CN103305456B - Construction method and ultralow temperature freezing and storing method of sinocyclocheilus grahami saccus olfactorius cell line - Google Patents
Construction method and ultralow temperature freezing and storing method of sinocyclocheilus grahami saccus olfactorius cell line Download PDFInfo
- Publication number
- CN103305456B CN103305456B CN201310291001.1A CN201310291001A CN103305456B CN 103305456 B CN103305456 B CN 103305456B CN 201310291001 A CN201310291001 A CN 201310291001A CN 103305456 B CN103305456 B CN 103305456B
- Authority
- CN
- China
- Prior art keywords
- cell
- nutrient solution
- concentration
- posterity
- culture
- 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.)
- Active
Links
Abstract
The invention relates to a construction method and ultralow temperature freezing and storing method of a sinocyclocheilus grahami saccus olfactorius cell line, and belongs to the technical field of cell culture and ultralow temperature freezing and storing method of the biological cell of freshwater aquatic life. A sinocyclocheilus grahami saccus olfactorius tissue is used as a raw material to be cultured in an L-15 culture solution at the pH (Potential of Hydrogen) value of 7.0-7.2 and comprising fetal calf serum by means of a tissue explant and subculture is carried out by means of trypsin digestion. The method specifically comprises the preparation of a cell culture solution, primary culture and subculture. The construction method disclosed by the invention has the following beneficial effects: (1) primary culture takes short time, and cell mass is large and can be applied to chromosome analysis; (2) the constructed sinocyclocheilus grahami saccus olfactorius cell line is in fiber like morphology, can be subcultured continuously and directly applied to a research on biological characteristics, and satisfies the demands of the storage, the theoretical research and the application of sinocyclocheilus grahami germplasm resources; (3) the construction method is also applicable to the construction of saccus olfactorius cell lines of other fishes.
Description
Technical field:
The present invention relates to a kind of method of utilizing sinocyclocheilus grahami olfactory sac tissue to set up clone and superfreeze preservation, belong to the technical field that fresh water hydrobiont cell cultures and superfreeze are preserved.
Background technology:
Fish cell is cultivated and is arised from the sixties in 20th century, is developed so far and has formed a set of cell culture system that comprises substratum, microbiotic, former culture and the comparatively perfects such as cultural method that go down to posterity, and up to now, has set up more than 280 strain clones.China's fish cell is cultivated and is gone through more than 30 years, has also only set up more than 50 strain fish cell systems, and the kind relating to is had an appointment 30 kinds, mainly take ocean kind as main.From above example, can find out, the cell culture technology flow process of a set of maturation is not the successful essential condition of a strain Establishment of Cell Line, proven technique flow process can not make cell cultures succeed, cell cultures still need to be from providing the biological characteristics of primary cell species itself to start with, culture condition to cell culture system is adjusted, through arduous field study and a large amount of laboratory experiments, find optimum culture condition, just can make cell cultures succeed.Nearly 600 kinds of Yunnan original inhabitants freshwater fish, accounts for 50% of Freshwater Fishes In China, and therefore, Yunnan original inhabitants' fish germ plasma resource is preserved and seemed particularly important, but the rarely seen report of cell culture studies.
Sinocyclocheilus grahami Sinocyclocheilus grahami(Regan; 1904) be under the jurisdiction of Cypriniformes (Cypriniformes) Cyprinidae (Cyprinidae) gold thread Barb and belong to (Sinocyclocheilus); be the endemic species of Dianchi Lake Basin, national II level watches for animals.Sinocyclocheilus grahami disappears at lake, Dian Chi body at present, now exists only in streams, lake and dragon's pool.Although sinocyclocheilus grahami artificial propagation techniques has been researched and developed successfully, and the planned Dian Chi of releasing back, but the limitation due to cultured fishes itself, the pool is supported artificial propagation under environment and is easily caused chromosome abnormalty, as polyploidization and different times of change, need cell Short-term Culture for karyotyping to detect the quality of artificial propagation kind, guarantee to release individual surviving rate and breeding potential.And less for this individual relative of sinocyclocheilus grahami, the fish that blood flow volume is less, traditional blood Short-term Culture can not meet the needs of karyotyping; On the other hand, because the suitableeest existence temperature of sinocyclocheilus grahami is 22 ℃ of left and right, the culture temperature that has determined cell is not high, and temperature is the key factor that affects Growth of Cells speed, therefore the former culture of sinocyclocheilus grahami fin ray clone continues two months nearly, therefore be necessary to find the method that can obtain fast cell, with the Short-term Culture that meets sinocyclocheilus grahami cell for karyotyping.
By literature search, have no and identical report of the present invention.
Summary of the invention:
The construction process that the object of this invention is to provide a kind of sinocyclocheilus grahami olfactory sac clone easy to operation, to meet the needs to the quick karyotyping of sinocyclocheilus grahami, germ plasm resource preservation and theoretical investigation.
The construction process of sinocyclocheilus grahami olfactory sac clone of the present invention, step is as follows:
1 preparation HBSS thimerosal and cell culture fluid
HBSS thimerosal: add microbiotic in HBSS, making sulphuric acid kanamycin concentration is 100 μ g/ml, and gentamicin concentration is 20 μ g/ml, and amphotericin B concentration is 20 μ g/ml;
Basic culture solution: add foetal calf serum in L-15 nutrient solution, make foetal calf serum volume account for 10% of cumulative volume;
Primary nutrient solution: add foetal calf serum and microbiotic in L-15 nutrient solution, make foetal calf serum volume account for 20% of cumulative volume, sulphuric acid kanamycin concentration is 100 μ g/ml, and gentamicin concentration is 20 μ g/ml, and amphotericin B concentration is 20 μ g/ml;
Nutrient solution goes down to posterity: in L-15 nutrient solution, add foetal calf serum and microbiotic, make foetal calf serum volume account for 20% of cumulative volume, sulphuric acid kanamycin concentration is 50 μ g/ml, and gentamicin concentration is 10 μ g/ml, and amphotericin B concentration is 10 μ g/ml;
Regulating the pH value of above-mentioned nutrient solution is 7.0-7.2, is positioned in 4 ℃ of refrigerators and saves backup.
2 former cultures
First with 8 mg/L potassium permanganate, soak sinocyclocheilus grahami 10 min, fish is carried out after overall disinfection, again with 75% alcohol disinfecting fish body, be placed in Bechtop, with aseptic disscting instrument, get its olfactory sac, be placed in 12 orifice plates, add HBSS medicining liquid dipping olfactory sac tissue; Soak after 15 min, olfactory sac tissue is cleaned 5 times with HBSS thimerosal, be cut into and organize fritter, and be inoculated in 25 cm
2in Tissue Culture Flask, in 20 ℃ of incubators, be inverted and spend the night, add primary nutrient solution 3 ml next day again, in 20 ℃ of incubators, start former culture, every three and half amounts are changed nutrient solution, and within the 4th day, cell starts to move from tissue block, within 20 days, grows up to cell monolayer.
3 cultivations of going down to posterity
At the bottom of being paved with bottle, primary olfactory sac cell starts to go down to posterity after 80%.Going down to posterity, it is as follows to cultivate concrete grammar, primary nutrient solution in first sucking-off culturing bottle, add trypsinase-EDTA solution 1 ml of 0.13%, digestion 2min, after cell rounding, add go down to posterity nutrient solution 1 ml with in and pancreatin reaction, gently the piping and druming bottle end, attached cell is come off, go down to posterity first and pass by 1:1, after this by 1:2, go down to posterity, in 20 ℃ of incubators, continue to cultivate; Within every 5 days, go down to posterity once, while reaching for the 5th generation, cell culture fluid changes basic culture solution into, now, and Establishment of Cell Line success.At present, passage reached for 30 generations.
4 cell cryopreservations and recovery
(a) preparation cell cryopreservation protection liquid: frozen storing liquid comprises foetal calf serum and DMSO, mixes matching while using by the volume ratio of 9:1.
(b) cell cryopreservation: the cell in the vegetative period of taking the logarithm, after above-mentioned trysinization, centrifugal 8 min of cell suspension 1200 rpm, discard supernatant liquor.To the cells frozen storing liquid that adds preparation in cell precipitation, resuspended, make the concentration of cell be about 1 * 10
6individual/ml, is transferred to 1 ml cell suspension in 1.8 ml cryopreservation tubes, and cryopreservation tube is placed in to program temperature reduction box, and-80 ℃ are spent the night, and then puts into the medium-term and long-term preservation of liquid nitrogen.
(c) cell recovery: above-mentioned cryopreservation tube is taken out from liquid nitrogen, put into 37 ℃ of water-baths and rock to thawing fast.Then in aseptic operating platform, the cell that will thaw is transferred in 15 ml centrifuge tubes, and adds equivalent basic culture solution, and centrifugal 8 min of 1000 rpm, except supernatant liquor.Use basic culture solution re-suspended cell, and be transferred in Tissue Culture Flask, in 20 ℃ of incubators, cultivate, within 7 days, cell grows up to individual layer.
Unusual effect of the present invention is:
1, easy to operation.
2,, with respect to the blood Short-term Culture for karyotyping, in olfactory sac tissue block, just migration cell out has activity, and cell concentration is large, can be used for chromosome analysis.
3, compare with fin ray clone, the former culture of olfactory sac clone is consuming time short, and does not need somatomedin.
4, the sinocyclocheilus grahami olfactory sac clone form building is for becoming fiber-like, can continuous passage and directly apply to biological characteristic research, met the needs to the preservation of sinocyclocheilus grahami germ plasm resource and theoretical investigation and application;
5, this construction process is also applicable to the clone that other fish build olfactory sac.
Embodiment:
1 preparation HBSS thimerosal and cell culture fluid
HBSS thimerosal: add microbiotic in HBSS, making sulphuric acid kanamycin concentration is 100 μ g/ml, and gentamicin concentration is 20 μ g/ml, and amphotericin B concentration is 20 μ g/ml.
Basic culture solution: add foetal calf serum in L-15 nutrient solution, make foetal calf serum volume account for 10% of cumulative volume;
Primary nutrient solution: add foetal calf serum and microbiotic in L-15 nutrient solution, make foetal calf serum volume account for 20% of cumulative volume, sulphuric acid kanamycin concentration is 100 μ g/ml, and gentamicin concentration is 20 μ g/ml, and amphotericin B concentration is 20 μ g/ml;
Nutrient solution goes down to posterity: in L-15 nutrient solution, add foetal calf serum and microbiotic, make foetal calf serum volume account for 20% of cumulative volume, sulphuric acid kanamycin concentration is 50 μ g/ml, and gentamicin concentration is 10 μ g/ml, and amphotericin B concentration is 10 μ g/ml;
Regulating the pH value of above-mentioned nutrient solution is 7.0-7.2, is positioned in 4 ℃ of refrigerators and saves backup.
2 former cultures
First with 8 mg/L potassium permanganate, soak sinocyclocheilus grahami 10 min, fish is carried out after overall disinfection, again with 75% alcohol disinfecting fish body, be placed in Bechtop, with aseptic disscting instrument, get its olfactory sac, be placed in 12 orifice plates, add HBSS medicining liquid dipping olfactory sac tissue; Soak after 15 min, olfactory sac tissue is cleaned 5 times with HBSS thimerosal, be cut into and organize fritter, and be inoculated in 25 cm
2in Tissue Culture Flask, in 20 ℃ of incubators, be inverted and spend the night, add primary nutrient solution 3 ml next day again, in 20 ℃ of incubators, start former culture, every three and half amounts are changed nutrient solution, and within the 4th day, cell starts to move from tissue block, within 20 days, grows up to cell monolayer.
3 cultivations of going down to posterity
At the bottom of being paved with bottle, primary olfactory sac cell starts to go down to posterity after 80%.Going down to posterity, it is as follows to cultivate concrete grammar, primary nutrient solution in first sucking-off culturing bottle, add trypsinase-EDTA solution 1 ml of 0.13%, digestion 2min, after cell rounding, add go down to posterity nutrient solution 1 ml with in and pancreatin reaction, gently the piping and druming bottle end, attached cell is come off, go down to posterity first and pass by 1:1, after this by 1:2, go down to posterity, in 20 ℃ of incubators, continue to cultivate; Within every 5 days, go down to posterity once, while reaching for the 5th generation, cell culture fluid changes basic culture solution into, now, and Establishment of Cell Line success.At present, passage reached for 30 generations.
4 cell cryopreservations and recovery
(a) preparation cell cryopreservation protection liquid: frozen storing liquid comprises foetal calf serum and DMSO, mixes matching while using by the volume ratio of 9:1.
(b) cell cryopreservation: the cell in the vegetative period of taking the logarithm, after above-mentioned trysinization, centrifugal 8 min of cell suspension 1200 rpm, discard supernatant liquor.To the cells frozen storing liquid that adds preparation in cell precipitation, resuspended, make the concentration of cell be about 1 * 10
6individual/ml, is transferred to 1 ml cell suspension in 1.8 ml cryopreservation tubes, and cryopreservation tube is placed in to program temperature reduction box, and-80 ℃ are spent the night, and then puts into the medium-term and long-term preservation of liquid nitrogen.
(c) cell recovery: above-mentioned cryopreservation tube is taken out from liquid nitrogen, put into 37 ℃ of water-baths and rock to thawing fast.Then in aseptic operating platform, the cell that will thaw is transferred in 15 ml centrifuge tubes, and adds equivalent basic culture solution, and centrifugal 8 min of 1000 rpm, except supernatant liquor.Use basic culture solution re-suspended cell, and be transferred in Tissue Culture Flask, in 20 ℃ of incubators, cultivate, within 7 days, cell grows up to individual layer.
Claims (1)
1. a construction process for sinocyclocheilus grahami olfactory sac clone, is characterized in that the step of this construction process is as follows:
(1) prepare HBSS thimerosal and cell culture fluid
HBSS thimerosal: add microbiotic in HBSS, making sulphuric acid kanamycin concentration is 100 μ g/ml, and gentamicin concentration is 20 μ g/ml, and amphotericin B concentration is 20 μ g/ml;
Basic culture solution: add foetal calf serum in L-15 nutrient solution, make foetal calf serum volume account for 10% of cumulative volume;
Primary nutrient solution: add foetal calf serum and microbiotic in L-15 nutrient solution, make foetal calf serum volume account for 20% of cumulative volume, sulphuric acid kanamycin concentration is 100 μ g/ml, and gentamicin concentration is 20 μ g/ml, and amphotericin B concentration is 20 μ g/ml;
Nutrient solution goes down to posterity: in L-15 nutrient solution, add foetal calf serum and microbiotic, make foetal calf serum volume account for 20% of cumulative volume, sulphuric acid kanamycin concentration is 50 μ g/ml, and gentamicin concentration is 10 μ g/ml, and amphotericin B concentration is 10 μ g/ml;
Regulating the pH value of above-mentioned nutrient solution is 7.0-7.2, is positioned in 4 ℃ of refrigerators and saves backup;
(2) former culture
First with 8mg/L potassium permanganate, soak sinocyclocheilus grahami 10min, fish is carried out after overall disinfection, again, with 75% alcohol disinfecting fish body, be placed in Bechtop, with aseptic disscting instrument, get its olfactory sac, be placed in 12 orifice plates, add HBSS medicining liquid dipping olfactory sac tissue, soak after 15min, olfactory sac tissue is cleaned 5 times with HBSS thimerosal, be cut into and organize fritter, and be inoculated in 25cm
2in Tissue Culture Flask, in 20 ℃ of incubators, be inverted and spend the night, add primary nutrient solution 3ml next day again, in 20 ℃ of incubators, start former culture, every three and half amounts are changed nutrient solution, and within the 4th day, cell starts to move from tissue block, within 20 days, grows up to cell monolayer;
(3) cultivation of going down to posterity
At the bottom of being paved with bottle, primary olfactory sac cell starts to go down to posterity after 80%, going down to posterity, it is as follows to cultivate concrete grammar, and the primary nutrient solution in first sucking-off culturing bottle adds trypsinase-EDTA solution 1ml of 0.13%, digestion 2min, after cell rounding, add go down to posterity nutrient solution 1ml with in and pancreatin reaction, a piping and druming bottle end, attached cell is come off, go down to posterity first and pass by 1:1, after this by 1:2, go down to posterity, in 20 ℃ of incubators, continue to cultivate; Within every 5 days, go down to posterity once, while reaching for the 5th generation, cell culture fluid changes basic culture solution into, now, and Establishment of Cell Line success;
(4) cell cryopreservation and recovery
(a) preparation cell cryopreservation protection liquid: frozen storing liquid comprises foetal calf serum and DMSO, mixes matching while using by the volume ratio of 9:1;
(b) cell cryopreservation: the cell in the vegetative period of taking the logarithm, after above-mentioned trysinization, the centrifugal 8min of cell suspension 1200rpm, discards supernatant liquor, and to the cells frozen storing liquid that adds preparation in cell precipitation, resuspended, the concentration that makes cell is 1 * 10
6individual/ml, is transferred to 1ml cell suspension in 1.8ml cryopreservation tube, and cryopreservation tube is placed in to program temperature reduction box, and-80 ℃ are spent the night, and then puts into the medium-term and long-term preservation of liquid nitrogen;
(c) cell recovery: above-mentioned cryopreservation tube is taken out from liquid nitrogen, putting into 37 ℃ of water-baths rocks fast to thawing, then in aseptic operating platform, the cell that will thaw is transferred in 15ml centrifuge tube, and adds equivalent basic culture solution, the centrifugal 8min of 1000rpm, except supernatant liquor, use basic culture solution re-suspended cell, and be transferred in Tissue Culture Flask, in 20 ℃ of incubators, cultivate, within 7 days, cell grows up to individual layer.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310291001.1A CN103305456B (en) | 2013-07-11 | 2013-07-11 | Construction method and ultralow temperature freezing and storing method of sinocyclocheilus grahami saccus olfactorius cell line |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310291001.1A CN103305456B (en) | 2013-07-11 | 2013-07-11 | Construction method and ultralow temperature freezing and storing method of sinocyclocheilus grahami saccus olfactorius cell line |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103305456A CN103305456A (en) | 2013-09-18 |
| CN103305456B true CN103305456B (en) | 2014-11-19 |
Family
ID=49131178
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310291001.1A Active CN103305456B (en) | 2013-07-11 | 2013-07-11 | Construction method and ultralow temperature freezing and storing method of sinocyclocheilus grahami saccus olfactorius cell line |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103305456B (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104818239B (en) * | 2015-04-27 | 2018-10-26 | 中国科学院昆明动物研究所 | A kind of construction method of dead color lip fish liver cell system |
| CN104805052B (en) * | 2015-04-27 | 2020-03-31 | 中国科学院昆明动物研究所 | Method for constructing muscle cell line of neolabeo fasciatus |
| CN105349484B (en) * | 2015-12-01 | 2019-03-08 | 中国科学院昆明动物研究所 | A kind of construction method of rhinoceros horn gold thread Barb heart cell system |
| CN105309420B (en) * | 2015-12-01 | 2017-10-27 | 中国科学院昆明动物研究所 | A kind of sinocyclocheilus grahami sperm super-low temperature freezing store method |
| CN105331576B (en) * | 2015-12-01 | 2019-03-08 | 中国科学院昆明动物研究所 | A kind of construction method for pacifying water gold thread Barb spleen cell system |
| CN106479960B (en) * | 2016-10-27 | 2020-03-31 | 中国科学院昆明动物研究所 | Construction method of spinibarbus grahami fin cell line |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102757933B (en) * | 2012-08-07 | 2013-10-23 | 中国科学院昆明动物研究所 | Construction method of muscle cell line of anabarilius grahami |
| CN102757934B (en) * | 2012-08-07 | 2013-05-29 | 中国科学院昆明动物研究所 | Construction method of fin cell line of anabarilius grahami |
| CN102757932A (en) * | 2012-08-07 | 2012-10-31 | 中国科学院昆明动物研究所 | Construction method of sinocyclocheilus grahami grahami fin cell line |
-
2013
- 2013-07-11 CN CN201310291001.1A patent/CN103305456B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN103305456A (en) | 2013-09-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103305456B (en) | Construction method and ultralow temperature freezing and storing method of sinocyclocheilus grahami saccus olfactorius cell line | |
| CN102919127B (en) | Method for building bamboo reed tissue culture system | |
| CN103355237B (en) | Culture method for promoting river crabs to shell and delaying gonad development | |
| CN103409365B (en) | Method for establishing schizothorax wangchiachii heart cell line and performing ultralow temperature cryopreservation on schizothorax wangchiachii heart cell line | |
| CN101773041B (en) | Inoculation method of cistanche tubulosa by using light medium-mesh bag container | |
| CN105532527A (en) | Ecological breeding method for Hippocampus erectus | |
| CN101647391A (en) | Culture method of oriental lily test tube bulbs | |
| CN103436490B (en) | A kind of structure of Schizothorax grahami fin clone and cryopreservation method | |
| CN101796924B (en) | Method for improving annular stalk growing rate of oriental lily test tube bulbs | |
| CN102757933B (en) | Construction method of muscle cell line of anabarilius grahami | |
| CN103039366B (en) | Industrial production method of mycorrhizal seedlings of Changbai Mountain rhododendron plants | |
| CN104488723B (en) | A kind of Herba Epimedii tissue culture quick propagation culturing method | |
| CN101984043A (en) | Open cultivation method of diatom | |
| CN102613051A (en) | Method for plant rapid propagation through air culture | |
| CN102757932A (en) | Construction method of sinocyclocheilus grahami grahami fin cell line | |
| CN101870962A (en) | Method for constructing Tibetan antelope skin fibroblast line | |
| CN107691226A (en) | The regeneration culture medium of lotus somatic embryo development ways and its application | |
| CN103766039B (en) | Method for breaking dormancy of nervilis fordii schlecht bulb | |
| CN102187786A (en) | Method for asexual propagation of wild aweto fungi | |
| CN104542296A (en) | Open rooting method for sugarcane tissue culture seedlings | |
| CN102450216B (en) | Hormone induction propagation technology for potamogeton maackianus | |
| CN106577130A (en) | Apple tree seedling growing method by cutting | |
| CN102422812A (en) | Preparation method of medium for rapid breeding of Photinia fraseri | |
| CN112806264A (en) | Tissue culture and rapid propagation method for Acer catalpa | |
| CN1817111A (en) | Yangxicai breeding technology |
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 |