing experiment reaction sufficiently completely.
4, the liquid outlet of outlet tube is 2~3cm at a distance from open slot, is closer, and be can effectively avoid after dripping under liquid
Splashing and influence freeze thawing effect.
5, the speed control can limit the revolving speed of second motor, so that the gear uniform rotation, thus band
The piston rod is moved at the uniform velocity to move.
6, the diameter of the opening of open slot is 3cm, and the liquid that ensure that outlet tube instils fully enters in open slot；It is described
Open slot eliminates the visual field interference range at cylindrical grooves bottom right angle, then makes open slot in up big and down small round table-like
The suction operation of observation and glass capillary while more volume of liquid can be accepted more conducively under microscope.
7, the spring clip is fixed on the axially different position of the outlet tube, the adjustable outlet tube goes out
The distance between liquid mouth and accommodating plate.
8, inner wall of the outside wall surface of piston and the piston rod with the outlet tube fits, and improves outlet tube and pushes away
The stability of fluid injection body.
9, the center of circle that salient point can be used for calibrating the central axis and the cyclotron oscillation pallet of the liquid outlet aligns, and is convenient for
Naked-eye observation and operation.
10, the center point of the open slot slot bottom is equipped with mark point, and the mark point can be true referring to first salient point
Initial placement position of the fixed accommodating plate on the limiting groove, is convenient for naked-eye observation and operation.
11, the basic bottom liquid of freezing liquid and thawing solution select HTF (people's Culture in oviduct liquid) then be consider its ratio it is existing
HEPES the or MOPS buffer system of technology is closer to the intracorporal natural physiological environment of people.
Detailed description of the invention
Fig. 1 is the structural schematic diagram of device of the present invention in an embodiment；
When Fig. 2 is using the existing liquid of glass freezing in the world (formula 2 specially in background technique), permeability freezing
The graph of relation of protective agent concentration and reaction time；
When Fig. 3 is that 1 described device of application drawing carries out refrigeration operation, the pass of permeability cryoprotector concentration and reaction time
It is curve graph；
When Fig. 4 is using the existing thawing solution of vitrifying in the world (formula 2 specially described in background technology), permeability
The graph of relation of cryoprotector concentration and reaction time；
When Fig. 5 is that 1 described device of application drawing carries out defrosting operation, the pass of permeability cryoprotector concentration and reaction time
It is curve graph.
Wherein, in Fig. 2~5, the t of horizontal axis indicates the time, and (V/V, the %) of the longitudinal axis indicates volume fraction.
Specific embodiment
It is clearly and completely described below in conjunction with technical solution of the attached drawing to the invention patent, it is clear that described
Embodiment is a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field is common
Technical staff's every other embodiment obtained without making creative work belongs to the model that the present invention protects
It encloses.
In the description of the present invention, it should be noted that such as occur term " center ", "upper", "lower", "left", "right",
The orientation or positional relationship of the instructions such as "vertical", "horizontal", "inner", "outside" is to be based on the orientation or positional relationship shown in the drawings,
Be merely for convenience of description of the present invention and simplification of the description, rather than the device or element of indication or suggestion meaning must have it is specific
Orientation, be constructed and operated in a specific orientation, therefore be not considered as limiting the invention.In addition, such as there is term "
One ", " second ", " third " are used for descriptive purposes only and cannot be understood as indicating or suggesting relative importance.
In the description of the present invention, it should be noted that unless otherwise clearly defined and limited, term " peace such as occur
Dress ", " connected ", " connection " shall be understood in a broad sense, for example, it may be being fixedly connected, may be a detachable connection, or integrally
Connection；It can be mechanical connection, be also possible to be electrically connected；Can be directly connected, can also indirectly connected through an intermediary,
It can be the connection inside two elements.For the ordinary skill in the art, above-mentioned art can be understood with concrete condition
The concrete meaning of language in the present invention.
Referring to attached drawing, the present embodiment provides a kind of human oocytes and embryo vitrifying freeze thawing device, described device packets
Horizontally disposed chassis 27 is included, first motor 21 is fixed on the chassis 27, the output shaft 211 of the first motor 21 is perpendicular
Directly upwardly extends and be connected with vibration section 22；The vibration section 22 includes cyclotron oscillation pallet 25, the cyclotron oscillation pallet 25
Lower surface and the output shaft 211 of the first motor 21 be fixedly linked, to drive the cyclotron oscillation pallet 25 to rotate, and institute
First motor 21 is stated to be connected with the first power supply 22 and control the first switch 24 of 21 start and stop of first motor；
Specifically, the output shaft of the first motor is central axial parallel with vertical direction, the first motor 21
Rotation can drive the cyclotron oscillation pallet 25 to rotate synchronously.
The limiting groove 26 for accommodating accommodating plate 3, the limit are offered on the upper surface of the cyclotron oscillation pallet 25
The central axis of position groove 26 is conllinear with the central axis of output shaft 211 of the first motor 21；The accommodating plate 3 is eccentrically set on
In the limiting groove 26, the centre of the accommodating plate 3 offers the open slot 32 for holding liquid, and the open slot 32
Notch be open upwards；
Specifically, the accommodating plate 3 is transparent.
Specifically, can promote described when the cyclotron oscillation pallet 25 rotates under the drive of the first motor 21
Plate 3 is accommodated to swing.And the accommodating plate 3 is eccentrically set in the limiting groove 26, i.e., the central axis of the described accommodating plate 3 and institute
The central axis for stating cyclotron oscillation pallet 25 deviates, for example, the central axis of the cyclotron oscillation pallet 25 deviates in the horizontal direction
The central axis 2mm of the cyclotron oscillation pallet 25, so that rotating diameter amplitude of the accommodating plate 3 on convolution concussion pallet 25 is
4mm, so that the accommodating plate 3, which has, preferably rocks effect, so that the liquid in the open slot 32 be promoted uniformly to mix.
The side on the chassis 27 is equipped with the bracket 8 extended straight up, is fixed on the bracket 8 horizontal-extending
Extend folder 81；And the fixing end for extending folder 81 is fixed on the bracket 8, the free end for extending folder 81 is to the bottom
The upper horizontal of disk 27 extends；
Specifically, the bracket 8 is in elongated rod shape, the fixing end fixation for extending folder 81 is set in the bracket 8
Between on position.
The through-hole penetrated through along the vertical direction is offered on the free end for extending folder 81, outlet tube 1 slidably extends through
The through-hole is simultaneously fixed on the extension folder 81 by clamp assemblies；The bottom end of the outlet tube 1 offers liquid outlet 13, institute
The top that liquid outlet 13 is located at the opening of the open slot 32 is stated, to inject the liquid into the open slot 32；And it is described
Outlet tube 1 is located at the tube body for extending 81 lower section of folder and is equipped with scale；
Specifically, the central axis of the liquid outlet 13 is conllinear with the central axis of the limiting groove 26.
Specifically, the capacity of the outlet tube 1 is 1ml, wherein the scale 11 includes the label graduation mark of volume and right
The scale registration answered.The scale registration is gradually increased upwards from the liquid outlet 13 of liquid outlet 13, and the appearance of adjacent two graduation mark
Product moment value is 0.1ml.
In the present embodiment, the scale 11 that outlet tube 1 is equipped with facilitates experimenter to start preceding standard in freezing or operation of thawing
Quantitative liquid (freezing liquid or equilibration buffer) is really drawn, avoids liquid while guaranteeing experiment reaction sufficiently completely
Waste.
In the present embodiment, the liquid outlet 13 of outlet tube 1 is 2~3cm at a distance from open slot 32, is closer, can have
Effect avoids the splashing after dripping under liquid and influences freeze thawing effect.
Being connected in the top of the outlet tube 1 can move up and down along the outlet tube 1 with described for aspirating or injecting
The driving portion of liquid, the driving portion include that can slide up and down to the piston being arranged in the outlet tube 1, institute along the vertical direction
The top for stating piston is connected by connecting rod 12 with piston rod；The piston rod can axially slidably setting along the outlet tube 1
It sets in the outlet tube 1, the piston rod runs through the top end opening of the outlet tube 1, and the piston rod is towards the branch
The side 122 of frame is equipped with the rack gear extended along the vertical direction, and the rack and pinion 5 is meshed；
The gear 5 is arranged on bracket 8 by support plate 7, the fixed output for being set in the second motor 6 of the gear 5
On axis 61, second motor 6 is fixed in the support plate 7, and the support plate 7 is fixed on the bracket 8, and the branch
Fagging 7 is located at the top for extending folder 81；
Specifically, the output shaft 61 and the piston rod of second motor 6 are perpendicular.Second motor 6 drives institute
The rotation of gear 5 is stated, the gear 5 drives the rack gear being meshed in the course of rotation, so that moving down on the piston rod
It is dynamic, to aspirate or inject the liquid.
Second motor 6 is connected with second source 62, the second switch 65 of control 6 start and stop of the second motor and control
The speed control 63 of the revolving speed of second motor 6.
Specifically, the speed control 63 controls the revolving speed of second motor 6, the liquid discharge can control
The flow of liquid or feed liquor.The speed control 63 can limit the revolving speed of second motor 6, so that the gear 5 at the uniform velocity turns
It is dynamic, to drive the piston rod at the uniform velocity to move, and the liquid speed degree that goes out of liquid is 100ul/min.
Further, the first motor 21 is fixed on the chassis 27 by motor support plate 23.
Specifically, 21 slow rotation of first motor, and the revolving speed of the first motor is 100rpm.
Further, described device further includes timer 64, and the timer 64 is fixed in the support plate 7.
Further, the area of the opening of the open slot 32 is greater than the area of slot bottom.
Specifically, the open slot 32 is in up big and down small round table-like, wherein the diameter of the opening of the open slot 32 is
3cm, the groover rot diameter of the open slot 32 are 2cm, and the groove depth of the open slot 32 is 1cm.
In embodiment, the diameter of the opening of open slot 32 is 3cm, and the liquid that ensure that outlet tube instils fully enters out
In mouth slot 32；The open slot 32 eliminates the visual field interference at cylindrical grooves bottom right angle in up big and down small round table-like
Area, then enable the suction of observation and glass capillary of the open slot 32 while accepting more volume of liquid more conducively under microscope
Extract operation.
Further, the clamp assemblies are specially spring clip 811, and the outlet tube 1 is by being located at extension folder 81
Above and below spring clip 811 be fixed on extension folder 81.
Specifically, the spring clip 811 is clamped in outside the outlet tube 1, and the spring clip 811 be coarser than it is described logical
Hole is slided to limit the outlet tube 1 along the outlet tube 1.The spring clip 811 is fixed on the outlet tube 1 not
On coaxial position, the distance between liquid outlet 13 and accommodating plate 3 of the adjustable outlet tube 1.
Further, the outer wall of the piston rod and the inner wall of the outlet tube 1 fit.
Specifically, inner wall of the outside wall surface of the piston and the piston rod with the outlet tube fits, improve
Outlet tube injects the stability of liquid.Wherein, the piston rod is equipped with along the vertical direction towards the side 122 of the bracket 8
The rack gear of extension, the other side 121 of the piston rod are smooth surface.
Further, 25 the center point of cyclotron oscillation pallet is labeled with salient point 251.
Specifically, the salient point 251 can be used for calibrating the central axis and the cyclotron oscillation pallet 25 of the liquid outlet 13
The center of circle align, be convenient for naked-eye observation and operation.
Further, the center point of 32 slot bottom of open slot is equipped with mark point 31.The mark point 31 is referring to described
One salient point 251 is convenient for naked-eye observation and behaviour with initial placement position of the determination accommodating plate 3 on the limiting groove 26
Make.7, the liquid is glass freezing liquid or thawing solution, wherein the glass freezing liquid is equilibration buffer, single concentration
Permeability frozen solution or single concentration impermeability frozen solution.
Wherein: the ingredient of the equilibration buffer is people's Culture in oviduct liquid that volume fraction is 95% and volume fraction is
The mixed liquor of 5% human serum substitute.
The permeability frozen solution of the single concentration is people's Culture in oviduct liquid that volume fraction is 55%, volume point
Count the mixed of the human serum substitute for 5%, the dimethyl sulfoxide that volume fraction is 20% and the ethylene glycol that volume fraction is 20%
Close object.
The impermeability frozen solution of the single concentration is the sucrose that volume fraction is 95% and volume fraction is 5%
Human serum substitute mixture.
The thawing solution be volume fraction be 65% people's Culture in oviduct liquid, volume fraction be 30% sucrose and volume
The mixture for the human serum substitute that score is 5%.
Specifically, people's Culture in oviduct liquid is selected from human tubal fluid HTF1024 (SAGE, USA), human tubal fluid
HTF1023 (SAGE, USA)；The human serum substitute is selected from human serum albumin HSA (Vitrolife, Sweden)；It is described
Dimethyl sulfoxide, ethylene glycol are permeability cryoprotector, are respectively selected from dimethyl sulfoxide DMSO (SIGMA, USA) and second
Glycol EG (SIGMA, USA)；The sucrose is impermeability cryoprotector, is selected from sucrose Sucrose (SIGMA, USA).
In the present embodiment, it is to consider that the basic bottom liquid of freezing liquid and thawing solution, which selects HTF (people's Culture in oviduct liquid) then,
HEPES the or MOPS buffer system of the prior art of its ratio is closer to the intracorporal natural physiological environment of people.
The present embodiment also provides the application method of a kind of human oocytes and embryo vitrifying freeze thawing device, including following
Step:
Step 1: outlet tube by driving portion extract 700ul the single concentration permeability frozen solution, and to
The equilibration buffer that 300ul is added in the open slot 32 of plate 3 is accommodated, the permeability frozen solution of the single concentration is made
Balance 30min is stood at 25 DEG C with the equilibration buffer；
Step 2: adjusting 1 position of outlet tube, make that it keeps vertical with chassis 27 and liquid outlet 13 is located at cyclotron oscillation pallet
At the 2~3cm of surface in 25 centers of circle, then accommodating plate 3 is placed in the limiting groove 26 of cyclotron oscillation pallet 25；
Step 3: starting to carry out glass freezing, the egg mother cell to be freezed is added into the open slot 32 of accommodating plate 3
Or embryo, the first switch 24 of first motor 21 is opened, the cyclotron oscillation pallet 25 is rotated, and revolving speed is 100rpm, from
And drive accommodating 3 circular motion of plate；
After step 4:1min, the second switch 65 of the second motor 6 is opened, so that second motor 6 is with 5 turns of moving gear
Dynamic, gear 5 is meshed with rack gear, and piston rod pushes down on piston, by the permeability frozen solution of the single concentration with
The flow velocity of 100ul/min is released from liquid outlet 13 and is injected in open slot 32, while opening timing device 64, timing 6min；
Step 5: when timer 64 issues the prompt sound, closing out second switch 65, and with suction pipe to the open slot
The impermeability frozen solution of the single concentration of 400ul is added in 32；
After step 6:1min, close first switch 24, with glass capillary by open slot 32 egg mother cell or embryo
It is transferred to glass freezing to carry on bar, and is stored in the liquid nitrogen of -196 DEG C of investment rapidly；
Step 7: extracting the equilibration buffer of 1ml with the piston rod in outlet tube 1, and be added into open slot 32
The thawing solution of 300ul makes the equilibration buffer stand balance 30min under room temperature environment, and will fill the defrosting
The accommodating plate 3 of liquid, which is placed in 37 DEG C of incubator, stands balance 30min；
Step 8: adjusting the position of outlet tube 1, keep its holding perpendicular with chassis 27, and liquid outlet 13 is located at cyclotron oscillation
At the 2~3cm of surface in 25 center of circle of pallet；
Step 9: starting to carry out vitrifying defrosting, accommodating plate 3 is taken out out of incubator and is placed on station, then rapidly
The egg mother cell to be thawed is taken out from -196 DEG C of liquid nitrogen or embryo is placed into the open slot 32 for filling the thawing solution
It is interior, pass through micro- sem observation and confirm that egg mother cell or embryo carry from freezing and unloads and entered in open slot 32 on bar, then
Accommodating plate 3 is placed in the limiting groove 26 of the cyclotron oscillation pallet 25, and opens first switch 24, makes cyclotron oscillation pallet
25 are rotated, and rotation speed is 100rpm；
After step 10:2min, the second switch 66 of the second motor 6 is opened, by the equilibration buffer with 100ul/min's
Flow velocity instills in the open slot 32, while opening timing device, timing 9min；
Step 11: when timer issues the prompt sound, closing second switch 66 and first switch 24, use glass capillary
Egg mother cell or embryo are transferred in gamete culture solution or spilting of an egg culture solution out of open slot 32, and are placed in 37 DEG C, CO2It is dense
In the constant temperature and humidity incubator that degree is 6%, in case subsequent operation.
Pass through the comparison of Fig. 3 and Fig. 2, it can be seen that freezed compared with existing vitrifying using device of the present invention
It freezes (Fig. 2), the increasing gradual in continuity with the passage in reaction time of the concentration of permeability frozen solution DMSO and EG
High (Fig. 3)；And pass through the comparison of Fig. 5 and Fig. 4, it can be seen that thaw compared with existing glass using device of the present invention
(Fig. 4) is frozen in neutralizing, the concentration of the impermeability frozen solution sucrose drop gradual in continuity with the passage in reaction time
Low (Fig. 5), entire vitro reactions environment are softer.
The embodiment of the present application at least has the following technical effect that
1, device of the present invention passes through the collective effect of outlet tube and cyclotron oscillation disk, so that egg mother cell/embryo exists
The external environment for carrying out glass freezing and a freezing liquid or thawing solution concentration continuity gradual change are in during thawing, and
The operation that experimenter aspirates repeatedly to embryo, blows and beats, shifts, stirs etc. is eliminated, vitro and artificial is effectively reduced
Operate to embryo stress sexual stimulus influence, be beneficial at a specified future date development and the filial generation safety of embryo.Experiment people is also mitigated simultaneously
The working strength of member, other work in the off time expansion laboratory during enabling personnel to efficiently use freeze-thaw, mentions
High working efficiency.
2, the accommodating plate is eccentrically set in the limiting groove, i.e., the central axis of the described accommodating plate and the convolution are shaken
The central axis for swinging pallet deviates, so that the accommodating plate, which has, preferably rocks effect, to promote in the open slot
Liquid uniformly mixes.
3, the scale that outlet tube is equipped with facilitates experimenter accurately to draw quantitative liquid before freezing or operation of thawing start
Body (freezing liquid or equilibration buffer) avoids the waste of liquid while guaranteeing experiment reaction sufficiently completely.
4, the liquid outlet of outlet tube is 2~3cm at a distance from open slot, is closer, and be can effectively avoid after dripping under liquid
Splashing and influence freeze thawing effect.
5, the speed control can limit the revolving speed of second motor, so that the gear uniform rotation, thus band
The piston rod is moved at the uniform velocity to move.
6, the diameter of the opening of open slot is 3cm, and the liquid that ensure that outlet tube instils fully enters in open slot；It is described
Open slot eliminates the visual field interference range at cylindrical grooves bottom right angle, then makes open slot in up big and down small round table-like
The suction operation of observation and glass capillary while more volume of liquid can be accepted more conducively under microscope.
7, the spring clip is fixed on the axially different position of the outlet tube, the adjustable outlet tube goes out
The distance between liquid mouth and accommodating plate.
8, inner wall of the outside wall surface of piston and the piston rod with the outlet tube fits, and improves outlet tube and pushes away
The stability of fluid injection body.
9, the center of circle that salient point can be used for calibrating the central axis and the cyclotron oscillation pallet of the liquid outlet aligns, and is convenient for
Naked-eye observation and operation.
10, the center point of the open slot slot bottom is equipped with mark point, and the mark point can be true referring to first salient point
Initial placement position of the fixed accommodating plate on the limiting groove, is convenient for naked-eye observation and operation.
11, the basic bottom liquid of freezing liquid and thawing solution select HTF (people's Culture in oviduct liquid) then be consider its ratio it is existing
HEPES the or MOPS buffer system of technology is closer to the intracorporal natural physiological environment of people.
Content described in this specification embodiment is only enumerating to the way of realization of inventive concept, protection of the invention
Range should not be construed as being limited to the specific forms stated in the embodiments, and protection scope of the present invention also includes art technology
Personnel conceive according to the present invention it is conceivable that equivalent technologies mean.
Claims (9)
1. a kind of human oocytes and embryo vitrifying freeze thawing device, it is characterised in that: described device includes horizontally disposed
Chassis is fixed with first motor on the chassis, and the output shaft of the first motor extends straight up and is connected with vibration
Portion；The vibration section includes cyclotron oscillation pallet, the lower surface of the cyclotron oscillation pallet and the output shaft of the first motor
It is fixedly linked, to drive the cyclotron oscillation pallet to rotate, and the first motor is connected with the first power supply and controls described the
The first switch of one motor start and stop；
The limiting groove for accommodating accommodating plate is offered on the upper surface of the cyclotron oscillation pallet, in the limiting groove
Mandrel is conllinear with the central axis of the output shaft of the first motor；The accommodating plate is eccentrically set in the limiting groove, institute
The centre for stating accommodating plate offers open slot for holding liquid, and the notch of the open slot is open upwards；
The side on the chassis is equipped with the bracket extended straight up, and horizontal-extending extension folder is fixed on the bracket；
And the fixing end for extending folder is fixed on the bracket, the free end for extending folder is prolonged to the upper horizontal on the chassis
It stretches；
The through-hole penetrated through along the vertical direction is offered on the free end for extending folder, outlet tube slidably extends through the through-hole
And it is fixed on the extension folder by clamp assemblies；The bottom end of the outlet tube offers liquid outlet, and the liquid outlet is located at
The top of the opening of the open slot, to inject the liquid into the open slot；And the outlet tube is located at the extension
The tube body of folder lower section is equipped with scale；
Being connected in the top of the outlet tube can move up and down along the outlet tube for aspirating or injecting the liquid
Driving portion, the driving portion include that can slide up and down to the piston being arranged in the outlet tube, the piston along the vertical direction
Top be connected with piston rod by connecting rod；The piston rod can along the outlet tube be axially slidably disposed in it is described go out
In liquid pipe, the piston rod runs through the top end opening of the outlet tube, and the piston rod is equipped with towards the side of the bracket
The rack gear extended along the vertical direction, and the rack and pinion is meshed；
The gear is arranged on bracket by support plate, and the gear fixation is set on the output shaft of the second motor, described
Second motor is fixed in the support plate, and the support plate is fixed on the bracket, and the support plate is located at described prolong
Stretch the top of folder；
Second motor is connected with second source, the second switch of control the second motor start and stop and control second electricity
The speed control of the revolving speed of machine.
2. a kind of human oocytes as described in claim 1 and embryo vitrifying freeze thawing device, it is characterised in that: described the
One motor is fixed on the chassis by motor support plate.
3. a kind of human oocytes as described in claim 1 and embryo vitrifying freeze thawing device, it is characterised in that: described in institute
Device further includes timer, and the timer is fixed in the support plate.
4. a kind of human oocytes as described in claim 1 and embryo vitrifying freeze thawing device, it is characterised in that: described to open
The area of the opening of mouth slot is greater than the area of slot bottom.
5. a kind of human oocytes as described in claim 1 and embryo vitrifying freeze thawing device, it is characterised in that: the folder
Holding component is specially spring clip, and the outlet tube is fixed on described prolong by being located at the spring clip extended above and below folder
It stretches on folder.
6. a kind of human oocytes as described in claim 1 and embryo vitrifying freeze thawing device, it is characterised in that: the work
The outer wall of stopper rod and the inner wall of the outlet tube fit.
7. a kind of human oocytes as described in claim 1 and embryo vitrifying freeze thawing device, it is characterised in that: described time
Rotation oscillating tray the center point is labeled with red salient point.
8. a kind of human oocytes as described in claim 1 and embryo vitrifying freeze thawing device, it is characterised in that: the liquid
Body is glass freezing liquid or thawing solution, wherein the glass freezing liquid is that equilibration buffer, the permeability of single concentration are cold
Freeze the impermeability frozen solution of protection liquid or single concentration.
Wherein: the ingredient of the equilibration buffer is people's Culture in oviduct liquid that volume fraction is 95% and volume fraction is 5%
Human serum substitute mixed liquor.
The permeability frozen solution of the single concentration be volume fraction be 55% people's Culture in oviduct liquid, volume fraction be
The mixture for the ethylene glycol that the dimethyl sulfoxide and volume fraction that 5% human serum substitute, volume fraction are 20% are 20%.
The people that the impermeability frozen solution of the single concentration is the sucrose that volume fraction is 95% and volume fraction is 5%
The mixture of serum substitute.
The thawing solution be volume fraction be 65% people's Culture in oviduct liquid, volume fraction be 30% sucrose and volume fraction
For the mixture of 5% human serum substitute.
9. the user of a kind of human oocytes as claimed in any one of claims 1 to 8 and embryo vitrifying freeze thawing device
Method, which comprises the following steps:
Step 1: outlet tube 1 extracts the permeability frozen solution of the single concentration of 700ul by driving portion, and to accommodating
The equilibration buffer of 300ul is added in the open slot of plate, and makes permeability frozen solution and the institute of the single concentration
It states equilibration buffer and stands balance 30min at 25 DEG C；
Step 2: adjust outlet tube position, make its be located at the chassis vertical and liquid outlet of holding the cyclotron oscillation pallet center of circle just on
At 2~3cm of side, then accommodating plate is placed in the limiting groove of cyclotron oscillation pallet；
Step 3: start to carry out glass freezing, the egg mother cell to be freezed or embryo are added into the open slot of accommodating plate,
The first switch for opening first motor rotates the cyclotron oscillation pallet, and revolving speed is 100rpm, to drive accommodating plate circle
Zhou Yundong；
After step 4:1min, the second switch of the second motor is opened, so that second motor driven gear rotates, gear and tooth
Item is meshed, and piston rod pushes down on piston, by the permeability frozen solution of the single concentration with the flow velocity of 100ul/min
It releases and is injected in open slot from liquid outlet, while opening timing device, timing 6min；
Step 5: when timer issues the prompt sound, closing out second switch, and be added into the open slot with suction pipe
The impermeability frozen solution of the single concentration of 400ul；
After step 6:1min, close first switch, with glass capillary by open slot egg mother cell or embryo transfer to glass
Glassization freezing carries on bar, and is stored in the liquid nitrogen of -196 DEG C of investment rapidly；
Step 7: start carry out vitrifying defrosting before, in outlet tube piston rod extract 1ml the equilibration buffer, and to
Thawing solution described in 300ul is added in open slot, the equilibration buffer is made to stand balance 30min at 25 DEG C, and institute will be filled
The accommodating plate for stating thawing solution is placed in 37 DEG C of the interior standing balance 30min of incubator；
Step 8: adjusting the position of outlet tube, keep its holding perpendicular with chassis, and liquid outlet is located at the cyclotron oscillation pallet center of circle
2~3cm of surface at；
Step 9: starting to carry out vitrifying defrosting, accommodating plate is taken out out of incubator and is placed on station, then rapidly from -196
DEG C liquid nitrogen in take out the egg mother cell to be thawed or embryo is placed into the open slot for filling the thawing solution, by showing
Micro mirror is observed and is confirmed that egg mother cell or embryo carry from freezing and unloads and entered in open slot on bar, then accommodating plate is placed in
In the limiting groove of the cyclotron oscillation pallet, and first switch is opened, rotates cyclotron oscillation pallet, and rotation speed
For 100rpm；
After step 10:2min, the second switch of the second motor is opened, the equilibration buffer is dripped with the flow velocity of 100ul/min
Enter in the open slot, while opening timing device, timing 9min；
Step 11: when timer issues the prompt sound, second switch and first switch are closed, it is with glass capillary that ovum is female thin
Born of the same parents or embryo are transferred in gamete culture solution or spilting of an egg culture solution out of open slot, and are placed in 37 DEG C, CO2Concentration is 6%
In constant temperature and humidity incubator, in case subsequent operation.
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| CN201811365778.7A CN109497040B (en) | 2018-11-16 | 2018-11-16 | Human oocyte and embryo vitrification freezing and thawing device and application method thereof |
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| CN201811365778.7A CN109497040B (en) | 2018-11-16 | 2018-11-16 | Human oocyte and embryo vitrification freezing and thawing device and application method thereof |
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