CN102428912A - Non-toxic cell refrigerating liquid containing cholesterol and preparation method thereof - Google Patents
Non-toxic cell refrigerating liquid containing cholesterol and preparation method thereof Download PDFInfo
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- CN102428912A CN102428912A CN2011103455486A CN201110345548A CN102428912A CN 102428912 A CN102428912 A CN 102428912A CN 2011103455486 A CN2011103455486 A CN 2011103455486A CN 201110345548 A CN201110345548 A CN 201110345548A CN 102428912 A CN102428912 A CN 102428912A
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Abstract
The invention discloses a non-toxic cell refrigerating liquid containing cholesterol. A preparation method of the non-toxic cell refrigerating liquid comprises the following steps of: dissolving self-made CLC (Cholesterol-Loaded Cyclodextrin) and EDTA (Ethylene Diamine Tetraacetic Acid) in m199 cell culture liquid, stirring and uniformly mixing the liquid, and filtering the liquid to remove bacteria through a filter membrane to obtain the non-toxic cell refrigerating liquid. The contract that cholesterol can not be dissolved in the culture liquid is solved, and the action of protection cell membranes is exerted.
Description
Technical field
The present invention relates to a kind of cell freezing agent, the cell that belongs in the biology is preserved the field.
Background technology
Be used for the frozen of cell at present, mostly use DMSO to be used as the protectant of cell, DMSO Chinese dimethyl sulfoxide (DMSO) is a kind of organic compound of sulfur-bearing, molecular formula (CH3)
2SO, normal temperature is the transparency liquid of no color or smell down, is a kind of hygroscopic flammable liquid, has high polarity, high boiling point, Heat stability is good, the characteristic miscible with water can be dissolved in most of organic matters such as ethanol, propyl alcohol, benzene and chloroform, is described as " alembroth ".But research shows that there is certain toxic action in DMSO, and hydrophobic group has an effect with protein; Cause protein denaturation; Have blood vessel toxicity and liver renal toxicity, can skin ambustion and make the skin tingle, suck high volatilization concentration and can cause headache and dizzy; The most common symptom for feel sick, vomiting and fash, send garlic, onion, oyster flavor in the gas of exhalation.
Summary of the invention
Therefore, there is great demand in people to nontoxic cell freezing liquid, do not find also that up to now the inventor gropes finally to work out nontoxic cytoprotection freezing liquid, thereby accomplished the present invention through a large amount of development tests about relevant report of the present invention.
The object of the invention just provides a kind of nontoxic cell freezing liquid with protection cytosis.
Another object of the present invention just provides the preparation method of this cell freezing liquid.
Scheme of the present invention be with CLC with by the cholesterol of beta-schardinger dextrin-ring bag and EDTA as the protectant Main Ingredients and Appearance of cell freezing, substituting DMSO, thereby bring into play the pair cell protective effect.EDTA wherein, the chinesization formal name used at school: ethylenediamine tetra-acetic acid, outward appearance are white crystalline powder; Be good complexing agent; Be mainly used in water softening, effectively the multiple metal ion in the chelating hard water is controlled polymerization rate; Reduce the toxic action of metal ion pair cell, in the residing liquid environment of cell, can protect by pair cell.Beta-schardinger dextrin-is white crystalline powder water-soluble, irreducibility.Be that cyclodextrin glycan translocase acts on starch after the product that the hydrolysis cyclization forms; Be tubular structure; Its two ends are hydrophily with the outside; And the inside of tube is hydrophobicity, borrows the electrical attraction of Van der Waals force utilization that some sizes and the suitable drug molecule (like halogen, volatilization wet goods) of shape are contained in the circulus.It plays clathration among the present invention.Cholesterol, formal name used at school are 5-cholene-3-β-alcohol.On the C-3 of steroids nuclear, connect monohydroxy, and on the C-17 position, be connected to the aliphatic lateral chain of one eight carbon or more carbon atoms.Be to synthesize through the condensation reaction of isoprene unit, can be used as the precursor of cholic acid and steroid hormone by acetyl coenzyme A.Its dissolubility and fats are seemingly, and be water insoluble, is soluble in ether, chloroform equal solvent.Cholesterol is the indispensable important substance of animal tissue cell, and it not only participates in forming cell membrane, and in the flowability of regulating film, increases the stability of film and the permeability aspect of reduction water-soluble substances and play an important role.
Embodiment
The present invention is formulated by following reagent
CLC 200mg: self-control crystal.Final concentration is 2 mg/ml in cell culture fluid.
CLC (by the cholesterol of beta-schardinger dextrin-ring bag) is a kind of self-control crystal: processed by following article: cholesterol 200mg, beta-schardinger dextrin-1mg, chloroform 1ml, methyl alcohol 2ml.Concrete manufacturing process is following: earlier the 200mg cholesterol is dissolved in the 1ml chloroform, the 1mg beta-schardinger dextrin-is dissolved in the 2ml methyl alcohol, and after mixing, evaporating obtains crystal.This crystal and EDTA are joined in the cell culture fluid by a certain percentage, just obtained new cell freezing liquid.
EDTA 150mg: company provides by Shanghai, Shanghai space biological reagent.Final concentration is 1.5mg/ml in cell culture fluid.
M199 cell culture fluid 100ml: provide by the lucky promise bio tech ltd in Shanghai.
Compound method of the present invention:
1, earlier the 100mlm199 cell culture fluid is put into clean container;
2, again with homemade CLC 200 mg and EDTA 150 mg, be dissolved in the 100 mlm199 cell culture fluids and mix, make the CLC final concentration reach 2mg/ml, the EDTA final concentration reaches 1.5 mg/ml.
3, with 0.2 μ m membrane filtration degerming, promptly process nontoxic cell freezing liquid.
The present invention selects materials accurately; Reasonable mixture ratio, EDTA can form complex with metal ion, and after beta-schardinger dextrin-encapsulates cholesterol; Solved the contradiction that cholesterol is insoluble to culture fluid; Thereby combine with cell membrane smoothly, the protective effect of performance cell membrane is further set forth effect of the present invention below in conjunction with the contrast experiment.
Contrast experiment's data sees the following form:
Warp repeatedly repeated experiments shows; With cell cryopreservation 2 months, the anabiosis rate nearly 10 percentage point of cell can be improved in recovery back and the DMSO contrast contrast of adding 10% with freezing liquid of the present invention; And freezing liquid of the present invention is nontoxic; Harmless, compare with the DMSO of routine, certain application and promotional value are arranged.
Claims (3)
1. nontoxic cell freezing liquid that contains cholesterol is characterized in that: it comprise homemade CLC by the cholesterol 200mg of beta-schardinger dextrin-ring bag and EDTA150mg and be dissolved in the cell culture fluid of 100mlm199 nontoxic cell freezing liquid.
2. the preparation method of nontoxic cell freezing liquid according to claim 1: it is characterized in that: it comprises the following steps:
(1) earlier the 100mlm199 cell culture fluid is put into clean container;
(2) homemade CLC 200mg and EDTA150mg are dissolved in the cell culture fluid of 100mlm199 mix again, make the CLC final concentration reach 2mg/ml, the EDTA final concentration reaches 1.5mg/ml;
(3) with 0.2 μ m membrane filtration degerming, promptly process nontoxic cell freezing liquid.
3. freezing liquid according to claim 1, wherein said CLC is a kind of self-control crystal by the cholesterol of beta-schardinger dextrin-ring bag: processed by following article: cholesterol 200mg, beta-schardinger dextrin-1mg; Chloroform 1ml; Methyl alcohol 2ml, concrete manufacturing process is following: earlier the 200mg cholesterol is dissolved in the 1ml chloroform, the 1mg beta-schardinger dextrin-is dissolved in the 2ml methyl alcohol; Mixing and stirring promptly obtains crystal after evaporating.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011103455486A CN102428912A (en) | 2011-11-05 | 2011-11-05 | Non-toxic cell refrigerating liquid containing cholesterol and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011103455486A CN102428912A (en) | 2011-11-05 | 2011-11-05 | Non-toxic cell refrigerating liquid containing cholesterol and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102428912A true CN102428912A (en) | 2012-05-02 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2011103455486A Pending CN102428912A (en) | 2011-11-05 | 2011-11-05 | Non-toxic cell refrigerating liquid containing cholesterol and preparation method thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113215085A (en) * | 2021-05-07 | 2021-08-06 | 澳门大学 | Lipid substance additive and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN87104158A (en) * | 1986-05-15 | 1988-02-17 | 赛尔系统有限公司 | Biological cryoprotection |
| WO2004026255A2 (en) * | 2002-09-20 | 2004-04-01 | La Jolla Biosciences | Compositions and methods for preventing infection |
-
2011
- 2011-11-05 CN CN2011103455486A patent/CN102428912A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN87104158A (en) * | 1986-05-15 | 1988-02-17 | 赛尔系统有限公司 | Biological cryoprotection |
| WO2004026255A2 (en) * | 2002-09-20 | 2004-04-01 | La Jolla Biosciences | Compositions and methods for preventing infection |
Non-Patent Citations (2)
| Title |
|---|
| 《中国奶牛》 20070220 陈军 等 beta-环糊精胆固醇包合物对牛性控精子品质的影响 , 第2期 * |
| 陈军 等: "β-环糊精胆固醇包合物对牛性控精子品质的影响", 《中国奶牛》, no. 2, 20 February 2007 (2007-02-20) * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113215085A (en) * | 2021-05-07 | 2021-08-06 | 澳门大学 | Lipid substance additive and application thereof |
| CN113215085B (en) * | 2021-05-07 | 2024-05-10 | 澳门大学 | Lipid substance additive and application thereof |
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Application publication date: 20120502 |