CN102492650A - Construction method for Hexagrammos otakii cell line - Google Patents
Construction method for Hexagrammos otakii cell line Download PDFInfo
- Publication number
- CN102492650A CN102492650A CN201110377593XA CN201110377593A CN102492650A CN 102492650 A CN102492650 A CN 102492650A CN 201110377593X A CN201110377593X A CN 201110377593XA CN 201110377593 A CN201110377593 A CN 201110377593A CN 102492650 A CN102492650 A CN 102492650A
- Authority
- CN
- China
- Prior art keywords
- cell
- concentration
- nutrient solution
- hexagrammos otakii
- cell line
- 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.)
- Pending
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a construction method for a Hexagrammos otakii cell line. The construction method comprises the following steps: 1, selecting a DMEM/F12 medium, adding a fetal calf serum, a human basic fibroblast growth factor, an I type insulin-like growth factor and chondroitin sulfate to prepare a cell culture solution, and storing the cell culture solution in a 4DEG C environment; 2, carrying out double resisting and rinsing processing on tissues of the Hexagrammos otakii on a super-clean workbench, and carrying out primary culture in a culture bottle; and 3, preparing a cell suspension after cells grow into a single layer, and adding the culture solution to subculture. The method of the invention has the advantages of simple step and easy operation; the morphology of the constructed tissue cell line of Hexagrammos otakii fins (HOFs), Hexagrammos otakii lips (HOLs) and Hexagrammos otakii kidneys (HOKs) likes a fiber, the serial passage of the cell line can be realized, and the Hexagrammos otakii cell line can be directly applied to pathogenic characteristic researches, vaccine developments and function gene researches, so needs for application researches of theoretical researches, virus vaccine developments and the like of Hexagrammos otakii are satisfied; and the construction method is also suitable for constructing cell lines of fins, lips and kidneys of other fishes.
Description
Technical field
The present invention relates to a kind of construction process of clone, particularly a kind of construction process of greenling clone.
Background technology
Greenling
Hexagrammos otakiiClaim European Greenling again, the popular name yellow croaker belongs to the cold warm nature demersal fish in coastal waters, and, Bohai Sea yellow in China and Japan, Korea and Russian Far East Zhu Hai have distribution, and its meat flavour is delicious, and fine and tender taste has the title of " northern lithosporic ".The greenling adaptive faculty is strong, and is low temperature resistant, and the fast biological property of early sexual maturity and digenesis is arranged again, inhabits near coastal waters cay and the island, and the migration scope of activity is little, therefore is the desirable fingerling that culture in the reef gulf.In recent years, the market requirement of greenling is increased day by day, wild greenling population quantity can not satisfy supply far away, and its mariculture industry remains development.The foundation of greenling clone can be satisfied the needs of greenling virusology, pathology, toxicology, immunology, genetic breeding and applied researcies such as research of quality saving scheduling theory and virus vaccines development.
Summary of the invention
The construction process that the purpose of this invention is to provide simple, the easy-operating greenling clone of a kind of step is to satisfy the needs to applied researcies such as greenling theoretical investigation and virus vaccines developments.
The construction process of greenling clone of the present invention comprises the steps:
⑴, preparation cell culture fluid
Select the DMEM/F12 substratum; In substratum, add foetal calf serum, rh-bFGF, I type rhIGF-1 and CHS respectively; Make foetal calf serum account for 20% of TV; The concentration of rh-bFGF is 5ng/ml, and the concentration of I type rhIGF-1 is 40ng/ml, and the concentration of CHS is 20ug/ml;
Under 4 ℃ environment, deposit;
, former be commissioned to train foster
On Bechtop, get the tissue of greenling, carry out immersion treatment with the mixed solution of penicillium mould and Streptomycin sulphate, the concentration of penicillium mould is 100IU/ml, and the concentration of Streptomycin sulphate is 100ug/ml, and the treatment time is 30 minutes; After using the PBS rinsing again, 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 tissue block of above-mentioned processing
2In the Tissue Culture Flask, add nutrient solution 3 ml, at 25 ℃ of CO
2Start in the incubator former be commissioned to train foster;
⑶, the cultivation of going down to posterity
After treating that cell grows up to individual layer, the nutrient solution in the sucking-off culturing bottle, the trypsin solution 1ml of adding 0.25%; Digestion 1min behind the cell rounding, inhales and removes trypsin solution; With the old nutrient solution add-back of sucking-off before, at the bottom of suction pipe piping and druming culturing bottle, process cell suspension; In cell suspension, mend new nutrient solution, the volume ratio of cell suspension and nutrient solution is 1:2, is inoculated in then in two culturing bottles; At 25 ℃ of CO
2Cultivate in the incubator.After treating that cell grows up to individual layer once more, still go down to posterity according to the method described above.
The construction process of greenling clone of the present invention, the wherein said former foster step of being commissioned to train also needs unite digestion 30min to massive texture with 0.5% Unidasa and 0.2%II Collagen Type VI enzyme.
The construction process of greenling clone of the present invention, process step is simple, processing ease; Constructed greenling fin, kiss end and renal tissue clone (HOF, HOL, HOK) form are fiber-like; Can continuous passage and directly apply to pathogenic characteristic research, vaccine development and functional gene research, satisfied needs to applied researcies such as greenling theoretical investigation and virus vaccines developments; This construction process is applicable to that also other fish make up the clone of fin, kiss end and kidney.
Embodiment
Embodiment 1:
⑴, preparation cell culture fluid
Select the DMEM/F12 substratum of Hyclone company; In substratum, add foetal calf serum, rh-bFGF, I type rhIGF-1 and CHS respectively; Make foetal calf serum account for 20% of TV; The concentration of rh-bFGF is 5ng/ml, and the concentration of I type rhIGF-1 is 40ng/ml, and the concentration of CHS is 20ug/ml;
Under 4 ℃ environment, deposit;
, former be commissioned to train foster
On Bechtop, get the fin tissue of greenling, carry out immersion treatment with the mixed solution of penicillium mould and Streptomycin sulphate, the concentration of penicillium mould is 100IU/ml, and the concentration of Streptomycin sulphate is 100ug/ml, and the treatment time is 30 minutes; After using the PBS rinsing again, in a small amount of 5%FBS-DMEM/F12 (Ph7.2), be cut into 0.8～1.2mm
3Bulk, with 0.5% Unidasa and 0.2%II Collagen Type VI enzyme block fin tissue is united digestion 30min again;
Tissue block through above-mentioned processing evenly is inoculated in 25cm
2In the Tissue Culture Flask, add nutrient solution 3 ml, at 25 ℃ of CO
2Start in the incubator former be commissioned to train foster;
⑶, the cultivation of going down to posterity
After treating that cell grows up to individual layer, the nutrient solution in the sucking-off culturing bottle, the trypsin solution 1ml of adding 0.25%; Digestion 1min behind the cell rounding, inhales and removes trypsin solution; With the old nutrient solution add-back of sucking-off before, at the bottom of suction pipe piping and druming culturing bottle, process cell suspension; In cell suspension, mend new nutrient solution, the volume ratio of cell suspension and nutrient solution is 1:2, is inoculated in then in two culturing bottles; At 25 ℃ of CO
2Cultivate in the incubator.After treating that cell grows up to individual layer once more, still go down to posterity according to the method described above.
Embodiment 2:
⑴, preparation cell culture fluid
Select the DMEM/F12 substratum of Hyclone company; In substratum, add foetal calf serum, rh-bFGF, I type rhIGF-1 and CHS respectively; Make foetal calf serum account for 20% of TV; The concentration of rh-bFGF is 5ng/ml, and the concentration of I type rhIGF-1 is 405ng/ml, and the concentration of CHS is 20ug/ml;
Under 4 ℃ environment, deposit;
, former be commissioned to train foster
On Bechtop, get the kiss end tissue of greenling, carry out immersion treatment with the mixed solution of penicillium mould and Streptomycin sulphate, the concentration of penicillium mould is 100IU/ml, and the concentration of Streptomycin sulphate is 100ug/ml, and the treatment time is 30 minutes; After using the PBS rinsing again, in a small amount of 5%FBS-DMEM/F12 (Ph7.2), be cut into 0.8～1.2mm
3Bulk, with 0.5% Unidasa and 0.2%II Collagen Type VI enzyme block fin tissue is united digestion 30min again;
Tissue block through above-mentioned processing evenly is inoculated in 25cm
2In the Tissue Culture Flask, add nutrient solution 3 ml, at 25 ℃ of CO
2Start in the incubator former be commissioned to train foster;
⑶, the cultivation of going down to posterity
After treating that cell grows up to individual layer, the nutrient solution in the sucking-off culturing bottle, the trypsin solution 1ml of adding 0.25%; Digestion 1min behind the cell rounding, inhales and removes trypsin solution; With the old nutrient solution add-back of sucking-off before, at the bottom of suction pipe piping and druming culturing bottle, process cell suspension; In cell suspension, mend new nutrient solution, the volume ratio of cell suspension and nutrient solution is 1:2, is inoculated in then in two culturing bottles; At 25 ℃ of CO
2Cultivate in the incubator.After treating that cell grows up to individual layer once more, still go down to posterity according to the method described above.
Embodiment 3:
⑴, preparation cell culture fluid
Select the DMEM/F12 substratum of Hyclone company; In substratum, add foetal calf serum, rh-bFGF, I type rhIGF-1 and CHS respectively; Make foetal calf serum account for 20% of TV; The concentration of rh-bFGF is 5ng/ml, and the concentration of I type rhIGF-1 is 405ng/ml, and the concentration of CHS is 20ug/ml;
Under 4 ℃ environment, deposit;
, former be commissioned to train foster
On Bechtop, get the renal tissue of greenling, with being cut into 0.8～1.2mm after 5%FBS-DMEM/F12 (Ph7.2) rinsing once
3Bulk; (viscera tissue can twoly resist and the associating digestion process).
Tissue block through above-mentioned processing evenly is inoculated in 25cm
2In the Tissue Culture Flask, add nutrient solution 3 ml, at 25 ℃ of CO
2Start in the incubator former be commissioned to train foster;
⑶, the cultivation of going down to posterity
After treating that cell grows up to individual layer, the nutrient solution in the sucking-off culturing bottle, the trypsin solution 1ml of adding 0.25%; Digestion 1min behind the cell rounding, inhales and removes trypsin solution; With the old nutrient solution add-back of sucking-off before, at the bottom of suction pipe piping and druming culturing bottle, process cell suspension; In cell suspension, mend new nutrient solution, the volume ratio of cell suspension and nutrient solution is 1:2, is inoculated in then in two culturing bottles; At 25 ℃ of CO
2Cultivate in the incubator.After treating that cell grows up to individual layer once more, still go down to posterity according to the method described above.
Cell cryopreservation and recovery to above-mentioned structure:
Configuration 1ml cell cryopreservation protection liquid: get 0.2mlFBS respectively, 0.6ml DMEM/F12 and 0.2mlDMSO, mixing place in 4 ℃ of refrigerators subsequent use.
Cell cryopreservation: get the cell that is in logarithmic phase of same algebraically, harvested cell after above-mentioned trysinization, the centrifugal 10min of 1000rpm discards supernatant.In cell precipitation, add configured the frozen protection liquid of 1ml, resuspended, adjustment cell concn to 1 * 10
6Individual/ml, cell suspension is transferred in the aseptic frozen pipe, and clicked programmed cooling: place 30min ,-20 ℃ of refrigerators placement 1h ,-80 ℃ of refrigerator overnight in 4 ℃ of refrigerators, put into the medium-term and long-term preservation of liquid nitrogen (196 ℃) at last.
Cell recovery: the water-bath temperature is transferred to 40 ℃ and 25 ℃ respectively, from liquid nitrogen container, take out the cell cryopreservation pipe rapidly, put into 40 ℃ of water-baths, treat to change in 25 ℃ of water-baths after cells frozen storing liquid melts.Under the aseptic condition cell suspension in the cell cryopreservation pipe is transferred in the 10ml centrifuge tube then, and adds equivalent DMEM/F12 nutrient solution, the centrifugal 10min of 1000rpm removes supernatant, collecting cell.Use the nutrient solution re-suspended cell, and transfer in the Tissue Culture Flask, 25 ℃ of CO
2Cultivate in the incubator, full dose is changed nutrient solution behind the 12h, proceeds to cultivate, and cell can grow up to individual layer after 4-5 days.
Claims (2)
1. the construction process of a greenling clone is characterized in that: comprise the steps:
⑴, preparation cell culture fluid
Select the DMEM/F12 substratum; In substratum, add foetal calf serum, rh-bFGF, I type rhIGF-1 and CHS respectively; Make foetal calf serum account for 20% of TV; The concentration of rh-bFGF is 5ng/ml, and the concentration of I type rhIGF-1 is 40ng/ml, and the concentration of CHS is 20ug/ml;
Under 4 ℃ environment, deposit;
, former be commissioned to train foster
On Bechtop, get the tissue of greenling, carry out immersion treatment with the mixed solution of penicillium mould and Streptomycin sulphate, the concentration of penicillium mould is 100IU/ml, and the concentration of Streptomycin sulphate is 100ug/ml, and the treatment time is 30 minutes; After using the PBS rinsing again, be cut into 0.8～1.2mm
3Bulk, evenly be inoculated in 25cm through the tissue block of above-mentioned processing
2In the Tissue Culture Flask, add nutrient solution 3 ml, at 25 ℃ of CO
2Start in the incubator former be commissioned to train foster;
⑶, the cultivation of going down to posterity
After treating that cell grows up to individual layer, the nutrient solution in the sucking-off culturing bottle, the trypsin solution 1ml of adding 0.25%; Digestion 1min behind the cell rounding, inhales and removes trypsin solution; With the old nutrient solution add-back of sucking-off before, at the bottom of suction pipe piping and druming culturing bottle, process cell suspension; In cell suspension, mend new nutrient solution, the volume ratio of cell suspension and nutrient solution is 1:2, is inoculated in then in two culturing bottles; At 25 ℃ of CO
2Cultivate in the incubator;
After treating that cell grows up to individual layer once more, still go down to posterity according to the method described above.
2. the construction process of greenling clone according to claim 1 is characterized in that: the described former foster step of being commissioned to train also needs unite digestion 30min to massive texture with 0.5% Unidasa and 0.2%II Collagen Type VI enzyme.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110377593XA CN102492650A (en) | 2011-11-24 | 2011-11-24 | Construction method for Hexagrammos otakii cell line |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110377593XA CN102492650A (en) | 2011-11-24 | 2011-11-24 | Construction method for Hexagrammos otakii cell line |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102492650A true CN102492650A (en) | 2012-06-13 |
Family
ID=46184513
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201110377593XA Pending CN102492650A (en) | 2011-11-24 | 2011-11-24 | Construction method for Hexagrammos otakii cell line |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102492650A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN103436490A (en) * | 2013-09-05 | 2013-12-11 | 中国科学院昆明动物研究所 | Construction and ultralow temperature freezing preservation method of fin cell line of schizothorax grahami |
| CN103937736A (en) * | 2014-04-04 | 2014-07-23 | 山西农业大学 | Method for establishing fish gill cell line |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101372682A (en) * | 2008-10-15 | 2009-02-25 | 中国海洋大学 | Construction method of Epinephelus fuscoguttatus fin cell line |
| CN101451121A (en) * | 2008-12-26 | 2009-06-10 | 中国海洋大学 | Construction method of Epinephelus fuscoguttatus heart cell line |
-
2011
- 2011-11-24 CN CN201110377593XA patent/CN102492650A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101372682A (en) * | 2008-10-15 | 2009-02-25 | 中国海洋大学 | Construction method of Epinephelus fuscoguttatus fin cell line |
| CN101451121A (en) * | 2008-12-26 | 2009-06-10 | 中国海洋大学 | Construction method of Epinephelus fuscoguttatus heart cell line |
Non-Patent Citations (1)
| Title |
|---|
| 王文君 等: "大泷六线鱼脾脏细胞体外培养的研究", 《中国海洋大学学报》, vol. 40, no. 8, 31 August 2010 (2010-08-31), pages 93 - 97 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN102766595B (en) * | 2012-08-07 | 2013-11-20 | 中国科学院昆明动物研究所 | Construction method for anabarilius grahami cardiac cell line |
| CN103436490A (en) * | 2013-09-05 | 2013-12-11 | 中国科学院昆明动物研究所 | Construction and ultralow temperature freezing preservation method of fin cell line of schizothorax grahami |
| CN103436490B (en) * | 2013-09-05 | 2015-10-14 | 中国科学院昆明动物研究所 | A kind of structure of Schizothorax grahami fin clone and cryopreservation method |
| CN103937736A (en) * | 2014-04-04 | 2014-07-23 | 山西农业大学 | Method for establishing fish gill cell line |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102757934B (en) | Construction method of fin cell line of anabarilius grahami | |
| CN101429492B (en) | Mature method for in vitro culture of porcine oocyte | |
| CN102660494A (en) | Method for building a goat mammary epithetical cell line | |
| CN105766707B (en) | The breeding method of high temperature resistant sea urchin | |
| CN102487866B (en) | Large-scale natural propagation method for artificially culturing siganus guttatus parents | |
| CN101407784B (en) | Separation and purification method, as well as preservation method for ruminant galactophore epithelial cell | |
| CN102119655A (en) | Natural light rapid breeding method for dendrobium officinale | |
| CN107653225A (en) | A kind of culture medium and its amplification method for being used to expand human mesenchymal stem cell | |
| CN103275925A (en) | Construction method of mandarin fish brain cell system | |
| CN110463627A (en) | A kind of annual artificial fecundation method of mandarin fish | |
| CN101627737B (en) | Method for establishing blue crab family and selecting improved breed | |
| CN102757933B (en) | Construction method of muscle cell line of anabarilius grahami | |
| CN102492650A (en) | Construction method for Hexagrammos otakii cell line | |
| CN105360109A (en) | Ultralow-temperature freezing and storing method for sperms of yellow catfishes | |
| CN105850729A (en) | Verbena bonariensis cultivation method | |
| CN114317419A (en) | Method for constructing muscle cell line of Gymnocypris przewalskii | |
| CN105475129A (en) | Tissue-culture rapid propagation method for arundina graminifolia | |
| CN102757932A (en) | Construction method of sinocyclocheilus grahami grahami fin cell line | |
| CN102766595B (en) | Construction method for anabarilius grahami cardiac cell line | |
| CN103891661B (en) | The collecting method of high-quality Patinopecten yessoensis seminal fluid | |
| CN102925407B (en) | In-vitro culture method of triploid loach fin cells | |
| CN103289950A (en) | Methods for in vitro separation, culture, preservation and recovery of milk cow omasum epithelial cells | |
| CN104542296A (en) | Open rooting method for sugarcane tissue culture seedlings | |
| CN103436490A (en) | Construction and ultralow temperature freezing preservation method of fin cell line of schizothorax grahami | |
| CN105039241A (en) | Pelodiscus sinensis heart cell continuous cell line and establishing method and ultra-low-temperature cryopreservation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20120613 |