CN110074097A - A kind of cryopreservation resuscitation solution system saving ovarian activity - Google Patents
A kind of cryopreservation resuscitation solution system saving ovarian activity Download PDFInfo
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- CN110074097A CN110074097A CN201910478788.XA CN201910478788A CN110074097A CN 110074097 A CN110074097 A CN 110074097A CN 201910478788 A CN201910478788 A CN 201910478788A CN 110074097 A CN110074097 A CN 110074097A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
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Abstract
A kind of cryopreservation resuscitation solution system of preservation ovarian activity of the invention, be using concentration be 10~100g/L glycerin gelatine compound water solution as basis solution, glycerol in glycerin gelatine compound: the mass ratio of gelatin is 10: 1, monarda glycoside, growth factor, palmityl tripeptides -1, heparin poloxamer and vitamin C are added in base soln, ovary is placed in the solution system and is frozen, the biological activity after Cryopreservation recovery can be improved.
Description
Technical field
The present invention relates to a kind of method of Cryopreservation activity recovery, in particular to freezing for ovary saves solution and its makes
Use method.
Background technique
Ovary is the reproductive organs of jenny.Ovary microenvironment refers to the tissue of the situation of reaching maturity of ovary, ovary
Can the quantity for the primordial follicle that whether eucaryotic cell structure is complete, contains in ovary, primordial follicle reach maturity and ovulate, ovary is starched
Can the thickness and mature egg of film layer be smoothly discharged, the inflammation of pelvic cavity and adhesion situation, ovarian hormonal secretion cell function, body
The level and ratio of interior sex hormone, nerve ending function and adjust situation, Microvasculature function and blood supply (supply oxygen
Gas, nutriment exclude products of cellular metabolism) general designation of situation etc., these factors collectively form ovary microenvironment.Ovary micro-loop
Border affects whether discharge ovarian follicle is mature, and conditioning ovary microenvironment helps to improve discharge ovarian follicle quality, improves pregnancy chance.
Ovary tissue Cryopreservation Technology is most potential and of greatest concern one of the technology for guaranteeing female fertility.It is freezing
In the process, it is the bottleneck for being most difficult to capture that maintenance ovary, which is microenvironment,.The ovary cryop reservation liquid having been reported is frequently with dimethyl
The cryoprotectors such as sulfoxide (DMSO), propylene glycol, sucrose, trehalose, glycerol, animal blood serum, but this kind of cryoprotector effect
It is undesirable and have cytotoxicity (such as DMSO) or potential safety issue (such as animal blood serum).Therefore, study it is a kind of nontoxic and
The high cryoprotector of cell survival rate, with regard to becoming an ovarian freezing field problem urgently to be resolved.With non-toxic and guarantor
Deposit cell activity, it has also become cryoprotector area research hot spot in recent years.
Cryoprotector is avoiding the freezing injury in human ovarian tissue refrigerating process from playing a significant role, but its own
There are histocyte toxicity, and during freezing pre-equilibration, ovary tissue (30-90 minutes) is placed in cryoprotection for a long time
In agent, due to the overlong time of balance, cryoprotector often makes ovary tissue by cytotoxic damage, causes ovum female thin
The decline of born of the same parents' developmental potency.
Although by freezen protective ovary carry out embryo IVC succeeded, thaw after egg mother cell quantity and
Quality sharp fall, causes the significant wastage of egg mother cell, and oocyte maturation rate and Embryo viability are lower.
Although having developed a variety of Cryosreservation solutions at present, its holding time and recovery activity are undesirable, are badly in need of
Develop a kind of preservation solution system of biological activity that can keep ovary for a long time.
In order to meet the needs of clinical treatment, the ideal cryopreservation resuscitation solution system needs for saving ovarian activity meet with
Lower requirement: (1) be conducive to safeguard the microenvironment of ovary；(2) formulation components are highly-safe, and preparation is convenient, cooperate freezen protective side
Formula application, holding time are long；(3) it is easy cleaned removal, side effect will not be generated to body, and do not influence the normal function of ovary
Energy and bioactivity；(4) the ovarian activity recovery frozen is fast, and egg mother cell quantity and quality are guaranteed after defrosting.
Have not yet to see the report for meeting the ovary cryop reservation solution system of all of above requirement.
Summary of the invention
It is (i.e. existing to freeze that protect solution not to be able to maintain ovary long that the purpose of the present invention is to overcome the shortcomings of the existing technology
Phase freeze after biological activity, the problem of influencing egg mother cell quantity and quality), the cryopreservation resuscitation for saving ovarian activity is provided
Solution system to keep biological activity after ovary Long-term Cryopreservation to provide maximum microenvironment guarantee for a long time, while meeting reason
The requirements of the cryopreservation resuscitation solution system for the preservation ovarian activity thought, for realize clinical treatment safely, effectively, it is convenient,
Economy lays the foundation.
A kind of cryopreservation resuscitation solution system of preservation ovarian activity of the invention is the glycerol for being 10~100g/L with concentration
Gelatin-compounded object aqueous solution is used as basic solution, glycerol in glycerin gelatine compound: the mass ratio of gelatin is 10: 1, base soln
Middle addition monarda glycoside, growth factor, palmityl tripeptides -1, heparin poloxamer and vitamin C are fragrant in the base soln
The concentration of beeweed glycosides is 0.001g/L~0.5g/L, the concentration of growth factor is 0.001g/L~0.05g/L, palmityl tripeptides -1
Concentration be 0.001g/L~0.5g/L, concentration 10g/L~60g/L of heparin poloxamer, ascorbic concentration 0.001g/L
~0.5g/L.
Above-mentioned solution system be using concentration be 20~50g/L glycerin gelatine compound water solution as basis solution,
In the base soln concentration of monarda glycoside be 0.005g/L~0.01g/L, the concentration of growth factor be 0.005g/L~
0.01g/L, palmityl tripeptides -1 concentration be 0.01g/L~0.2g/L, concentration 15g/L~30g/L of heparin poloxamer, dimension
Concentration 0.05g/L~0.2g/L of raw element C.
Polyalcohol can further be added in above-mentioned base soln, comprising: ethylene glycol, propylene glycol, polyethylene glycol, sweet dew
Alcohol, sorbierite, maltitol, xylitol, lactitol.
Sugar can further be added in above-mentioned base soln, comprising: sucrose, lactose, galactolipin, ribose, deoxyribose,
Maltose, glucose, fructose, trehalose, sorbose, xylose, raffinose, mannose, starch, dextrin.
Above-mentioned growth factor, comprising: gonad granulocyte hormone, vascular endothelial growth factor, fibroblastic growth
The factor, neurotrophic factor, transforming growth factor.
The preferred basic fibroblast growth factor of above-mentioned growth factor.
Above-mentioned freezes the refrigerating process referred to using 0 DEG C of following temperature.
A kind of cryopreservation resuscitation solution system of above-mentioned preservation ovarian activity the preparation method comprises the following steps:
(1) gelatin is placed in container, 2 times of gelatin quality of water is added and impregnates, is sufficiently swollen in 100 DEG C of water-baths of postposition and adds
Heat makes to dissolve, and glycerol is added after completely dissolution, stirs evenly, and temperature is down to 15 DEG C, forms glycerin gelatine compound water solution；
(2) heparin poloxamer is dissolved in the water of 4 times of quality, and monarda glycoside, growth factor and palmityl tripeptides-are added
1, stir evenly, glycerin gelatine compound water solution be added, is added vitamin C after mixing, mix save ovarian activity
Cryopreservation resuscitation solution system.
The common osmotic pressure of pharmacy can further be added in the cryopreservation resuscitation solution system of above-mentioned preservation ovarian activity
Regulator, electrolyte, antioxidant, bacteriostatic agent.
The cryopreservation resuscitation solution system of above-mentioned preservation ovarian activity saves in 0~4 DEG C of light protected environment.
A kind of cryopreservation resuscitation solution system of preservation ovarian activity of the invention is compared with existing Ovary storage solution, tool
It has the advantage that 1. without using any reagents for having potential security risk such as DMSO, animal blood serums, will not generate because reagent remains
Caused toxicity；2. being conducive to safeguard the microenvironment of ovary；3. formulation components are highly-safe, preparation is convenient, cooperation freezing
Preserving type application, holding time are long；4. being easy cleaned removal, side effect will not be generated to body, and do not influence ovary
Normal function and bioactivity；5. the ovarian activity recovery frozen is fast, and egg mother cell quantity and quality are guaranteed after defrosting.
Specific embodiment
The specific embodiment of the invention is discussed in detail below.It should be noted that technology described in following embodiments is special
The combination of sign or technical characteristic is not construed as isolated, they can be combined with each other, to reach better skill
Art effect.
Embodiment 1 saves the preparation of the cryopreservation resuscitation solution system of ovarian activity
According to the component ratio of table 1, the cryopreservation resuscitation solution system for saving ovarian activity is prepared.
Save ovarian activity cryopreservation resuscitation solution system the preparation method is as follows:
(1) gelatin for weighing specified amount, is placed in appropriate vessel, and 2 times of gelatin quality of water is added and impregnates, sufficiently after swelling
Setting heating in 100 DEG C of water-baths makes to dissolve, and glycerol is added after completely dissolution, stirs evenly, and temperature is down to 15 DEG C, forms glycerin gelatine
Compound water solution；
(2) heparin poloxamer is dissolved in the water of 4 times of quality, and monarda glycoside, growth factor and palmityl tripeptides-are added
1, stir evenly, glycerin gelatine compound water solution be added, is added vitamin C after mixing, mix save ovarian activity
Cryopreservation resuscitation solution system.
Each experimental group be according to the component and proportional arrangement in the claim of this application item protection scope, and it is each right
It is the sample that a certain component missing, replacement or concentration of component exceed the claim of this application item protection scope according to group.
Table 1 saves the experimental group and control group composition of the cryopreservation resuscitation solution system of ovarian activity
Note: "/" represents this component and is not present, and " * " represents this component (column name component) and replaced by the component in table
It changes.
The Cryopreservation activity rating of 2 sheep ovary of embodiment
Each experimental group and control group solution prepared by embodiment 1 freezes and resuscitation process by following, then measurement recovery
Sheep ovarian activity afterwards, the application effect appraisal of the cryopreservation resuscitation solution system for saving ovarian activity.
Freeze: it is cold that the good sheep ovary of separating treatment is put into the 50mL equipped with promotion Cryopreservation activity recovery solution respectively
Freeze in bag, sequencing is refrigerated to -90 DEG C, keeps 5min, then stores freezing in bag investment liquid nitrogen equipped with ovary.
Recovery: each group sample takes out from liquid nitrogen after a week, is placed in 37 DEG C of water-baths and melts to mixture of ice and water, the moment shakes straight
Melt to complete.
Determination of activity: according to document [Fundamentals of cryobiology in reproductive
Medicine.Reprod Biomed Online.2004,9 (6): 680-691. and Whole sheep ovary
Cryopreservation:evaluation of a slow freezing protocol with
Dimethylsulphoxide.J Assist Reprod Genet.2011,28 (1): 7-14.], by ovary tissue form,
The indexs such as primordial follicle counts, apoptotic cell dyes, evaluation save the cryopreservation resuscitation solution application effect of ovarian activity.With normal
Ovary is control (indices full marks), is scored to each group, and effect is higher, and scoring is higher, and full marks 10 divide.Experimental group and control group
The cryopreservation resuscitation solution system of preservation ovarian activity 2 the results are shown in Table for the application effect appraisal of sheep ovary.
The sheep ovary application effect appraisal result of 2 experimental group of table and control group
Tissue morphology observation indicate that, egg mother cell is spherical in shape in the sheep ovary of experimental group, includes the thin of complete circle
Karyon, the monolayer of particles cell wrapping being around evenly distributed.And ovarian follicle shows as karyopyknosis in the sheep ovary of control group,
Basilar memebrane is imperfect, neighboring particles cell arrangement disorder.
Primordial follicle count results show that original Follicle number is more in the sheep ovary of experimental group, normal morphology primordial follicle
Ratio is higher than 90%, close to 94% ratio normally organized.And original Follicle number is few in the sheep ovary of control group, normal morphology
Primordial follicle ratio is lower than 80%, hence it is evident that is different from experimental group.
Apoptotic cell coloration result further shows that the apoptosis rate of the sheep ovary cortex part of experimental group is obviously low
In control group, show that group subassembly of the invention is conducive to safeguard ovary microenvironment.
In terms of comprehensive score, 2 groups and 8 groups of effect is preferable in experimental group, followed by 7 groups, 6 groups and 1 group.And control group is bright
Aobvious to be inferior to experimental group, wherein 4 groups of effect is worst, followed by 9,10,12,17,18 groups, remaining control group result is close.
Above-mentioned detailed description is illustrating for the possible embodiments invented, and the embodiment is not to limit this hair
Bright the scope of the patents, it is all without departing from equivalence enforcement or change of the invention, it should all be contained in the scope of the patents of the invention.Separately
Outside, those skilled in the art can also do each in other forms and details in the claims in the present invention scope of disclosure and spirit
Kind modifies, adds and replaces.Certainly, the variation such as these spirit is done according to the present invention various modifications, addition and replacements, is all answered
Comprising within scope of the present invention.
Claims (10)
1. a kind of cryopreservation resuscitation solution system for saving ovarian activity, be primarily characterized in that: the solution system is with dense
The glycerin gelatine compound water solution that degree is 10~100g/L is used as basic solution, glycerol in the glycerin gelatine compound:
The mass ratio of gelatin is 10: 1, and monarda glycoside, growth factor, palmityl tripeptides -1, heparin pool are added in the base soln
Luo Shamu and vitamin C, in the base soln concentration of monarda glycoside be 0.001g/L~0.5g/L, growth factor it is dense
Degree is 0.001g/L~0.05g/L, the concentration of palmityl tripeptides -1 is 0.001g/L~0.5g/L, the concentration of heparin poloxamer
10g/L~60g/L, ascorbic concentration 0.001g/L~0.5g/L.
2. a kind of cryopreservation resuscitation solution system for saving ovarian activity according to claim 1, is primarily characterized in that: described
Solution system be using concentration be 20~50g/L glycerin gelatine compound water solution as basis solution, the base soln
The concentration of middle monarda glycoside is 0.005g/L~0.01g/L, the concentration of growth factor is 0.005g/L~0.01g/L, palmityl
The concentration of tripeptides -1 is 0.01g/L~0.2g/L, concentration 15g/L~30g/L of heparin poloxamer, ascorbic concentration
0.05g/L~0.2g/L.
3. a kind of cryopreservation resuscitation solution system for saving ovarian activity according to claim 1, it is characterized in that: the basis is molten
Polyalcohol can further be added in liquid, comprising: ethylene glycol, propylene glycol, polyethylene glycol, mannitol, sorbierite, maltitol,
Xylitol, lactitol.
4. a kind of cryopreservation resuscitation solution system for saving ovarian activity according to claim 1, it is characterized in that: the basis is molten
Sugar can further be added in liquid, comprising: sucrose, lactose, galactolipin, ribose, deoxyribose, maltose, glucose, fructose,
Trehalose, sorbose, xylose, raffinose, mannose, starch, dextrin.
5. it is according to claim 1 it is a kind of save ovarian activity cryopreservation resuscitation solution system, it is characterized in that: the growth because
Son, comprising: gonad granulocyte hormone, fibroblast growth factor, neurotrophic factor, turns vascular endothelial growth factor
Change growth factor.
6. it is according to claim 5 it is a kind of save ovarian activity cryopreservation resuscitation solution system, it is characterized in that: the growth because
The preferred basic fibroblast growth factor of son.
7. the cryopreservation resuscitation solution system of a kind of preservation ovarian activity according to claim 1~6, it is characterized in that: the jelly
Deposit the refrigerating process referred to using 0 DEG C of following temperature.
8. the cryopreservation resuscitation solution system preparation method of the preservation ovarian activity of a kind of claim 1~6, it is characterized in that: preparation
Method are as follows:
(1) gelatin is placed in container, 2 times of gelatin quality of water is added and impregnates, being sufficiently swollen to heat in 100 DEG C of water-baths of postposition makes
Dissolution, glycerol is added after completely dissolution, stirs evenly, and temperature is down to 15 DEG C, forms glycerin gelatine compound water solution；
(2) heparin poloxamer is dissolved in the water of 4 times of quality, and monarda glycoside, growth factor and palmityl tripeptides -1 are added, stir
Mix uniformly, glycerin gelatine compound water solution be added, is added vitamin C after mixing, mix save freezing for ovarian activity
Recovery solution system.
9. a kind of cryopreservation resuscitation solution system preparation method for saving ovarian activity according to claim 8, it is characterized in that: described
Preservation ovarian activity cryopreservation resuscitation solution system in the common osmotic pressure regulator of pharmacy, electrolysis can further be added
Matter, antioxidant, bacteriostatic agent.
10. a kind of cryopreservation resuscitation solution system preparation method for saving ovarian activity according to claim 8, it is characterized in that: institute
The cryopreservation resuscitation solution system for the preservation ovarian activity stated saves in 0~4 DEG C of light protected environment.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110352951A (en) * | 2018-11-15 | 2019-10-22 | 崔磊 | A kind of serum-free is without DMSO tissue engineered bone frozen stock solution and its preparation and cryopreservation methods |
| CN111919835A (en) * | 2020-08-06 | 2020-11-13 | 温州医科大学 | Preservation solution for maintaining activity of red blood cells |
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| WO2004019679A1 (en) * | 2001-05-29 | 2004-03-11 | I.M.T. Interface Multigrad Technology Ltd | Methods of preserving the functionality of an organ. |
| CN100999722A (en) * | 2007-01-08 | 2007-07-18 | 安徽省立医院 | In-vitro cultivating matural process of immatural ovocyte in ovarium organized block |
| CN108753683A (en) * | 2018-05-26 | 2018-11-06 | 温州医科大学 | A kind of solution system promoting cryopreserved tissue organ and cell activity recovery |
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2019
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Patent Citations (4)
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| JPH05112401A (en) * | 1991-10-17 | 1993-05-07 | Kyodo Shiryo Kk | Preservation of mammalian ovarium |
| WO2004019679A1 (en) * | 2001-05-29 | 2004-03-11 | I.M.T. Interface Multigrad Technology Ltd | Methods of preserving the functionality of an organ. |
| CN100999722A (en) * | 2007-01-08 | 2007-07-18 | 安徽省立医院 | In-vitro cultivating matural process of immatural ovocyte in ovarium organized block |
| CN108753683A (en) * | 2018-05-26 | 2018-11-06 | 温州医科大学 | A kind of solution system promoting cryopreserved tissue organ and cell activity recovery |
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| Title |
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| AYAKA TANAKA等: "Effect of sustained release of basic fibroblast growth factor using biodegradable gelatin hydrogels on frozen-thawed human ovarian tissue in a xenograft model", 《THE JOURNAL OF OBSTETRICS AND GYNAECOLOGY RESEARCH》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110352951A (en) * | 2018-11-15 | 2019-10-22 | 崔磊 | A kind of serum-free is without DMSO tissue engineered bone frozen stock solution and its preparation and cryopreservation methods |
| CN111919835A (en) * | 2020-08-06 | 2020-11-13 | 温州医科大学 | Preservation solution for maintaining activity of red blood cells |
| CN111919835B (en) * | 2020-08-06 | 2022-02-08 | 温州医科大学 | Preservation solution for maintaining activity of red blood cells |
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