CN106818707A - Pet testis tissue cryopreservation resuscitation reagent and cryopreservation resuscitation method - Google Patents
Pet testis tissue cryopreservation resuscitation reagent and cryopreservation resuscitation method Download PDFInfo
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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
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- 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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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- 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
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Abstract
The present invention provides a kind of pet testis tissue and freezes reagent set, including：(1) reagent 1 is frozen：2~7W/V% of LBP-X, 0.5~5W/V% of cholesterol, 6~14V/V% of dimethyl sulfoxide (DMSO), 80~90V/V% of propane diols 2~7V/V%, MEM culture medium；(2) reagent 2 is frozen：8~10W/V% of LBP-X, 0.5~5W/V% of cholesterol, 6~20V/V% of dimethyl sulfoxide (DMSO), 70~80V/V% of propane diols 5~15V/V%, MEM culture medium；(3) reagent 3 is frozen：15~20W/V% of LBP-X, 0.5~5W/V% of cholesterol, 6~20V/V% of dimethyl sulfoxide (DMSO), 70~80V/V% of propane diols 5~15V/V%, MEM culture medium.The present invention also provides a kind of pet testis tissue recovery reagent set and pet testis tissue freezes and method for resuscitation.
Description
Technical field
The present invention relates to testis tissue freezen protective, more particularly it relates to pet testis tissue freezes and answers
Soviet Union's reagent and pet testis 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 castration of pet refers to the testis and its affiliated group's enucleation of male pet.For individual pets, castration
Operation can reduce the probability that male pet occurs hyperplasia of prostate and infection, reduce the incidence of perineocele.
However, after Testectomy, at present all abandoning testis 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, and can also increase the incidence of osteosarcoma.
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, testis tissue Cryopreservation Technology, spermatogonium Differentiation Induction in vitro technology and interstitial tissue[of testis] are thin
Born of the same parents' Vitro Culture Techniques are evolving.Preferable mode is to preserve fecundity by Cryopreservation Technology, optional when being necessary
The vitro differentiation of selecting property goes out sperm to recover fecundity, and amplification interstitial cell can be used for autologous feedback and recover hormonal readiness.Pet
The such Cryopreservation Technology of 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 testis 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 testis tissue recovery reagent comprising pet testis tissue, and definite ingredients stabilization is protected
The matter phase is long, and freezing and recovering for pet testis 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 first organize glass frozen preservation, effect is remote in pet testis tissue freezes
Better than traditional sucrose (SUC)；For testis tissue characteristic, freeze and recovery reagent in add cholesterol (CHO), larger journey
The activity for protecting spermatogonium and sperm in testis tissue of degree.
The first aspect of the present invention, there is provided pet testis tissue freezes reagent set.
The pet testis tissue freezes reagent set to be included：
(1) reagent 1 is frozen：2~7W/V% of LBP-X, 0.5~5W/V% of cholesterol, 6~14V/ of dimethyl sulfoxide (DMSO)
V%, 80~90V/V% of propane diols 2~7V/V%, MEM culture medium；
(2) reagent 2 is frozen：8~10W/V% of LBP-X, 0.5~5W/V% of cholesterol, 6~20V/ of dimethyl sulfoxide (DMSO)
V%, 70~80V/V% of propane diols 5~15V/V%, MEM culture medium；
(3) reagent 3 is frozen：15~20W/V% of LBP-X, 0.5~5W/V% of cholesterol, 6~20V/ of dimethyl sulfoxide (DMSO)
V%, 70~80V/V% of propane diols 5~15V/V%, MEM culture medium.
Preferably, the reagent set that freezes includes：
(1) reagent 1 is frozen：2~7W/V% of LBP-X, 0.5~5W/V% of cholesterol, 6~14V/ of dimethyl sulfoxide (DMSO)
V%, propane diols 2~7V/V%, MEM 80~90V/V% of culture medium, 0.1~0.5W/V% of polyvinylpyrrolidone, glucose 1
~10W/V%；
(2) reagent 2 is frozen：8~10W/V% of LBP-X, 0.5~5W/V% of cholesterol, 6~20V/ of dimethyl sulfoxide (DMSO)
V%, propane diols 5~15V/V%, MEM 70~80V/V% of culture medium, 0.1~0.5W/V% of polyvinylpyrrolidone, glucose 1
~10W/V%；
(3) reagent 3 is frozen：15~20W/V% of LBP-X, 0.5~5W/V% of cholesterol, 6~20V/ of dimethyl sulfoxide (DMSO)
V%, propane diols 5~15V/V%, MEM 70~80V/V% of culture medium, 0.1~0.5W/V% of polyvinylpyrrolidone, glucose 1
~10W/V%.
Preferably, the reagent set that freezes includes：
(1) reagent 1 is frozen：LBP-X 6W/V%, cholesterol 2.5W/V%, dimethyl sulfoxide (DMSO) 10V/V%, propane diols
5V/V%, MEM culture medium 85V/V%；
(2) reagent 2 is frozen：LBP-X 9W/V%, cholesterol 2.5W/V%, dimethyl sulfoxide (DMSO) 12V/V%, propane diols
13V/V%, MEM culture medium 75V/V%；
(3) reagent 3 is frozen：LBP-X 18W/V%, cholesterol 2.5W/V%, dimethyl sulfoxide (DMSO) 12V/V%, propane diols
13V/V%, MEM culture medium 75V/V%.
More excellent, the reagent set that freezes includes：
(1) reagent 1 is frozen：LBP-X 6W/V%, cholesterol 2.5W/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%, cholesterol 2.5W/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%, cholesterol 2.5W/V%, dimethyl sulfoxide (DMSO) 12V/V%, propane diols
13V/V%, MEM culture medium 75V/V%, polyvinylpyrrolidone 0.3W/V%, glucose 5W/V%.
The second aspect of the present invention, there is provided pet testis tissue recovery reagent set, the recovery reagent set includes：
(1) recovery reagent 1：35~55W/V% of LBP-X, 0.5~5W/V% of cholesterol, vitamin E 0.05~
99.65~99.95V/V% of 0.35V/V%, MEM culture medium；
(2) recovery reagent 2：25~45W/V% of LBP-X, 0.5~5W/V% of cholesterol, vitamin E 0.05~
99.65~99.95V/V% of 0.35V/V%, MEM culture medium；
(3) recovery reagent 3：15~35W/V% of LBP-X, 0.5~5W/V% of cholesterol, vitamin E 0.05~
99.65~99.95V/V% of 0.35V/V%, MEM culture medium；
(4) recovery reagent 4：5~25W/V% of LBP-X, 0.5~5W/V% of cholesterol, vitamin E 0.05~
99.65~99.95V/V% of 0.35V/V%, MEM culture medium.
Preferably, the recovery reagent set includes：
(1) recovery reagent 1：35~55W/V% of LBP-X, 0.5~5W/V% of cholesterol, vitamin E 0.05~
0.35V/V%, MEM 99.65~99.95V/V% of culture medium, 0.1~0.5W/V% of polyvinylpyrrolidone, glucose 1~
10W/V%；
(2) recovery reagent 2：25~45W/V% of LBP-X, 0.5~5W/V% of cholesterol, vitamin E 0.05~
0.35V/V%, MEM 99.65~99.95V/V% of culture medium, 0.1~0.5W/V% of polyvinylpyrrolidone, glucose 1~
10W/V%；
(3) recovery reagent 3：15~35W/V% of LBP-X, 0.5~5W/V% of cholesterol, vitamin E 0.05~
0.35V/V%, MEM 99.65~99.95V/V% of culture medium, 1~10W/V% of glucose；
(4) recovery reagent 4：5~25W/V% of LBP-X, 0.5~5W/V% of cholesterol, vitamin E 0.05~
0.35V/V%, MEM 99.65~99.95V/V% of culture medium, 1~10W/V% of glucose.
Preferably, the recovery reagent set includes：
(1) recovery reagent 1：LBP-X 40W/V%, cholesterol 2.5W/V%, vitamin E 0.2V/V%, MEM culture medium
99.8%；
(2) recovery reagent 2：LBP-X 30W/V%, cholesterol 2.5W/V%, vitamin E 0.2V/V%, MEM culture medium
99.8%；
(3) recovery reagent 3：LBP-X 20W/V%, cholesterol 2.5W/V%, vitamin E 0.2V/V%, MEM culture medium
99.8%；
(4) recovery reagent 4：LBP-X 10W/V%, cholesterol 2.5W/V%, vitamin E 0.2V/V%, MEM culture medium
99.8%.
More excellent, the recovery reagent set includes：
(1) recovery reagent 1：LBP-X 40W/V%, cholesterol 2.5W/V%, vitamin E 0.2V/V%, MEM culture medium
99.8%, polyvinylpyrrolidone 0.3W/V%, glucose 5W/V%；
(2) recovery reagent 2：LBP-X 30W/V%, cholesterol 2.5W/V%, vitamin E 0.2V/V%, MEM culture medium
99.8%, polyvinylpyrrolidone 0.3W/V%, glucose 5W/V%；
(3) recovery reagent 3：LBP-X 20W/V%, cholesterol 2.5W/V%, vitamin E 0.2V/V%, MEM culture medium
99.8%, glucose 5W/V%；
(4) recovery reagent 4：LBP-X 10W/V%, cholesterol 2.5W/V%, vitamin E 0.2V/V%, MEM culture medium
99.8%, glucose 5W/V%.
The third aspect of the invention is to provide a kind of pet testis 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 testis tissue cryopreservation methods, including：By in vitro pet testis
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 testis tissue method for resuscitation, including：Will be in vitro through what is frozen
Pet testis 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 testis tissue short-term (about a week), recover reaches
To more than 80%, long-term (about three years) freeze, the motility rate recovered reaches more than 55%, can be good at freezen protective fecundity,
To the chance of pet second selecting.LBP-X is introduced freeze field first by inventor, and vitamin E is introduced into recovery examination first
Agent, achieves the effect of highly significant.Additionally, for the physiological conditions containing abundant cholesterol in spermatogonium and sperm,
Cholesterol is added in frozen stock solution and resuscitation fluid prevents intracellular cholesterol from losing and influenceing cytoactive.Three kinds of material collaborations
Effect, achieves the synergy of highly significant for being frozen for pet testis tissue, being recovered.Agent formulations of the present invention are clear and definite,
It is convenient to prepare, beneficial to large-scale production.
All the time, tissue glass frozen preservation field focuses on, for different cell and tissue, finding toxicity smaller
Vitrification solution mix proportion scheme and improve drop, the method for rewarming rate.By being combined two kinds of cell-protectings two in the present invention
Methyl sulfoxide 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 testis tissue freezes the preparation of reagent
(1) so that 100ml freezes the preparation of 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), cholesterol (purchased from SIGMA), ((abbreviation PVP) is purchased polyvinylpyrrolidone
From SIGMA), during glucose (be purchased from SIGMA) adds above-mentioned bottle, rock mixing.With 0.22 μm of filter after solid all dissolves
(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), and 4 DEG C of preservations are standby
With；
(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), cholesterol (purchased from SIGMA), polyvinylpyrrolidone (purchased from SIGMA),
Glucose (being purchased from SIGMA) is added in above-mentioned bottle, rocks mixing.(it is purchased from 0.22 μm of filter after after solid all dissolving
MILLIPORE) filtration sterilization, to by autoclaved 100ml indigo plant mouths bottle (being purchased from another name for Sichuan Province ox), 4 DEG C save backup for filtering；
(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), cholesterol (purchased from SIGMA), polyvinylpyrrolidone (purchased from SIGMA),
Glucose (being purchased from SIGMA) is added in above-mentioned bottle, rocks mixing.(it is purchased from 0.22 μm of filter after after solid all dissolving
MILLIPORE) filtration sterilization, to by autoclaved 100ml indigo plant mouths bottle (being purchased from another name for Sichuan Province ox), 4 DEG C save backup for filtering.
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 testis 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, measure vitamin E (oily liquids,
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), and jog is mixed
It is even；Weigh LBP-X (biological purchased from Si Nuote), cholesterol (purchased from SIGMA), polyvinylpyrrolidone (purchased from SIGMA),
Glucose (being purchased from SIGMA) is added in above-mentioned bottle, rocks mixing.(it is purchased from 0.22 μm of filter after after solid all dissolving
MILLIPORE) filtration sterilization, 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.Weigh LBP-X (purchase
It is biological from Si Nuote), cholesterol (being purchased from SIGMA), polyvinylpyrrolidone (being purchased from SIGMA), glucose (being purchased from SIGMA) plus
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) 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；Weigh LBP-X (purchase
It is biological from Si Nuote), during cholesterol (being purchased from SIGMA), glucose (being purchased from SIGMA) adds above-mentioned bottle, rock mixing.Treat solid
All filtered to by autoclaved 100ml indigo plant mouth bottles with 0.22 μm of filter (being purchased from MILLIPORE) filtration sterilization after dissolving
(being purchased from another name for Sichuan Province ox), 4 DEG C save backup；
(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；Weigh LBP-X (purchase
It is biological from Si Nuote), during cholesterol (being purchased from SIGMA), glucose (being purchased from SIGMA) adds above-mentioned bottle, rock mixing.Treat solid
All filtered to by autoclaved 100ml indigo plant mouth bottles with 0.22 μm of filter (being purchased from MILLIPORE) filtration sterilization after dissolving
(being purchased from another name for Sichuan Province ox), 4 DEG C save backup.
Pet testis 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：Except freeze and recovery reagent in addition to Cholesterol removal, other conditions and embodiment 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：Reagent is frozen to be cultivated including 95%DMEM
Base, 5%DMSO；Recovery reagent is DMEM culture mediums, and concrete operation step is shown in that (in clean, all things on earth tiny stream, Yin Mei Jun waits to freeze to document
Grow [J] China andrology magazine, 2008,22 (12) of androgone after preservation Immature testicle tissue transplantation:15-
18.).Table 3：
| Control A groups | Control B groups | Control C groups | ||
| Freeze reagent 1 | DMSO(ml) | 10 | 10 | 10 |
| Propane diols (ml) | 5 | 5 | 5 | |
| MEM culture mediums (ml) | 85 | 85 | 85 | |
| LBP-X/sucrose (g) | 6 (LBP-Xs) | 6 (sucrose) | 6 (sucrose) | |
| Cholesterol (g) | - | - | 2.5 | |
| PVP(g) | 0.3 | 0.3 | 0.3 | |
| Glucose (g) | 5 | 5 | 5 | |
| Freeze reagent 2 | DMSO(ml) | 12 | 12 | 12 |
| Propane diols (ml) | 13 | 13 | 13 | |
| MEM culture mediums (ml) | 75 | 75 | 75 | |
| LBP-X/sucrose (g) | 9 (LBP-Xs) | 9 (sucrose) | 9 (sucrose) | |
| Cholesterol (g) | - | - | 2.5 | |
| PVP(g) | 0.3 | 0.3 | 0.3 | |
| Glucose (g) | 5 | 5 | 5 | |
| Freeze reagent 3 | DMSO(ml) | 12 | 12 | 12 |
| Propane diols (ml) | 13 | 13 | 13 | |
| MEM culture mediums (ml) | 75 | 75 | 75 | |
| LBP-X/sucrose (g) | 18 (LBP-Xs) | 18 (sucrose) | 18 (sucrose) | |
| Cholesterol (g) | - | - | 2.5 | |
| PVP(g) | 0.3 | 0.3 | 0.3 | |
| Glucose (g) | 5 | 5 | 5 | |
| Recovery reagent 1 | Vitamin E (ml) | 0.2 | 0.2 | 0.2 |
| MEM culture mediums (ml) | 99.8 | 99.8 | 99.8 | |
| LBP-X/sucrose (g) | 40 (LBP-Xs) | 40 (sucrose) | 40 (sucrose) | |
| Cholesterol (g) | - | - | 2.5 | |
| PVP(g) | 0.3 | 0.3 | 0.3 | |
| Glucose (g) | 5 | 5 | 5 | |
| Recovery reagent 2 | Vitamin E (ml) | 0.2 | 0.2 | 0.2 |
| MEM culture mediums (ml) | 99.8 | 99.8 | 99.8 | |
| LBP-X/sucrose (g) | 30 (LBP-Xs) | 30 (sucrose) | 30 (sucrose) | |
| Cholesterol (g) | - | - | 2.5 | |
| PVP(g) | 0.3 | 0.3 | 0.3 | |
| Glucose (g) | 5 | 5 | 5 | |
| Recovery reagent 3 | Vitamin E (ml) | 0.2 | 0.2 | 0.2 |
| MEM culture mediums (ml) | 99.8 | 99.8 | 99.8 | |
| LBP-X/sucrose (g) | 20 (LBP-Xs) | 20 (sucrose) | 20 (sucrose) | |
| Cholesterol (g) | - | - | 2.5 | |
| PVP(g) | - | - | - | |
| Glucose (g) | 5 | 5 | 5 | |
| Recovery reagent 4 | Vitamin E (ml) | 0.2 | 0.2 | 0.2 |
| MEM culture mediums (ml) | 99.8 | 99.8 | 99.8 | |
| LBP-X/sucrose (g) | 10 (LBP-Xs) | 10 (sucrose) | 10 (sucrose) | |
| Cholesterol (g) | - | - | 2.5 | |
| PVP(g) | - | - | - | |
| Glucose (g) | 5 | 5 | 5 |
Effect test：
1. pet testis tissue is frozen, the short run effect of recovery reagent is verified
(1) acquisition of testis tissue：In the case of pet master, pet doctor's informed consent, 6~August age is collected respectively
Pet dog carries out the testis 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 testis tissue：Testis 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) testis tissue freezes and recovery：The random testis 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 testosterone and tissue activity's OD values：By each embodiment in (3), in control group and (2)
The histotomy of fresh control group be respectively implanted in 6 orifice plate of cell culture, 1 hole and cultivate, 2ml10%DMEM culture mediums, 37
DEG C, 5%CO2Cultivated 48 hours in cell culture incubator.500ul supernatants are collected, (is purchased from using testosterone ELISA detection kit
CAYMAN testis tissue testosterone secretion amount) is detected.Histotomy is cut into 1mmX2mmX2mm sizes, inserts in 96 orifice plate, 1 hole,
100ul10%DMEM culture mediums are added, the enhanced CCK-8 solution of 10ul (being purchased from the green skies), 37 DEG C, 5%CO are added per hole2Carefully
Cultivated 1 hour in born of the same parents' incubator.Using multi-function microplate reader, 450nm wavelength is chosen, read OD values.Each experiment repeats three respectively
It is secondary, average as experimental result, experimental result as shown in table 4, the reflection of table 4 freeze 1 week after resuscitation team testosterone secretion work(
Tissue activity's OD values after energy situation and short-term cryopreservation resuscitation.No matter supernatant testosterone secretion situation or tissue activity's OD values, it is real
The result after a cryopreservation resuscitation is applied all close to fresh control group, and each embodiment result is superior to control A groups, control B groups, right
According to C groups and traditional control group, LBP-X of the invention, cholesterol, vitamin E synergy are illustrated, cryopreservation resuscitation is lived
The raising of rate has remarkable result.
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 testis that 6~August age pet cat cut off during sterilization operation using same method
Ball 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, testosterone secretion amount position 12.1nmol/L is as a result shown, tissue activity OD values are 1.61.
2. pet testis tissue is frozen, the long-term effect of recovery reagent is verified
(1) acquisition of testis tissue：In the case of pet master, pet doctor's informed consent, 6~August age is collected respectively
Pet dog carries out the testis 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 testis tissue：Testis 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) testis tissue freezes and recovery：The testis 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 95%DMEM culture mediums, 5%DMSO；Recovery reagent be DMEM culture mediums, concrete operation step see document (in
Clean, all things on earth tiny stream, Yin Mei Jun waits [J] China man that grows of androgone after freezen protective Immature testicle tissue transplantations
Scientific Magazine, 2008,22 (12):15-18.).
(4) detection of tissue culture supernatant testosterone and tissue activity's OD values：The tissue of each embodiment and traditional control group is cut
Piece is cultivated in being respectively implanted 6 orifice plate of cell culture, 1 hole, 2ml 10%DMEM culture mediums, 37 DEG C, in 5%CO2 cell culture incubators
Culture 48 hours.500ul supernatants are collected, testosterone ELISA detection kit (being purchased from CAYMAN) detection testis tissue testosterone is used
Secretory volume.Histotomy is cut into 1mm × 2mm × 2mm sizes, inserts in 96 orifice plate, 1 hole and adds 100ul10%DMEM cultures
Base, the enhanced CCK-8 solution of 10ul (being purchased from the green skies), 37 DEG C, 5%CO are added per hole2Cultivated 1 hour in cell culture incubator.
Using multi-function microplate reader, 450nm wavelength is chosen, read OD values.Each time point is randomly provided 3 tissue samples.Experiment knot
Really as shown in table 5, resuscitation team testosterone secretion function situation and Long-term Cryopreservation recovery after the reflection of table 5 freezes 0.5,1,2,3 years
Tissue activity OD values afterwards.Experimental group effect of the present invention is much better than traditional control group under long-term storage conditions as shown in Table 5, tissue
Preservation effect is relatively stable after being 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, 0.5~5W/V% of cholesterol, dimethyl sulfoxide (DMSO) 6~14V/V%, third
80~90V/V% of glycol 2~7V/V%, MEM culture medium；
(2) reagent 2 is frozen：8~10W/V% of LBP-X, 0.5~5W/V% of cholesterol, dimethyl sulfoxide (DMSO) 6~20V/V%, third
70~80V/V% of glycol 5~15V/V%, MEM culture medium；
(3) reagent 3 is frozen：15~20W/V% of LBP-X, 0.5~5W/V% of cholesterol, 6~20V/V% of dimethyl sulfoxide (DMSO),
70~80V/V% of propane diols 5~15V/V%, MEM culture medium.
2. one kind freezes reagent set, including：
(1) reagent 1 is frozen：2~7W/V% of LBP-X, 0.5~5W/V% of cholesterol, dimethyl sulfoxide (DMSO) 6~14V/V%, third
Glycol 2~7V/V%, MEM 80~90V/V% of culture medium, 0.1~0.5W/V% of polyvinylpyrrolidone, 1~10W/ of glucose
V%；
(2) reagent 2 is frozen：8~10W/V% of LBP-X, 0.5~5W/V% of cholesterol, dimethyl sulfoxide (DMSO) 6~20V/V%, third
Glycol 5~15V/V%, MEM 70~80V/V% of culture medium, 0.1~0.5W/V% of polyvinylpyrrolidone, 1~10W/ of glucose
V%；
(3) reagent 3 is frozen：15~20W/V% of LBP-X, 0.5~5W/V% of cholesterol, 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, glucose 1~
10W/V%.
3. purposes of the reagent set described in claim 1 or 2 in pet testis tissue freezes.
4. a kind of recovery reagent set, including：
(1) recovery reagent 1：35~55W/V% of LBP-X, 0.5~5W/V% of cholesterol, 0.05~0.35V/ of vitamin E
99.65~99.95V/V% of V%, MEM culture medium；
(2) recovery reagent 2：25~45W/V% of LBP-X, 0.5~5W/V% of cholesterol, 0.05~0.35V/ of vitamin E
99.65~99.95V/V% of V%, MEM culture medium；
(3) recovery reagent 3：15~35W/V% of LBP-X, 0.5~5W/V% of cholesterol, 0.05~0.35V/ of vitamin E
99.65~99.95V/V% of V%, MEM culture medium；
(4) recovery reagent 4：5~25W/V% of LBP-X, 0.5~5W/V% of cholesterol, 0.05~0.35V/V% of vitamin E,
99.65~99.95V/V% of MEM culture mediums.
5. a kind of recovery reagent set, including：
(1) recovery reagent 1：35~55W/V% of LBP-X, 0.5~5W/V% of cholesterol, 0.05~0.35V/ of vitamin E
V%, MEM 99.65~99.95V/V% of culture medium, 0.1~0.5W/V% of polyvinylpyrrolidone, 1~10W/V% of glucose；
(2) recovery reagent 2：25~45W/V% of LBP-X, 0.5~5W/V% of cholesterol, 0.05~0.35V/ of vitamin E
V%, MEM 99.65~99.95V/V% of culture medium, 0.1~0.5W/V% of polyvinylpyrrolidone, 1~10W/V% of glucose；
(3) recovery reagent 3：15~35W/V% of LBP-X, 0.5~5W/V% of cholesterol, 0.05~0.35V/ of vitamin E
V%, MEM 99.65~99.95V/V% of culture medium, 1~10W/V% of glucose；
(4) recovery reagent 4：5~25W/V% of LBP-X, 0.5~5W/V% of cholesterol, 0.05~0.35V/V% of vitamin E,
MEM 99.65~99.95V/V% of culture medium, 1~10W/V% of glucose.
6. the reagent set described in claim 4 or 5 pet testis 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 testis tissue cryopreservation methods, including：In vitro pet testis 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 testis tissue method for resuscitation, including：In vitro pet testis 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, kept for more than 10 minutes at room temperature.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111345284A (en) * | 2020-04-09 | 2020-06-30 | 四川大学华西第二医院 | Human testicular tissue suspension cryoprotectant and preparation method thereof |
| CN111919836A (en) * | 2020-08-18 | 2020-11-13 | 吉林大学 | Serum-free cryopreservation protective agent for bovine testicular tissues, cryopreservation method and application |
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2017
- 2017-01-16 CN CN201710028220.9A patent/CN106818707A/en active Pending
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| CN111345284A (en) * | 2020-04-09 | 2020-06-30 | 四川大学华西第二医院 | Human testicular tissue suspension cryoprotectant and preparation method thereof |
| CN111919836A (en) * | 2020-08-18 | 2020-11-13 | 吉林大学 | Serum-free cryopreservation protective agent for bovine testicular tissues, cryopreservation method and application |
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