CN102925407A - In-vitro culture method of triploid loach fin cells - Google Patents
In-vitro culture method of triploid loach fin cells Download PDFInfo
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- CN102925407A CN102925407A CN2012104776084A CN201210477608A CN102925407A CN 102925407 A CN102925407 A CN 102925407A CN 2012104776084 A CN2012104776084 A CN 2012104776084A CN 201210477608 A CN201210477608 A CN 201210477608A CN 102925407 A CN102925407 A CN 102925407A
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Abstract
The invention discloses an in-vitro culture method of triploid loach fin cells. The method includes selecting DMEM/F12 culture medium, and adding fetal bovine serum (FBS), human basic fibroblast growth factor (hbFGF), type-I insulin-like growth factor, epidermal growth factor (EGF) and chondroitin sulfate to prepare cell culture solution; rinsing triploid loach fin with povidone-iodine (PVP-I) and penicillin-streptomycin on a working bench; performing primary culture in a cell culture bottle until a single layer is formed; preparing into a cell suspension; adding culture medium, and performing subculture. The procedure is easy and simple in operation. The cultured triploid loach fin cell is in fibrous morphology, can be sub-cultured successively and directly applied to multiploid fish biological research, virus separation and proliferation, vaccine development and functional gene research.
Description
Technical field
The present invention relates to the extracorporeal culturing method of loach fin cell, particularly a kind of simple to operate, can be used for the research of Polyploid fish quasi-biology, the viral extracorporeal culturing method that separates the triploid loach fin cell of amplification, vaccine development and functional gene research.
Background technology
Loach (
Misgurnus anguillicaudatus)In taxonomy, belong to Cypriniformes (Cypriniformes), Cobitidae (Cobitidae), flower loach subfamily (Cobitinae), loach genus (Misgurnus), be the distinctive wild Small Freshwater Fishes in Asia, be distributed widely in most areas and Vietnam, Korea and Japan on the south China Liaohe River.Each freshwater of China all has distribution such as lake, pond, He Xi, ditch, rice field etc.Loach fine and tender taste, delicious, nutritious, the title of " ginseng in the water " is arranged, except higher edibleness, it also has fixed nourishing medicinal efficacy, is one of special aquatic products product salable on the market.Except having Important Economic value, the research material of the aspects such as loach or a kind of good cytology, genetics.The triploid loach has that growth is fast, the characteristics of strong resistance, and triploid loach fin cell can satisfy the needs that the biological study of triploid loach, virus separated the applied researcies such as amplification, functional gene research and toxicology.But, because the easily problem such as floating, external difficult growth of microbial contamination, cell in the fin cell cultures, occurs easily, up to now also not about the relevant report of vitro culture triploid loach fin cell.
Summary of the invention
The present invention is in order to solve the existing above-mentioned technical problem of prior art, provide a kind of simple to operate, can be used for the extracorporeal culturing method that the research of Polyploid fish quasi-biology, virus separate the triploid loach fin cell of amplification, vaccine development and functional gene research.
Technical solution of the present invention is: a kind of extracorporeal culturing method of triploid loach fin cell is characterized in that carrying out as follows:
A. prepare cell culture fluid
Get the DMEM/F12 substratum, add respectively foetal calf serum, rh-bFGF, I type rhIGF-1, epithelical cell growth factor and chondroitin sulfate in the substratum and prepare cell culture fluid, wherein foetal calf serum accounts for 20% of cumulative volume, the concentration of rh-bFGF is 10ng/ml, and I type rhIGF-1 concentration is that the concentration of 20ng/ml, epithelical cell growth factor is that the concentration of 10ng/ml, chondroitin sulfate is 10ug/ml; Under 4 ℃ environment, deposit;
B. former culture
Getting the fin tissue of triploid loach at worktable, is that 10% Iodophor soaked 15 minutes with mass concentration; Use the mixed liquid dipping 30 minutes of penicillin and Streptomycin sulphate, penicillin concn is 500IU/ml again, and the concentration of Streptomycin sulphate is 500ug/ml; After using again the PBS rinsing, in 5%FBS-DMEM/F12(pH7.2) in be cut into 0.8 ~ 1.2mm
3Bulk; Fin tissue block through above-mentioned processing evenly is inoculated in 25cm
2In the Tissue Culture Flask, add nutrient solution to the 5ml/ bottle after in 25 ℃ of CO2 incubators, just putting dry doubling 24h, start former culture to cell and grow up to individual layer;
C. the cultivation of going down to posterity
Nutrient solution in the sucking-off culturing bottle, the trypsin solution 1ml of adding 0.25% sucks trypsin solution after 5 seconds, so that the bottle end forms one deck Trypsin enzyme membrane, digestion 5min gently knocks Tissue Culture Flask.With the nutrient solution add-back of before sucking-off, with suction pipe piping and druming, make cell suspension; Cell suspension is assigned in two new culturing bottles with volume ratio 1:1; In two culturing bottles, add a step gained nutrient solution to 5ml; In 25 ℃ of CO2 incubators, be cultured to cell and grow up to individual layer.
Step of the present invention is simple, easy to operate, overcome and occurred easily the problems such as microbial contamination, cell are floating, external difficult growth in the fin cell cultures, the form of the triploid loach fin cell of cultivating is fiber-like, can continuous passage and directly apply to the Polyploid fish biological study, virus is separated amplification, vaccine development and functional gene research.
Description of drawings
Fig. 1 is that the embodiment of the invention is through passing the triploid loach fin cell mirror figure below in 10 generations.
Fig. 2 is embodiment of the invention institute cultured cells dyeing somascope figure below.
Embodiment
Embodiment 1:
A. prepare cell culture fluid
Select the DMEM/F12 substratum of Hyclone company, add respectively foetal calf serum, rh-bFGF, I type rhIGF-1, epithelical cell growth factor and chondroitin sulfate in the substratum and prepare cell culture fluid, wherein foetal calf serum accounts for 20% of cumulative volume, the concentration of rh-bFGF is 10ng/ml, and I type rhIGF-1 concentration is that the concentration of 20ng/ml, epithelical cell growth factor is that the concentration of 10ng/ml, chondroitin sulfate is 10ug/ml; Under 4 ℃ environment, deposit;
B. former culture
With high two anti-sterilization water aeration (the 1000IU/mL penicillin of loach, 1000 μ g/mL Streptomycin sulphate) after supporting 16 ~ 24h temporarily in, with containing Gao Shuankang (1000IU/mL penicillin, 1000 μ g/mL Streptomycin sulphate) gets the fin tissue of triploid loach at Bechtop after 1 ‰ the phenylcarbinol anesthesia, use the Iodophor immersion treatment, Iodophor concentration is 10%, and the treatment time is 15 minutes; Mixed solution with penicillin and Streptomycin sulphate carries out immersion treatment again, and penicillin concn is 500IU/ml, and the concentration of Streptomycin sulphate is 500ug/ml, and the treatment time is 30 minutes; After using again the PBS rinsing, in a small amount of 5%FBS-DMEM/F12 (Ph7.2), be cut into 0.8 ~ 1.2mm
3Bulk evenly be inoculated in 25cm through the fin tissue block of above-mentioned processing
2In the Tissue Culture Flask, add nutrient solution to the 5ml/ bottle after in 25 ℃ of CO2 incubators, just putting dry doubling 24h, start former culture to cell and grow up to individual layer;
C. the cultivation of going down to posterity
Nutrient solution in the sucking-off culturing bottle, the trypsin solution 1ml of adding 0.25% sucks trypsin solution after 5 seconds, so that the bottle end forms one deck Trypsin enzyme membrane, digestion 5min gently knocks Tissue Culture Flask.With the nutrient solution add-back of before sucking-off, with suction pipe piping and druming, make cell suspension; Cell suspension is assigned in two new culturing bottles with volume ratio 1:1; In two culturing bottles, add a step gained nutrient solution to 5ml; In 25 ℃ of CO2 incubators, be cultured to cell and grow up to individual layer.
Can after cell grows up to individual layer, repeat the cultivation of repeatedly going down to posterity of c step, pass the triploid loach fin cell in 10 generations as shown in Figure 1.
Experiment:
Embodiment of the invention institute cultured cells is carried out chromosome analysis:
Get one bottle in the fin cell that embodiment 1 cultivates, add colchicine, make its final concentration reach 1 μ g/ml, after 2 ~ 4h is cultivated in 25 ℃ of continuation, be made into cell suspension according to the method in the c step, and be collected in the centrifuge tube, abandon supernatant behind the centrifugal 10min of 1000rpm, the 0.075mol/mlKCl solution that adds the 3ml preheating is made suspension at 25 ℃ of lower hypotonic 30min with suction pipe piping and druming, abandon supernatant behind the centrifugal 5min of 1500rpm, the Carnoy stationary liquid is fixed 3 times, each 15min, and put into-20 ℃ of refrigerator overnight, again with the centrifugal 5min of 1500rpm once after, be fixed liquid 1ml, drip sheet and overdo, buckle with the Giemsa dye liquor and dye.To the metacinesis phase in the chromosome sectioning, carry out the chromosome number statistics, mirror figure below shows that chromosome number is 75 as shown in Figure 2.
The result proves: institute's cultured cells is triploid loach cell.
Frozen and the recovery to embodiment 1 cultured cells:
Configuration 1ml cell cryopreservation protection liquid: get respectively 0.2mlFBS, 0.6mlDMEM/F12 and 0.2mlDMSO, mixing place 4 ℃ of refrigerators for subsequent use.
Cell cryopreservation: get the cell that is in logarithmic phase of cultivating, harvested cell behind 0.25% tryptic digestion is abandoned supernatant behind the centrifugal 10min of 1000rpm; In precipitation, add the 1ml cell cryopreservation protection liquid that disposes, re-suspended cell concentration to 1 ⅹ 10
6Individual/ml, cell suspension is transferred in the aseptic cryopreservation tube, and by following programmed cooling: place 30min in 4 ℃ of refrigerators ,-20 ℃ of refrigerators are placed 1h ,-80 ℃ of refrigerator overnight, put at last that liquid nitrogen (196 ℃) is medium-term and long-term to be preserved.
Cell recovery: the water-bath temperature is transferred to respectively 40 ℃ and 25 ℃, rapidly cell cryopreservation tube is taken out from liquid nitrogen container, put into 40 ℃ of water-baths, after cells frozen storing liquid dissolves, change in 25 ℃ of water-baths; Then under the aseptic condition cell suspension in the cell cryopreservation tube is changed in the centrifuge tube, and add the DMEM/F12 nutrient solution of equivalent, the centrifugal 10min of 1000rpm removes supernatant, collecting cell.Use the nutrient solution re-suspended cell, and be transferred in the Tissue Culture Flask, cultivate in 25 ℃ of CO2 incubators, full dose is changed nutrient solution behind the 12h, continues to cultivate, and cell can grow up to individual layer after 4 ~ 5 days.The result shows: cell still can continued growth behind cryopreservation resuscitation.
Claims (1)
1. the extracorporeal culturing method of a triploid loach fin cell is characterized in that carrying out as follows:
A. prepare cell culture fluid
Get the DMEM/F12 substratum, add respectively foetal calf serum, rh-bFGF, I type rhIGF-1, epithelical cell growth factor and chondroitin sulfate in the substratum and prepare cell culture fluid, wherein foetal calf serum accounts for 20% of cumulative volume, the concentration of rh-bFGF is 10ng/ml, and I type rhIGF-1 concentration is that the concentration of 20ng/ml, epithelical cell growth factor is that the concentration of 10ng/ml, chondroitin sulfate is 10ug/ml; Under 4 ℃ environment, deposit;
B. former culture
Getting the fin tissue of triploid loach at worktable, is that 10% Iodophor soaked 15 minutes with mass concentration; Use the mixed liquid dipping 30 minutes of penicillin and Streptomycin sulphate, penicillin concn is 500IU/ml again, and the concentration of Streptomycin sulphate is 500ug/ml; After using again the PBS rinsing, in 5%FBS-DMEM/F12(pH7.2) in be cut into 0.8 ~ 1.2mm
3Bulk; Fin tissue block through above-mentioned processing evenly is inoculated in 25cm
2In the Tissue Culture Flask, add nutrient solution to the 5ml/ bottle after in 25 ℃ of CO2 incubators, just putting dry doubling 24h, start former culture to cell and grow up to individual layer;
C. the cultivation of going down to posterity
Nutrient solution in the sucking-off culturing bottle, the trypsin solution 1ml of adding 0.25% sucks trypsin solution after 5 seconds, so that the bottle end forms one deck Trypsin enzyme membrane, digestion 5min gently knocks Tissue Culture Flask; With the nutrient solution add-back of before sucking-off, with suction pipe piping and druming, make cell suspension; Cell suspension is assigned in two new culturing bottles with volume ratio 1:1; In two culturing bottles, add a step gained nutrient solution to 5ml; In 25 ℃ of CO2 incubators, be cultured to cell and grow up to individual layer.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103525749A (en) * | 2013-11-04 | 2014-01-22 | 大连海洋大学 | Triploid loach fin cell line TRMF and establishing method thereof |
| CN103555657A (en) * | 2013-11-04 | 2014-02-05 | 大连海洋大学 | Tetraploid loach fin cell line TEMF and establishment method thereof |
| CN103651195A (en) * | 2013-11-22 | 2014-03-26 | 大连海洋大学 | Cold shock induction method for paramisgurnus dabryanus androgenesis haploid |
| CN109136170A (en) * | 2018-08-20 | 2019-01-04 | 东北农业大学 | A kind of serum free medium and its application suitable for carp three times somatic growth |
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| CN1793338A (en) * | 2005-11-17 | 2006-06-28 | 中国海洋大学 | Process for structuring large brill fin clone |
| CN101372682A (en) * | 2008-10-15 | 2009-02-25 | 中国海洋大学 | Construction method of Epinephelus fuscoguttatus fin cell line |
| CN102757934A (en) * | 2012-08-07 | 2012-10-31 | 中国科学院昆明动物研究所 | Construction method of fin cell line of anabarilius grahami |
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2012
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| CN1793338A (en) * | 2005-11-17 | 2006-06-28 | 中国海洋大学 | Process for structuring large brill fin clone |
| CN101372682A (en) * | 2008-10-15 | 2009-02-25 | 中国海洋大学 | Construction method of Epinephelus fuscoguttatus fin cell line |
| CN102757934A (en) * | 2012-08-07 | 2012-10-31 | 中国科学院昆明动物研究所 | Construction method of fin cell line of anabarilius grahami |
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| 付思思等: "锦鲤组织细胞体外培养的初步研究", 《中国海洋大学学报》, vol. 42, no. 3, 31 March 2012 (2012-03-31), pages 44 - 49 * |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103525749A (en) * | 2013-11-04 | 2014-01-22 | 大连海洋大学 | Triploid loach fin cell line TRMF and establishing method thereof |
| CN103555657A (en) * | 2013-11-04 | 2014-02-05 | 大连海洋大学 | Tetraploid loach fin cell line TEMF and establishment method thereof |
| CN103525749B (en) * | 2013-11-04 | 2015-07-01 | 大连海洋大学 | Triploid loach fin cell line TRMF and establishing method thereof |
| CN103651195A (en) * | 2013-11-22 | 2014-03-26 | 大连海洋大学 | Cold shock induction method for paramisgurnus dabryanus androgenesis haploid |
| CN109136170A (en) * | 2018-08-20 | 2019-01-04 | 东北农业大学 | A kind of serum free medium and its application suitable for carp three times somatic growth |
| CN109136170B (en) * | 2018-08-20 | 2022-02-18 | 东北农业大学 | Serum-free medium suitable for carp triploid cell growth and application thereof |
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