CN101253856A - Vitrification frozen stock solution - Google Patents
Vitrification frozen stock solution Download PDFInfo
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- CN101253856A CN101253856A CNA2008100273535A CN200810027353A CN101253856A CN 101253856 A CN101253856 A CN 101253856A CN A2008100273535 A CNA2008100273535 A CN A2008100273535A CN 200810027353 A CN200810027353 A CN 200810027353A CN 101253856 A CN101253856 A CN 101253856A
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Abstract
The invention discloses a vitrification frozen stock solution. Each 100 milliliters of the solution contains 0.1-5g of polyvinyl alcohol, 1-30g of dimethyl sulfoxide, 1-30g of 1, 2-propylene glycol and water for the rest. The inventive frozen stock solution contains a penetrability and a non-penetrability cryoprotective protective agents, particularly the polyvinyl alcohol which is taken as the non-penetrability frozen stock solution component, has obvious effects of preventing the ice crystal growth and restraining the recrystallization and is capable of effectively forming the vitrification frozen stock at low temperature. Given the factors including the toxicity of the protective agent and the penetrability, the vitrification property and stability as well as the speeds of warming and cooling, etc. the inventive frozen stock solution is subject to the vitrification state in the cooling process and further guarantees the stability of the vitrification solution in the warming process. The inventive frozen stock solution has simple and all-purpose effects and is applicable to the vitrification frozen stock of various cells, tissues and organs.
Description
Technical field
The present invention relates to a kind of tissue and organ and preserve the vitrification frozen stock solution of usefulness.
Background technology
Low temperature is preserved biomaterial, and particularly tissue and organ are one of important goals of cryobiology area research.At present, the low temperature of simple tissues such as cell and embryo is preserved and has been obtained obvious effects, but also there is limitation to a certain degree in the preservation of complex organization and organ.Reason is when cold service during in organism, freezing of water takes place in its inside inevitably, freeze in the cell or the extracellular freezes no matter be, the capital causes the forfeiture of cytoactive and function, and in-15--50 ℃ this temperature range, cause cell inactivation thereby very easily form a large amount of ice crystals inside and outside the cell.In freezing and rewarming process, cell must pass through this dangerous temperature interval twice.Therefore, effectively protecting cell safety by this warm area, avoid ice crystal to form the damage that causes, is the key that biomaterial is successfully preserved.
Vitrifying is meant that liquid state changes amorphous solidification process into.Method for vitrification is exactly a kind of method of preserving biomaterial under complete vitrifying state.Normal molecule and ion distribution can be avoided the physical injury that biomaterial is caused because of freezing when glassy solids can keep liquid.Under attainable rate of temperature fall, the vitrifying method need use multiple antifreezing agent to be made into the vitrification solution of high concentration, and after temperature reduced, whole solution becomes got very thickness, finally enters a kind of vitrifying state.Method for vitrification is simple to operate fast, is a kind of very promising low temperature store method.Can method for vitrification successfully be used for the preservation of simple tissues such as embryo, but can't determine finally solve the preservation of tissue and organ at present.
Cryoprotective agent is divided into permeability and impermeability two big classes.Mostly the permeability antifreezing agent is some low-molecular-weight neutral substances, and they can infiltrate cell interior, reduces electrolytical concentration in freezing point, the formation of minimizing ice crystal, the dilute solution, reduces solute damage, the inside and outside osmotic pressure of statocyte and alleviates cell shrink degree.Permeability antifreezing agent commonly used mainly contains little molecule polyhydroxy-alcohols such as glycerine, dimethyl sulfoxide (DMSO), ethylene glycol and propane diols.The molecular weight of impermeability antifreezing agent is bigger, can not enter cell interior, and they reduce the solute damage by the outer electrolytical concentration of diluting cells, by reducing the extracellular free water content to reach the damage that reduces the outer ice crystal of born of the same parents.Impermeability antifreezing agent commonly used has sucrose, glucose, trehalose, bovine serum albumin, albumin, Sodium Hyaluronate and HES etc.Single antifreezing agent respectively has pluses and minuses, can not satisfy many-sided requirement that biomaterial low temperature is preserved, and generally adopts two or more to mix and uses.The permeability antifreezing agent generally has toxic action, and the impermeability antifreezing agent can cause that cell transition shrinks, and the two mixes use can remedy above-mentioned shortcoming, improve preservation effect, can also impel the change of some thermodynamic parameter of solution, raise vitrifying desired concn reduction etc. such as vitrification point.These change for vitrifying preservation biomaterial is very useful.
Summary of the invention
The objective of the invention is to overcome the shortcoming that prior art exists, a kind of permeability and impermeability cryoprotective agent of containing simultaneously is provided, can effectively form the vitrification frozen stock solution that low temperature glass thaws and deposits.
To achieve these goals, the present invention adopts following technical scheme:
A kind of vitrification frozen stock solution, contain in per 100 ml solns:
Polyvinyl alcohol 0.1～5 gram
Dimethyl sulfoxide (DMSO) 1～30 gram
1,2-propane diols 1～30 gram
Surplus is a water.
Its preferred ratio is to contain in per 100 ml solns:
Polyvinyl alcohol 1～5 gram
Dimethyl sulfoxide (DMSO) 10～20 grams
1,2-propane diols 10～20 grams
Surplus is a water.
The present invention selects dimethyl sulfoxide (DMSO) (DMSO) and 1,2-propane diols (PE) is the permeability antifreezing agent, selecting to antifreeze protein polyvinyl alcohol (PVA) similar, that can stop ice-crystal growth and restrain the recrystallization characteristics to be arranged is the impermeability antifreezing agent, forms a kind of multi-component vitrification frozen stock solution.This multi-component glass frozen preservation liquid system has stronger vitrifying ability, shows to be easy in the temperature-fall period enter the vitrifying state, and has only the seldom water formation crystal of part.Need also simultaneously to guarantee the stability of vitrification solution in temperature-rise period, show and go vitrifying or recrystallization to postpone in the temperature-rise period that tangible recrystallization rejection characteristic is arranged.
Compared with prior art, the present invention has following beneficial effect:
1. compare with disclosed multicomponent cryopreserving liquid; cryopreserving liquid of the present invention contains permeability and impermeability cryoprotective agent simultaneously; especially adopting polyvinyl alcohol is impermeability antifreezing agent component; have tangible prevention ice-crystal growth and restrain recrystallization, can effectively form low temperature glass and thaw and deposit effect.
2. the multicomponent glass of the present invention liquid storage system of thawing has been considered factors such as protectant toxicity, permeability, vitrifying ability, vitrifying stability and intensification rate of temperature fall simultaneously.So not only in temperature-fall period, be easy to enter the vitrifying state, and can also guarantee the stability of vitrification solution in temperature-rise period.And vitrification solution is realized from the steady rewarming of low temperature preservation state to the normal temperature state than difficult in the existing frozen technology.
3. multicomponent cryopreserving liquid of the present invention has simple and general effect, is suitable for the glass frozen preservation of various cells, tissue and organ.
Embodiment
Embodiment 1
With the polyvinyl alcohol 0.1g for preparing in advance as the impermeability antifreezing agent, with 1,2-propane diols 30g and dimethyl sulfoxide (DMSO) 30g be as the permeability antifreezing agent, three components mix the PVA/DMSO/PE system, add entry and be made into 100ml solution, be vitrification frozen stock solution to this system.
Embodiment 2
With the polyvinyl alcohol 1g for preparing in advance as the impermeability antifreezing agent, with 1,2-propane diols 20g and dimethyl sulfoxide (DMSO) 20g be as the permeability antifreezing agent, three components mix the PVA/DMSO/PE system, add entry and be made into 100ml solution, be vitrification frozen stock solution to this system.
Embodiment 3
With the polyvinyl alcohol 2g for preparing in advance as the impermeability antifreezing agent, with 1,2-propane diols 10g and dimethyl sulfoxide (DMSO) 10g be as the permeability antifreezing agent, three components mix the PVA/DMSO/PE system, add entry and be made into 100ml solution, be vitrification frozen stock solution to this system.
Embodiment 4
With the polyvinyl alcohol 2g for preparing in advance as the impermeability antifreezing agent, with 1,2-propane diols 5g and dimethyl sulfoxide (DMSO) 25g be as the permeability antifreezing agent, three components mix the PVA/DMSO/PE system, add entry and be made into 100ml solution, be vitrification frozen stock solution to this system.
Embodiment 5
With the polyvinyl alcohol 3g for preparing in advance as the impermeability antifreezing agent, with 1,2-propane diols 25g and dimethyl sulfoxide (DMSO) 5g be as the permeability antifreezing agent, three components mix the PVA/DMSO/PE system, add entry and be made into 100ml solution, be vitrification frozen stock solution to this system.
Embodiment 6
With the polyvinyl alcohol 4g for preparing in advance as the impermeability antifreezing agent, with 1,2-propane diols 5g and dimethyl sulfoxide (DMSO) 5g be as the permeability antifreezing agent, three components mix the PVA/DMSO/PE system, add entry and be made into 100ml solution, be vitrification frozen stock solution to this system.
Embodiment 7
With the polyvinyl alcohol 5g for preparing in advance as the impermeability antifreezing agent, with 1,2-propane diols 1g and dimethyl sulfoxide (DMSO) 1g be as the permeability antifreezing agent, three components mix the PVA/DMSO/PE system, add entry and be made into 100ml solution, be vitrification frozen stock solution to this system.
Embodiment 8
With the polyvinyl alcohol 5g for preparing in advance as the impermeability antifreezing agent, with 1,2-propane diols 30g and dimethyl sulfoxide (DMSO) 10g be as the permeability antifreezing agent, three components mix the PVA/DMSO/PE system, add entry and be made into 100ml solution, be vitrification frozen stock solution to this system.
Claims (2)
1. vitrification frozen stock solution is characterized in that containing in per 100 ml solns:
Polyvinyl alcohol 0.1～5 gram
Dimethyl sulfoxide (DMSO) 1～30 gram
1,2-propane diols 1～30 gram
Surplus is a water.
2. vitrification frozen stock solution as claimed in claim 1 is characterized in that containing in per 100 ml solns:
Polyvinyl alcohol 1～5 gram
Dimethyl sulfoxide (DMSO) 10～20 grams
1,2-propane diols 10～20 grams
Surplus is a water.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2008100273535A CN101253856A (en) | 2008-04-11 | 2008-04-11 | Vitrification frozen stock solution |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2008100273535A CN101253856A (en) | 2008-04-11 | 2008-04-11 | Vitrification frozen stock solution |
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| CN101253856A true CN101253856A (en) | 2008-09-03 |
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| CNA2008100273535A Pending CN101253856A (en) | 2008-04-11 | 2008-04-11 | Vitrification frozen stock solution |
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Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101897330A (en) * | 2010-08-31 | 2010-12-01 | 湖南光琇高新生命科技有限公司 | Low-toxicity vitrified frozen solution of human embryonic stem cells (hESCs) and method of using same |
| CN102106342A (en) * | 2009-12-29 | 2011-06-29 | 山东省齐鲁干细胞工程有限公司 | Method for storing mesenchymal stem cells and method for culturing mesenchymal stem cells |
| CN102258008A (en) * | 2011-05-31 | 2011-11-30 | 成都大学 | Vitrification ultra-low temperature preserving method for effectively preserving bitter buckwheat callus |
| CN102295782A (en) * | 2011-07-04 | 2011-12-28 | 四川大学 | Gelating resistant polyvinyl alcohol aqueous solution and preparation method thereof |
| CN102550541A (en) * | 2010-12-23 | 2012-07-11 | 潘峰 | Organ vitrification freezing and preserving fluid |
| CN103421693A (en) * | 2013-09-05 | 2013-12-04 | 黑龙江省科学院微生物研究所 | Bradyrhizobium japonicum vitrified cryopreservation liquid and method for vitrified cryopreservation of bradyrhizobium japonicum |
| CN105638643A (en) * | 2016-02-16 | 2016-06-08 | 云南省畜牧兽医科学院 | Freezing diluent for seminal fluid of livestock |
| CN106993606A (en) * | 2017-04-20 | 2017-08-01 | 苏州新赛美生物科技有限公司 | A kind of cells frozen storing liquid without albumen and serum and preparation method thereof |
| CN108990964A (en) * | 2018-08-09 | 2018-12-14 | 华东理工大学 | Cells frozen storing liquid |
| CN109392891A (en) * | 2018-10-26 | 2019-03-01 | 银丰生物工程集团有限公司 | A kind of methods and applications freezing human umbilical tissue according to layer of structure system |
| CN110495447A (en) * | 2019-09-10 | 2019-11-26 | 湖南思为康医药有限公司 | A kind of method immunocyte glass frozen preservation protection liquid and freeze immunocyte |
| CN111789101A (en) * | 2019-04-09 | 2020-10-20 | 北京大学第三医院 | Application of PVA-based cryopreservation liquid in cryopreservation of oocytes or embryos |
| CN113518554A (en) * | 2019-02-07 | 2021-10-19 | 细胞物质股份公司 | Composition for cryopreservation of biological materials |
-
2008
- 2008-04-11 CN CNA2008100273535A patent/CN101253856A/en active Pending
Cited By (20)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102106342A (en) * | 2009-12-29 | 2011-06-29 | 山东省齐鲁干细胞工程有限公司 | Method for storing mesenchymal stem cells and method for culturing mesenchymal stem cells |
| CN102106342B (en) * | 2009-12-29 | 2013-08-28 | 山东省齐鲁干细胞工程有限公司 | Method for storing mesenchymal stem cells and method for culturing mesenchymal stem cells |
| CN101897330A (en) * | 2010-08-31 | 2010-12-01 | 湖南光琇高新生命科技有限公司 | Low-toxicity vitrified frozen solution of human embryonic stem cells (hESCs) and method of using same |
| CN102550541A (en) * | 2010-12-23 | 2012-07-11 | 潘峰 | Organ vitrification freezing and preserving fluid |
| CN102550541B (en) * | 2010-12-23 | 2016-02-17 | 潘峰 | Organ vitrificated cryopreserration liquid |
| CN102258008A (en) * | 2011-05-31 | 2011-11-30 | 成都大学 | Vitrification ultra-low temperature preserving method for effectively preserving bitter buckwheat callus |
| CN102258008B (en) * | 2011-05-31 | 2013-08-21 | 成都大学 | Vitrification ultra-low temperature preserving method for effectively preserving bitter buckwheat callus |
| CN102295782A (en) * | 2011-07-04 | 2011-12-28 | 四川大学 | Gelating resistant polyvinyl alcohol aqueous solution and preparation method thereof |
| CN103421693B (en) * | 2013-09-05 | 2019-10-15 | 黑龙江省科学院微生物研究所 | The method of rihizobium japonicum vitrification frozen stock solution and glass frozen preservation rihizobium japonicum |
| CN103421693A (en) * | 2013-09-05 | 2013-12-04 | 黑龙江省科学院微生物研究所 | Bradyrhizobium japonicum vitrified cryopreservation liquid and method for vitrified cryopreservation of bradyrhizobium japonicum |
| CN105638643A (en) * | 2016-02-16 | 2016-06-08 | 云南省畜牧兽医科学院 | Freezing diluent for seminal fluid of livestock |
| CN105638643B (en) * | 2016-02-16 | 2018-01-12 | 云南省畜牧兽医科学院 | A kind of sheep sperm freezing dilution liquid |
| CN106993606A (en) * | 2017-04-20 | 2017-08-01 | 苏州新赛美生物科技有限公司 | A kind of cells frozen storing liquid without albumen and serum and preparation method thereof |
| CN108990964A (en) * | 2018-08-09 | 2018-12-14 | 华东理工大学 | Cells frozen storing liquid |
| CN108990964B (en) * | 2018-08-09 | 2022-01-28 | 华东理工大学 | Cell cryopreservation liquid |
| CN109392891A (en) * | 2018-10-26 | 2019-03-01 | 银丰生物工程集团有限公司 | A kind of methods and applications freezing human umbilical tissue according to layer of structure system |
| CN113518554A (en) * | 2019-02-07 | 2021-10-19 | 细胞物质股份公司 | Composition for cryopreservation of biological materials |
| CN111789101A (en) * | 2019-04-09 | 2020-10-20 | 北京大学第三医院 | Application of PVA-based cryopreservation liquid in cryopreservation of oocytes or embryos |
| CN111789101B (en) * | 2019-04-09 | 2022-09-06 | 北京大学第三医院 | Application of PVA-based cryopreservation liquid in cryopreservation of oocytes or embryos |
| CN110495447A (en) * | 2019-09-10 | 2019-11-26 | 湖南思为康医药有限公司 | A kind of method immunocyte glass frozen preservation protection liquid and freeze immunocyte |
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Open date: 20080903 |