CN105039241A - Pelodiscus sinensis heart cell continuous cell line and establishing method and ultra-low-temperature cryopreservation method thereof - Google Patents
Pelodiscus sinensis heart cell continuous cell line and establishing method and ultra-low-temperature cryopreservation method thereof Download PDFInfo
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Abstract
The invention relates to a pelodiscus sinensis heart cell continuous cell line which is preserved in the common microorganism center of China Committee for Culture Collection of Microorganisms and has a preservation number of CGMCC No.10597, and further relates to an establishing method and ultra-low-temperature cryopreservation method of the pelodiscus sinensis heart cell continuous cell line. The establishing method includes the following steps of firstly, conducting primary culture, wherein a pelodiscus sinensis heart is cut into tissue small pieces after being preprocessed, trypsin is inoculated into a culture bottle after being digested at the room temperature, and subculture is not started until cells are fully laid on 90% or more of the bottle bottom; secondly, conducting subculture, wherein air is blown on the bottle bottom through a trypsin digesting method to make adherent cells fall down so that subculture can be conducted. By means of the established pelodiscus sinensis heart cell continuous cell line, the research on viral diseases of pelodiscus sinensis and other turtle animals and the research on related therapy drugs for instance the analysis of chromosome and the research of iridescent virus can be conducted on the level of molecular cells, and the requirements for pelodiscus sinensis germplasm resource conservation and theoretical research and application can be met.
Description
Technical field
The present invention relates to biomass cells culture technique field, particularly relate to a kind of Trionyx sinensis (Wiegmann) heart cell continuous cell line and construction process thereof and cryopreservation method.
Background technology
Trionyx sinensis (Wiegmann) (Pelodiscussinensis) has another name called water fish, soft-shelled turtle, the soft-shelled turtle, be subordinate to reptilia, Chelonia, Trionychidae, all having wild Trionyx sinensis (Wiegmann) to distribute in China, Japan, North Vietnam, Korea S and Russian east, is the hydrocoles that a kind of preciousness, economic worth are very high.In recent years, the cultivation amount of Trionyx sinensis (Wiegmann) has increases trend year by year, but, due to the natural life habit of breeding way the changes Trionyx sinensis (Wiegmann) such as artificially controlling temperature, high-density be intensive, frequently, various disease frequently occurs, and the serious threat of soft-shelled turtle disease China and supported soft-shelled turtle industry in Trionyx sinensis (Wiegmann) commodity and seedling transaction simultaneously, year financial loss reaches several hundred million unit, constrains further developing of foster soft-shelled turtle industry.Therefore, diseases prevention and treatment are important research directions of Trionyx sinensis (Wiegmann) aquaculture.
The research of Trionyx sinensis (Wiegmann) disease, starts late, and fundamental research is weak, and the disease of such animal has, and latent period is long, the course of disease is long, easily cause the features such as complication, seriously hinders carrying out of Trionyx sinensis (Wiegmann) diseases prevention and treatment.The Trionyx sinensis (Wiegmann) disease in succession reported in recent years has Red neck disease, reddish macules, furunculosis, aeromonas hydrophila disease, putrid skin disease, septicemia, blister sore, bubble disease, shothole disease, ichthyophthiriasis, iridescent virus disease etc., cause the factor of these diseases, existing viral also have bacteroidal, wherein virus be some diseases as mucus rhinitis, necrotic enteritis and various pneumonia and hepatitis protopathy because of one of, be herpes like virus (Herpesvirus) as caused the virus of ichthyophthiriasis; Red neck disease virus is rhabdovirus (Rhabdovirus); Red Globe virus is similar with adenovirus (Adenovirius); White abdominal shell disease has two-strain, a kind of similar adenovirus, and another kind of similar reovirus (Reovirus), parasitizes in spleen and kidney respectively.
Clone refers to that primary cell culture is through the cell colony of breeding successfully that goes down to posterity first, wherein the cell of continuous passage can be called continuous cell line or infinite cell line, and the clone of cultured continuously can not be called finite cell lines.At present, the study general of the viral infectious of testudinate animal selects the clones such as grass carp gonad cell as Study of Support, does not also have suitable Trionyx sinensis (Wiegmann) clone as carrier to study Trionyx Sinensis Virus disease.Research finds, the tissue and the cell that infect the organ such as liver,spleen,kidney, the heart, intestines, lung of viral Trionyx sinensis (Wiegmann) all have impaired to some extent, and the visible heart is one of important target tissue of infecting of Trionyx Sinensis Virus.The nucleus of normal heart cell is bar-shaped, has a small amount of protoplasma around it, and the Trionyx sinensis (Wiegmann) heart sections infected after virus is visible: heart cell hypochromatosis, myofiber arrangement disorder, fracture, in distortion with downright bad change.Visible, Trionyx sinensis (Wiegmann) heart clone is the comparatively ideal carrier for studying Trionyx Sinensis Virus disease, but, current Trionyx sinensis (Wiegmann) heart cell culture studies rarely has report, commercial Trionyx sinensis (Wiegmann) heart clone is not introduced so far, Trionyx sinensis (Wiegmann) heart cell continuous cell line is not reported so far especially, therefore the research of Trionyx sinensis (Wiegmann) cell becomes the bottleneck of restriction Trionyx Sinensis Virus disease deep structure research, and research Trionyx sinensis (Wiegmann) heart cell continuous cell line is significant to the research bottleneck breaking Trionyx Sinensis Virus disease.
Summary of the invention
First technical problem to be solved by this invention provides a kind of Trionyx sinensis (Wiegmann) heart cell continuous cell line, and this clone can be used as the carrier of Trionyx Sinensis Virus disease research.
Second technical problem to be solved by this invention is the construction process providing a kind of simple above-mentioned Trionyx sinensis (Wiegmann) heart cell continuous cell line for prior art.
3rd technical problem to be solved by this invention is the cryopreservation method providing a kind of effective preservation above-mentioned Trionyx sinensis (Wiegmann) heart cell continuous cell line for prior art.
The present invention solves the technical scheme that first technical problem adopt: a kind of Trionyx sinensis (Wiegmann) heart cell continuous cell line, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, the preservation time is on May 18th, 2015, and deposit number is CGMCCNo.10597.
The present invention solves the technical scheme that second technical problem adopt: the construction process of above-mentioned Trionyx sinensis (Wiegmann) heart cell continuous cell line comprises the following steps:
(1) original cuiture
Be placed in Bechtop after cleaning, sterilization Trionyx sinensis (Wiegmann) body, get its heart and cleaning removing clot tissue and reticular tissue, heart is cut into 1mm
3and following organize fritter, the trypsinase room temperature digestion 20min ~ 25min of 0.125% ~ 0.25%, then postdigestive tissue block is inoculated in culturing bottle, add original cuiture liquid upside down to cultivate in 30 DEG C of incubators, overturn culturing bottle after 24h and add original cuiture liquid to appropriate, every 3d changes a nutrient solution, after about one week, cell starts to move from tissue block, the cell of moving out after about two weeks merges into individual layer around tissue block, now remove tissue block, when at the bottom of cell is paved with bottle to more than 90% time, start Secondary Culture;
(2) Secondary Culture
Original cuiture liquid in sucking-off culturing bottle, add the trypsin-EDTA solutions that 2 ~ 3ml trypsinase concentration is 0.125% ~ 0.25%, digestion 10 ~ 30s, until cell and surrounding tissue link occurs loosen after, add stop digestion cultivation liquid with in pancreatin react; At the bottom of piping and druming bottle, attached cell is come off, and the centrifugal 8 ~ 10min of 800rpm ~ 1000rpm adds original cuiture liquid and goes down to posterity, and the 1:1 by volume that goes down to posterity first passes, and after this 1:2 or 1:3 goes down to posterity by volume, continues to cultivate in 30 DEG C of incubators; Every 5d ~ 7d goes down to posterity once, when reaching for the 10th generation, cell culture fluid is changed into Secondary Culture liquid, Establishment of Cell Line success.
As preferably, the layoutprocedure of described original cuiture liquid is: in MEM nutrient solution, add foetal calf serum, cell growth factor EGF and microbiotic, foetal calf serum volume is made to account for 15% of cumulative volume, EGF concentration is 10ng/ml, penicillin concn is 100IU/ml, Streptomycin sulphate concentration is 100 μ g/ml, and amphotericin B concentration is 20 μ g/ml, and pH value is 7.0 ~ 7.2.
As preferably, the layoutprocedure of described Secondary Culture liquid is: in MEM nutrient solution, add foetal calf serum and microbiotic, foetal calf serum volume is made to account for 15% of cumulative volume, mycin concentration is 100IU/ml, Streptomycin sulphate concentration is 100 μ g/ml, amphotericin B concentration is 20 μ g/ml, and pH value is 7.0 ~ 7.2.
The present invention solves the 3rd technical scheme that technical problem adopts: the cryopreservation method of above-mentioned Trionyx sinensis (Wiegmann) heart cell continuous cell line comprises the following steps:
(1) prepare cell cryopreservation protection liquid: by foetal calf serum and DMSO by volume 9:1 mix, mix obtained cell cryopreservation and protect liquid;
(2) cell cryopreservation: the cell in vegetative period of taking the logarithm, after the trypsin-EDTA solutions digestion that trypsinase concentration is 0.25%, cell suspension is in the centrifugal 8min ~ 10min of 800rpm ~ 1000rpm, discard supernatant liquor, the cells frozen storing liquid of preparation is added in cell precipitation, resuspended, make the concentration of cell reach 1 × 10
6individual/ml, is transferred to cell suspension in cryopreservation tube, cryopreservation tube is placed in program temperature reduction box, and-80 DEG C are spent the night, and then puts into the medium-term and long-term preservation of liquid nitrogen.
Compared with prior art, the invention has the advantages that: the present invention is directed to Trionyx sinensis (Wiegmann) heart tissue characteristic, adopt trypsinization, avoid conventional organization plant block method exist cell move out difficulty etc. problem, the normal adherent growth of the primary cardiac cell energy also increment fast obtained, continuous passage is also successfully built, this heart cell system is after continuous passage, still can keep the biological characteristics of Trionyx sinensis (Wiegmann) heart cell, therefore this clone can carry out the research of the virus disease of Trionyx sinensis (Wiegmann) and other testudinate animals in molecular cell level, the research of prevention and associated treatment medicine, such as chromosome analysis, irido virus research etc., and the needs that also can meet Trionyx sinensis (Wiegmann) Germ-plasma resources protection and theoretical investigation and application, construction process in the present invention is except being applicable to Trionyx sinensis (Wiegmann) heart extracellular, also other aquatic products are applicable to, the structure of reptile heart cell system.
Accompanying drawing explanation
Fig. 1 is the cellular form figure (10 × 10) in the present invention after Trionyx sinensis (Wiegmann) heart cell continuous cell line original cuiture 7d;
Fig. 2 be in the present invention Trionyx sinensis (Wiegmann) heart cell continuous cell line primary cell first time go down to posterity before cellular form figure (10 × 10);
Fig. 3 is the cellular form figure (10 × 10) in the present invention after Trionyx sinensis (Wiegmann) heart cell continuous cell line Secondary Culture to the cryopreservation in the 10th generation;
Fig. 4 be in the present invention Trionyx sinensis (Wiegmann) heart cell continuous cell line Secondary Culture to the cellular form figure (10 × 10) in the 33rd generation;
Fig. 5 is the cellular form figure (10 × 10) of Trionyx sinensis (Wiegmann) heart cell continuous cell line after phenodin-eosin staining procedures process in the present invention;
Fig. 6 is the growth curve chart of Trionyx sinensis (Wiegmann) heart cell continuous cell line in the present invention.
Preservation illustrates: Classification And Nomenclature: Trionyx sinensis (Wiegmann) heart cell continuous cell line, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, and the preservation time is on May 18th, 2015, and deposit number is CGMCCNo.10597.
Embodiment
Below in conjunction with accompanying drawing embodiment, the present invention is described in further detail.
Embodiment 1: the structure of Trionyx sinensis (Wiegmann) heart cell continuous cell line
The preparation of 1.1PBS thimerosal and cell culture fluid
PBS thimerosal: add microbiotic in PBS, makes penicillin concn be 100IU/ml, and Streptomycin sulphate concentration is 100 μ g/ml, and amphotericin B concentration is 20 μ g/ml.
Original cuiture liquid: add foetal calf serum, cell growth factor EGF and microbiotic in MEM nutrient solution, foetal calf serum volume is made to account for 15% of cumulative volume, EGF concentration is 10ng/ml, penicillin concn is 100IU/ml, Streptomycin sulphate concentration is 100 μ g/ml, amphotericin B concentration is 20 μ g/ml, and the pH value regulating nutrient solution is 7.0 ~ 7.2, is positioned in 4 degree of refrigerators and saves backup.
Secondary Culture liquid: add foetal calf serum and microbiotic in MEM nutrient solution, makes foetal calf serum volume account for 15% of cumulative volume, and mycin concentration is 100IU/ml, and Streptomycin sulphate concentration is 100 μ g/ml, and amphotericin B concentration is 20 μ g/ml; Stop digestion nutrient solution: in MEM nutrient solution, add foetal calf serum, make foetal calf serum volume account for 10% of cumulative volume, the pH value regulating nutrient solution is 7.0 ~ 7.2, is positioned in 4 degree of refrigerators and saves backup.
1.2 original cuiture
With the potassium permanganate liquid soaking disinfection Trionyx sinensis (Wiegmann) body 30min of 0.1%, with the alcohol wipe soft-shelled turtle surface of 75% after cleaning, be placed in Bechtop.Get its heart with aseptic disscting instrument, be placed in sterile petri dish, PBS thimerosal cleaning removing clot tissue and reticular tissue.Then, with aseptic apparatus, heart is cut into 1mm
3size organizes fritter, and with 0.25% trypsinase room temperature digestion 20min, afterwards postdigestive tissue block is inoculated in 25cm
2in Tissue Culture Flask, add original cuiture liquid 2ml upside down and start original cuiture in 30 DEG C of incubators, overturn Tissue Culture Flask after 24h and add original cuiture liquid to 10ml.After this, every 3d changes a nutrient solution, and 7d cell starts to move from tissue block, and 15d merges into individual layer around tissue block, now removes tissue block, and 20d grows up to cell monolayer.
1.3 Secondary Culture
When primary Trionyx sinensis (Wiegmann) heart cell to be paved with at the bottom of Tissue Culture Flask bottle 90%, start to go down to posterity.First, original cuiture liquid in sucking-off culturing bottle, add the trypsin-EDTA solutions of 2ml0.25%, digestion 10 ~ 20s, until cell and surrounding tissue link occur loosen after, add 4ml stop digestion cultivate liquid (foetal calf serum) with in and pancreatin reaction, piping and druming bottle at the bottom of, attached cell is come off, and the centrifugal 10min of 800rpm adds 10ml primary cell culture liquid and goes down to posterity.1:1 goes down to posterity by volume first, and after this 1:2 goes down to posterity by volume, continues to cultivate in 30 DEG C of incubators; Every 7d goes down to posterity once, when reaching for the 10th generation, cell culture fluid is changed into Secondary Culture liquid, now, and Establishment of Cell Line success.
Embodiment 2: the structure of Trionyx sinensis (Wiegmann) heart cell continuous cell line
2.1 original cuiture
With the potassium permanganate liquid soaking disinfection Trionyx sinensis (Wiegmann) body 30min of 0.1%, with the alcohol wipe soft-shelled turtle surface of 75% after cleaning, be placed in Bechtop.Get its heart with aseptic disscting instrument, be placed in sterile petri dish, PBS thimerosal cleaning removing clot tissue and reticular tissue.Then, with aseptic apparatus, heart is cut into 0.8mm
3size organizes fritter, and with 0.125% trypsinase room temperature digestion 25min, afterwards postdigestive tissue block is inoculated in 25cm
2in Tissue Culture Flask, add original cuiture liquid 2ml upside down and start original cuiture in 30 DEG C of incubators, overturn Tissue Culture Flask after 24h and add original cuiture liquid to 10ml.After this, every 3d changes a nutrient solution, and 7d cell starts to move from tissue block, and 15d merges into individual layer around tissue block, now removes tissue block, and 20d grows up to cell monolayer.
2.2 Secondary Culture
When primary Trionyx sinensis (Wiegmann) heart cell to be paved with at the bottom of Tissue Culture Flask bottle 90%, start to go down to posterity.First, original cuiture liquid in sucking-off culturing bottle, add the trypsin-EDTA solutions that 3ml trypsinase concentration is 0.125%, digestion 20 ~ 30s, until cell and surrounding tissue link occur loosen after, add 4ml stop digestion cultivate liquid (foetal calf serum) with in and pancreatin reaction, piping and druming bottle at the bottom of, attached cell is come off, and the centrifugal 8min of 1000rpm adds 10ml primary cell culture liquid and goes down to posterity.1:1 goes down to posterity by volume first, and after this 1:3 goes down to posterity by volume, continues to cultivate in 30 DEG C of incubators; Every 5d goes down to posterity once, when reaching for the 10th generation, cell culture fluid is changed into Secondary Culture liquid, now, and Establishment of Cell Line success.
Embodiment 3: cell cryopreservation and recovery
3.1 cell cryopreservation
Foetal calf serum and DMSO are pressed the volume ratio mixing of 9:1, after mixing, obtain cell cryopreservation protection liquid.The cell of taking the logarithm vegetative period, after the trypsin-EDTA solutions digestion of 0.25%, the centrifugal 8min of cell suspension 1000rpm, discards supernatant liquor, adds the cells frozen storing liquid of preparation in cell precipitation, resuspended, makes the concentration of cell be 1 × 10
6individual/ml, being transferred to by 1ml cell suspension in 1.8ml cryopreservation tube and cryopreservation tube is placed in program temperature reduction box ,-80 DEG C are spent the night, and then put into the medium-term and long-term preservation of liquid nitrogen.
3.2 cell recovery
Above-mentioned cryopreservation tube is taken out from liquid nitrogen, put into rapidly 37 DEG C of water-baths to rock fast to thawing, then in aseptic operating platform, the cell that will thaw is transferred in 15ml centrifuge tube, and add equivalent primary cell culture liquid, the centrifugal 8min of 1000rpm, except supernatant liquor, with Secondary Culture liquid re-suspended cell, and be transferred in Tissue Culture Flask, cultivate in 30 DEG C of incubators, change whole nutrient solution after 1d, namely 7d cell grows up to individual layer.
In above-described embodiment 2, PBS thimerosal is identical with embodiment 1 with the compound method of cell culture fluid, embodiment 1, the Trionyx sinensis (Wiegmann) heart cell of preparation continuous passage 14 months in vitro in 2, reached for 53 generations at present, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, the preservation time is on May 18th, 2015, deposit number is CGMCCNo.10597, Fig. 5 is the cellular form figure of Trionyx sinensis (Wiegmann) heart cell after phenodin-eosin staining procedures (hematoxylin-eosinstaining) process, visible Trionyx sinensis (Wiegmann) heart cell body is larger, the weak basophilia of kytoplasm, the larger ovalize of karyon, in blueness, tenuigenin takes on a red color (in figure, non-Show Color is by means of only text description) to nucleus.As shown in Figure 1, after Trionyx sinensis (Wiegmann) heart cell primary cultivates 7d, cell is moved out around tissue block, and converges around tissue block gradually and form individual layer; As shown in Figure 2, before primary cell goes down to posterity for the first time, cell confluency becomes individual layer, cell body is large, in fusiform or star, nucleus is in the oval of rule, and cell outline is unclear, has projection, there is typical inoblast form, Fig. 6 is the growth curve chart of Trionyx sinensis (Wiegmann) heart cell, and as can be seen from Figure 6, this cell 1d after going down to posterity is residence time, 2d starts to enter exponential phase, 5d starts to enter plateau, and 8d enters decline phase, and selecting 5d ~ 7d to go down to posterity is the result considering cell quantity and proliferative conditions; As shown in Figure 3, after Trionyx sinensis (Wiegmann) heart cell continuous cell line Secondary Culture to the cryopreservation in the 10th generation, cell is homogeneous, and after recovery, cell state is good; As shown in Figure 4, Trionyx sinensis (Wiegmann) heart cell continuous cell line Secondary Culture is to the cellular form and in the past and indifference in the 33rd generation, and to breed stability better for this continuous cell line as seen.
Claims (5)
1. a Trionyx sinensis (Wiegmann) heart cell continuous cell line, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is CGMCCNo.10597.
2. a construction process for Trionyx sinensis (Wiegmann) heart cell continuous cell line as claimed in claim 1, is characterized in that comprising the following steps:
(1) original cuiture
Be placed in Bechtop after cleaning, sterilization Trionyx sinensis (Wiegmann) body, get its heart and cleaning removing clot tissue and reticular tissue, heart is cut into 1mm
3and following organize fritter, the trypsinase room temperature digestion 20min ~ 25min of 0.125% ~ 0.25%, then postdigestive tissue block is inoculated in culturing bottle, add original cuiture liquid upside down to cultivate in 30 DEG C of incubators, overturn culturing bottle after 24h and add original cuiture liquid to appropriate; After this, every 3d changes a nutrient solution, and cell starts to move from tissue block after about one week, and the cell of moving out after about two weeks merges into individual layer around tissue block, now removes tissue block, when at the bottom of cell is paved with bottle to more than 90% time, start Secondary Culture;
(2) Secondary Culture
Original cuiture liquid in sucking-off culturing bottle, add the trypsin-EDTA solutions that 2 ~ 3ml trypsinase concentration is 0.125% ~ 0.25%, digestion 10 ~ 30s, until cell and surrounding tissue link occurs loosen after, add stop digestion cultivation liquid with in pancreatin react; At the bottom of piping and druming bottle, attached cell is come off, and the centrifugal 8 ~ 10min of 800rpm ~ 1000rpm adds original cuiture liquid and goes down to posterity, and 1:1 goes down to posterity by volume first, and after this 1:2 or 1:3 goes down to posterity by volume, continues to cultivate in 30 DEG C of incubators; Every 5d ~ 7d goes down to posterity once, when reaching for the 10th generation, cell culture fluid is changed into Secondary Culture liquid, Establishment of Cell Line success.
3. the construction process of Trionyx sinensis (Wiegmann) heart cell continuous cell line as claimed in claim 2, it is characterized in that: the layoutprocedure of described original cuiture liquid is: in MEM nutrient solution, add foetal calf serum, cell growth factor EGF and microbiotic, foetal calf serum volume is made to account for 15% of cumulative volume, EGF concentration is 10ng/ml, penicillin concn is 100IU/ml, Streptomycin sulphate concentration is 100 μ g/ml, and amphotericin B concentration is 20 μ g/ml, and pH value is 7.0 ~ 7.2.
4. the construction process of Trionyx sinensis (Wiegmann) heart cell continuous cell line as claimed in claim 2, it is characterized in that: the layoutprocedure of described Secondary Culture liquid is: in MEM nutrient solution, add foetal calf serum and microbiotic, foetal calf serum volume is made to account for 15% of cumulative volume, mycin concentration is 100IU/ml, Streptomycin sulphate concentration is 100 μ g/ml, amphotericin B concentration is 20 μ g/ml, and pH value is 7.0 ~ 7.2.
5. a cryopreservation method for Trionyx sinensis (Wiegmann) heart cell continuous cell line as claimed in claim 1, is characterized in that comprising the following steps:
(1) prepare cell cryopreservation protection liquid: by foetal calf serum and DMSO by volume 9:1 mix, mix obtained cell cryopreservation and protect liquid;
(2) cell cryopreservation: the cell in vegetative period of taking the logarithm, after the trypsin-EDTA solutions digestion that trypsinase concentration is 0.25%, cell suspension is in the centrifugal 8min ~ 10min of 800rpm ~ 1000rpm, discard supernatant liquor, the cells frozen storing liquid of preparation is added in cell precipitation, resuspended, make the concentration of cell reach 1 × 10
6individual/ml, is transferred to cell suspension in cryopreservation tube, cryopreservation tube is placed in program temperature reduction box, and-80 DEG C are spent the night, and then puts into the medium-term and long-term preservation of liquid nitrogen.
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| CN107858323A (en) * | 2017-11-08 | 2018-03-30 | 广东省生物资源应用研究所 | A kind of red ear tortoise embryonic fibroblasts cell line and its construction method |
| CN109792984A (en) * | 2019-02-01 | 2019-05-24 | 北京健坤禾润科技有限公司 | It is a kind of for the cell cryopreservation culture medium of cell injuring model and its application |
| CN109792983A (en) * | 2018-12-17 | 2019-05-24 | 浙江万里学院 | Spermatozoon in soft-shelled turtle Trionyx sinensis low temperature dilution based on Shelled Turtle Trionyx Sinensis hibernation serum |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107858323A (en) * | 2017-11-08 | 2018-03-30 | 广东省生物资源应用研究所 | A kind of red ear tortoise embryonic fibroblasts cell line and its construction method |
| CN107858323B (en) * | 2017-11-08 | 2020-10-09 | 广东省生物资源应用研究所 | Tortoise embryo fibroblast line and construction method thereof |
| CN109792983A (en) * | 2018-12-17 | 2019-05-24 | 浙江万里学院 | Spermatozoon in soft-shelled turtle Trionyx sinensis low temperature dilution based on Shelled Turtle Trionyx Sinensis hibernation serum |
| CN109792984A (en) * | 2019-02-01 | 2019-05-24 | 北京健坤禾润科技有限公司 | It is a kind of for the cell cryopreservation culture medium of cell injuring model and its application |
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| CN105039241B (en) | 2018-12-18 |
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