CN100567480C - Method for milk cow skin freezing preservation - Google Patents
Method for milk cow skin freezing preservation Download PDFInfo
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- CN100567480C CN100567480C CNB2006100386089A CN200610038608A CN100567480C CN 100567480 C CN100567480 C CN 100567480C CN B2006100386089 A CNB2006100386089 A CN B2006100386089A CN 200610038608 A CN200610038608 A CN 200610038608A CN 100567480 C CN100567480 C CN 100567480C
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- milk cow
- preservation
- freezing
- cow skin
- skin
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Abstract
Method for milk cow skin freezing preservation relates to fields such as germ plasm resource preservation, transgenosis, cryogenic freezing preservation, organizational project.The milk cow skin tissue is placed frozen solution, after being refrigerated to protection liquid freezes with the dry ice fumigation and steaming method, shift in the refrigerator of putting-80 ℃.The present invention has studied toxicity and the protectiveness of refrigerant to the milk cow skin energy for growth, sets up the somatic novel method of freezing preservation by research.By with aforesaid method, the higher milk cow skin freezing tissue of back surviving rate can obtain to thaw.After thawing, skin histology can grow inoblast.The present invention need not special freezing instrument requirement, simple operation easily, and the collection of somatic tissue's sample is very convenient, is used for the preservation of milk cow germ plasm resource, cloned animal etc., also is suitable for the prolonged preservation of rare animal or precious cell.
Description
Technical field
The present invention relates to fields such as germ plasm resource preservation, transgenosis, cryogenic freezing preservation, organizational project.
Technical background
The birth of nuclear transfer technology is produced transgenic animal and the recovery and the resource preservation of extinct species have brought new hope.Nuclear transfer technology needs the support of two cell systems, and the one, come the ovocyte of autoreceptor ovary tissue, another is from the embryonic stem cell of donor or somatocyte.The fetus that present scientists successfully can get the live body sampling and the somatocyte of adult animals are in vitro culture, and the acquisition of ovocyte usually is subjected to the restriction in female livestock breed cycle, can occur clinically supplying and the not corresponding to contradiction that takes time.Ovary tissue and somatic freezing preservation at present has been widely used in the clinical study and improvement of breed of species such as comprising people, mouse, rabbit, sheep, ox, pig, marmoset and elephant.Usually the chemical substance that can be used as the cryoprotectant application has: dimethyl sulfoxide (DMSO) (DMSO), glycerine (GLY), ethylene glycol (EG), propylene glycol (PROH), methyl alcohol etc.But they are bigger to the adaptability difference of different plant species and different cells.Related organization's cell is detected in people, ox ovary tissue and ox fetal skin cell to the research of all kinds of protectant tolerances and preservation effect.
Summary of the invention
The object of the invention is to invent a kind of method for milk cow skin freezing preservation, to set up the somatic novel method of freezing preservation.
The present invention places frozen solution with the milk cow skin tissue, after being refrigerated to protection liquid freezes with the dry ice fumigation and steaming method, shifts in the refrigerator of putting-80 ℃.
Concrete grammar can have three kinds:
Frozen solution is that concentration is 10%～30% glycerine, freezing before, be balance 20～25min under 4 ℃ of conditions earlier in envrionment temperature.
Or frozen solution is that concentration is 10%～30% propylene glycol, freezing before, be balance 20～25min under 22 ℃ of conditions in envrionment temperature earlier.
Or frozen solution is that concentration is 10%～30% dimethyl sulfoxide (DMSO), freezing before, be balance 20～25min under 4 ℃ of conditions in envrionment temperature earlier.
The present invention has studied toxicity and the protectiveness of three kinds of refrigerants to the milk cow skin energy for growth, sets up the somatic novel method of freezing preservation by research.By with aforesaid method, the higher milk cow skin freezing tissue of back surviving rate can obtain to thaw.After thawing, skin histology can grow inoblast.The present invention need not special freezing instrument requirement, simple operation easily, and the collection of somatic tissue's sample is very convenient, is used for the preservation of milk cow germ plasm resource, cloned animal etc., also is suitable for the prolonged preservation of rare animal or precious cell.
Specific embodiment
1, the fresh skin sample obtains
Alcohol disinfecting the ears of an ox or cow with 75% is cut 1～2cm rapidly with sample-pickup tongs
2The skin chunk of size is placed on the sample of gathering and preserves and transport to the laboratory in the dry ice in 0.5～1h.At first with 75% the alcohol the ears of an ox or cow of sterilizing rapidly, use aseptic PBS rinsing more repeatedly, unhairing is peeled off epiderm skin, collects subcutis and also is cut into 2mm
3The tissue block of size.
2, the toxicity test of skin histology
Get 12～15 milk cow skin tissues, 4 ℃ with two different conditions of room temperature under place three kinds of different cryoprotectant glycerine, propylene glycol, the dimethyl sulfoxide (DMSO) of three gradient concentrations (10%, 20%, 30%) respectively.Be under 4 ℃ and the 22 ℃ of conditions behind balance 20～25min in envrionment temperature respectively, the rinsing three times respectively of the fresh medium of preheating.Be attached to 25mm with the EDTA-Ox blood plasma
2Add nutrient solution on the culturing bottle inwall and directly cultivate Hoechst fluorescent dyeing identification of cell growing state.
The toxicity test result shows: milk cow skin is organized between each concentration with 10% glycerine (GLY), and 20% propylene glycol (PROH) growth result is best, and significant difference (P＜0.05) between other concentration.Total result is: better than equilibrated under 22 ℃ of conditions in equilibrated skin histology growth rate under 4 ℃ of conditions.But be organized under 4 ℃ of equilibrium conditionss, be exposed to the tissue growth rate height in glycerine (GLY) and the dimethyl sulfoxide (DMSO) (DMSO).Under 22 ℃ of equilibrium conditionss, the tissue growth rate that is exposed in the propylene glycol (PROH) is higher.
3, skin histology freezing and thawing
Process freezing and that thaw is as follows: get 3～5 skin histology pieces at random and put in the freeze pipe that the different protection of 0.5m1 different concns liquid is housed in advance, be divided into four groups then and handle.First group of balance 20～25min at room temperature, second group without balance in contrast.Third and fourth group of equal balance 20～25min at room temperature.The 3rd group through cooling and freezing preservation on dry ice after the balance, and the 4th group is then directly dropped in the liquid nitrogen and preserve, and fishes for afterwards and is kept under-80 ℃ of conditions.The dry ice temperature-fall period is: freeze pipe is placed on to be cooled at a slow speed on the support of dry ice face top shifts in the refrigerator of putting-80 ℃ after nutrient solution freezes.Behind the cryopreservation through 1～2 week, freeze pipe is taken out the 1-1.5min that thaws in the water-bath that directly is put into 37 ℃.With the DMEM rinsing of the fresh preheating that contains 10%FCS 3 times, remove cryoprotectant.Through after thawing, these tissues are attached to 25mm with the EDTA-Ox blood plasma
2Adding nutrient solution on the culturing bottle inwall cultivates.
4, the cultivation of freezing back skin histology
Milk cow skin tissue culture medium consists of beta-mercaptoethanol, 1% non-essential amino acid, 1% VITAMIN, 100U/ml penicillin and the 100mg/ml Streptomycin sulphate that contains 10%FCS, 2mM glutamine, 0.1mM.Hoechst dyeing identification of cell growing state.
Skin histology is put in the culturing bottle, renews bright nutrient solution every day and cultivates.
Cultivating in 11 days identical time, the ears of an ox or cow skin histology can grow a large amount of cells, expansion easily, growth rate average out to 65.6%.
Milk cow skin is organized the growing state after freezing: with the dry ice cooling, there are significant difference (p＜0.05) in 10%GLY, 20%GLY, 10%PROH and 20%PROH between organizing as protectant growing state and control group and other.Tissue with liquid nitrogen cooling there is no growth in a organized way.
Claims (4)
1, method for milk cow skin freezing preservation is characterized in that: first balance 20～25min under 4～22 ℃ of temperature, then milk cow skin tissue is placed frozen solution, with the dry ice fumigation and steaming method be refrigerated to protect liquid to freeze after, transfer is put in-80 ℃ the refrigerator.
2, according to the described method for milk cow skin freezing preservation of claim 1, it is characterized in that described frozen solution is that concentration is 10%～30% glycerine, freezing preceding equilibrium temperature is 4 ℃.
3, according to the described method for milk cow skin freezing preservation of claim 1, it is characterized in that described frozen solution is that concentration is 10%～30% propylene glycol, freezing preceding equilibrium temperature is 22 ℃.
4, according to the described method for milk cow skin freezing preservation of claim 1, it is characterized in that described frozen solution is that concentration is 10%～30% dimethyl sulfoxide (DMSO), freezing preceding equilibrium temperature is 4 ℃.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2006100386089A CN100567480C (en) | 2006-03-02 | 2006-03-02 | Method for milk cow skin freezing preservation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2006100386089A CN100567480C (en) | 2006-03-02 | 2006-03-02 | Method for milk cow skin freezing preservation |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1821390A CN1821390A (en) | 2006-08-23 |
| CN100567480C true CN100567480C (en) | 2009-12-09 |
Family
ID=36922964
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2006100386089A Expired - Fee Related CN100567480C (en) | 2006-03-02 | 2006-03-02 | Method for milk cow skin freezing preservation |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN100567480C (en) |
-
2006
- 2006-03-02 CN CNB2006100386089A patent/CN100567480C/en not_active Expired - Fee Related
Non-Patent Citations (4)
| Title |
|---|
| 马皮肤成纤维细胞的体外培养与冷冻保存. 刘春霞.中国畜牧兽医，第34卷第3期. 2007 |
| 马皮肤成纤维细胞的体外培养与冷冻保存. 刘春霞.中国畜牧兽医，第34卷第3期. 2007 * |
| 鸡胚胎原始生殖细胞对不同冷冻体系适应性的研究. 李碧春等.解剖学报，第36卷第5期. 2005 |
| 鸡胚胎原始生殖细胞对不同冷冻体系适应性的研究. 李碧春等.解剖学报，第36卷第5期. 2005 * |
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| Publication number | Publication date |
|---|---|
| CN1821390A (en) | 2006-08-23 |
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Granted publication date: 20091209 Termination date: 20130302 |