CN103305456A - 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
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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 cultivates and to arise from the sixties in 20th century, and development has formed the cell culture system that a cover comprises the comparatively perfects such as substratum, microbiotic, former culture and the cultural method that goes down to posterity so far, up to now, has set up more than 280 strain clones.China's fish cell is cultivated and was gone through more than 30 years, has also only set up more than 50 strain fish cells system, and the kind that relates to is had an appointment 30 kinds, mainly take the ocean kind as main.Can find out from above example, the ripe cell culture technology flow process of one cover is not the essential condition of a strain Establishment of Cell Line success, the proven technique flow process can not make cell cultures succeed, cell cultures still needs to start with from the biological characteristics that primary cell species itself are provided, culture condition to cell culture system is adjusted, through arduous field study and a large amount of laboratory experiments, find optimum culture condition, cell cultures is succeeded.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 the dragon's pool.Although the sinocyclocheilus grahami artificial propagation techniques has been researched and developed successfully, and the planned Dian Chi of releasing back, but because the limitation of cultured fishes itself, artificial propagation easily causes chromosome abnormalty under the foster environment in the pool, such as polyploidization and different times of change, need the cell Short-term Culture to be used for karyotyping to detect the quality of artificial propagation kind, guarantee 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 satisfy the needs of karyotyping; On the other hand, because the suitableeest existence temperature of sinocyclocheilus grahami is about 22 ℃, 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 seek the method that can obtain fast cell, be used for karyotyping with the Short-term Culture that satisfies the sinocyclocheilus grahami cell.
By literature search, have no and identical report of the present invention.
Summary of the invention:
The construction process that the purpose of this invention is to provide a kind of sinocyclocheilus grahami olfactory sac clone easy to operation is preserved and the needs of theoretical investigation the quick karyotyping of sinocyclocheilus grahami, germ plasm resource to satisfy.
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
The 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: in the L-15 nutrient solution, add foetal calf serum, make the foetal calf serum volume account for 10% of cumulative volume;
Former culture liquid: add foetal calf serum and microbiotic in the L-15 nutrient solution, make the 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: add foetal calf serum and microbiotic in the L-15 nutrient solution, make the 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;
The pH value of regulating above-mentioned nutrient solution is 7.0-7.2, is positioned in 4 ℃ of refrigerators to save backup.
2 former cultures
Soak sinocyclocheilus grahami 10 min with 8 mg/L potassium permanganate first, fish is carried out overall disinfection after, again with 75% alcohol disinfecting fish body, place Bechtop, get its olfactory sac with aseptic disscting instrument, place 12 orifice plates, add HBSS medicining liquid dipping olfactory sac tissue; After soaking 15 min, the olfactory sac tissue is cleaned 5 times with the HBSS thimerosal, be cut into and organize fritter, and it is inoculated in 25 cm
2In the Tissue Culture Flask, be inverted in 20 ℃ of incubators and spend the night, add former culture liquid 3 ml next day again, start former culture in 20 ℃ of incubators, per three and half amounts are changed nutrient solutions, and cell began to move from tissue block in the 4th day, grow up to cell monolayer in 20 days.
3 cultivations of going down to posterity
Began to go down to posterity after 80% at the bottom of former generation, the olfactory sac cell was paved with bottle.Going down to posterity, it is as follows to cultivate concrete grammar, former culture liquid in elder generation's sucking-off culturing bottle, the trypsinase of adding 0.13%-EDTA solution 1 ml, digestion 2min is behind the cell rounding, add go down to posterity nutrient solution 1 ml with in and the pancreatin reaction, gently the piping and druming bottle end, attached cell is come off, going down to posterity first passes by 1:1, after this go down to posterity by 1:2, in 20 ℃ of incubators, continue to cultivate; Went down to posterity once in per 5 days, when reaching for the 5th generation, cell culture fluid changes basic culture solution into, at this moment, and the 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, presses the volume ratio of 9:1 and mixes matching while using.
(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.In cell precipitation, add the cells frozen storing liquid of preparing, resuspended, make the concentration of cell be about 1 * 10
6Individual/ml, 1 ml cell suspension is transferred in the 1.8 ml cryopreservation tubes, cryopreservation tube is placed program temperature reduction box ,-80 ℃ are spent the night, and then put 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, in the cell transfer to 15 of will the thawing ml centrifuge tube, and add the equivalent basic culture solution, centrifugal 8 min of 1000 rpm are except supernatant liquor.Use the basic culture solution re-suspended cell, and be transferred in the Tissue Culture Flask, cultivate in 20 ℃ of incubators, cell namely grew up to individual layer in 7 days.
Unusual effect of the present invention is:
1, easy to operation.
2, with respect to the blood Short-term Culture that is used for karyotyping, the cell that has just moved out in the olfactory sac tissue block 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 that makes up is for becoming fiber-like, can continuous passage and directly apply to biological characteristic research, satisfied the needs to the preservation of sinocyclocheilus grahami germ plasm resource and theoretical investigation and application;
5, this construction process also is applicable to the clone that other fish make up olfactory sac.
Embodiment:
1 preparation HBSS thimerosal and cell culture fluid
The 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: in the L-15 nutrient solution, add foetal calf serum, make the foetal calf serum volume account for 10% of cumulative volume;
Former culture liquid: add foetal calf serum and microbiotic in the L-15 nutrient solution, make the 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: add foetal calf serum and microbiotic in the L-15 nutrient solution, make the 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;
The pH value of regulating above-mentioned nutrient solution is 7.0-7.2, is positioned in 4 ℃ of refrigerators to save backup.
2 former cultures
Soak sinocyclocheilus grahami 10 min with 8 mg/L potassium permanganate first, fish is carried out overall disinfection after, again with 75% alcohol disinfecting fish body, place Bechtop, get its olfactory sac with aseptic disscting instrument, place 12 orifice plates, add HBSS medicining liquid dipping olfactory sac tissue; After soaking 15 min, the olfactory sac tissue is cleaned 5 times with the HBSS thimerosal, be cut into and organize fritter, and it is inoculated in 25 cm
2In the Tissue Culture Flask, be inverted in 20 ℃ of incubators and spend the night, add former culture liquid 3 ml next day again, start former culture in 20 ℃ of incubators, per three and half amounts are changed nutrient solutions, and cell began to move from tissue block in the 4th day, grow up to cell monolayer in 20 days.
3 cultivations of going down to posterity
Began to go down to posterity after 80% at the bottom of former generation, the olfactory sac cell was paved with bottle.Going down to posterity, it is as follows to cultivate concrete grammar, former culture liquid in elder generation's sucking-off culturing bottle, the trypsinase of adding 0.13%-EDTA solution 1 ml, digestion 2min is behind the cell rounding, add go down to posterity nutrient solution 1 ml with in and the pancreatin reaction, gently the piping and druming bottle end, attached cell is come off, going down to posterity first passes by 1:1, after this go down to posterity by 1:2, in 20 ℃ of incubators, continue to cultivate; Went down to posterity once in per 5 days, when reaching for the 5th generation, cell culture fluid changes basic culture solution into, at this moment, and the 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, presses the volume ratio of 9:1 and mixes matching while using.
(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.In cell precipitation, add the cells frozen storing liquid of preparing, resuspended, make the concentration of cell be about 1 * 10
6Individual/ml, 1 ml cell suspension is transferred in the 1.8 ml cryopreservation tubes, cryopreservation tube is placed program temperature reduction box ,-80 ℃ are spent the night, and then put 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, in the cell transfer to 15 of will the thawing ml centrifuge tube, and add the equivalent basic culture solution, centrifugal 8 min of 1000 rpm are except supernatant liquor.Use the basic culture solution re-suspended cell, and be transferred in the Tissue Culture Flask, cultivate in 20 ℃ of incubators, cell namely grew up to individual layer in 7 days.
Claims (1)
1. structure and the cryopreservation method of a sinocyclocheilus grahami olfactory sac clone is characterized in that the step of this construction process is as follows:
(1) preparation HBSS thimerosal and cell culture fluid
The 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: in the L-15 nutrient solution, add foetal calf serum, make the foetal calf serum volume account for 10% of cumulative volume;
Former culture liquid: add foetal calf serum and microbiotic in the L-15 nutrient solution, make the 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: add foetal calf serum and microbiotic in the L-15 nutrient solution, make the 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;
The pH value of regulating above-mentioned nutrient solution is 7.0-7.2, is positioned over to save backup in 4 ℃ of refrigerators;
(2) former culture
Soak sinocyclocheilus grahami 10 min with 8 mg/L potassium permanganate first, after fish carried out overall disinfection, again with 75% alcohol disinfecting fish body, place Bechtop, get its olfactory sac with aseptic disscting instrument, place 12 orifice plates, add HBSS medicining liquid dipping olfactory sac tissue, soak 15 min after, with olfactory sac tissue usefulness HBSS thimerosal cleaning 5 times, be cut into and organize fritter, and it is inoculated in 25 cm
2In the Tissue Culture Flask, be inverted in 20 ℃ of incubators and spend the night, add former culture liquid 3 ml next day again, start former culture in 20 ℃ of incubators, per three and half amounts are changed nutrient solutions, and cell began to move from tissue block in the 4th day, grow up to cell monolayer in 20 days;
(3) cultivation of going down to posterity
Began to go down to posterity after 80% at the bottom of former generation, the olfactory sac cell was paved with bottle, going down to posterity, it is as follows to cultivate concrete grammar, the former culture liquid in the first sucking-off culturing bottle, the trypsinase of adding 0.13%-EDTA solution 1 ml, digestion 2min, behind the cell rounding, add go down to posterity nutrient solution 1 ml with in and the pancreatin reaction, a piping and druming bottle end, attached cell is come off, going down to posterity first passes by 1:1, after this goes down to posterity by 1:2, continues to cultivate in 20 ℃ of incubators; Went down to posterity once in per 5 days, when reaching for the 5th generation, cell culture fluid changes basic culture solution into, at this moment, and the 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, presses the volume ratio of 9:1 and mixes matching while using;
(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, add the cells frozen storing liquid of preparing in cell precipitation, and resuspended, the concentration that makes cell is 1 * 10
6Individual/ml, 1 ml cell suspension is transferred in the 1.8 ml cryopreservation tubes, cryopreservation tube is placed program temperature reduction box ,-80 ℃ are spent the night, and then put 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, in the cell transfer to 15 of will the thawing ml centrifuge tube, and add the equivalent basic culture solution, centrifugal 8 min of 1000 rpm, except supernatant liquor, use the basic culture solution re-suspended cell, and be transferred in the Tissue Culture Flask, cultivate in 20 ℃ of incubators, cell namely grew up to individual layer in 7 days.
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| CN104805052A (en) * | 2015-04-27 | 2015-07-29 | 中国科学院昆明动物研究所 | Method for building neolissochilus banasi muscle cell line |
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| CN105309420A (en) * | 2015-12-01 | 2016-02-10 | 中国科学院昆明动物研究所 | Ultralow-temperature cryopreservation method for sperms of sinocyclocheilus grahami |
| CN105349484A (en) * | 2015-12-01 | 2016-02-24 | 中国科学院昆明动物研究所 | Method for constructing sinocyclocheilus rhinocerous heart cell line |
| CN106479960A (en) * | 2016-10-27 | 2017-03-08 | 中国科学院昆明动物研究所 | A kind of construction method of smooth agnail fin clone |
| 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 |
| CN115261324A (en) * | 2022-07-13 | 2022-11-01 | 华中农业大学 | Culture method of fish olfactory neurons |
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| CN104805052A (en) * | 2015-04-27 | 2015-07-29 | 中国科学院昆明动物研究所 | Method for building neolissochilus banasi muscle cell line |
| CN104818239A (en) * | 2015-04-27 | 2015-08-05 | 中国科学院昆明动物研究所 | Method for establishing liver cell lines of semilabeo obscurus |
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| CN105309420A (en) * | 2015-12-01 | 2016-02-10 | 中国科学院昆明动物研究所 | Ultralow-temperature cryopreservation method for sperms of sinocyclocheilus grahami |
| CN105349484A (en) * | 2015-12-01 | 2016-02-24 | 中国科学院昆明动物研究所 | Method for constructing sinocyclocheilus rhinocerous heart cell line |
| 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 |
| 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 |
| CN106479960A (en) * | 2016-10-27 | 2017-03-08 | 中国科学院昆明动物研究所 | A kind of construction method of smooth agnail fin clone |
| 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 |
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| CN115261324B (en) * | 2022-07-13 | 2024-05-17 | 华中农业大学 | Culture method of fish olfactory neurons |
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