CN106689118A - Pet ovarian tissue cryopreservation resuscitation reagent and cryopreservation resuscitation method - Google Patents

Pet ovarian tissue cryopreservation resuscitation reagent and cryopreservation resuscitation method Download PDF

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CN106689118A
CN106689118A CN201710028226.6A CN201710028226A CN106689118A CN 106689118 A CN106689118 A CN 106689118A CN 201710028226 A CN201710028226 A CN 201710028226A CN 106689118 A CN106689118 A CN 106689118A
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reagent
lbp
recovery
pet
culture medium
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朱梅花
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Shanghai Still Biotechnology Co Ltd
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Shanghai Still Biotechnology Co Ltd
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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
    • 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
    • A01N1/0278—Physical preservation processes
    • A01N1/0284—Temperature processes, i.e. using a designated change in temperature over time
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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
    • C12N5/0681—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
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/34—Sugars
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/38—Vitamins
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/50—Soluble polymers, e.g. polyethyleneglycol [PEG]
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/165—Vascular endothelial growth factor [VEGF]

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Abstract

The invention provides a pet ovarian tissue cryopreservation resuscitation reagent which comprises: (1) a cryopreservation resuscitation reagent 1 including 2-7W/V% of lycium barbarum polysaccharide, 6-14V/V% of dimethyl sulfoxide, 2-7V/V% of propylene glycol and 80-90V/V% of an MEM (Memminimum Essential Medium) culture medium; (2) a cryopreservation resuscitation reagent 2 including 8-10W/V% of lycium barbarum polysaccharide, 6-20V/V% of dimethyl sulfoxide, 5-15V/V% of propylene glycol and 70-80V/V% of an MEM culture medium; (3) a cryopreservation resuscitation reagent 3 including 15-20W/V% of lycium barbarum polysaccharide, 6-20V/V% of dimethyl sulfoxide, 5-15V/V% of propylene glycol and 70-80V/V% of an MEM culture medium. The invention further provides a pet ovarian tissue cryopreservation resuscitation reagent group and a pet ovarian tissue cryopreservation and resuscitation method.

Description

Pet ovary tissue cryopreservation resuscitation reagent and cryopreservation resuscitation method
Technical field
The present invention relates to ovary tissue freezen protective, more particularly it relates to pet ovary tissue freezes and answers Soviet Union's reagent and pet ovary tissue freeze and method for resuscitation.
Background technology
Pet (referring mainly to as the dog and cat of companion animals) can accompany the mankind, alleviate the pressure in daily life, pleased Happy mood, promotes human health, as a member in family.With China's expanding economy, the exacerbation of aging degree, to doting on The demand of thing is increasing.While pet quantity increases, the industry such as pet food, pet supplies, pet training has obtained fluffy Vigorous development.Pet health domain variability is paid close attention to by enough, also not yet forms specification.Compared to human health market, dote on Thing health market has very big space to wait to excavate.Love pet, pays close attention to pet health.
General recommendation pet carries out sterilization or castration operation in 6 to August age both at home and abroad.Major advantage includes avoiding a lot With the generation of reproductive hormone relevant disease, time cost of the pet owner in terms of pet reproduction is saved, control Population.
The sterilization of pet refers to the oophorectomy or oophorohysterectomy of female pet.For individual pets, Sterilization operation can reduce the risk that female pet suffers from breast cancer, and eliminate the risk of pyrometra, it is to avoid ovarian cyst, difficult labour etc. The generation of disease.
However, after excision ovary, at present all abandoning ovary tissue, pet not only loses fecundity, internal Hormonal readiness has also suffered destruction.Long-term hormonal readiness imbalance can cause that the pet state of mind is dispirited, abnormal behavior, with year The growth in age often leads to the metabolic diseases such as obesity.The kinematic system development of pet can be also affected, and articular ligament is developed not Good, the onset risk such as osteoporosis increases.
Current processing mode cannot give the chance of pet second selecting, and sterilization means that forfeiture fecundity swashs with destruction Plain level.Compared to human health field, ovary freezes and is evolving with autotransplanting technology.Preferable mode is by freezing Technology preserves fecundity, and autotransplantation is optionally carried out when being necessary, recovers fecundity and hormonal readiness.Pet is good for The such technology in health field does not obtain development and application still.
The content of the invention
The invention technical problem to be solved
It is an object of the invention to provide a kind of pet ovary tissue rapidly and efficiently freeze with recovery reagent and freeze with Method for resuscitation.
Scheme for solving above-mentioned technical problem
The present invention freezes reagent and pet ovary tissue recovery reagent comprising pet ovary tissue, and definite ingredients stabilization is protected The matter phase is long, and freezing and recovering for pet ovary tissue is may be directly applied to after preparation, easy to use, and tissue recovery activity is high.This Invention relies on tissue glass frozen preservation (Rapid-Freezing Method directly enters liquid nitrogen from normal temperature) theory and technology, and to freeze combine in reagent makes With two kinds of cryoprotective agent dimethyl sulfoxide (DMSO)s (DMSO), propane diols (PROH), protective agent is lowered while improving vitrifying efficiency To the toxicity of cell, LBP-X (LBP) is used to organize glass frozen preservation first, effect is remote in pet ovary tissue freezes Better than traditional sucrose (SUC)；Add vitamin E (VE) and blood vessel endothelium first for ovary tissue characteristic, in recovery reagent Growth factor (VEGF), largely improves the damage that the oxidative stress in resuscitation process is caused to cell.
The first aspect of the present invention, there is provided pet ovary tissue freezes reagent set,
The pet ovary tissue freezes reagent set to be included：
(1) reagent 1 is frozen：2~7W/V% of LBP-X, 6~14V/V% of dimethyl sulfoxide (DMSO), 2~7V/V% of propane diols, 80~90V/V% of MEM culture mediums；
(2) reagent 2 is frozen：8~10W/V% of LBP-X, 6~20V/V% of dimethyl sulfoxide (DMSO), 5~15V/ of propane diols 70~80V/V% of V%, MEM culture medium；
(3) reagent 3 is frozen：15~20W/V% of LBP-X, 6~20V/V% of dimethyl sulfoxide (DMSO), 5~15V/ of propane diols 70~80V/V% of V%, MEM culture medium.
Preferably, the reagent set that freezes includes：
(1) reagent 1 is frozen：2~7W/V% of LBP-X, 6~14V/V% of dimethyl sulfoxide (DMSO), 2~7V/V% of propane diols, MEM 80~90V/V% of culture medium, 0.1~0.5W/V% of polyvinylpyrrolidone, 1~10W/V% of glucose；
(2) reagent 2 is frozen：8~10W/V% of LBP-X, 6~20V/V% of dimethyl sulfoxide (DMSO), 5~15V/ of propane diols V%, MEM 70~80V/V% of culture medium, 0.1~0.5W/V% of polyvinylpyrrolidone, 1~10W/V% of glucose；
(3) reagent 3 is frozen：15~20W/V% of LBP-X, 6~20V/V% of dimethyl sulfoxide (DMSO), 5~15V/ of propane diols V%, MEM 70~80V/V% of culture medium, 0.1~0.5W/V% of polyvinylpyrrolidone, 1~10W/V% of glucose.
Preferably, the reagent set that freezes includes：
(1) reagent 1 is frozen：LBP-X 6W/V%, dimethyl sulfoxide (DMSO) 10V/V%, propane diols 5V/V%, MEM culture medium 85V/V%；
(2) reagent 2 is frozen：LBP-X 9W/V%, dimethyl sulfoxide (DMSO) 12V/V%, propane diols 13V/V%, MEM culture medium 75V/V%；
(3) reagent 3 is frozen：LBP-X 18W/V%, dimethyl sulfoxide (DMSO) 12V/V%, propane diols 13V/V%, MEM culture Base 75V/V%.
More excellent, the reagent set that freezes includes：
(1) reagent 1 is frozen：LBP-X 6W/V%, dimethyl sulfoxide (DMSO) 10V/V%, propane diols 5V/V%, MEM culture medium 85V/V%, polyvinylpyrrolidone 0.3W/V%, glucose 5W/V%；
(2) reagent 2 is frozen：LBP-X 9W/V%, dimethyl sulfoxide (DMSO) 12V/V%, propane diols 13V/V%, MEM culture medium 75V/V%, polyvinylpyrrolidone 0.3W/V%, glucose 5W/V%；
(3) reagent 3 is frozen：LBP-X 18W/V%, dimethyl sulfoxide (DMSO) 12V/V%, propane diols 13V/V%, MEM culture Base 75V/V%, polyvinylpyrrolidone 0.3W/V%, glucose 5W/V%.
The second aspect of the present invention, there is provided pet ovary tissue recovery reagent set, the recovery reagent set includes：
(1) recovery reagent 1：35~55W/V% of LBP-X, vitamin E 0.05~0.35V/V%, MEM culture medium 99.65~99.95V/V%；
(2) recovery reagent 2：25~45W/V% of LBP-X, vitamin E 0.05~0.35V/V%, MEM culture medium 99.65~99.95V/V%；
(3) recovery reagent 3：15~35W/V% of LBP-X, vitamin E 0.05~0.35V/V%, MEM culture medium 99.65~99.95V/V%；
(4) recovery reagent 4：5~25W/V% of LBP-X, vitamin E 0.05~0.35V/V%, MEM culture medium 99.65~99.95V/V%, the μ g/L of VEGF 1~10.
Preferably, the recovery reagent set includes：
(1) recovery reagent 1：35~55W/V% of LBP-X, vitamin E 0.05~0.35V/V%, MEM culture medium 99.65~99.95V/V%, 0.1~0.5W/V% of polyvinylpyrrolidone, 1~10W/V% of glucose；
(2) recovery reagent 2：25~45W/V% of LBP-X, vitamin E 0.05~0.35V/V%, MEM culture medium 99.65~99.95V/V%, 0.1~0.5W/V% of polyvinylpyrrolidone, 1~10W/V% of glucose；
(3) recovery reagent 3：15~35W/V% of LBP-X, vitamin E 0.05~0.35V/V%, MEM culture medium 99.65~99.95V/V%, 1~10W/V% of glucose；
(4) recovery reagent 4：5~25W/V% of LBP-X, vitamin E 0.05~0.35V/V%, MEM culture medium 99.65~99.95V/V%, 1~10W/V% of glucose, the μ g/L of VEGF 1~10.
Preferably, the recovery reagent set includes：
(1) recovery reagent 1：LBP-X 40W/V%, vitamin E 0.2V/V%, MEM culture medium 99.8%；
(2) recovery reagent 2：LBP-X 30W/V%, vitamin E 0.2V/V%, MEM culture medium 99.8%；
(3) recovery reagent 3：LBP-X 20W/V%, vitamin E 0.2V/V%, MEM culture medium 99.8%；
(4) recovery reagent 4：LBP-X 10W/V%, vitamin E 0.2V/V%, MEM culture medium 99.8%, Ink vessel transfusing The μ g/L of skin growth factor 1.
More excellent, the recovery reagent set includes：
(1) recovery reagent 1：LBP-X 40W/V%, vitamin E 0.2V/V%, MEM culture medium 99.8%, polyethylene Pyrrolidones 0.3W/V%, glucose 5W/V%；
(2) recovery reagent 2：LBP-X 30W/V%, vitamin E 0.2V/V%, MEM culture medium 99.8%, polyethylene Pyrrolidones 0.3W/V%, glucose 5W/V%；
(3) recovery reagent 3：LBP-X 20W/V%, vitamin E 0.2V/V%, MEM culture medium 99.8%, glucose 5W/V%；
(4) recovery reagent 4：LBP-X 10W/V%, vitamin E 0.2V/V%, MEM culture medium 99.8%, glucose 5W/V%, the μ g/L of VEGF 1.
The third aspect of the invention is to provide a kind of pet ovary tissue cryopreservation resuscitation reagent set, including above-mentioned first The recovery reagent set for freezing reagent set and second aspect of aspect.
The fourth aspect of the invention is to provide a kind of pet ovary tissue cryopreservation methods, including：By in vitro pet ovary Tissue immerse successively above-mentioned first aspect freeze reagent 1, freeze reagent 2, freeze reagent 3 in keep more than 5 minutes after, liquid Nitrogen is stored.
The fifth aspect of the invention is to provide a kind of pet ovary tissue method for resuscitation, including：Will be in vitro through what is frozen Pet ovary tissue immerses in the recovery reagent 1 of above-mentioned second aspect, recovery reagent 2 successively, is respectively kept for 2 minutes at 37 ± 2 DEG C More than, afterwards, move into recovery reagent 3, the recovery reagent 4 of above-mentioned second aspect, respectively kept for more than 10 minutes at room temperature.
Invention effect
The motility rate that cryopreservation resuscitation reagent set of the invention can freeze pet ovary tissue short-term (about a week), recover reaches To more than 90%, long-term (about three years) freeze, the motility rate recovered reaches more than 70%, can be good at freezen protective fecundity. LBP-X is introduced freeze field first by inventor, and vitamin E is introduced into recovery reagent, the recovery reagent of final step first In add micro VEGF first, three kinds of materials synergies, achieve frozen for pet ovary tissue, The synergy of the highly significant of recovery.Clearly, it is convenient to prepare, beneficial to large-scale production for agent formulations of the present invention.
All the time, tissue glass frozen preservation area research focuses on, for different cell and tissue, finding toxicity Less vitrification solution mix proportion scheme and raising drop, the method for rewarming rate.By being combined two kinds of cytoprotections in the present invention Agent dimethyl sulfoxide (DMSO) and propane diols, the toxicity for controlling cell-protecting to cause cell while in order to be effective.
The present invention freezes reagent set and includes 3 kinds of reagents, and recovery reagent set includes 4 kinds of reagents, and gradient distribution raises and reduce Chinese holly The concentration of Qi polysaccharide is excellent compared with one-step method effect.
Specific embodiment
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention Rather than limitation the scope of the present invention.
The experimental technique of unreceipted actual conditions in the following example, generally according to normal condition or according to institute of manufacturer The condition of suggestion.Unless otherwise indicated, otherwise percentage and number is calculated by weight.Unless otherwise defined, used in text All specialties are identical with meaning familiar to one skilled in the art institute with scientific words.Additionally, any similar to described content Or during the method and material of equalization all can be applied to the present invention.Preferable implementation described in text only presents a demonstration it with material With.
Embodiment 1-5：Pet ovary tissue freezes the preparation of reagent
(1) by taking the preparation that 100ml freezes reagent 1 as an example：Respectively according to the amount shown in table 1, measure dimethyl sulfoxide (DMSO) and (be purchased from SIGMA), propane diols (being purchased from SIGMA), MEM culture mediums (being purchased from GIBCO) are put into 250ml indigo plant mouths bottle (purchased from another name for Sichuan Province ox), and jog is mixed It is even, weigh LBP-X (biological purchased from Si Nuote), polyvinylpyrrolidone ((abbreviation PVP) is purchased from SIGMA), glucose (purchase From SIGMA) add above-mentioned bottle, rock mixing.With 0.22 μm of filter (being purchased from MILLIPORE) filtering after solid all dissolves Degerming, to by autoclaved 100ml indigo plant mouths bottle (being purchased from another name for Sichuan Province ox), 4 DEG C save backup for filtering；
(2) so that 100ml freezes the preparation of reagent 2 as an example：Respectively according to the amount shown in table 1, measure dimethyl sulfoxide (DMSO) and (be purchased from SIGMA), propane diols (being purchased from SIGMA), MEM culture mediums (being purchased from GIBCO) are put into 250ml indigo plant mouths bottle (purchased from another name for Sichuan Province ox), and jog is mixed It is even, weigh LBP-X (biological purchased from Si Nuote), polyvinylpyrrolidone (purchased from SIGMA), glucose (purchased from SIGMA) and add Enter in above-mentioned bottle, rock mixing.With 0.22 μm of filter (being purchased from MILLIPORE) filtration sterilization, filtering after solid all dissolves To by autoclaved 100ml indigo plant mouths bottle (being purchased from another name for Sichuan Province ox), 4 DEG C save backup；
(3) so that 100ml freezes the preparation of reagent 3 as an example：Respectively according to the amount shown in table 1, measure dimethyl sulfoxide (DMSO) and (be purchased from SIGMA), propane diols (being purchased from SIGMA), MEM culture mediums (being purchased from GIBCO) are put into 250ml indigo plant mouths bottle (purchased from another name for Sichuan Province ox), and jog is mixed It is even, weigh LBP-X (biological purchased from Si Nuote), polyvinylpyrrolidone (purchased from SIGMA), glucose (purchased from SIGMA) and add Enter in above-mentioned bottle, rock mixing.With 0.22 μm of filter (being purchased from MILLIPORE) filtration sterilization, filtering after solid all dissolves To by autoclaved 100ml indigo plant mouths bottle (being purchased from another name for Sichuan Province ox), 4 DEG C save backup.
Pet testis tissue of the invention freezes reagent set includes three kinds of reagents, and LBP-X concentration therein increases successively Greatly.
Table 1：Freeze the preparation of reagent
Embodiment 6-10：The preparation of pet ovary tissue recovery reagent
(1) by taking the preparation of 100ml recoveries reagent 1 as an example：Respectively according to the amount shown in table 2, vitamin E (oily liquid is measured Body, purity mark >=98%) (be purchased from SIGMA), MEM culture mediums (being purchased from GIBCO) are put into 250ml indigo plant mouths bottle (purchased from another name for Sichuan Province ox), gently It is shaken to mixed even, weighs LBP-X (biological purchased from Si Nuote), polyvinylpyrrolidone (purchased from SIGMA), glucose and (be purchased from SIGMA) add in above-mentioned bottle, rock mixing.Filtered after being crossed with 0.22 μm of filter (being purchased from MILLIPORE) after solid all dissolving Bacterium, to by autoclaved 100ml indigo plant mouths bottle (being purchased from another name for Sichuan Province ox), 4 DEG C save backup for filtering；
(2) by taking the preparation of 100ml recoveries reagent 2 as an example：Respectively according to the amount shown in table 2, measure vitamin E and (be purchased from SIGMA), MEM culture mediums (being purchased from GIBCO) are put into 250ml indigo plant mouths bottle (purchased from another name for Sichuan Province ox), and jog is mixed, and weigh LBP-X (purchase It is biological from Si Nuote), during polyvinylpyrrolidone (being purchased from SIGMA), glucose (being purchased from SIGMA) adds above-mentioned bottle, rock mixed It is even.With 0.22 μm of filter (being purchased from MILLIPORE) filtration sterilization after solid all dissolves, filtering is to by autoclaved 100ml indigo plant mouths bottle (being purchased from another name for Sichuan Province ox), 4 DEG C save backup；
(3) by taking the preparation of 100ml recoveries reagent 3 as an example：Respectively according to the amount shown in table 2, measure vitamin E and (be purchased from SIGMA), MEM culture mediums (being purchased from GIBCO) are put into 250ml indigo plant mouths bottle (purchased from another name for Sichuan Province ox), and jog is mixed, and weigh LBP-X (purchase It is biological from Si Nuote), during glucose (being purchased from SIGMA) adds above-mentioned bottle, rock mixing.With 0.22 μm after solid all dissolves Filter (being purchased from MILLIPORE) filtration sterilization, filtering is to by autoclaved 100ml indigo plant mouths bottle (being purchased from another name for Sichuan Province ox), 4 DEG C of preservations It is standby；
(4) by taking the preparation of 100ml recoveries reagent 4 as an example：Respectively according to the amount shown in table 2, measure vitamin E and (be purchased from SIGMA), MEM culture mediums (being purchased from GIBCO) are put into 250ml indigo plant mouths bottle (purchased from another name for Sichuan Province ox), and jog is mixed, and weigh LBP-X (purchase It is biological from Si Nuote), during glucose (being purchased from SIGMA), VEGF (being purchased from SIGMA) add above-mentioned bottle, rock Mix.With 0.22 μm of filter (being purchased from MILLIPORE) filtration sterilization after solid all dissolves, filtering is to by autoclaved 100ml indigo plant mouths bottle (being purchased from another name for Sichuan Province ox), 4 DEG C save backup.
Pet ovary tissue recovery reagent set of the invention includes four kinds of reagents, and LBP-X concentration therein drops successively It is low.
Table 2：The preparation of recovery reagent
Reference examples
Control A groups：In addition to removing vitamin E and VEGF in recovery reagent, other conditions and reality Apply example 5 and embodiment 10 is identical.
Control B groups：LBP-X is changed the sucrose of equivalent on the basis of experiment contrast A groups into, other conditions are constant.
Control C groups：Except will freeze and recovery reagent in LBP-X change equivalent into sucrose in addition to, other conditions and reality Apply example 5 and embodiment 10 is identical.
Traditional control group, is frozen using reference reagent and method, is summarized as follows：Freezing reagent includes 2mol/L dimethyl Sulfoxide, 2mol/L propane diols, the sucrose of various concentrations, include 0.5~0.125mol/L sucrose, concrete operations step in recovery reagent (Li Yubin, Zhou Canquan, Yang Guofen wait two kinds of Cryopreservation sides of human ovarian tissues to the rapid glass frozen preservation scheme seen in document Research [J] the Zhongshan University journal medical science version of method, 2006,27 (6):704-708.).
Table 3：
Effect test：
1. pet ovary tissue is frozen, the short run effect of recovery reagent is verified
(1) acquisition of ovary tissue：In the case of pet master, pet doctor's informed consent, 6~August age is collected respectively Pet dog carries out the ovary tissue cut off during sterilization operation.In 20 minutes, unnecessary fat, manadesma is rejected, it is trimmed to 1~ The tissue of 2cm thickness, is immersed in the DMEM culture mediums containing 10% serum (purchased from GIBCO) (purchased from GIBCO), is placed in ice On.
(2) section of ovary tissue：Ovary tissue piece is further pruned with knife blade, thickness control 0.8~ 1.2mm, length is controlled in 5~10mm, and width control system is in 2~5mm.Cut operation whole process is grasped on ice in 10% serum DMEM Make, in completion in 30 minutes.
Reserved part histotomy does not carry out follow-up cryopreservation resuscitation operation, and be used as follow-up check experiment does not freeze group Knit section (hereinafter referred to as " fresh control group ").
(3) ovary tissue freezes and recovery：The random ovary tissue taken out in (2) is cut into slices 3, is inhaled using sterile gauze Clean excessive moisture.Histotomy immersion embodiment 1 is frozen into reagent 1, normal temperature 10 minutes.After histotomy takes out, after Continuous to blot net excessive moisture using sterile gauze, immigration is frozen in reagent 2, normal temperature 20 minutes.Same operation is moved into and freezes examination In agent 3, normal temperature 30 minutes.Histotomy is taken out, is positioned on metal copper mesh bar, net excessive moisture is blotted using sterile gauze. Immediately in the input aseptic liquid nitrogen of preprepared, histotomy prior precooling can be transferred to together with copper mesh bar after 5 minutes In cryopreservation tube, liquid nitrogen storage.After 1 week, prepare recovery cryopreserved tissue, 37 DEG C of water baths preheat the recovery reagent 1 of embodiment 6, answer Soviet Union's reagent 2.In liquid nitrogen take out histotomy inserted rapidly in recovery reagent 1 together with copper mesh bar, jog, 37 DEG C 2 minutes, it is seen that group Section is knitted to be come off from copper mesh bar, during histotomy moved into recovery reagent 2,37 DEG C 5 minutes.Then histotomy is moved successively In entering recovery reagent 3, recovery reagent 4, each 10 minutes of room temperature (referred to as embodiment 1+6).
Embodiment 2+7, embodiment 3+8, embodiment 4+9, embodiment 5+10, control A groups, control are carried out under conditions of same B groups, control C groups, the experiment of traditional control group.
(4) detection of tissue culture supernatant estradiol and normal primordial follicle are counted：By each embodiment, control group in (3) with And the histotomy of the fresh control group in (2) is respectively implanted culture in 6 orifice plate of cell culture, 1 hole, 2ml 10%DMEM trainings Support base, 37 DEG C, 5%CO2Cultivated 48 hours in cell culture incubator.500ul supernatants are collected, estradiol (E2) detection kit is used (being purchased from IMMUNOCLONE) detection ovary tissue estradiol production amount.Histotomy makes HE and dyes after being embedded with routine paraffin wax Section, counts 100 primordial follicles under microscope, count normal primordial follicle quantity.Each experiment in triplicate, is averaged respectively Value as experimental result, experimental result as shown in table 4, the reflection of table 4 freeze 1 week after resuscitation team estradiol production function situation with And the normal primordial follicle accounting situation of short-term cryopreservation resuscitation.No matter supernatant estradiol production situation or normal primordial follicle are accounted for Than the result after embodiment cryopreservation resuscitation is all close to fresh control group, and each embodiment result is better than control A groups, control B Group, control C groups and traditional control group, illustrate that LBP-X of the invention, vitamin E, VEGF collaboration are made With there is remarkable result to the raising of cryopreservation resuscitation motility rate.
Table 4：Freeze, the short run effect of recovery reagent
Note：Embodiment 1+6 represents that the recovery agent combination for freezing reagent+embodiment 6 of embodiment 1 is used, similarly hereinafter.
Additionally, the present invention collects the ovum that 6~August age pet cat cut off during sterilization operation using same method Nest tissue, has carried out freezing and experiment of recovering for above-mentioned (1)-(4), and freezing reagent is embodiment 5, and recovery reagent is embodiment 10, estradiol production amount is as a result shown for 58pmol/L, normal primordial follicle percentage is 92%.
2. pet ovary tissue is frozen, the long-term effect of recovery reagent is verified
(1) acquisition of ovary tissue：In the case of pet master, pet doctor's informed consent, 6~August age is collected respectively Pet dog carries out the ovary tissue cut off during sterilization operation.In 20 minutes, unnecessary fat, manadesma is rejected, it is trimmed to 1~ The tissue of 2cm thickness, is immersed in the DMEM culture mediums containing 10% serum (purchased from GIBCO) (purchased from GIBCO), is placed in ice On.
(2) section of ovary tissue：Ovary tissue piece is further pruned with knife blade, thickness control 0.8~ 1.2mm, length is controlled in 5~10mm, and width control system is in 2~5mm.Cut operation whole process is grasped on ice in 10% serum DMEM Make, in completion in 30 minutes.
(3) ovary tissue freezes and recovery：The ovary tissue taken out in (2) is cut into slices 18, is randomly divided into 6 groups, every group 3 Piece, net excessive moisture is blotted using sterile gauze.Take that 5 groups of histotomies immerse 5 groups of embodiments respectively freezes reagent 1, often Temperature 10 minutes.After histotomy takes out, it is continuing with sterile gauze and blots net excessive moisture, immigration is frozen in reagent 2, normal temperature 20 Minute.Same operation immigration is frozen in reagent 3, normal temperature 30 minutes.Histotomy is taken out, is positioned on metal copper mesh bar, made Net excessive moisture is blotted with sterile gauze.Immediately in the input aseptic liquid nitrogen of preprepared, can be by histotomy after 5 minutes It is transferred in the cryopreservation tube of prior precooling together with copper mesh bar, liquid nitrogen storage.Different time point resuscitation teams are set, and time point is set It is 0.5 year, 1 year, 2 years, 3 years.When preparing recovery cryopreserved tissue, 37 DEG C of water baths preheat recovery reagent 1, recovery reagent 2.Liquid nitrogen Middle taking-up tissue is inserted rapidly in recovery reagent 1 together with copper mesh bar, jog, 37 DEG C 2 minutes, it is seen that histotomy is from copper mesh bar On come off, during tissue cut into immigration recovery reagent 2,37 DEG C 5 minutes.Then histotomy moved into recovery reagent 3 successively, answered In Soviet Union's reagent 4, each 10 minutes of room temperature.
Taking 1 group of histotomy carries out the experiment of traditional control group, is frozen using reference reagent and method, is summarized as follows：Freeze Depositing reagent includes 2mol/L dimethyl sulfoxide (DMSO)s, 2mol/L propane diols, the sucrose of various concentrations, in recovery reagent comprising 0.5~ 0.125mo l/L sucrose, (Li Yubin, Zhou Canquan, Yang Guofen wait to the glass frozen preservation scheme that concrete operation step is shown in document The two kinds of research of Cryopreservation method [J] Zhongshan University journal medical science versions of human ovarian tissue, 2006,27 (6):704- 708.)。
(4) detection of tissue culture supernatant estradiol and normal primordial follicle are counted：By each embodiment and traditional control group Histotomy is cultivated in being respectively implanted 6 orifice plate of cell culture, 1 hole, 2ml 10%DMEM culture mediums, 37 DEG C, 5%CO2Cell is trained Cultivated 48 hours in foster case.500u l supernatants are collected, estradiol (E2) detection kit (being purchased from IMMUNOCLONE) detection is used Ovary tissue estradiol production amount.Tissue makes HE stained slices after being embedded with routine paraffin wax, and 100 originals are counted under microscope Beginning ovarian follicle, counts normal primordial follicle quantity.Each time point is randomly provided 3 tissue samples.Experimental result is as shown in table 5, The reflection of table 5 freeze 0.5,1,2,3 years after resuscitation team estradiol production function situation and the normal original ovum of Long-term Cryopreservation recovery Bubble accounting situation.As shown in Table 5, embodiment of the present invention group effect is much better than traditional control group under long-term storage conditions, and Each group froze after effect was varied slightly at 1 year or so and tends towards stability, and illustrated that the quality of glass frozen preservation technology is mainly reflected in group Protection during into liquid nitrogen is woven, preservation effect is relatively stable after tissue is stored in liquid nitrogen.
Table 5：Freeze, the long-term effect of recovery reagent
Below the preferred embodiment to the invention is illustrated, but the invention be not limited to it is described Embodiment, those of ordinary skill in the art can also make a variety of equivalent on the premise of without prejudice to the invention spirit Modification or replacement, these equivalent modifications or replacement are all contained in the application claim limited range.

Claims (9)

1. one kind freezes reagent set, including：
(1) reagent 1 is frozen：2~7W/V% of LBP-X, 6~14V/V% of dimethyl sulfoxide (DMSO), propane diols 2~7V/V%, MEM training Support 80~90V/V% of base；
(2) reagent 2 is frozen：8~10W/V% of LBP-X, 6~20V/V% of dimethyl sulfoxide (DMSO), propane diols 5~15V/V%, MEM 70~80V/V% of culture medium；
(3) reagent 3 is frozen：15~20W/V% of LBP-X, 6~20V/V% of dimethyl sulfoxide (DMSO), 5~15V/V% of propane diols, 70~80V/V% of MEM culture mediums.
2. one kind freezes reagent set, including：
(1) reagent 1 is frozen：2~7W/V% of LBP-X, 6~14V/V% of dimethyl sulfoxide (DMSO), propane diols 2~7V/V%, MEM training Support base 80~90V/V%, 0.1~0.5W/V% of polyvinylpyrrolidone, 1~10W/V% of glucose；
(2) reagent 2 is frozen：8~10W/V% of LBP-X, 6~20V/V% of dimethyl sulfoxide (DMSO), propane diols 5~15V/V%, MEM 70~80V/V% of culture medium, 0.1~0.5W/V% of polyvinylpyrrolidone, 1~10W/V% of glucose；
(3) reagent 3 is frozen：15~20W/V% of LBP-X, 6~20V/V% of dimethyl sulfoxide (DMSO), 5~15V/V% of propane diols, MEM 70~80V/V% of culture medium, 0.1~0.5W/V% of polyvinylpyrrolidone, 1~10W/V% of glucose.
3. purposes of the reagent set described in claim 1 or 2 in pet ovary tissue freezes.
4. a kind of recovery reagent set, including：
(1) recovery reagent 1：35~55W/V% of LBP-X, vitamin E 0.05~0.35V/V%, MEM culture medium 99.65~ 99.95V/V%；
(2) recovery reagent 2：25~45W/V% of LBP-X, vitamin E 0.05~0.35V/V%, MEM culture medium 99.65~ 99.95V/V%；
(3) recovery reagent 3：15~35W/V% of LBP-X, vitamin E 0.05~0.35V/V%, MEM culture medium 99.65~ 99.95V/V%；
(4) recovery reagent 4：5~25W/V% of LBP-X, vitamin E 0.05~0.35V/V%, MEM culture medium 99.65~ 99.95V/V%, the μ g/L of VEGF 1~10.
5. a kind of recovery reagent set, including：
(1) recovery reagent 1：35~55W/V% of LBP-X, vitamin E 0.05~0.35V/V%, MEM culture medium 99.65~ 99.95V/V%, 0.1~0.5W/V% of polyvinylpyrrolidone, 1~10W/V% of glucose；
(2) recovery reagent 2：25~45W/V% of LBP-X, vitamin E 0.05~0.35V/V%, MEM culture medium 99.65~ 99.95V/V%, 0.1~0.5W/V% of polyvinylpyrrolidone, 1~10W/V% of glucose；
(3) recovery reagent 3：15~35W/V% of LBP-X, vitamin E 0.05~0.35V/V%, MEM culture medium 99.65~ 99.95V/V%, 1~10W/V% of glucose；
(4) recovery reagent 4：5~25W/V% of LBP-X, vitamin E 0.05~0.35V/V%, MEM culture medium 99.65~ 99.95V/V%, 1~10W/V% of glucose, the μ g/L of VEGF 1~10.
6. the reagent set described in claim 4 or 5 pet ovary tissue recovery in purposes.
7. a kind of cryopreservation resuscitation reagent set, including freezing described in reagent set and claim 4 or 5 described in claim 1 or 2 Recovery reagent set.
8. a kind of pet ovary tissue cryopreservation methods, including：In vitro pet ovary tissue is immersed into the institute of claim 1 or 2 successively State freeze reagent 1, freeze reagent 2, freeze reagent 3 in keep more than 5 minutes after, liquid nitrogen store.
9. a kind of pet ovary tissue method for resuscitation, including：In vitro pet ovary tissue through freezing is immersed into right successively will Ask in recovery reagent 1, the recovery reagent 2 described in 4 or 5, respectively kept at 37 ± 2 DEG C more than 2 minutes, afterwards, move into claim 4 Or in recovery reagent 3, the recovery reagent 4 described in 5, it is each at room temperature to be kept for more than 10 minutes.
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RU2678106C2 (en) * 2018-06-08 2019-01-23 Федеральное государственное бюджетное учреждение "Национальный медицинский исследовательский центр радиологии" Министерства здравоохранения Российской Федерации (ФГБУ "НМИЦ радиологии" Минздрава России) Method of vitrification of ovarial tissue
CN113207869A (en) * 2018-03-26 2021-08-06 首都医科大学附属北京妇产医院 Cryopreservation protective solution for ovarian tissues

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108641999A (en) * 2018-03-26 2018-10-12 阮祥燕 A kind of ovary tissue freeze and method for resuscitation
CN113207869A (en) * 2018-03-26 2021-08-06 首都医科大学附属北京妇产医院 Cryopreservation protective solution for ovarian tissues
CN113207869B (en) * 2018-03-26 2022-06-07 首都医科大学附属北京妇产医院 Cryopreservation protective solution for ovarian tissues
RU2678106C2 (en) * 2018-06-08 2019-01-23 Федеральное государственное бюджетное учреждение "Национальный медицинский исследовательский центр радиологии" Министерства здравоохранения Российской Федерации (ФГБУ "НМИЦ радиологии" Минздрава России) Method of vitrification of ovarial tissue

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