CN110352949A - A kind of ovary tissue freezes protection and resuscitation fluid - Google Patents
A kind of ovary tissue freezes protection and resuscitation fluid Download PDFInfo
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- CN110352949A CN110352949A CN201810254362.1A CN201810254362A CN110352949A CN 110352949 A CN110352949 A CN 110352949A CN 201810254362 A CN201810254362 A CN 201810254362A CN 110352949 A CN110352949 A CN 110352949A
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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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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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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- 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
- C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
- C12N5/0682—Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle cells
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- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/32—Amino acids
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- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/32—Amino acids
- C12N2500/33—Amino acids other than alpha-amino carboxylic acids, e.g. beta-amino acids, taurine
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- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/34—Sugars
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/38—Vitamins
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/999—Small molecules not provided for elsewhere
Abstract
Protection and resuscitation fluid are frozen the invention discloses a kind of ovary tissue, wherein freezing protection liquid includes Leibovitz L-15 culture medium, 8-12V/V%DMSO and 0.5-2V/V%HSA and/or 8-10V/V%SSS.Resuscitation fluid includes: 80-90V/V%DPBS, 10-20V/V%FBS and/or 10-20V/V%HSA, 0.13mol/L-0.8mol/L sucrose.The present invention also provides ovary tissue cryopreservation methods and method for resuscitation.It is provided by the invention to freeze protection liquid and recovery protection liquid is used cooperatively, and using method of the invention can Cryopreservation tissue rapidly and efficiently, and can be improved the ovary tissue motility rate after recovery.
Description
Technical field
The invention belongs to ovary tissue freezen protective fields, specifically, be related to a kind of ovary tissue freeze protection with
Resuscitation fluid further further relates to the cryopreservation methods and method for resuscitation of ovary tissue.
Background technique
Chinese annual new cancer patient 4,300,000 or so, and cancer patient's five-year survival rate can be up to 80-90% with
On, 70% or more survivor has urgent fertility demand, but chemicotherapy but results in while saving cancer patient's life
The damage of 70%-100% ovarian function makes it lose fecundity, greatly influences the physical and mental health and happy family life of patient；
Due to the extremely early decline of ovarian function, lead to the various chronic diseases of patient such as cardiovascular disease, diabetes, osteoporosis, presenile
Dull-witted rate dead early obviously increases, and increases the huge medical burden of country.Therefore, ovarian function is protected before chemicotherapy
It is extremely important.
Have at present to the method for fecundity protection several: ovum freezes, embryo freezing and ovary tissue freeze.Embryo freezing
For the routine techniques of test-tube baby, ovum is frozen, and current China is limited to infertile IVF patient and takes essence failure or Ovarian hyper thorn
Swash the patient for abandoning the period, be not suitable for preadolescence girl and be badly in need of the cancer patient of chemicotherapy mostly, there is great limitation
Property.
And it is current fecundity protection side newest, most promising in the world that ovary tissue, which freezes with implantation technique again,
Method.It takes part ovary tissue is specially treated to freeze before chemicotherapy, transplants in ex vivo after its clinical recovery, not only may be used
To restore the fecundity of patient, the endocrine function of its ovary and the hot topic of anti-aging can also be restored.In America and Europe
Developed country, ovary tissue freezes has been used as conventional fecundity protection technique to apply for more than ten years with implantation technique again, and at me
State there is no life birth to report.
In order to guarantee the activity after ovary tissue is transplanted back again in owner's body, the reagent for freezing and recovering of ovary tissue
It is most important with method.Freezing for ovary tissue directly affects the activity after transplanting again with resuscitation fluid and method, and therefore, it is necessary to mention
It can be suitable for that ovary tissue freezes, recovers for one kind, improve ovary tissue and transplant active reagent and its method again.
In view of this present invention is specifically proposed.
Summary of the invention
The technical problem to be solved in the present invention is that overcome the deficiencies of the prior art and provide a kind of ovary tissue freezes guarantor
Shield and resuscitation fluid, including freeze protection liquid and resuscitation fluid, can Cryopreservation tissue rapidly and efficiently, and can be improved recovery
Ovary tissue motility rate afterwards.
In order to solve the above technical problems, the present invention is using the basic conception of technical solution:
What the first object of the present invention was to provide a kind of ovary tissue freezes protection and resuscitation fluid, including freezes protection liquid,
The protection liquid that freezes includes Leibovitz L-15 culture medium, 8-10V/V%DMSO and 0.5-1.0V/V%HSA or 8-
10V/V%SSS.
The protection liquid that freezes of ovary tissue provided by the invention can be good at freezen protective fecundity.It freezes in protection liquid
Ingredient is simply clear, permeability protection liquid dimethyl sulfoxide (DMSO) and basal medium in addition to being not possible to substitution at present
LeibovitzL-15 culture medium also contains SSS or HAS.Wherein, the addition concentration of HAS is very low, can either be as infiltration
Property protection liquid, and side reaction can be reduced.And SSS, that is, haemocyanin substitute content is higher, can either play stable protein
Effect, and can be avoided the pollution problem of haemocyanin.
Further embodiment, the protection liquid that freezes includes Leibovitz L-15 culture medium, 10V/V%DMSO, 1V/
V%HSA,
Or freezing protection liquid includes Leibovitz L-15 culture medium, 10V/V%DMSO, 10V/V%SSS.
Further embodiment, the protection liquid that freezes further includes taurine, and the content of the taurine is 0.5-
5mM；
Preferably, the content of taurine is 0.5-1mM；It is furthermore preferred that the content of taurine is 0.5mM.
Taurine, which is added to, to be frozen in protection liquid, is able to maintain the stability of membrane structure, is adjusted osmotic pressure, adjusts into the cell
Calcium ion level is conducive to the activity for keeping ovary tissue under ultralow temperature, is conducive to the motility rate for improving the ovary tissue after recovery.
Further embodiment, the protection liquid that freezes further includes nitrogen acetylcysteine, the nitrogen mucolyticum
The content of acid is 0.5-5mM；
Preferably, the content of nitrogen acetylcysteine is 3-5mM；It is furthermore preferred that the content of nitrogen acetylcysteine is
5mM。
Nitrogen acetylcysteine, that is, NAC can be used as a kind of antioxidant, first is that can remove in free environment
Hydroxy radical, hydrogen peroxide and hypochlorous acid can improve the microenvironment of cell.Meanwhile nitrogen acetylcysteine can also quilt
It is changed into cysteine, phase structure in stabilizing cell membrane and film.When in use, nitrogen acetylcysteine can be cooperateed with taurine
Effect, not only improves microenvironment, additionally it is possible to increase the tolerance reduced to temperature, protect cell, the cell after improving recovery is living
Rate.
Further embodiment, the protection liquid that freezes further includes vitamin E, and the content of the vitamin E is 0.1-
10mM；
Preferably, the content of vitamin E is 3-7mM；It is furthermore preferred that the content of vitamin E is 5mM.
Vitamin E is used alone or is used cooperatively respectively with nitrogen acetylcysteine and taurine, can be improved cell
Motility rate promotes growth, and then it can be improved and produces effect, increases culture medium stability, utmostly reduces the poly- of toxic ammonia
Collection reduces cytotoxicity and grows to cell advantageous.
Further embodiment, the protection liquid that freezes further includes vitamin C, and the ascorbic content is 0.5-
2.0mM；
Preferably, ascorbic content is 2.0M.
A kind of ovary tissue of the invention freezes protection and resuscitation fluid, further includes resuscitation fluid, and the resuscitation fluid includes:
80-90V/V%DPBS, 10-20V/V%HAS and 0.13mol/L-0.8mol/L sucrose.
Further embodiment, the resuscitation fluid further include taurine, and the content of the taurine is 0.5-5mM；
Preferably, the content of taurine is 0.5-1mM；It is furthermore preferred that the content of taurine is 0.5mM.
Further embodiment, the described protection liquid that freezes further includes nitrogen acetylcysteine, at least one in vitamin E
Kind；The content of the nitrogen acetylcysteine is 0.5-5mM；The content of vitamin E is 0.1-10mM；
Preferably, the content of nitrogen acetylcysteine is 3-5mM, and the content of vitamin E is 3-7mM；
It is furthermore preferred that the content of nitrogen acetylcysteine is 5mM, the content of vitamin E is 5mM.
Further embodiment, the resuscitation fluid further include vitamin C, and the ascorbic content is 0.5-
2.0mM；
Preferably, ascorbic content is 2.0M.
Further embodiment, the resuscitation fluid include:
(1) resuscitation fluid 1:90V/V%DPBS, 10V/V%HAS, 0.5-0.8mol/L sucrose；
(2) resuscitation fluid 2:90V/V%DPBS, 10V/V%HAS, 0.3-0.5mol/L sucrose；
(3) resuscitation fluid 3:90V/V%DPBS, 10V/V%HAS, 0.12-0.3mol/L sucrose；
Preferably, the resuscitation fluid includes:
(1) resuscitation fluid 1:90V/V%DPBS, 10V/V%HAS, 0.75mol/L sucrose；
(2) resuscitation fluid 2:90V/V%DPBS, 10V/V%HAS, 0.375mol/L sucrose；
(3) resuscitation fluid 3:90V/V%DPBS, 10V/V%HAS, 0.125mol/L sucrose.
Further embodiment, the resuscitation fluid include:
(1) resuscitation fluid 1:90V/V%DPBS, 10V/V%HAS, 5mM nitrogen acetylcysteine and/or 5mM taurine and/
Or 5mM vitamin E, 0.5-0.8mol/L sucrose；
(2) resuscitation fluid 2:90V/V%DPBS, 10V/V%HAS, 5mM nitrogen acetylcysteine and/or 5mM taurine and/
Or 5mM vitamin E, 0.3-0.5mol/L sucrose；
(3) resuscitation fluid 3:90V/V%DPBS, 10V/V%HAS, 5mM nitrogen acetylcysteine and/or 5mM taurine and/
Or 5mM vitamin E, 0.13-0.3mol/L sucrose；
Preferably, the resuscitation fluid includes:
(1) resuscitation fluid 1:90V/V%DPBS, 10V/V%HAS, 5mM nitrogen acetylcysteine, 0.75mol/L sucrose；
(2) resuscitation fluid 2:90V/V%DPBS, 10V/V%HAS, 5mM nitrogen acetylcysteine, 0.375mol/L sucrose；
(3) resuscitation fluid 3:90V/V%DPBS, 10V/V%HAS, 5mM nitrogen acetylcysteine, 0.125mol/L sucrose.
It preferably, further include vitamin C in resuscitation fluid 3, ascorbic content is 0.5mM.
The second object of the present invention be to provide a kind of ovary tissue freeze and method for resuscitation, wherein including cryopreservation methods,
The cryopreservation methods the following steps are included:
(1) it pre-processes: ovary tissue being placed in transfer liquid, medullary substance is removed, retain cortex and be cut to tissue；
(2) osmotic equilibrium: tissue is transferred to and is filled in the sample box for freezing protection liquid, is placed in ice and balances；
(3) freeze: be added in cryopreservation tube it is a certain amount of freeze protection liquid, tissue block is put into cryopreservation tube, is then gradually dropped
Temperature carries out liquid nitrogen cryopreservation.
Further embodiment, in cryopreservation methods, the process that the gradually cooling carries out liquid nitrogen cryopreservation includes:
(1) 2 DEG C of balance, maintains 15min；(2) -2 DEG C are down to the speed of 6 DEG C/min；(3) it is dropped with the speed of 4 DEG C/min
To -4 DEG C；(4) -7.5 DEG C are down to the speed of 2 DEG C/min；(5) -8 DEG C are down to the speed of 0.5 DEG C/min；(6) with 0.5 DEG C/
The speed of min is down to -11.5 DEG C；(7) -13 DEG C are down to the speed of 0.1 DEG C/min；(8) be down to the speed of 0.3 DEG C/min-
42℃；(9) -100 DEG C are down to the speed of 50 DEG C/min.
The gradually cooling used in the cryopreservation methods of ovary tissue of the invention carries out the process of liquid nitrogen cryopreservation, is divided into nine
Grade, each stage are cooled down with specific rate.It is frozen, in resuscitation process in cooling, the membrane structure of gonad cell can be because of substance
Phase transformation reason is damaged by different degrees of solute, ice crystal.The cooling frozen storage process multistage of the application changes, in different coolings
Stage using suitable cooling rate, can slowly gradually form very tiny ice crystal, while guaranteeing in cryopreservation tube everywhere
The uniformity of crystallization can play the effect of extraordinary protection membrane structure.It is specific:
In step (1), cryopreservation tube is first in 2 DEG C of maintenance 15min, to terminate equilibration time, solution and tissue in cryopreservation tube
Block reaches relatively stable state.Next it cools in the temperature course for planting freezing point, the application uses gradient and gradually cools down
Mode.- 2 DEG C are down to the speed of 6 DEG C/min first, during being down to -2 DEG C for 2 DEG C, due to freezing the ingredient of protection liquid
And the reason of concentration, crystallization condition is not achieved, can be cooled down with biggish fast speed, to ovary tissue substantially without shadow
It rings.Next, being down to -4 DEG C again with the speed of 4 DEG C/min；- 7.5 DEG C are down to the speed of 2 DEG C/min；Cooling rate is increasingly
Slowly, basic constant gradient cools down at a slow speed, avoids generating nucleus and local rapid crystallization inside cryopreservation tube, makes to still maintain in cryopreservation tube
Solution state waits homogeneously crystallized.And after temperature reaches -7.5 DEG C, during being down to -8 DEG C by -7.5 DEG C, with 0.5 DEG C/min's
Speed is down to -8 DEG C, and rate of temperature fall is minimum, to guarantee accurately to reach the temperature for planting freezing point, guarantees seeded crystallization
(seeding) accurate temperature when.At -8 DEG C, the solution in cryopreservation tube reaches the critical point of crystallization, can reach after seeding
Homogeneously crystallized effect.Continue to cool down after crystallization, during -8 DEG C to -11,5 DEG C, can be made with the speed cooling of 0.5 DEG C/min
Nucleus is evenly distributed everywhere in cryopreservation tube, avoids the formation of local non-uniform phenomenon, avoids the damage of ovary tissue.And-
During 11.5 DEG C Dao -13 DEG C, rate of temperature fall is further decreased, can be formed as much as possible after ice crystal inoculation uniform, tiny
Ice crystal, reduce damage to ovary tissue as far as possible, while the quick dissolution in resuscitation process can be conducive to, be conducive to improve
Activity after ovary tissue recovery.
In addition, the application's freezes in protection liquid in addition to Leibovitz L-15 culture medium, 10V/V%DMSO, 1V/V%
HAS or 10V/V%SSS also contains taurine, nitrogen acetylcysteine propylhomoserin, vitamin E and vitamin C, can play guarantor
The effect of membrane structure is protected, the protection liquid that freezes of formation is also more suitable for reaching critical-temperature at -8 DEG C, at such a temperature
Critical state can be reached, when seeding is conducive to quickly form crystallization.
It so, it is possible Cryopreservation tissue rapidly and efficiently, reduce the damage to ovary tissue；Cooperate corresponding freeze simultaneously
Deposit protection liquid and recovery protection liquid, the activity of the ovary tissue after recovery can be further increased.
Further, ovary tissue is derived from sluggish ovary, and the region of corpus luteum is had without being taken from.Because that
The low and weak tissue of the ovarian follicle density of partial cortical, which is easy to rupture, is unfavorable for follow-up work.
Further embodiment, during gradually cooling carries out liquid nitrogen cryopreservation, temperature reaches plant freezing point when being down to -8 DEG C, this
When dip liquid nitrogen and be placed at the liquid level for freezing tube wall, crystallization starting.
Further embodiment, in cryopreservation methods, the cortex of ovary tissue cut into 4 ± 0.5mm × 8 ± 0.6mm × 1 ±
0.2mm。
Further embodiment, in cryopreservation methods, the protection liquid that freezes includes Leibovitz L-15 culture medium, 8-
10V/V%DMSO and 0.5-1.0V/V%HSA or 8-10V/V%SSS.
Further embodiment, the protection liquid that freezes further includes in taurine, nitrogen acetylcysteine and vitamin E
It is at least one, wherein the content of taurine is 0.5-5mM, and the content of nitrogen acetylcysteine is 0.5-5mM, and vitamin E contains
Amount is 0.1-10mM；
Preferably, the content of taurine is 0.5mM, and the content of nitrogen acetylcysteine is 5mM, and the content of vitamin E is
3-5mM。
A kind of ovary tissue provided by the invention freeze and method for resuscitation, further include method for resuscitation, the recovery side
Method the following steps are included:
(1) cryopreservation tube equipped with ovary tissue is removed from liquid nitrogen, is dissolved；
(2) ovary tissue is taken out from cryopreservation tube, sequentially adds in the solution of resuscitation fluid 1, resuscitation fluid 2, resuscitation fluid 3 and divides
Not Bao Chi certain time, place into eluent and elute, complete recovery.
Further embodiment, in method for resuscitation, resuscitation fluid 1, resuscitation fluid 2, resuscitation fluid 3 are respectively as follows:
(1) resuscitation fluid 1:90V/V%DPBS, 10V/V%HAS, 0.5-0.8mol/L sucrose；
(2) resuscitation fluid 2:90V/V%DPBS, 10V/V%HAS, 0.3-0.5mol/L sucrose；
(3) resuscitation fluid 3:90V/V%DPBS, 10V/V%HAS, 0.12-0.3mol/L sucrose；
Further embodiment further includes taurine, nitrogen acetylcysteine, dimension in resuscitation fluid 1, resuscitation fluid 2 and resuscitation fluid 3
At least one of raw element E；The content of the taurine is 0.5-5mM；The content of the nitrogen acetylcysteine is
0.5-5mM；The content of vitamin E is 0.1-10mM；
Preferably, the content of nitrogen acetylcysteine is 3-5mM, and the content of vitamin E is 3-7mM；
It is furthermore preferred that the content of nitrogen acetylcysteine is 5mM, the content of vitamin E is 5mM.
Further embodiment, the time that ovary tissue is kept in resuscitation fluid 1, resuscitation fluid 2 and resuscitation fluid 3 successively reduce；
Preferably, the time that ovary tissue is kept in resuscitation fluid 1, resuscitation fluid 2, resuscitation fluid 3 is respectively 15min.
Further embodiment, the eluent include 90V/V%DPBS, 10V/V%FBS；It is taken out from resuscitation fluid 3
Ovary tissue cleans twice in eluent, successively keeps 10min and 5min respectively in eluent；
Further embodiment, eluent further include at least one of taurine, nitrogen acetylcysteine, vitamin E；Institute
The content for the taurine stated is 0.5-5mM；The content of the nitrogen acetylcysteine is 0.5-5mM；The content of vitamin E is
0.1-10mM。
After adopting the above technical scheme, compared with the prior art, the invention has the following beneficial effects:
1, the work for freezing protection liquid and resuscitation fluid and ovary tissue capable of being made to freeze, recover of ovary tissue provided by the invention
Rate reaches 85% or more；It can be good at freezen protective fecundity.It is simply clear to freeze ingredient in protection liquid, in addition to there is no at present
Permeability protection liquid dimethyl sulfoxide (DMSO) of method substitution and basal medium Leibovitz L-15 culture medium, also contain
SSS or HSA.Wherein, the addition concentration of HSA is very low, can either protect liquid as permeability, and can reduce side reaction.And
SSS, that is, haemocyanin substitute content is higher, can either play the role of stable protein, and can be avoided the dirt of haemocyanin
Dye problem.
2, the protection liquid that freezes in the present invention further includes having in taurine, nitrogen acetylcysteine and vitamin E at least
One kind can be improved oxidation resistance, correspondingly, also adding identical ingredient in resuscitation fluid, freezes protection liquid and resuscitation fluid can
To play synergistic effect, the motility rate of gonad cell after cryopreservation resuscitation is further improved.
3, the gradually cooling used in the cryopreservation methods of ovary tissue of the invention carries out the process of liquid nitrogen cryopreservation, is divided into nine
Grade, each stage are cooled down with specific rate.Before temperature is down to -7.5 DEG C, cooled down with biggish rate of temperature fall,
Low temperature degree is not relatively high at this time, and the influence to ovary tissue is smaller.It is -7.5 DEG C when being down to -8 DEG C, slow with minimum rate of temperature fall
It reduces, can accurately reach the temperature for planting freezing point, guarantee accurate temperature when crystallization (seeding).After crystallization, then with more
Small rate of temperature fall is down to -42 DEG C, guarantees that the damage of ovary tissue minimizes.After -42 DEG C, the activity of ovary tissue is substantially steady
It is fixed, then extremely -100 DEG C of fast cooling, under the premise of guaranteeing that ovary tissue is active, greatly improves and freeze efficiency, avoid outer bound pair
The influence of ovary tissue.It so, it is possible Cryopreservation tissue rapidly and efficiently, reduce the damage to ovary tissue；Cooperate simultaneously
Protection liquid and recovery protection liquid, the activity of the ovary tissue after recovery can be further increased are frozen accordingly.
4, in method for resuscitation of the present invention, using 3 kinds of resuscitation fluids, 2 elutions, gradient distribution reduces the concentration of carbohydrate,
Resuscitation effect more optimizes.
A specific embodiment of the invention is described in further detail with reference to the accompanying drawing.
Detailed description of the invention
Attached drawing is as a part of the invention, and for providing further understanding of the invention, of the invention is schematic
Examples and descriptions thereof are used to explain the present invention, but does not constitute an undue limitation on the present invention.Obviously, the accompanying drawings in the following description
Only some embodiments to those skilled in the art without creative efforts, can be with
Other accompanying drawings can also be obtained according to these attached drawings.In the accompanying drawings:
Fig. 1 is the cytological map of the microscopically observation of ovary tissue Activity determination in test example 1 of the present invention；
Wherein, figure A1, B1, C1, D1 and E1 is the observation result after Calcein-AM dyeing under fluorescence microscope；
A2, B2, C2, D2 and E2 are in the observation of common light microscopic under the same visual field as a result, A1 and A2 is control group, and B1 and B2 are test
Group 1, C1 and C2 is test group 2, and D1 and D2 are test group 3, and E1 and E2 are test group 4；
Fig. 2 is the cytological map of the microscopically observation of 2 mesostroma Apoptosis assay of test example of the present invention；
Wherein, it is the stroma cell of apoptosis in left figure a1-e1, is the stroma cell of non-apoptosis, right figure in middle figure a2-e2
A3-e3 is the superposition of left figure and middle figure.The above picture is the same visual field, amplification factor × 400；
Figure a1 and a2 is control group, and figure b1 and b2 is test group 1, and figure c1 and c2 is test group 2, and figure d1 and d2 is test group
3 (NAC 5mM), figure e1 and e2 are test group 4；
Fig. 3 is that the reactive oxygen species of ovary tissue in test example 3 of the present invention compare figure；
Fig. 4 is that the total antioxidant capacity level of ovary tissue in test example 4 of the present invention compares figure.
It should be noted that these attached drawings and verbal description are not intended to the design model limiting the invention in any way
It encloses, but illustrates idea of the invention by referring to specific embodiments for those skilled in the art.
Specific embodiment
In order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below in conjunction with the embodiment of the present invention
In attached drawing, the technical solution in embodiment is clearly and completely described, the following examples are intended to illustrate the invention, but
It is not intended to limit the scope of the invention.
Embodiment 1-6 ovary tissue freezes the preparation of protection liquid
By taking 100ml freezes the preparation of protection liquid as an example: respectively according to the amount of each ingredient in table 1, measuring Leibovitz
L-15 culture medium, DMSO, HAS, SSS are added in reagent bottle, and jog mixes, with 0.22um filter filtration sterilization, filter paper warp
It crosses in autoclaved reagent bottle, 4 DEG C save backup, or -20 DEG C freeze, and it is spare that when use moves to 4 DEG C of refrigerators.
Table 1 freezes the ingredient 1 of protection liquid
Embodiment 7-17 ovary tissue freezes the preparation of protection liquid
By taking 100ml freezes the preparation of protection liquid as an example: respectively according to the amount of each ingredient in table 2, measuring Leibovitz
L-15 culture medium, DMSO, HAS, SSS are added in reagent bottle, and jog mixes, and taurine, the nitrogen acetyl of corresponding amount is then added
Cysteine, vitamin E, vitamin C and heparin.The filtration sterilization of 0.22um filter, filter paper are used after solid is completely dissolved
By in autoclaved reagent bottle, 4 DEG C are saved backup, or -20 DEG C freeze, it is spare that when use, moves to 4 DEG C of refrigerators.
Table 2 freezes the ingredient 2 of protection liquid
The preparation of embodiment 18-20 ovary tissue resuscitation fluid
By taking 100ml freezes the preparation of protection liquid as an example: respectively according to the amount of each ingredient in table 2, measuring Leibovitz
L-15 culture medium, DMSO, HAS, SSS are added in reagent bottle, and jog mixes, or adds the taurine of corresponding amount, nitrogen second
Acyl cysteine, vitamin E, vitamin C.The filtration sterilization of 0.22um filter is used after solid is completely dissolved, filter paper is through excessively high
In the reagent bottle for pressing sterilizing, 4 DEG C are saved backup, or -20 DEG C freeze, and it is spare that when use moves to 4 DEG C of refrigerators.
The ingredient of 3 resuscitation fluid of table
The cryopreservation methods of 28 ovary tissue of embodiment
1, material prepares: tweezers, scalpel, knife handle, Punch, 1.8ml cryopreservation tube, cryopreservation tube lid, big culture dish, 110ml
Sample box, frozen stock solution shift liquid, slab, and foam box freezes pipe support, 1000 microlitres of sample loading guns, 100 microlitres of pipette tips, the culture of four holes
Plate, culture solution (AIM-V)；Wherein, transfer liquid can be custodiol；
In addition, frozen stock solution can be the frozen stock solution of any scheme in embodiment 1-26, it is preferred to use embodiment 1,2,4 with
And embodiment 7-13, the scheme of embodiment 17, it is preferred that using the scheme for being embodiment 7 or embodiment 8 or embodiment 9.
2, preparation
(1) prepare cryopreservation tube；Tweezers, knife handle, needle holder disinfection；Punch, 1.8ml cryopreservation tube, cryopreservation tube lid are big to cultivate
Ware, 110ml sample box, 1000 microlitres of sample loading guns, 100 microlitres of pipette tips etc. are put into superclean bench, disinfection by ultraviolet light.
(2) operating slab, frozen stock solution, by -20 degree to move to+4 DEG C of refrigerators spare, shifts 2 parts of liquid.
(3)CO2Case: 4 well culture plates add 600ul AIM-V culture solution (serum-free cell culture medium)；DPBS (can be reserved for 28
It).
3, ovary tissue is handled
1) the box-packed ice of foam by frozen stock solution, freezes pipe support, in transfer liquid insertion ice, stand-by on one side.
2) superclean bench is put into after 4 DEG C of slab disinfections.
3) it by 2 culture dishes openings, is placed on slab, a transfer liquid of right side culture dish addition, in two culture ware lids
Respectively plus moiety shifts liquid.
4) tweezers, scalpel, Punch are ready to, are placed in the culture dish lid of upper right side.
5) tissue samples box is taken out, tissue is poured into the culture dish of left side together with transfer liquid.
6) sterile gloves are replaced.
7) preliminary treatment tissue removes medullary substance, pays attention to protecting the complete and wet of cortex.The good cortex of preliminary treatment is moved
Into right side culture dish.
8) by the cortex tissue as much as possible for being cut to 4*8*1mm.
9) 110ml sample box is taken, about 7ml frozen stock solution is added, covers cassette bottom.The tissue cut is put into sample box,
It is inserted into ice, sufficiently balances.
10) 1.7ml frozen stock solution is added in each cryopreservation tube, is placed in and freezes pipe support, is inserted into cooling box and is pre-chilled.
12) shift to an earlier date 5 minutes transfer tissues: tissue being quickly put into corresponding cryopreservation tube.Cryopreservation tube is inserted directly into foam box
In ice, it is ready for liquid nitrogen cryopreservation.
4, tissue freezing
(1) 2 DEG C of balance, maintains 15min；(2) -2 DEG C are down to the speed of 6 DEG C/min；(3) it is dropped with the speed of 4 DEG C/min
To -4 DEG C；(4) -7.5 DEG C are down to the speed of 2 DEG C/min；(5) -8 DEG C are down to the speed of 0.5 DEG C/min；(6) with 0.5 DEG C/
The speed of min is down to -11.5 DEG C；(7) -13 DEG C are down to the speed of 0.1 DEG C/min；(8) be down to the speed of 0.3 DEG C/min-
42℃；(9) -100 DEG C are down to the speed of 50 DEG C/min.
Wherein, when temperature reaches -8 DEG C, reach and plant freezing point (seeding), take a small amount of liquid nitrogen, dip liquid nitrogen with long cotton swab and set
At the liquid level for freezing tube wall, it is seen that crystallization starting.Since nethermost cryopreservation tube, two long cotton swab alternately makes Seeding
With.Temperature slightly rises after Seeding, indicates successfully.
After temperature drops to -100 DEG C or less, quickly cryopreservation tube is moved into liquid nitrogen with long forceps and is saved.
The method for resuscitation of 29 ovary tissue of embodiment
1,1,2,3, W1, W2 are indicated on 6 well culture plates, cell strainer is placed in No. 1 hole, and syringe extracts resuscitation fluid 1, answers
Soviet Union liquid 2, resuscitation fluid 3, are successively separately added into corresponding hole, and eluent is added in W1, W2.
Wherein, as a preferred option, resuscitation fluid is used cooperatively with protection liquid is frozen；Resuscitation fluid and the volume for freezing protection liquid
Outer adding ingredient is consistent substantially.
For example, when using when freezing protection liquid, resuscitation fluid uses the resuscitation fluid of embodiment 18 in embodiment 1-6；When making
With embodiment 7 when freezing protection liquid, using the resuscitation fluid of embodiment 19；When using embodiment 8 freeze protection liquid when, use
The resuscitation fluid of embodiment 21；When using embodiment 9 freeze protection liquid when, using the resuscitation fluid of embodiment 22；Implement when using
When freezing protection liquid of example 10, using the resuscitation fluid of embodiment 20；When using embodiment 11 freeze protection liquid when, using implementation
The resuscitation fluid of example 23；When using embodiment 13 freeze protection liquid when, using the resuscitation fluid of embodiment 24.
2, opening water bath, 37 DEG C of target temperature；
3, liquid nitrogen is taken in foam box, the cryopreservation tube that clamping needs is placed in liquid nitrogen；
4, reach 37 degree to water bath temperature, press from both sides out cryopreservation tube, be put into 110ml sample box in room-temperature dissolution 30 seconds, after put
Enter water bath, keeps it to be immersed in the water about 2 minutes with tweezers, unscrew cryopreservation tube for tissue and pour into culture dish, then sandwich 1 with tweezers
Number hole.
6, hereafter start timing, 1,2, No. 3 hole is respectively 15 minutes, and W1 is 10 minutes, and W2 is 5 minutes.Tweezer should be used in time
Sub-folder takes culture hole where cell strainer replacement tissue.
7, freeze thawing and washing after, for transplanting tissue for transplant or Activity determination.If for detecting activity,
Tissue is put into the AIM-V culture solution got ready in advance, digestion in time, detection activity.
1 ovary tissue Activity determination of test example
The method of ovary tissue Activity determination uses Calcein-AM decoration method, in fluorescence microscopy microscopic observation gonad cell
With with common om observation gonad cell situation and counted under the same visual field.
Wherein, control group freezes protection liquid, the resuscitation fluid of embodiment 18 using embodiment 1, that is, does not add nitrogen second
Acyl cysteine (NAC) freezes ovary tissue using the cryopreservation methods of embodiment 28 and the method for resuscitation of embodiment 29
And recovery, then carry out Activity determination.Test group uses the resuscitation fluid for freezing protection liquid, embodiment 21 of embodiment 8, that is,
Liquid and resuscitation fluid are protected using freezing for nitrogen acetylcysteine is added to, utilizes the cryopreservation methods and embodiment 29 of embodiment 28
Method for resuscitation, ovary tissue is frozen and is recovered, Activity determination is then carried out.
Wherein, test group includes test group 1- test group 4, and the difference of test group 1-4 is nitrogen acetylcysteine (NAC)
Concentration difference, other components and content are all the same.Specifically, freezing nitrogen acetyl in protection liquid and resuscitation fluid in test group 1
The concentration of cysteine (NAC) is 0.5mM, and the concentration of nitrogen acetylcysteine (NAC) is 1mM in test group 2；Test group
The concentration of nitrogen acetylcysteine (NAC) is 5mM in 3；The concentration of nitrogen acetylcysteine (NAC) is in test group 4
25mM。
As a result as shown in fig. 1, wherein figure A1, B1, C1, D1 and E1 are the fluorescence microscope after Calcein-AM dyeing
Under observation result；A2, B2, C2, D2 and E2 are observation under the same visual field in common light microscopic as a result, amplification factor × 200.
Wherein figure A1 and A2 is control group as a result, not containing NAC；Scheming B1 and B2 is test group 1 as a result, NAC concentration is 0.5mM；
Scheming C1 and C2 is test group 2 as a result, i.e. NAC concentration is 1.0Mm；Scheming D1 and D2 is test group 3 as a result, i.e. NAC concentration is
5mM；Scheming E1 and E2 is test group 4 as a result, i.e. NAC concentration is 25mM.
As can be seen from the above results, control group freeze protection liquid and resuscitation fluid is able to maintain the activity of ovarian follicle, the visual field
Middle living cells quantity is more；And it is added in the test group of the NAC of various concentration, living cells in the visual field of test group 1- test 3
Quantity has increased slightly, and is able to maintain the activity of ovarian follicle；And the follicular activity of test group 4 is minimum, through statistical analysis p < 0.05, has
Significant difference has statistical significance.
Therefore, freezing for the NAC that the application contains concentration 0.5-5mM protects liquid and resuscitation fluid to can be improved cryopreservation resuscitation mistake
The activity of ovarian follicle in journey, wherein concentration is that freezing for the NAC of 5mM protects liquid and resuscitation fluid to be capable of the more apparent work for improving ovarian follicle
Property.
The test of 2 stroma cell apoptosis of test example
Detected using the test method of common detection stroma cell apoptosis ovary tissue stroma cell living cells and
Dead cell, it is for statistical analysis.In addition, the control group of this test example, test group 1-4 are identical with test example 1.
The testing result of this test example mesostroma Apoptosis is as shown in Figure 2, wherein left figure a1-e1 Green is to wither
Die stroma cell (due in attached drawing be black and white, color whiten be apoptosis stroma cell)；Blue is in middle figure a2-e2
The stroma cell of non-apoptosis (due to being black and white, the dotted stroma cell for non-apoptosis of light color in attached drawing)；Right figure a3-e3
For the superposition of left figure and middle figure.The above picture is the same visual field, amplification factor × 400.
Wherein, figure a1 and a2 is control group, does not contain NAC, and figure b1 and b2 is test group 1 (NAC 0.1mM), schemes c1 and c2
It is test group 3 (NAC 5mM) for test group 2 (NAC 1mM), figure d1 and d2, figure e1 and e2 is test group 4 (NAC 25mM).
Control group major part stroma cell apoptosis, it is little with the Apoptosis difference of control group in test group 1- test group 3,
But it is slightly higher.Specifically, the apoptosis rate median and interquartile range (%) of test group and test group indicate are as follows: control group-
NAC0mM:2.721(1.103,6.308)；Test group 1-NAC 0.5mM:2.737 (1.969,8.986)；Test group 2-NAC
1mM:2.222(1.575,7.528)；Test group 3-NAC 5mM:2.621 (1.477,6.060)；Test group 4-NAC 25mM:
0.043(0.000,2.588).The stroma cell apoptosis number of test group 4 is minimum.
The test of 3 reactive oxygen species of test example
Specific reactive oxygen species test (Reactive oxygen species (ROS) level) in accordance with the following methods into
Row:
Test equipment: historrhexis's instrument, centrifuge, pipettor, multi-functional plate reader, 96 orifice plates
Test reagent: Tris-Hcl, DCHF-DA, BCA protein quantification kit
Test procedure:
1, the control group of this test example, test group 1-4 are identical with test example 1.
2, ovary tissue cracks: 200ul protein extract (50mM Tris-HCl at pH is added in each tissue
7.5).Sample is as on ice with historrhexis's instrument high-speed homogenization 15S (homogenate 5S, stop 5S).3000g is centrifuged under the conditions of 4 DEG C
10min, separation supernatant are to be measured.
3, DCHF-DA solution is prepared: DMSO dissolves DCHF-DA to final concentration 10mM, stores for future use.
4, ROS is detected: taking the Tissue lysates of 20ul to add the diluted DCHF-DA of 20ul, (50mM Tris-HCl is diluted to
0.5mM), room temperature in 96 orifice plates is added after mixing to be kept in dark place 30 minutes.
5, signal detection: multi-functional/520 transmitting fluorescence letter of 480 excitation of plate reader detection of the M2 of Molecular Device
Number, obtain Ex480/Em520 numerical value.
6, determination of protein concentration:
1) BCA standard items dilute
4 BCA standard items of table dilute each group concentration
2) above-mentioned standard product dilution 25ul and diluted protein solution 25ul are separately added into 96 orifice plates.
3) measured according to sample size (every hole 200ul) and prepare working solution: 50 volume BCA reagent As add 1 volume BCA reagent B
(50:1) prepares appropriate BCA working solution, mixes well.
4) working fluid prepared in 3 is added in 96 orifice plates and is mixed, 37 DEG C are incubated for 30 minutes.
5) 562nM light absorption value is read, with protein content (μ g) for abscissa, light absorption value is ordinate, draws standard curve.
6) according to the light absorption value of institute's sample, corresponding protein content (μ g) can be checked on standard curve, divided by sample
Product dilution total volume (100 μ L) is sample actual concentrations (unit: μ g/ μ L) multiplied by sample extension rate.
7, ROS:Ex480/Em520/ protein concentration is calculated.
As a result as shown in figure 3, abscissa respectively represents each control group in figure and freezing for test group protects liquid and resuscitation fluid
The concentration of middle nitrogen acetylcysteine.As can be seen from Figure, with control group (control group), test group 1 (NAC 0.5mM),
It is compared with test group 2 (NAC 1mM), the reactive oxygen species (ROS is horizontal) of test group 3 (NAC 5mM) significantly reduce, through statistics
Analysis, p < 0.05 have statistical significance.Compared with test group 4 (NAC 25mM), ROS level also decreases, p > 0.05,
Illustrate that nitrogen acetylcysteine, which is added, can effectively reduce the reactive oxygen species after human ovarian tissue freezes/recovers.
4 total antioxidant capacity of test example is horizontal
Specific total antioxidant capacity hydraulic test (Reactive oxygen species (ROS) level) is according to following
Method carries out:
Test equipment: historrhexis's instrument, centrifuge, pipettor, multi-functional plate reader, 96 orifice plates
Test reagent: Tris.Hcl, ABTS, potassium persulphate, Trolox, BCA protein quantification reagent
Box；
Test procedure:
1, the control group of this test example, test group 1-4 are identical with test example 1.
2, ovary tissue cracks: 200ul protein extract (50mM Tris-HCl at pH is added in each tissue
7.5).Sample is as on ice with historrhexis's instrument high-speed homogenization 15S (homogenate 5S, stop 5S).3000g is centrifuged under the conditions of 4 DEG C
10min, separation supernatant are to be measured.
3, ABTS solution is prepared: 19.5mgABTS+3.3mg potassium persulphate+7ml 0.1mol/L
PBS (PH7.4) room temperature avoid light place 12 hours.
4, TAC is detected: PBS dilution (ratio of ABTS and PBS are about 1:80) above-mentioned solution arrives its 734nM light absorption value
1.0.Transparent 96 orifice plate is taken, every hole adds 15ul Tissue lysates+200ul ABTS solution to mix, and measurement 734nM light absorption value three divides
Clock.
5, signal detection: the multi-functional plate reader of the M2 of Molecular Device detects 734nM light absorption value.
6, determination of protein concentration: method is the same as the determination of protein concentration method in test example 3.
7, it is horizontal to calculate TAC: 734nM/ protein concentration
As a result as shown in figure 4, abscissa respectively represents each control group in figure and freezing for test group protects liquid and resuscitation fluid
The concentration of middle nitrogen acetylcysteine.As can be seen from Figure, with control group (control group), test group 2 (NAC 1mM) and
Test group 4 (NAC 25mM) is compared, and the total antioxidant capacity (TAC is horizontal) of test group 3 (NAC 5mM) significantly improves, through counting
Credit analysis, p < 0.05 have statistical significance.Compared with test group 1 (NAC 0.5mM), TAC level also increases, p >
0.05, illustrate to freeze and nitrogen acetylcysteine is added in protection liquid and resuscitation fluid can effectively be promoted after ovary tissue freezes/recover
Oxidation resistance.
On the basis for freezing protection liquid and resuscitation fluid of the control group of embodiment it can be seen from test example 3 and test example 4
On, the nitrogen acetylcysteine that concentration is 5mM is added, can effectively reduce the reactive oxygen species after human ovarian tissue freezes/recovers,
Promote oxidation resistance.
Test example 5
Liquid and resuscitation fluid is protected to be frozen freezing for use other compositions using the method for test example 1-4, recovery ovum
Nest tissue detects parameters respectively, and concrete outcome is as follows:
Control group: protection liquid-embodiment 1+ resuscitation fluid-embodiment 18 is frozen
Group 1: protection liquid-embodiment 7+ resuscitation fluid-embodiment 19 is frozen
Group 2: protection liquid-embodiment 8+ resuscitation fluid-embodiment 21 is frozen
Group 3: protection liquid-embodiment 9+ resuscitation fluid-embodiment 22 is frozen
Group 4: protection liquid-embodiment 10+ resuscitation fluid-embodiment 20 is frozen
Group 5: protection liquid-embodiment 11+ resuscitation fluid-embodiment 23 is frozen
Group 6: protection liquid-embodiment 12+ resuscitation fluid-embodiment 24 is frozen
Group 7: protection liquid-embodiment 14+ resuscitation fluid-embodiment 25 is frozen
Group 8: protection liquid-embodiment 16+ resuscitation fluid-embodiment 26 is frozen
Group 9: protection liquid-embodiment 17+ resuscitation fluid-embodiment 27 is frozen
Table 5 using difference freeze protection liquid and resuscitation fluid frozen, the parameters after recovery ovary tissue
As can be seen from the above table, of the invention to freeze protection liquid, resuscitation fluid cooperation cryopreservation methods and method for resuscitation, Neng Gouti
High ovary can Cryopreservation tissue rapidly and efficiently, and can be improved the ovary tissue motility rate and oxidation environment after recovery.
Wherein, be added to respectively taurine, nitrogen acetylcysteine, vitamin E freeze protection liquid, resuscitation fluid can significantly improve ovum
The oxidation environment of nest tissue improves oxidation resistance, while also can be improved ovary tissue motility rate.Any group of three of the above ingredient
After closing addition, synergistic effect can be played, ROS level is further decreased, it is horizontal to improve TAC.In addition, adding the above ingredient
On the basis of, vitamin C is further added, synergistic effect can be also played, ROS level is further decreased, it is horizontal to improve TAC.
The above is only presently preferred embodiments of the present invention, is not intended to limit the present invention in any form, though
So the present invention has been disclosed as a preferred embodiment, and however, it is not intended to limit the invention, any technology people for being familiar with this patent
Member without departing from the scope of the present invention, when the technology contents using above-mentioned prompt make it is a little change or be modified to
The equivalent embodiment of equivalent variations, but anything that does not depart from the technical scheme of the invention content, it is right according to the technical essence of the invention
Any simple modification, equivalent change and modification made by above embodiments, in the range of still falling within the present invention program.
Claims (10)
1. a kind of ovary tissue freezes protection and resuscitation fluid, which is characterized in that including freezing protection liquid, described freezes protection
Liquid includes Leibovitz L-15 culture medium, 8-10V/V%DMSO and 0.5-1.0V/V%HSA or 8-10V/V%SSS.
2. a kind of ovary tissue according to claim 1 freezes protection and resuscitation fluid, which is characterized in that described freezes
Protection liquid includes Leibovitz L-15 culture medium, 10V/V%DMSO, 1V/V%HSA,
Or freezing protection liquid includes Leibovitz L-15 culture medium, 10V/V%DMSO, 10V/V%SSS.
3. a kind of ovary tissue according to claim 1 or 2 freezes protection and resuscitation fluid, which is characterized in that described
Freezing protection liquid further includes taurine, and the content of the taurine is 0.5-5mM；
Preferably, the content of taurine is 0.5-1mM；It is furthermore preferred that the content of taurine is 0.5mM.
4. a kind of ovary tissue according to claim 1 to 3 freezes protection and resuscitation fluid, which is characterized in that described
The protection liquid that freezes further include nitrogen acetylcysteine, the content of the nitrogen acetylcysteine is 0.5-5mM；
Preferably, the content of nitrogen acetylcysteine is 3-5mM；It is furthermore preferred that the content of nitrogen acetylcysteine is 5mM.
5. a kind of ovary tissue according to claim 1 to 4 freezes protection and resuscitation fluid, which is characterized in that described
The protection liquid that freezes further include vitamin E, the content of the vitamin E is 0.1-10mM；
Preferably, the content of vitamin E is 3-7mM；It is furthermore preferred that the content of vitamin E is 5mM.
6. a kind of -5 any ovary tissues freeze protection and resuscitation fluid according to claim 1, which is characterized in that also wrap
Resuscitation fluid is included, the resuscitation fluid includes: 80-90V/V%DPBS, 10-20V/V%HAS and 0.13mol/L-0.8mol/L sugarcane
Sugar.
7. a kind of ovary tissue according to claim 6 freezes protection and resuscitation fluid, which is characterized in that the recovery
Liquid further includes taurine, and the content of the taurine is 0.5-5mM；
Preferably, the content of taurine is 0.5-1mM；It is furthermore preferred that the content of taurine is 0.5mM.
8. a kind of ovary tissue according to claim 1 or 2 freezes protection and resuscitation fluid, which is characterized in that described
Freezing protection liquid further includes at least one of nitrogen acetylcysteine, vitamin E；The content of the nitrogen acetylcysteine
For 0.5-5mM；The content of vitamin E is 0.1-10mM；
Preferably, the content of nitrogen acetylcysteine is 3-5mM, and the content of vitamin E is 3-7mM；
It is furthermore preferred that the content of nitrogen acetylcysteine is 5mM, the content of vitamin E is 5mM.
9. a kind of ovary tissue resuscitation fluid according to claim 6, which is characterized in that the resuscitation fluid includes:
(1) resuscitation fluid 1:90V/V%DPBS, 10V/V%HAS, 0.5-0.8mol/L sucrose；
(2) resuscitation fluid 2:90V/V%DPBS, 10V/V%HAS, 0.3-0.5mol/L sucrose；
(3) resuscitation fluid 3:90V/V%DPBS, 10V/V%HAS, 0.12-0.3mol/L sucrose；
Preferably, the resuscitation fluid includes:
(1) resuscitation fluid 1:90V/V%DPBS, 10V/V%HAS, 0.75mol/L sucrose；
(2) resuscitation fluid 2:90V/V%DPBS, 10V/V%HAS, 0.375mol/L sucrose；
(3) resuscitation fluid 3:90V/V%DPBS, 10V/V%HAS, 0.125mol/L sucrose.
10. a kind of ovary tissue resuscitation fluid according to claim 6, which is characterized in that the resuscitation fluid includes:
(1) resuscitation fluid 1:90V/V%DPBS, 10V/V%HAS, 5mM nitrogen acetylcysteine and/or 0.5mM taurine and/or
5mM vitamin E, 0.5-0.8mol/L sucrose；
(2) resuscitation fluid 2:90V/V%DPBS, 10V/V%HAS, 5mM nitrogen acetylcysteine and/or 0.5mM taurine and/or
5mM vitamin E, 0.3-0.5mol/L sucrose；
(3) resuscitation fluid 3:90V/V%DPBS, 10V/V%HAS, 5mM nitrogen acetylcysteine and/or 0.5mM taurine and/or
5mM vitamin E, 0.13-0.3mol/L sucrose；
Preferably, the resuscitation fluid includes:
(1) resuscitation fluid 1:90V/V%DPBS, 10V/V%HAS, 5mM nitrogen acetylcysteine, 0.75mol/L sucrose；
(2) resuscitation fluid 2:90V/V%DPBS, 10V/V%HAS, 5mM nitrogen acetylcysteine, 0.375mol/L sucrose；
(3) resuscitation fluid 3:90V/V%DPBS, 10V/V%HAS, 5mM nitrogen acetylcysteine, 0.125mol/L sucrose.
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| CN114514924A (en) * | 2021-03-23 | 2022-05-20 | 上海慧存医疗科技有限公司 | Ovarian tissue preservation method |
| CN115885971A (en) * | 2022-03-25 | 2023-04-04 | 同济大学 | Cryopreservation resuscitation method for ovarian tissues |
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| KR20230091584A (en) | 2021-12-16 | 2023-06-23 | 고려대학교 산학협력단 | Composition for cryopreservating ovarian comprising Klotho protein and N-acetylcysteine |
| CN115074315B (en) * | 2022-06-08 | 2023-10-31 | 四川省畜牧科学研究院 | High-quality pig ear tissue sampling and long-term high-efficiency preservation method |
| CN117099771B (en) * | 2023-07-27 | 2024-02-13 | 江苏太平洋美诺克生物药业股份有限公司 | Cell cryopreservation solution for high-throughput cryopreservation of Chinese hamster ovary cells |
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| CN113174362B (en) | 2022-07-15 |
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| CN113207869B (en) | 2022-06-07 |
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