CN102246745B - Antifreeze for cryopreservation of spermatogonial stem cells of animals and cryopreservation method thereof - Google Patents
Antifreeze for cryopreservation of spermatogonial stem cells of animals and cryopreservation method thereof Download PDFInfo
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- CN102246745B CN102246745B CN201110124683.8A CN201110124683A CN102246745B CN 102246745 B CN102246745 B CN 102246745B CN 201110124683 A CN201110124683 A CN 201110124683A CN 102246745 B CN102246745 B CN 102246745B
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- density lipoprotein
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Abstract
The invention discloses an antifreeze for cryopreservation of spermatogonial stem cells of animals and a cryopreservation method thereof. Low-density lipoprotein can be adopted, or 3mg/ml of low-density lipoprotein and 0.5mg/ml of trehalose can be mixed according to the volume ratio of 1:1, or 5mg/ml of low-density lipoprotein and 0.5mg/ml of lecithin can be mixed according to the volume ratio of 1:1. The recovery rate can be obviously improved.
Description
Technical field
The present invention relates to antifreeze and method thereof for the freezing preservation of animal stem spermatogonium, can make the anabiosis rate after animal stem spermatogonium freeze-thaw significantly improve.
Background technology
Stem spermatogonium (spermatogonial stem cells, SSCs) is the clone source that is forever divided into sperm, both can generate new stem cell by self, again can Proliferation, Differentiation forms the androgone in each stage until ripe sperm.For sperm freezing is preserved, the freezing preservation of spermatogonium has more advantage, because sperm has unique morphological structure, it is characterized in that core DNA highly concentrates and cytoplasmic loss, this makes it more responsive to the physical and chemical changes in Freezing-thawing process, thereby causes the death of part sperm.And stem spermatogonium is similar to normal somatic cell in morphological structure, it compared with sperm more ability be subject to Freezing-thawing program, thereby freezing preservation preferably.
Conventional antifreeze has dimethyl sulfoxide (DMSO) (DMSO), sucrose and glycerine etc. at present.DMSO and glycerine belong to permeability antifreeze, and sucrose is impermeability antifreeze.F.Izadyar etc., take 80%MEM+10%FCS as basic cryopreserving liquid, by changing antifreeze composition, have explored the frozen effect of different antifreezes to ox sperma-togonium A.Result shows, the sperma-togonium A motility rate of 10%FCS+10%DMSO or 10%FCS+1.4M glycerine is significantly higher than the cryopreserving liquid that only contains 10%FCS; And it is higher than the Cell viability of glycerinated cryopreserving liquid containing the cryopreserving liquid of DMSO; In the cryopreserving liquid based on 10%DMSO, FCS concentration is increased to 20% pair of survival after sperma-togonium A Cryopreservation and does not make significant difference; In the cryopreserving liquid based on 10%DMSO, the sucrose of interpolation 0.07,0.14,0.21M can significantly promote the cloning efficiency after the survival of ox sperma-togonium A, propagation and transplanting.They point out, use the cryopreserving liquid that contains MEM+10%FCS+10%DMSO+0.07M sucrose, adopt naturally frozen mode at a slow speed, are that the frozen one of ox spermatogonium is simple and effective method can obtain more than 70% Cell viability after Cryopreservation.
Summary of the invention
The object of the invention is to, a kind of antifreeze for the freezing preservation of animal stem spermatogonium and preparation and application are provided.Using low-density lipoprotein, trehalose and three kinds of glucides of lecithin as cryoprotector, explore best cryoprotection liquid system, to improve the anabiosis rate after animal stem spermatogonium freeze-thaw.
For achieving the above object, the present invention takes following technical scheme:
For the antifreeze of the freezing preservation of animal stem spermatogonium, described antifreeze is low-density lipoprotein.
Described antifreeze, described antifreeze is low-density lipoprotein and trehalose.
Described antifreeze, described antifreeze is low-density lipoprotein and lecithin.
Described antifreeze, the low-density lipoprotein that described antifreeze is 1mg/ml.
Described antifreeze, the low-density lipoprotein that described antifreeze is 3mg/ml and the trehalose of 0.5mg/ml were according to 1: 1 volume ratio compatibility.
Described antifreeze, the low-density lipoprotein that described antifreeze is 5mg/ml and the lecithin of 0.5mg/ml were according to 1: 1 volume ratio compatibility.
For the method for the freezing preservation of animal stem spermatogonium, in the cryovial of 0.5ml, add after the stem spermatogonium liquid of 0.2ml, add after above-mentioned single antifreeze 0.1ml or compatibility antifreeze (1: 1 volume) 0.2ml, ultra low temperature freezer and the liquid nitrogen container of putting into respectively-80 ℃ are freezing.
During the low-density lipoprotein 1d of 1mg/ml after-80 ℃ of freezing preservations of ultra low temperature freezer the resuscitation effect the best to spermatogonial stem cells into mouse, its anabiosis rate is up to 87.16%.The trehalose compatibility of 3mg/ml low-density lipoprotein and 0.5mg/ml, remarkable to the resuscitation effect of spermatogonial stem cells into mouse after-80 ℃ of freezing preservations of ultra low temperature freezer during 15d, anabiosis rate is up to 90.23%.The lecithin compatibility of 5mg/ml low-density lipoprotein and 0.5mg/ml, remarkable to the resuscitation effect of spermatogonial stem cells into mouse after-80 ℃ of freezing preservations of ultra low temperature freezer during 15d, anabiosis rate is up to 88.74%.
Accompanying drawing explanation
Figure 1A figure is 1mg/ml low-density lipoprotein stem spermatogonium after freeze-thaw in liquid nitrogen container.B figure is 1mg/ml low-density lipoprotein stem spermatogonium after freeze-thaw in-80 ℃ of ultra low temperature freezers.
Fig. 2 A figure is 3mg/ml low-density lipoprotein and 0.5mg/ml trehalose compatibility stem spermatogonium after freeze-thaw in liquid nitrogen container.B figure is the stem spermatogonium after freeze-thaw in 3mg/ml low-density lipoprotein and 0.5mg/ml trehalose compatibility-80 ℃ ultra low temperature freezer.
Fig. 3 A figure is 5mg/ml low-density lipoprotein and 0.5mg/ml lecithin compatibility stem spermatogonium after freeze-thaw in liquid nitrogen container.B figure is 5mg/ml low-density lipoprotein and 0.5mg/ml lecithin compatibility stem spermatogonium after freeze-thaw in-80 ℃ of ultra low temperature freezers.
Figure 41 mg/ml low-density lipoprotein antifreeze Cryopreservation rate figure.As seen from the figure, during the low-density lipoprotein 1d of 1mg/ml after-80 ℃ of freezing preservations of ultra low temperature freezer the resuscitation effect the best to spermatogonial stem cells into mouse, its anabiosis rate is up to 87.16%.
The trehalose compatibility antifreeze Cryopreservation rate figure of Fig. 5 .3mg/ml low-density lipoprotein and 0.5mg/ml.As seen from the figure, the trehalose compatibility of 3mg/ml low-density lipoprotein and 0.5mg/ml, remarkable to the resuscitation effect of spermatogonial stem cells into mouse after-80 ℃ of freezing preservations of ultra low temperature freezer during 15d, anabiosis rate is up to 90.23%.
The lecithin compatibility antifreeze Cryopreservation rate figure of Figure 65 mg/ml low-density lipoprotein and 0.5mg/ml.As seen from the figure, the lecithin compatibility of 5mg/ml low-density lipoprotein and 0.5mg/ml, remarkable to the resuscitation effect of spermatogonial stem cells into mouse after-80 ℃ of freezing preservations of ultra low temperature freezer during 15d, anabiosis rate is up to 88.74%.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.
The present embodiment arranges following cryoprotection scheme (each group arranges 6 repetitions, and wherein 3 are repeated to be placed in the ultra low temperature freezer of-80 ℃, and other 3 are repeated to insert in liquid nitrogen container, analyze the otherness of resuscitation effect under two kinds of conditions):
In the cryovial of blank group: 0.5ml, add the spermatogonial stem cells into mouse liquid of 0.2ml, 6 repetitions are set, by wherein 3 repeat to be placed in the freezing preservation of ultra low temperature freezer of-80 ℃, other 3 are repeated to be placed in the freezing preservation of liquid nitrogen container, then after thawing 20 minutes the 1st, 3,7,15,30 days time, calculate under the microscope its anabiosis rate in 37 ℃ of water-baths, analyze the otherness of resuscitation effect under two kinds of conditions.
In the cryovial of low-density lipoprotein group: 0.5ml, add after the spermatogonial stem cells into mouse liquid of 0.2ml, add again 1mg/ml third constellations density lipoprotein 0.1ml, 6 repetitions are set, by wherein 3 repeat to be placed in the freezing preservation of ultra low temperature freezer of-80 ℃, other 3 are repeated to be placed in the freezing preservation of liquid nitrogen container, then after thawing 20 minutes the 1st, 3,7,15,30 days time, calculate under the microscope its anabiosis rate in 37 ℃ of water-baths, analyze the otherness of resuscitation effect under two kinds of conditions.With reference to figure 1, visible after the freezing preservation of ultra low temperature freezer of-80 ℃, the form of stem spermatogonium is normal, and the highest anabiosis rate can reach 87.16%.
In the cryovial of low-density lipoprotein and trehalose compatibility: 0.5ml, add after the spermatogonial stem cells into mouse liquid of 0.2ml, add again the each 0.1ml of trehalose of 3mg/ml low-density lipoprotein and 0.5mg/ml, 6 repetitions are set, by wherein 3 repeat to be placed in the freezing preservation of ultra low temperature freezer of-80 ℃, other 3 are repeated to be placed in the freezing preservation of liquid nitrogen container, then after thawing 20 minutes the 1st, 3,7,15,30 days time, calculate under the microscope its anabiosis rate in 37 ℃ of water-baths, analyze the otherness of resuscitation effect under two kinds of conditions.With reference to figure 2, visible after the freezing preservation of ultra low temperature freezer of-80 ℃, the form of stem spermatogonium is normal, and the highest anabiosis rate can reach 90.23%.
In the cryovial of low-density lipoprotein and lecithin compatibility: 0.5ml, add after the stem spermatogonium liquid of 0.2ml, add again the each 0.1ml of lecithin of 5mg/ml low-density lipoprotein and 0.5mg/ml, 6 repetitions are set, by wherein 3 repeat to be placed in the freezing preservation of ultra low temperature freezer of-80 ℃, other 3 are repeated to be placed in the freezing preservation of liquid nitrogen container, then after thawing 20 minutes the 1st, 3,7,15,30 days time, calculate under the microscope its anabiosis rate in 37 ℃ of water-baths, analyze the otherness of resuscitation effect under two kinds of conditions.With reference to figure 3, visible after the freezing preservation of ultra low temperature freezer of-80 ℃, the form of stem spermatogonium is normal, and the highest anabiosis rate can reach 88.74%.
Should be understood that, for those of ordinary skills, can be improved according to the above description or convert, and all these improvement and conversion all should belong to the protection domain of claims of the present invention.
Claims (2)
1. for the antifreeze of the freezing preservation of animal stem spermatogonium, the low-density lipoprotein that described antifreeze is 3mg/ml and the trehalose of 0.5mg/ml were according to 1: 1 volume ratio compatibility.
2. for the method for the freezing preservation of animal stem spermatogonium, it is characterized in that, in the cryovial of 0.5ml, add after the stem spermatogonium liquid of 0.2ml, add described in claim 1 after antifreeze 0.2ml, put into respectively the ultra low temperature freezer of-80 ℃.
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| CN102907417B (en) * | 2012-10-22 | 2014-01-29 | 西北农林科技大学 | Freezing diluent formula for improving quality of Holstein bull frozen-thawed X-spermatozoa |
| KR101546487B1 (en) * | 2013-06-28 | 2015-08-25 | 대한민국 | Composition for cryopreservating sperm comprising LDL and anti-oxidant and uses thereof |
| CN103783034B (en) * | 2014-01-20 | 2015-10-21 | 广西大学 | One boar stem spermatogonium cryopreserving liquid and using method thereof |
| CN103999849B (en) * | 2014-05-07 | 2016-05-18 | 西北农林科技大学 | The antifreeze of the freezing preservation of a kind of mammal testis tissue and freeze-thaw method |
| CN106973889B (en) * | 2016-01-04 | 2018-08-24 | 南京三生生物技术股份有限公司 | A kind of cells frozen storing liquid of leukemia treating |
| CN107232186B (en) * | 2016-03-06 | 2018-09-21 | 郑州欧范医疗器械有限公司 | A kind of cells frozen storing liquid of human adipose-derived stem cell |
| CN106754724A (en) * | 2016-12-09 | 2017-05-31 | 西北农林科技大学 | A kind of ox stem spermatogonium system of immortalization and its construction method |
| CN108378019B (en) * | 2018-03-13 | 2021-03-02 | 诺赛联合(北京)生物医学科技有限公司 | Cryopreservation liquid for human spermatogonial stem cells |
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