CN103409365A - Method for establishing schizothorax wangchiachii heart cell line and performing ultralow temperature cryopreservation on schizothorax wangchiachii heart cell line - Google Patents
Method for establishing schizothorax wangchiachii heart cell line and performing ultralow temperature cryopreservation on schizothorax wangchiachii heart cell line Download PDFInfo
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Abstract
The invention relates to a method for establishing a schizothorax wangchiachii heart cell line and performing ultralow temperature cryoprservation on the schizothorax wangchiachii heart cell line, belonging to the technical field of freshwater aquatic organism cell culture and ultralow temperature cryopreservation. The method comprises the following steps of taking heart tissues of schizothorax wangchiachii as materials, and culturing the materials in an M199 culture solution by adopting a tissue block method and a two-enzyme digestion method, wherein the M199 culture solution contains fetal calf serum and cell growth factors and the pH value of the M199 culture solution is 7.0-7.2; and performing secondary culture by adopting a trypsase digestion method. The method specifically comprises the steps of preparing a cell culture solution, performing primary culture and performing secondary culture. The method has the beneficial effects as follows: 1, the operation is simple and convenient; 2, damages caused by a pure tissue block method to heart cells are reduced; 3, the form of the established schizothorax wangchiachii heart cell line is in the shape of fibroblasts, and the established schizothorax wangchiachii heart cell line grows well, can realize continuous passage culture, can be directly applied to biological characteristic researches, and meets requirements for storage, theoretical study and application of genetic resources of the schizothorax wangchiachii; and 4, the establishing method is also suitable for establishing heart cell lines of other types of fishes.
Description
Technical field:
The present invention relates to structure and cryopreservation method that a kind of short hairs schizothoracin heart cell is, belong to fresh water hydrobiont cell cultures and superfreeze Techniques of preserving field.
Background technology:
Fish cell is cultivated and is arised from the sixties in 20th century, and development has formed a set of cell culture system that 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 cell systems, and the kind related to is had an appointment 30 kinds, mainly take the ocean kind as main.From above example, can find out, the cell culture technology flow process of a set of maturation 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, through arduous field study and a large amount of laboratory experiments, culture condition to cell culture system is adjusted, and finds optimum culture condition, and cell cultures is succeeded.Nearly 600 kinds of Yunnan original inhabitants freshwater fish, account 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.
Short hairs schizothoracin Schizothorax wangchiachii(Fang, 1936) be under the jurisdiction of Cypriniformes (Cypriniformes), Cyprinidae (Cyprinidae), Schizothoracinae (Schizothoracinae), Schizothorax (Schizothorax), popular name, face fish, thin soft-shelled turtle, mainly be distributed in the Wujiang River, Jinsha jiang River and Yalongjiang River.The short hairs schizothoracin is ground fish, generally lives among the more anxious river, valley of current, is more common in the pool of bed scour in tributary, it is local famous and precious wild fish, in recent years investigation result shows, though also have some amount in its range of distribution, its output reduces greatly.Yunnan aquatic products institute artificial propagation success in 2007, but the subject matter existed is: fry and fingerling very easily infects ichthyophthiriasis and fish molds, and in case infect with regard to rapid spread, especially ichthyophthiriasis extremely refractory more, cause thus the seed mortality.Simultaneously, field is introduced a fine variety individual need for some time and could be adapted to the pool and support environment, and this laundering period is the outbreak period of various diseases, causes and introduces a fine variety individual death.Therefore, disease control is the key issue that the short hairs schizothoracin is propagated artificially, and cytobiology is the effective means of research virus function mechanism.On the other hand, due to the limitation of cultured fishes itself, the pool is supported artificial propagation under environment and is easily caused chromosome abnormalty, as polyploidization and different times of change, needs the cell Short-term Culture for the quality of karyotyping with detection artificial propagation kind.Therefore, need the construction process of a kind of short hairs schizothoracin clone of research, set up short hairs schizothoracin clone.
By literature search, have no and identical report of the present invention.
Summary of the invention:
The purpose of this invention is to provide structure and cryopreservation method that a kind of short hairs schizothoracin heart cell easy to operation is, to meet the needs to the preservation of short hairs schizothoracin germ plasm resource and theoretical investigation.
Structure and cryopreservation method that short hairs schizothoracin heart cell of the present invention is, its concrete steps are as follows:
(1) prepare PBS thimerosal and cell culture fluid
The PBS thimerosal: in PBS, add microbiotic, making penicillin concn is 200IU/ml, and Streptomycin sulphate concentration is 200 μ g/ml, and amphotericin B concentration is 20 μ g/ml;
Basic culture solution: add foetal calf serum in the M199 nutrient solution, make the foetal calf serum volume account for 10% of cumulative volume;
Primary nutrient solution: add foetal calf serum, cell growth factor bFGF and microbiotic in the M199 nutrient solution, make the foetal calf serum volume account for 15% of cumulative volume, bFGF concentration is 10ng/ml, penicillin concn is 150IU/ml, Streptomycin sulphate concentration is 150 μ g/ml, and amphotericin B concentration is 20 μ g/ml;
Nutrient solution goes down to posterity: add foetal calf serum, cell growth factor bFGF and microbiotic in the M199 nutrient solution, make the foetal calf serum volume account for 15% of cumulative volume, bFGF concentration is 6ng/ml, and penicillin concn is 100IU/ml, Streptomycin sulphate concentration is 100 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 and saves backup;
(2) former culture
Alcohol disinfecting healthy short hairs schizothoracin in 1 age fish body with 75%, be placed in Bechtop, with aseptic disscting instrument, get its heart, be placed in sterile petri dish, after PBS thimerosal cleaning heart tissue 3 times, with aseptic apparatus, be cut into and organize fritter, then with 2ml 0.2% trypsinase-EDTA, digest 6 min, with in the 0.2ml foetal calf serum, reacting with pancreatin, the centrifugal collection heart tissue of 1000rpm piece, in the heart tissue piece, add again 2ml 0.1% type Ⅳ collagen enzymic digestion 1h, 1200rpm centrifugal collection heart tissue piece and cell, primary nutrient solution is resuspended, and it is inoculated in to 25 cm
2in Tissue Culture Flask, in 18 ℃ of incubators, start former culture, every three and half amounts are changed nutrient solution, to remove not adherent tissue and dead cell,
(3) cultivation of going down to posterity
At the bottom of being paved with bottle, primary heart 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 0.1% trypsinase-EDTA solution 1 ml, digest 1 min, after cell rounding, add go down to posterity nutrient solution 1 ml with in and pancreatin reaction, a piping and druming bottle end, attached cell is come off, the 1:1 by volume that goes down to posterity first passes, and after this 1:2 goes down to posterity by volume, in 18 ℃ of incubators, continues to cultivate; Went down to posterity once in every 5 days, while reaching for the 5th generation, cell culture fluid changes basic culture solution into, now, and the Establishment of Cell Line success;
(4) cell cryopreservation and recovery
(a) preparation cell cryopreservation protection liquid: frozen storing liquid comprises the M199 nutrient solution, and foetal calf serum and DMSO press the volume ratio of 5:4.5:0.5 and mix, matching while using;
(b) cell cryopreservation: the cell in the vegetative period of taking the logarithm, after above-mentioned trysinization, centrifugal 6 min of cell suspension 1200 rpm, discard supernatant liquor, adds the cell cryopreservation of preparation to protect liquid in cell precipitation, and resuspended, the concentration that makes cell is 1 * 10
6Individual/ml, be 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: by above-mentioned cryopreservation tube from liquid nitrogen, taking out, putting into 37 ℃ of water-baths rocks fast to thawing, then in aseptic operating platform, the cell that will thaw is transferred in 15 ml centrifuge tubes, and adds the equivalent basic culture solution, centrifugal 6 min of 1200 rpm, except supernatant liquor, use the basic culture solution re-suspended cell, and be transferred in Tissue Culture Flask, in 18 ℃ of incubators, cultivate, cell namely grew up to individual layer in 5 days.
In each step of the present invention, per-cent used is volume percent
Unusual effect of the present invention is:
1, easy to operation.
2, reduced the damage of pure tissue block method to heart cell.
3, the short hairs schizothoracin heart cell built be form for becoming fiber-like, growth conditions is good, can continuous passage and directly apply to biological characteristic research, met the needs to the germ plasm resource preservation of short hairs schizothoracin and theoretical investigation and application;
4, this construction process also is applicable to other fish and builds heart cell system.
Embodiment:
Structure and the cryopreservation method of the short hairs schizothoracin heart cell system of the present embodiment, its concrete steps are as follows:
(1) prepare PBS thimerosal and cell culture fluid
The PBS thimerosal: in PBS, add microbiotic, making penicillin concn is 200IU/ml, and Streptomycin sulphate concentration is 200 μ g/ml, and amphotericin B concentration is 20 μ g/ml;
Basic culture solution: add foetal calf serum in the M199 nutrient solution, make the foetal calf serum volume account for 10% of cumulative volume;
Primary nutrient solution: add foetal calf serum, cell growth factor bFGF and microbiotic in the M199 nutrient solution, make the foetal calf serum volume account for 15% of cumulative volume, bFGF concentration is 10ng/ml, penicillin concn is 150IU/ml, Streptomycin sulphate concentration is 150 μ g/ml, and amphotericin B concentration is 20 μ g/ml;
Nutrient solution goes down to posterity: add foetal calf serum, cell growth factor bFGF and microbiotic in the M199 nutrient solution, make the foetal calf serum volume account for 15% of cumulative volume, bFGF concentration is 6ng/ml, and penicillin concn is 100IU/ml, Streptomycin sulphate concentration is 100 μ g/ml, and amphotericin B concentration is 10 μ g/ml;
The pH value of regulating above-mentioned nutrient solution is 7.0 or 7.2, is positioned in 4 ℃ of refrigerators and saves backup;
(2) former culture
Alcohol disinfecting healthy short hairs schizothoracin in 1 age fish body with 75%, be placed in Bechtop, with aseptic disscting instrument, get its heart, be placed in sterile petri dish, after PBS thimerosal cleaning heart tissue 3 times, with aseptic apparatus, be cut into and organize fritter, then with 2ml 0.2% trypsinase-EDTA, digest 6 min, with in the 0.2ml foetal calf serum, reacting with pancreatin, the centrifugal collection heart tissue of 1000rpm piece, in the heart tissue piece, add again 2ml 0.1% type Ⅳ collagen enzymic digestion 1h, 1200rpm centrifugal collection heart tissue piece and cell, primary nutrient solution is resuspended, and it is inoculated in to 25 cm
2in Tissue Culture Flask, in 18 ℃ of incubators, start former culture, every three and half amounts are changed nutrient solution, to remove not adherent tissue and dead cell, in former culture,
(3) cultivation of going down to posterity
At the bottom of being paved with bottle, primary heart 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 0.1% trypsinase-EDTA solution 1 ml, digest 1 min, after cell rounding, add go down to posterity nutrient solution 1 ml with in and pancreatin reaction, a piping and druming bottle end, attached cell is come off, the 1:1 by volume that goes down to posterity first passes, and after this 1:2 goes down to posterity by volume, in 18 ℃ of incubators, continues to cultivate; Went down to posterity once in every 5 days, while reaching for the 5th generation, cell culture fluid changes basic culture solution into, now, and the Establishment of Cell Line success;
(4) cell cryopreservation and recovery
(a) preparation cell cryopreservation protection liquid: frozen storing liquid comprises the M199 nutrient solution, and foetal calf serum and DMSO press the volume ratio of 5:4.5:0.5 and mix, matching while using;
(b) cell cryopreservation: the cell in the vegetative period of taking the logarithm, after above-mentioned trysinization, centrifugal 6 min of cell suspension 1200 rpm, discard supernatant liquor, adds the cell cryopreservation of preparation to protect liquid in cell precipitation, and resuspended, the concentration that makes cell is 1 * 10
6Individual/ml, be 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: by above-mentioned cryopreservation tube from liquid nitrogen, taking out, putting into 37 ℃ of water-baths rocks fast to thawing, then in aseptic operating platform, the cell that will thaw is transferred in 15 ml centrifuge tubes, and adds the equivalent basic culture solution, centrifugal 6 min of 1200 rpm, except supernatant liquor, use the basic culture solution re-suspended cell, and be transferred in Tissue Culture Flask, in 18 ℃ of incubators, cultivate, cell namely grew up to individual layer in 5 days.
(5) in each step, per-cent used is volume percent.
Claims (1)
1. structure and the cryopreservation method of short hairs schizothoracin heart cell system is characterized in that the step of the method is as follows:
(1) prepare PBS thimerosal and cell culture fluid
The PBS thimerosal: in PBS, add microbiotic, making penicillin concn is 200IU/ml, and Streptomycin sulphate concentration is 200 μ g/ml, and amphotericin B concentration is 20 μ g/ml;
Basic culture solution: add foetal calf serum in the M199 nutrient solution, make the foetal calf serum volume account for 10% of cumulative volume;
Primary nutrient solution: add foetal calf serum, cell growth factor bFGF and microbiotic in the M199 nutrient solution, make the foetal calf serum volume account for 15% of cumulative volume, bFGF concentration is 10ng/ml, penicillin concn is 150IU/ml, Streptomycin sulphate concentration is 150 μ g/ml, and amphotericin B concentration is 20 μ g/ml;
Nutrient solution goes down to posterity: add foetal calf serum, cell growth factor bFGF and microbiotic in the M199 nutrient solution, make the foetal calf serum volume account for 15% of cumulative volume, bFGF concentration is 6ng/ml, and penicillin concn is 100IU/ml, Streptomycin sulphate concentration is 100 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 and saves backup;
(2) former culture
Alcohol disinfecting healthy short hairs schizothoracin in 1 age fish body with 75%, be placed in Bechtop, with aseptic disscting instrument, get its heart, be placed in sterile petri dish, after PBS thimerosal cleaning heart tissue 3 times, with aseptic apparatus, be cut into and organize fritter, then with 2ml 0.2% trypsinase-EDTA, digest 6 min, with in the 0.2ml foetal calf serum, reacting with pancreatin, the centrifugal collection heart tissue of 1000rpm piece, in the heart tissue piece, add again 2ml 0.1% type Ⅳ collagen enzymic digestion 1h, 1200rpm centrifugal collection heart tissue piece and cell, primary nutrient solution is resuspended, and it is inoculated in to 25 cm
2in Tissue Culture Flask, in 18 ℃ of incubators, start former culture, every three and half amounts are changed nutrient solution, to remove not adherent tissue and dead cell,
(3) cultivation of going down to posterity
At the bottom of being paved with bottle, primary heart 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 0.1% trypsinase-EDTA solution 1 ml, digest 1 min, after cell rounding, add go down to posterity nutrient solution 1 ml with in and pancreatin reaction, a piping and druming bottle end, attached cell is come off, the 1:1 by volume that goes down to posterity first passes, and after this 1:2 goes down to posterity by volume, in 18 ℃ of incubators, continues to cultivate; Went down to posterity once in every 5 days, while reaching for the 5th generation, cell culture fluid changes basic culture solution into, now, and the Establishment of Cell Line success;
(4) cell cryopreservation and recovery
(a) preparation cell cryopreservation protection liquid: frozen storing liquid comprises the M199 nutrient solution, and foetal calf serum and DMSO press the volume ratio of 5:4.5:0.5 and mix, matching while using;
(b) cell cryopreservation: the cell in the vegetative period of taking the logarithm, after above-mentioned trysinization, centrifugal 6 min of cell suspension 1200 rpm, discard supernatant liquor, adds the cell cryopreservation of preparation to protect liquid in cell precipitation, and resuspended, the concentration that makes cell is 1 * 10
6Individual/ml, be 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: by above-mentioned cryopreservation tube from liquid nitrogen, taking out, putting into 37 ℃ of water-baths rocks fast to thawing, then in aseptic operating platform, the cell that will thaw is transferred in 15 ml centrifuge tubes, and adds the equivalent basic culture solution, centrifugal 6 min of 1200 rpm, except supernatant liquor, use the basic culture solution re-suspended cell, and be transferred in Tissue Culture Flask, in 18 ℃ of incubators, cultivate, cell namely grew up to individual layer in 5 days.
(5) in each step, per-cent used is volume percent.
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| CN104762252A (en) * | 2015-04-27 | 2015-07-08 | 上海海洋大学 | Grass goldfish pelvic fin cell line constructing method |
| CN105039241A (en) * | 2015-08-05 | 2015-11-11 | 浙江万里学院 | Pelodiscus sinensis heart cell continuous cell line and establishing method and ultra-low-temperature cryopreservation method thereof |
| CN106479959A (en) * | 2016-10-21 | 2017-03-08 | 中国长江三峡集团公司中华鲟研究所 | A kind of construction method of paddlefish heart cell system |
| CN106508891A (en) * | 2016-10-27 | 2017-03-22 | 中国科学院昆明动物研究所 | Construction method of kidney cell line of Schizothorax taliensis |
| CN110684709A (en) * | 2019-10-21 | 2020-01-14 | 江苏春之雨生物科技发展有限公司 | Haliotis discus hannai cell culture medium and cell line construction method |
| CN113184839A (en) * | 2021-05-12 | 2021-07-30 | 沈阳建筑大学 | Cell culture carrier capable of regulating cell growth state |
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| CN102766595A (en) * | 2012-08-07 | 2012-11-07 | 中国科学院昆明动物研究所 | Construction method for anabarilius grahami cardiac cell line |
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| CN1561704A (en) * | 2004-03-31 | 2005-01-12 | 马直科 | Artificial breeding method for schizothorax |
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| CN104762252A (en) * | 2015-04-27 | 2015-07-08 | 上海海洋大学 | Grass goldfish pelvic fin cell line constructing method |
| CN104762252B (en) * | 2015-04-27 | 2017-06-16 | 上海海洋大学 | A kind of construction method of grass goldfish abdomeinal fin cell line |
| CN105039241A (en) * | 2015-08-05 | 2015-11-11 | 浙江万里学院 | Pelodiscus sinensis heart cell continuous cell line and establishing method and ultra-low-temperature cryopreservation method thereof |
| CN105039241B (en) * | 2015-08-05 | 2018-12-18 | 浙江万里学院 | Shelled Turtle Trionyx Sinensis heart cell continuous cell line and its construction method and cryopreservation method |
| CN106479959A (en) * | 2016-10-21 | 2017-03-08 | 中国长江三峡集团公司中华鲟研究所 | A kind of construction method of paddlefish heart cell system |
| CN106508891A (en) * | 2016-10-27 | 2017-03-22 | 中国科学院昆明动物研究所 | Construction method of kidney cell line of Schizothorax taliensis |
| CN106508891B (en) * | 2016-10-27 | 2019-09-20 | 中国科学院昆明动物研究所 | A kind of construction method of Dali schizothoracin kidney cell system |
| CN110684709A (en) * | 2019-10-21 | 2020-01-14 | 江苏春之雨生物科技发展有限公司 | Haliotis discus hannai cell culture medium and cell line construction method |
| CN113184839A (en) * | 2021-05-12 | 2021-07-30 | 沈阳建筑大学 | Cell culture carrier capable of regulating cell growth state |
| CN117050944A (en) * | 2023-08-31 | 2023-11-14 | 中国长江三峡集团有限公司中华鲟研究所 | In-vitro culture method for hypothalamus cells of schizothorax |
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