CN104054695B - Biological tissue's cryoprotective agent and preparation, using method - Google Patents
Biological tissue's cryoprotective agent and preparation, using method Download PDFInfo
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- CN104054695B CN104054695B CN201410316055.3A CN201410316055A CN104054695B CN 104054695 B CN104054695 B CN 104054695B CN 201410316055 A CN201410316055 A CN 201410316055A CN 104054695 B CN104054695 B CN 104054695B
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Abstract
Biological tissue's cryoprotective agent and preparation, using method, technical field is for the present invention relates to a kind of biological tissue cryoprotective agent and preparation, using method.At present, traditional biotechnology carries out preservation to biological sample in liquid nitrogen environment or ultra low temperature freezer.But the catabolic enzyme in low temperature environment in biological tissue still can carry out dissolved destruction effect to tissue, cell and genetic material are constantly degraded.The object of this invention is to provide one and can keep flesh tissue stay-in-grade biological tissue's cryoprotective agent and preparation, using method for a long time.Biological tissue's cryoprotective agent, by (NH
4)
2sO
4, NaCl, Na
2sO4, NaNO
3, KBrO
3, ultra-pure water composition, provide the preparation method of biological tissue's cryoprotective agent and the using method of biological tissue's cryoprotective agent simultaneously, the present invention can be simultaneously compatible with corresponding sample disposal test method, can more efficientlyly study accordingly and subsequent operation biological sample; The present invention is used for effective preservation of biological sample.
Description
Technical field
The present invention relates to a kind of biological tissue cryoprotective agent and preparation, using method.
Background technology
After biological flesh tissue leaves live body or original living environment, the endogenous enzymes of organism starts genetic material of degrading, and its degradation speed and endogenous enzymes content and temperature have direct relation.At present, traditional biotechnology carries out preservation to biological sample in liquid nitrogen environment or ultra low temperature freezer.But the catabolic enzyme in low temperature environment in biological tissue still can carry out dissolved destruction effect to tissue, cell and genetic material are constantly degraded.
The preservation of biological sample is a permanent job, and the decades that need of preserving of sample even go up a century.The biological sample of degraded can not meet the needs of scientific research can not the existence attribute of System recover biology.So the preservation of bio-genetic material is an important content of biological sample preservation, only zoic hereditary information is able to completely effectively to preserve, and is only the preservation to the real meaning of species.
The method of preservation biological sample hereditary information traditional at present carries out low-temperature preservation, and the preservation of cryoprotective agent to sample is most important.Develop the preservation of nontoxic non-irritating biological tissue cryoprotective agent to biological sample hereditary information to play an important role.
Summary of the invention
The object of this invention is to provide one and can keep flesh tissue stay-in-grade biological tissue's cryoprotective agent and preparation, using method for a long time.
Above-mentioned object is realized by following technical scheme:
A kind of biological tissue cryoprotective agent, by (NH
4)
2sO
4, NaCl, Na
2sO4, NaNO
3, KBrO
3, ultra-pure water composition, described (NH
4)
2sO
4parts by weight be 300-400, the parts by weight of described NaCl are 2-3, described Na
2the parts by weight of SO4 are 18-25, described NaNO
3parts by weight be 0.2-0.3, described KBrO
3parts by weight be 0.1-0.2, the parts by weight of described ultra-pure water are 1000; The preparation method of biological tissue's cryoprotective agent, adopt ultra-pure water as medium, the first step takes (NH4)
2the parts by weight of SO4 are 300-400, add in the ultra-pure water of 600 parts by weight; The parts by weight that second step takes NaCL are 2-3, Na
2the parts by weight of SO4 are the parts by weight of 18-25 and NaNO3 is 0.2-0.3, adds in the ultra-pure water of 300 parts by weight successively; The parts by weight that 3rd step takes KBrO3 are 0.1-0.2, then add in the ultra-pure water of 100 parts by weight; Three kinds of reagent of above-mentioned three step gained are respectively stirred 1min with glass bar, ultrasonic echography 20min; Then second step, the 3rd step two kinds of reagent are slowly added successively in the reagent of the first step, limit edged glass bar stirs, and finally with ultrasonic ultrasonic 10min again, pours in Brown Glass Brown glass bottles and jars only, for subsequent use with 4 DEG C of Refrigerator stores.
Described biological tissue's cryoprotective agent, described (NH
4)
2sO
4parts by weight be 300, the parts by weight of described NaCl are 2, described Na
2the parts by weight of SO4 are 18, described NaNO
3parts by weight be 0.2, described KBrO
3parts by weight be 0.1.
Described (NH
4)
2sO
4parts by weight be 400, the parts by weight of described NaCl are 3, described Na
2the parts by weight of SO4 are 25, described NaNO
3parts by weight be 0.3, described KBrO
3parts by weight be 0.2.
Described (NH
4)
2sO
4parts by weight be 350, the parts by weight of described NaCl are 2.5, described Na
2the parts by weight of SO4 are 20, described NaNO
3parts by weight be 0.25, described KBrO
3parts by weight be 0.15.
The using method of described biological tissue's cryoprotective agent; first step biological sample organizes stripping and slicing; the biological sample cut at room temperature soaks with biological tissue's cryoprotective agent by second step; make solution infiltrate cell and make sample; sample is 4 DEG C in temperature and places 6 hours by the 3rd step, and the 4th step is preserved under sample is moved into-20 DEG C or-86 DEG C of environment for a long time.
Beneficial effect:
1. the present invention can infiltrate rapidly the protection that tissue carries out cell and genetic material (DNA and RNA); The protective effect rapidly of protection liquid ensure that biological sample is used as the accuracy of the gene expression analysis result of scientific research; Sample can digital preservation, even repeatedly multigelation, genetic material is also non-degradable; The effective long term storage of low temperature can be carried out to biological sample.
2. the present invention protects sample in low temperature environment, together comes to protect biological sample, really accomplish effective preservation of biological sample at cellular level and molecular level.
3. the measure that the present invention adopts is after biological tissue is carried out field acquisition, with biological tissue's cryoprotective agent process; The effect of various catabolic enzymes of effective control organism, simultaneously can not also the 26S Proteasome Structure and Function of disorganize cell and bio-genetic material.
4. the present invention can react with effective biological group of a lot of endogenous catabolic enzyme groups such as ()-SH or-OH, thus destroys the activated centre of endogenous enzymes, plays the effect of the available protecting to biological tissue's genetic material.
5. the present invention is the once innovation to biological flesh tissue store method, makes the Cord blood of biological sample more effective.
6. the present invention can preserve the cell structure of sample and genetic material (DNA and RNA) at low ambient temperatures for a long time, prevents degraded.
7. the present invention can be simultaneously compatible with corresponding sample disposal test method, can more efficientlyly study accordingly and subsequent operation biological sample; The biological specimen adding biological tissue's cryoprotective agent can be preserved for a long time under-20 DEG C or-80 DEG C of conditions; Tissue through this product protection can be used for all about molecular biological subsequent experimental, comprises the research of the aspects such as the clone of genes of interest, RNA field and DNA fingerprint technology.
Embodiment:
Embodiment 1:
A kind of biological tissue cryoprotective agent, its composition comprises: (NH
4)
2sO
4, NaCl, Na
2sO4, NaNO
3, KBrO
3, ultra-pure water, described (NH
4)
2sO
4parts by weight be 300-400, the parts by weight of described NaCl are 2-3, described Na
2the parts by weight of SO4 are 18-25, described NaNO
3parts by weight be 0.2-0.3, described KBrO
3parts by weight be 0.1-0.2, the parts by weight of described ultra-pure water are 1000.
Embodiment 2:
Biological tissue's cryoprotective agent described in embodiment 1, described (NH
4)
2sO
4parts by weight be 300, the parts by weight of described NaCl are 2, described Na
2the parts by weight of SO4 are 18, described NaNO
3parts by weight be 0.2, described KBrO
3parts by weight be 0.1.
Embodiment 3:
Biological tissue's cryoprotective agent described in embodiment 1, described (NH
4)
2sO
4parts by weight be 400, the parts by weight of described NaCl are 3, described Na
2the parts by weight of SO4 are 25, described NaNO
3parts by weight be 0.3, described KBrO
3parts by weight be 0.2.
Embodiment 4:
Biological tissue's cryoprotective agent described in embodiment 1, described (NH
4)
2sO
4parts by weight be 350, the parts by weight of described NaCl are 2.5, described Na
2the parts by weight of SO4 are 20, described NaNO
3parts by weight be 0.25, described KBrO
3parts by weight be 0.15.
Embodiment 5:
A preparation method for biological tissue's cryoprotective agent, adopts ultra-pure water as medium, the first step takes (NH
4)
2sO
4parts by weight be 300-400, add in the ultra-pure water of 600 parts by weight; The parts by weight that second step takes NaCL are 2-3, Na
2the parts by weight of SO4 are the parts by weight of 18-25 and NaNO3 is 2-3, adds in the ultra-pure water of 300 parts by weight successively; The parts by weight that 3rd step takes KBrO3 are 0.1-0.2, then add in the ultra-pure water of 100 parts by weight; Three kinds of reagent of above-mentioned three step gained are respectively stirred 1min with glass bar, ultrasonic echography 20min; Then second step, the 3rd step two kinds of reagent are slowly added successively in the reagent of the first step, limit edged glass bar stirs, and finally with ultrasonic ultrasonic 10min again, pours in Brown Glass Brown glass bottles and jars only, for subsequent use with 4 DEG C of Refrigerator stores.
Embodiment 6:
The using method of a kind of biological tissue cryoprotective agent; first step biological sample organizes stripping and slicing; the biological sample cut at room temperature soaks with biological tissue's cryoprotective agent by second step; make solution infiltrate cell and make sample; sample is 4 DEG C in temperature and places 6 hours by the 3rd step, and the 4th step is preserved under sample is moved into-20 DEG C of environment for a long time.
Embodiment 7:
The using method of the biological tissue's cryoprotective agent described in embodiment 6; the biological sample tissue block (each dimension size is all less than 0.5cm) cut only needs at room temperature to immerse simply in biological tissue's cryoprotective agent (such as, the sample of 0.5 needs about 2.5 low-temperature protection reagent) of about 5 times of volumes.Solution infiltrates cell, stabilate tissue.Then sample is placed 6 hours (under biological tissue's immersion protectant liquid level), under then tissue being moved into-20 DEG C (tissue can not freeze) or-86 DEG C of environment at 4 DEG C.Such biological tissue can preserve for a long time, if carry out laboratory operation, can be used as the sample just obtained and carries out processing.Great majority tissue directly can proceed to lysis buffer and carry out homogenization; successively through cryoprotective agent process and freezing sample; mortar can be used to grind; or the after image flesh tissue that thaws equally processes; because various biological decomposition enzyme is inactivated, do not worry the release of cell rupture Sum decomposition enzyme.
Embodiment 8:
Biological tissue's cryoprotective agent described in embodiment 1, described (NH
4)
2sO
4parts by weight be 370, the parts by weight of described NaCl are 2, described Na
2the parts by weight of SO4 are 22, described NaNO
3parts by weight be 0.2, described KBrO
3parts by weight be 0.1.
Embodiment 9:
Biological tissue's cryoprotective agent described in embodiment 1, described (NH
4)
2sO
4parts by weight be 330, the parts by weight of described NaCl are 3, described Na
2the parts by weight of SO4 are 20, described NaNO
3parts by weight be 0.3, described KBrO
3parts by weight be 0.2.
Embodiment 10:
The using method of the biological tissue's cryoprotective agent described in embodiment 6; first step biological sample organizes stripping and slicing; the biological sample cut at room temperature soaks with biological tissue's cryoprotective agent by second step; make solution infiltrate cell and make sample; sample is 4 DEG C in temperature and places 6 hours by the 3rd step, and the 4th step is preserved under sample is moved into-86 DEG C of environment for a long time.
Embodiment 11:
The preparation method of the biological tissue's cryoprotective agent described in embodiment 5, adopts ultra-pure water as medium, the first step takes (NH
4)
2sO
4parts by weight be 300, add in the ultra-pure water of 600 parts by weight; The parts by weight that second step takes NaCL are 2, Na
2the parts by weight of SO4 be 18 and the parts by weight of NaNO3 be 2, add in the ultra-pure water of 300 parts by weight successively; The parts by weight that 3rd step takes KBrO3 are 0.1, then add in the ultra-pure water of 100 parts by weight; Three kinds of reagent of above-mentioned three step gained are respectively stirred 1min with glass bar, ultrasonic echography 20min; Then second step, the 3rd step two kinds of reagent are slowly added successively in the reagent of the first step, limit edged glass bar stirs, and finally with ultrasonic ultrasonic 10min again, pours in Brown Glass Brown glass bottles and jars only, for subsequent use with 4 DEG C of Refrigerator stores.
Embodiment 12:
The preparation method of the biological tissue's cryoprotective agent described in embodiment 5, adopts ultra-pure water as medium, the first step takes (NH
4)
2sO
4parts by weight be 400, add in the ultra-pure water of 600 parts by weight; The parts by weight that second step takes NaCL are 3, Na
2the parts by weight of SO4 be 25 and the parts by weight of NaNO3 be 3, add in the ultra-pure water of 300 parts by weight successively; The parts by weight that 3rd step takes KBrO3 are 0.2, then add in the ultra-pure water of 100 parts by weight; Three kinds of reagent of above-mentioned three step gained are respectively stirred 1min with glass bar, ultrasonic echography 20min; Then second step, the 3rd step two kinds of reagent are slowly added successively in the reagent of the first step, limit edged glass bar stirs, and finally with ultrasonic ultrasonic 10min again, pours in Brown Glass Brown glass bottles and jars only, for subsequent use with 4 DEG C of Refrigerator stores.
Embodiment 13:
Biological tissue's cryoprotective agent described in embodiment 2, described (NH
4)
2sO
4parts by weight be 300g, the parts by weight of described NaCl are 2g, described Na
2the parts by weight of SO4 are 18g, and the parts by weight of described NaNO3 are 0.2g, and the parts by weight of described KBrO3 are 0.1g.
Claims (5)
1. biological tissue's cryoprotective agent, by (NH
4)
2sO
4, NaCl, Na
2sO4, NaNO
3, KBrO
3, ultra-pure water composition, it is characterized in that: described (NH
4)
2sO
4parts by weight be 300-400, the parts by weight of described NaCl are 2-3, described Na
2the parts by weight of SO4 are 18-25, described NaNO
3parts by weight be 0.2-0.3, described KBrO
3parts by weight be 0.1-0.2, the parts by weight of described ultra-pure water are 1000; The preparation method of biological tissue's cryoprotective agent, adopt ultra-pure water as medium, the first step takes (NH4)
2the parts by weight of SO4 are 300-400, add in the ultra-pure water of 600 parts by weight; The parts by weight that second step takes NaCL are 2-3, Na
2the parts by weight of SO4 are the parts by weight of 18-25 and NaNO3 is 0.2-0.3, adds in the ultra-pure water of 300 parts by weight successively; The parts by weight that 3rd step takes KBrO3 are 0.1-0.2, then add in the ultra-pure water of 100 parts by weight; Three kinds of reagent of above-mentioned three step gained are respectively stirred 1min with glass bar, ultrasonic echography 20min; Then second step, the 3rd step two kinds of reagent are slowly added successively in the reagent of the first step, limit edged glass bar stirs, and finally with ultrasonic ultrasonic 10min again, pours in Brown Glass Brown glass bottles and jars only, for subsequent use with 4 DEG C of Refrigerator stores.
2. biological tissue according to claim 1 cryoprotective agent, is characterized in that: described (NH
4)
2sO
4parts by weight be 300, the parts by weight of described NaCl are 2, described Na
2the parts by weight of SO4 are 18, described NaNO
3parts by weight be 0.2, described KBrO
3parts by weight be 0.1.
3. biological tissue according to claim 1 cryoprotective agent, is characterized in that: described (NH
4)
2sO
4parts by weight be 400, the parts by weight of described NaCl are 3, described Na
2the parts by weight of SO4 are 25, described NaNO
3parts by weight be 0.3, described KBrO
3parts by weight be 0.2.
4. biological tissue according to claim 1 cryoprotective agent, is characterized in that: described (NH
4)
2sO
4parts by weight be 350, the parts by weight of described NaCl are 2.5, described Na
2the parts by weight of SO4 are 20, described NaNO
3parts by weight be 0.25, described KBrO
3parts by weight be 0.15.
5. the using method of biological tissue according to claim 1 cryoprotective agent; it is characterized in that: first step biological sample organizes stripping and slicing; the biological sample cut at room temperature soaks with biological tissue's cryoprotective agent by second step; make solution infiltrate cell and make sample; sample is 4 DEG C in temperature and places 6 hours by the 3rd step, and the 4th step is preserved under sample is moved into-20 DEG C or-86 DEG C of environment for a long time.
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CN107041364A (en) * | 2017-05-27 | 2017-08-15 | 广州基赛生物科技有限公司 | A kind of biological tissue's stability protective agent and its preparation method and application |
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CN101919381A (en) * | 2010-09-06 | 2010-12-22 | 南方医科大学珠江医院 | Preserving fluid of hepatic cells for biological artificial liver and preparation method thereof |
CN103783031A (en) * | 2012-10-29 | 2014-05-14 | 四川新生命干细胞科技股份有限公司 | Cell preserving liquid |
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CN101919381A (en) * | 2010-09-06 | 2010-12-22 | 南方医科大学珠江医院 | Preserving fluid of hepatic cells for biological artificial liver and preparation method thereof |
CN103783031A (en) * | 2012-10-29 | 2014-05-14 | 四川新生命干细胞科技股份有限公司 | Cell preserving liquid |
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