CN104054695B - Biological tissue's cryoprotective agent and preparation, using method - Google Patents

Biological tissue's cryoprotective agent and preparation, using method Download PDF

Info

Publication number
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
Authority
CN
China
Prior art keywords
weight
parts
biological tissue
cryoprotective agent
ultra
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Fee Related
Application number
CN201410316055.3A
Other languages
Chinese (zh)
Other versions
CN104054695A (en
Inventor
覃东立
张昌盛
李林海
王俊杰
张晓萌
钟震宇
王玉涛
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Heilongjiang River Fisheries Research Institute of Chinese Academy of Fishery Sciences
Original Assignee
Heilongjiang River Fisheries Research Institute of Chinese Academy of Fishery Sciences
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Heilongjiang River Fisheries Research Institute of Chinese Academy of Fishery Sciences filed Critical Heilongjiang River Fisheries Research Institute of Chinese Academy of Fishery Sciences
Priority to CN201410316055.3A priority Critical patent/CN104054695B/en
Publication of CN104054695A publication Critical patent/CN104054695A/en
Application granted granted Critical
Publication of CN104054695B publication Critical patent/CN104054695B/en
Expired - Fee Related legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Landscapes

  • Sampling And Sample Adjustment (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)

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

Biological tissue's cryoprotective agent and preparation, using method
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.
CN201410316055.3A 2014-07-04 2014-07-04 Biological tissue's cryoprotective agent and preparation, using method Expired - Fee Related CN104054695B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201410316055.3A CN104054695B (en) 2014-07-04 2014-07-04 Biological tissue's cryoprotective agent and preparation, using method

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201410316055.3A CN104054695B (en) 2014-07-04 2014-07-04 Biological tissue's cryoprotective agent and preparation, using method

Publications (2)

Publication Number Publication Date
CN104054695A CN104054695A (en) 2014-09-24
CN104054695B true CN104054695B (en) 2016-02-17

Family

ID=51542857

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201410316055.3A Expired - Fee Related CN104054695B (en) 2014-07-04 2014-07-04 Biological tissue's cryoprotective agent and preparation, using method

Country Status (1)

Country Link
CN (1) CN104054695B (en)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106857502A (en) * 2017-03-03 2017-06-20 湖北新纵科病毒疾病工程技术有限公司 A kind of the Sample storage liquid and store method that prevent RNA from degrading
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

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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

Also Published As

Publication number Publication date
CN104054695A (en) 2014-09-24

Similar Documents

Publication Publication Date Title
US20240027309A1 (en) Simple fixation and stabilisation
CA2298841A1 (en) Methods and reagents for preserving rna in cell and tissue samples
JP2010524505A (en) Sample storage for life sciences
CN104054695B (en) Biological tissue's cryoprotective agent and preparation, using method
CN103820320A (en) Non-freezing type RNA (Ribonucleic Acid) protection fluid
CN109609498A (en) A kind of the sample preservation liquid and its store method of animal tissue RNA
CN107041364A (en) A kind of biological tissue's stability protective agent and its preparation method and application
Komatsu et al. Deutrium oxide, dimethylsulfoxide and heat shock confer protection against hydrostatic pressure damage in yeast
Parmaksiz et al. Decellularization of bovine small intestinal submucosa
Kim et al. Production of antifreeze proteins by cold-adapted yeasts
CN105039306A (en) Saliva protection agent
Kato et al. Recovery of cell nuclei from 15,000 years old mammoth tissues and its injection into mouse enucleated matured oocytes
CN104031909A (en) Efficient extraction method for river crab genome DNA after long-term storage in alcohol
Phillips et al. Preparation of cell extracts by cryogrinding in an automated freezer mill
CN103740746B (en) The prokaryotic expression carrier of paddy rice metallothionein gene OsMT-1-2a and application thereof
CN106857502A (en) A kind of the Sample storage liquid and store method that prevent RNA from degrading
Junior DNA and RNA stabilization
CN102559654A (en) DNA (deoxyribonucleic acid) preserving solvent and DNA preserving method
CN103173433A (en) DNA (Deoxyribonucleic Acid) low-temperature storage solvent and storage method thereof
Harlow et al. A novel, simplified technique for preservation and rapid isolation of total RNA from the toxic dinoflagellate Alexandrium catenella (Dinophyceae)
US20230183777A1 (en) Hypoosmotic stabilizer for genomic dna of gut microbiota, and preparation method and use thereof
JP2016537032A5 (en)
CN106635770A (en) Blood sampling tube capable of protecting and stabilizing virus RNA
Lopes et al. Frost Fighters: Unveiling the Potentials of Microbial Antifreeze Proteins in Biotech Innovation
Corriveau et al. Effect of eight storage modes on DNA preservation

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
C14 Grant of patent or utility model
GR01 Patent grant
CF01 Termination of patent right due to non-payment of annual fee

Granted publication date: 20160217

Termination date: 20200704

CF01 Termination of patent right due to non-payment of annual fee