CN104531608A - Anguilla anguilla liver cell line and construction method and application thereof - Google Patents
Anguilla anguilla liver cell line and construction method and application thereof Download PDFInfo
- Publication number
- CN104531608A CN104531608A CN201510013504.1A CN201510013504A CN104531608A CN 104531608 A CN104531608 A CN 104531608A CN 201510013504 A CN201510013504 A CN 201510013504A CN 104531608 A CN104531608 A CN 104531608A
- Authority
- CN
- China
- Prior art keywords
- cell
- anguilla
- eel
- cell line
- culture solution
- 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 an anguilla anguilla liver cell line and a construction method and application thereof, and belongs to the technical field of sea fish cell culture. The cell line is preserved in the Chinese typical culture preservation center on December 16, 2014, and the serial number of preservation registration is CCTCC NO: C2014239. The anguilla anguilla liver cell line is fibroblast cells, the continuous passage achieves more than 60 generations, the adherence after cryopreservation resuscitation is good, the growth is stable, the liver cell line can be directly used for research on aquatic virology, anguilla anguilla cell biological characteristics and immune resistance gene function, and the lever cell line has been proved to be sensitive to eels herpes virus. According to the anguilla anguilla liver cell line and the construction method and application thereof, the constructing method is simple and convenient to implement, the repeatability is high, and the cost is moderate. The anguilla anguilla liver cell line and the construction method and application thereof can also be applied to the preparation of other anguilla anguilla cell lines.
Description
Technical field
The present invention relates to a kind of European eel (
anguilla Anguilla) liver cell system and construction process thereof and application, belong to seawater fish technical field of cell culture.
Background technology
Fish cell is cultivated and is started from the sixties in 20th century, first Teleostei permanent cell system within 1962, is set up from Wolf etc.---since rainbow trout gonadal cell system RTG-2, more than 250 strain fish cell systems are established both at home and abroad, having more than 40 to plant virus adopts cell cultures to be separated successfully, and the success that fish embryo stem cell is cultivated effectively facilitates engineered development especially.Fish research experiment has higher requirement for the maintenance of cultivation place, equipment and water body environment, using Cultured Fish Cells as experimental model, have the advantage that live body fish is incomparable: (1) cost is low, the maintenance of clone does not need large-scale cultivation equipment and a large amount of changes water and inflation; (2) reproducible, experiment condition can accurately control; (3) sample, observe simple and easy to do, be beneficial to the analysis carrying out great amount of samples.Fish cell line model research development in decades rapidly; the each side such as etiology, pharmacology, physiology, environmental toxicology, cytobiology, oncology and conservation of resources are widely used in; in virological investigation, especially there is consequence, be regarded as the basis of viral pathogen separation, qualification and characteristic research.
Common eel aquaculture is the important pillar industry of China's water industry, and its annual production accounts for 2/3 of Gross World Product, and single product export value ranks first in fishery products.European eel (
anguilla Anguilla) genus Anguilliforme (
anguilliformes), Anguillidae (
anguillidae), Anguilla (
anguilla), originate in the Atlantic Ocean, introduced China first in 1993, and realize feedstuff matched production domesticization the same year; Since drawing and forming merit, Europe eel aquaculture develops rapidly, the end of the nineties, European eel annual production exceeded Japanese eel, become the most important cultured eels kind of China, its fine and tender taste, delicious, rich in proteins, multivitamin and unsaturated fatty acids, there is high nutritive value, have the title of " soft gold ".Due to developing rapidly of aquaculture, the problems such as cultivation layout is unreasonable, density is excessive, current are not smooth generally occur, the situation of throw something and feed rotten bait and Drug abuse is had when adding, cause fish body immunity degradation and water pollution, all kinds of disease frequently breaks out, wherein be no lack of viral case, and current existing European eel cell line model is few, far can not meet research needs.
Because the reproductive habit of river migration and the life history complicated and changeable fall in European eel, its artificial propagation is still the world-class technical barrier do not separated so far; And Europe eel embryo and the low age young be difficult to obtain, and the restriction imported and exported glass eel after being listed in endangered species, objectively increases the difficulty that European eel somatocyte system sets up, more highlights the importance that this needs the technological gap filled up badly.
Summary of the invention
The object of this invention is to provide a kind of European eel liver cell system and construction process thereof and application, to make up the deficiency in prior art and cell model storehouse, and meet application needs European eel being carried out to Etiology analysis and the research such as physiological property, functional genome.
The construction process of European eel liver cell system involved in the present invention, its step is as follows:
1) examination of European eel is front prepares: the black young eel of the healthy European eel young is supported 1 ~ 2 week temporarily in indoor in clean seawater, and period is feeding bait not;
2) acquisition of European eel liver organization: ready for step 1) young eel is placed in the capable frost anesthesia of trash ice, to fish body to physical stimulation stress behavior disappear; After removing mucus with 2% tincture of iodine cotton balls abundant wiping fish surface, again with 75% cotton ball soaked in alcohol wiping fish surface 2 ~ 3 times, cut off body wall by side, take out liver organization, be placed in 0 DEG C of precooling and containing penicillin and the dual anti-sterilizing of Streptomycin sulphate without calcium magnesium D-Hanks balanced salt solution, clean 5 ~ 6 times;
3) original cuiture: by step 2) the European eel liver organization that obtains shreds into 0.5mm
3~ 1mm
3the tissue block of size, with step 2) described sterilizing is without the rinsing of calcium magnesium D-Hanks balanced salt solution; Soak culturing bottle bottom surface with the L-15 substratum not containing serum, the tissue block after rinsing is moved in 25mL culturing bottle, evenly distributed with the spacing of 0.3cm ~ 0.5cm; Be inverted body after 20 DEG C of airtight cultivations make tissue block adherent in 6 ~ 8 hours, in culturing bottle, add primary and early stage Secondary Culture complete culture solution, slowly right body, make nutrient solution invade tissues block, under 20 DEG C of constant temperature, start original cuiture;
4) early stage Secondary Culture: collect well-grown primary culture and carry out early stage Secondary Culture, the tissue block discarding nutrient solution before going down to posterity and come off, with 0.25% pancreatin rinse cellular layer, discard, add 0.25% trypsin digestion cell again, pancreatin is discarded when cell is and shrinks and be separated situation, add primary and early stage Secondary Culture complete culture solution and stop digestion, come off to the dispersion of biological cells and tissues block major part with connector bend dropping tube piping and druming culture, collecting cell and suspensions of tissues, centrifugal 3 minutes of 1000rpm, the resuspended precipitation of complete culture solution is used after removing supernatant, at 27 DEG C, enriched cultivation is carried out with the ratio of 1:1 ~ 2:1 according to biological cells and tissues number of blocks, repeat above step until confirm that culture consecutive numbers is for pollution-free sign, after going down to posterity cell can be formed smoothly and to be paved with bottle at the bottom of individual layer,
5) routine passage is cultivated: cell carries out routine passage cultivation to it after stablizing formation individual layer, with microscopy, Continuous Observation is carried out to culturing cell, go down to posterity at once when uniform slight stacking phenomenon appears in cell, with 0.25% pancreatin rinse cellular layer, discard, add 0.25% trypsin digestion cell again 30 seconds to 1 minute, treat that upper strata cellular contraction becomes bowlder and discards pancreatin, add routine passage cultivation complete culture solution, softly blow and beat supreme confluent monolayer cells with connector bend dropping tube to come off and make cell suspension, whole cell suspension is moved into new bottle, routine passage cultivation is carried out under 27 DEG C of constant temperature,
6) the frozen and recovery of cell: after cell enters routine passage cultivation stage, carry out liquid nitrogen freezing preservation to it, frozen storing liquid consists of 70%L-15,20% foetal calf serum and 10%DMSO in batches; During recovery cell, to thaw rapidly sample with 37 DEG C of water-baths, mix with routine passage cultivation complete culture solution after sucking-off cell suspension and be inoculated in culturing bottle, after 12 hours, change training liquid, continue to cultivate after removing non-viable cells and DMSO composition.
Step 3) and 4) described primary and to go down to posterity with complete culture solution be in early days based on L-15 substratum, be the foetal calf serum of 15% containing final concentration, final concentration is the penicillin of 200U/mL, and final concentration is the complete culture solution of the Streptomycin sulphate of 200 μ g/mL.
Described in step 5), routine passage cultivation complete culture solution is based on L-15 substratum, is the foetal calf serum of 10% containing final concentration, does not contain the complete culture solution of microbiotic and other nutritional supplements.
Per-cent used in each step of the present invention is volume percent.
According to above-mentioned clone construction process, establish a strain European eel liver cell system EL, this cell lies in and is deposited in China typical culture collection center on December 16th, 2014, and preservation is registered on the books and is numbered CCTCC NO:C2014239.
Described European eel liver cell system EL, this clone has following biological characteristics:
1) this cell is typical inoblast form, and continuous passage 60 generation still can keep this morphological specificity in vitro.
2) this cell line proliferation ability is strong, and the survival time is long, and under replaced medium condition once weekly, survival in vitro time limit Absorbable organic halogens reaches 70 ~ 90 days, and the longest more than 150 days, continuous passage 60 generation still keeps this physiological characteristic in vitro.
The present invention also protects the application of described European eel liver cell system EL as aquatic animal virus host cells; the EL cell of taking the logarithm vegetative period; common eel simplexvirus HVA-II is inoculated under maintenance media environment; typical cytopathy (cytopathic effect, CPE) phenomenon can be produced.
Described maintenance substratum, based on L-15 substratum, comprises the foetal calf serum that final concentration is 2%.
Beneficial effect of the present invention is:
1) technical scheme of the present invention is simple and easy to do, screening conditions are simply distinct, agents useful for same is conventional articles for use the most frequently used in fish cell cultivation, any special nutrition composition need not be added, thus ensure that there is wide application space in constructed European eel liver cell system, effectively fill up the technological gap that European eel somatocyte system model is set up;
2) European eel liver cell system (EL) Growth of Cells constructed by is vigorous, form is homogeneous, for fibroblast-like cells, can be more than continuous passage 60 generation, and it is well adherent after cryopreservation resuscitation, growth is stable, can be directly used in the research of aquatic products virusology, European eel characteristics of cell biology and immune disease-resistance gene function, and has been proved common eel simplexvirus responsive;
3) this construction process is simple to operate, and repeatability is strong, and moderate cost, is also applicable to other European eel somatocyte systems.
Accompanying drawing explanation
Fig. 1: European eel liver cell original cuiture light microscopic photo: a & b, dissimilar cell are moved out from tissue block; C & d, cell form the outgrowth of different shape around tissue block.
Fig. 2: European eel liver cell Secondary Culture light microscopic photo: a, 2nd generation EL cell; B, the 5th generation EL cell; C, the 11st generation EL cell; D, the 21st generation EL cell; E, the 41st generation EL cell; F, the 70th generation EL cell.
Fig. 3: European eel liver cell in vitro long time-histories is cultivated: a, EL cell cultures the 5th day; B, EL cell cultures the 28th day; C, EL cell cultures the 111st day.
Fig. 4: European eel liver cell system EL cell growth curve.
Fig. 5: European eel liver cell system EL frozen rear recovery the 8th day light microscopic photo.
Fig. 6: European eel liver cell system EL chromosomal pattern is analyzed: a, EL clone modal number distribution plan; B, EL clone chromosomal pattern typical figure (2n=38).
Fig. 7: the European eel liver cell system EL light microscopic photo infecting common eel simplexvirus (HVA-II): a, infect and connect malicious EL cell in latter 192 hours; 192 hr-control EL cells after b, infection.
embodiment:
In order to explain the present invention better, will further illustrate technical scheme of the present invention by specific embodiment below, protection scope of the present invention should comprise the full content of claim, but is not limited thereto.
example 1: the original cuiture of European eel liver cell
laboratory animal:
Body is about 20cm, the healthy European eel juvenile fish of about body weight 20g.
reagent:
L-15(is purchased from Hyclone); 1000000U/10mg green grass or young crops-Streptomycin sulphate mixed solution, dehydrated alcohol, the tincture of iodine (purchased from the raw work in Shanghai); NaCl, Na2HPO
4, KCl, KH2PO
4, NaHCO
3, trypsin Trypsin) (purchased from Sigma); Foetal calf serum (Fetal bovine serum, FBS) (purchased from Gibco).
instrument:
Bechtop (Air Tech); Inverted fluorescence microscope (Nikon); Biochemical cultivation case (rich fast); Ultrapure water machine (Millipore).
consumptive material:
Dissection eye scissors, ophthalmic tweezers, Venus cut (six or six ophthalmology); 25mL, 50mL Tissue Culture Flask (BD Falcon); 15mL centrifuge tube (BD Falcon); 5mL elbow glass dropper.
step:
1) test prepares with before the examination of fish: the European eel young (black young eel) is supported 1 ~ 2 week temporarily in indoor in clean seawater, and period is feeding bait not.
2) damping fluid and Digestive system preparation: configure 20 times without calcium magnesium D-Hanks storing solution by standard recipe, working concentration is diluted to aseptic ultrapure water, configuration is based on this penicillin of 200U/mL containing final concentration, and final concentration is the working buffer liquid of the Streptomycin sulphate of 200 μ g/mL and contains 0.25% tryptic Trypsin Digestive system.
3) acquisition of European eel liver organization: above-mentioned young eel is placed in the capable frost anesthesia of trash ice, to fish body to physical stimulation stress behavior disappear; After removing mucus with 2% tincture of iodine cotton balls abundant wiping fish surface, again with 75% cotton ball soaked in alcohol wiping fish surface 2 ~ 3 times, cut off body wall by side, take out liver organization, be placed in 0 DEG C of precooling and containing penicillin and the dual anti-sterilizing of Streptomycin sulphate without calcium magnesium D-Hanks solution, clean 5 ~ 6 times.
4) primary and early stage Secondary Culture complete culture solution is prepared: prepare based on L-15 substratum, be the foetal calf serum of 15% containing final concentration, final concentration is the penicillin of 200U/mL, and final concentration is the primary of the Streptomycin sulphate of 200 μ g/mL and goes down to posterity in early days and use complete culture solution.
5) original cuiture: above-mentioned European eel liver organization is shredded into 0.5 mm
3~ 1mm
3the tissue block of size, with the rinsing of above-mentioned D-Hanks liquid, removes hemocyte, fatty tissue and reticular tissue as far as possible; Do not soak culturing bottle bottom surface containing the L-15 substratum of serum with a small amount of, above-mentioned small tissue blocks is moved in 25mL culturing bottle, evenly distributed with the spacing of 0.3cm ~ 0.5cm; Be inverted body after 20 DEG C of airtight cultivations make tissue block adherent in 6 ~ 8 hours, in culturing bottle, add nutrient solution described in 1mL step 4), slowly right body, make nutrient solution invade tissues block, start original cuiture in 20 DEG C of constant temperature; Slowly adding complete culture solution 1mL every 24 hours after Primary culture is about 3mL to final volume, within every 72 hours thereafter, changes the complete culture solution of 50%.
achievement:
After inoculation, within 48th ~ 72 hours, have cell to move out from tissue block (Fig. 1: a & b) as seen, within about two weeks, visible cell forms more radial outgrowth (Fig. 1: c & d) around tissue block.
example 2: the early stage Secondary Culture of European eel liver cell
reagent:
L-15(is purchased from Hyclone); 1000000U/10mg green grass or young crops-Streptomycin sulphate mixed solution (purchased from the raw work in Shanghai); NaCl, Na2HPO
4, KCl, KH2PO
4, NaHCO
3, trypsin Trypsin) (purchased from Sigma); Foetal calf serum (Fetal bovine serum, FBS) (purchased from Gibco)
instrument:
Bechtop (Air Tech); Inverted fluorescence microscope (Nikon); Biochemical cultivation case (rich fast); Ultrapure water machine (Millipore); Whizzer (Beckman)
consumptive material:
25mL, 50mL Tissue Culture Flask (BD Falcon); 15mL centrifuge tube (BD Falcon); 5mL elbow glass dropper
step:
1) primary and early stage Secondary Culture complete culture solution is prepared: prepare based on L-15 substratum, be the foetal calf serum of 15% containing final concentration, final concentration is the penicillin of 200U/mL, and final concentration is the primary of the Streptomycin sulphate of 200 μ g/mL and goes down to posterity in early days and use complete culture solution.
2) early stage Secondary Culture: collect well-grown, the primary culture forming larger area cellular layer carries out early stage Secondary Culture, the tissue block discarding nutrient solution before going down to posterity and come off, with a small amount of 0.25% pancreatin rinse cellular layer, discard, add enough trypsin digestion cells again, pancreatin is discarded when cell is and shrinks and be separated situation, add complete culture solution as described in step 1) and stop digestion, come off to the dispersion of biological cells and tissues block major part with connector bend dropping tube piping and druming culture, collecting cell/suspensions of tissues, centrifugal 3 minutes of 1000rpm, after removing supernatant, by the resuspended precipitation of complete culture solution, at 27 DEG C, enriched cultivation is carried out with the ratio of 1:1 ~ 2:1 according to biological cells and tissues number of blocks, repeat above program until confirm that culture consecutive numbers is for pollution-free sign, after going down to posterity cell can be formed smoothly and to be paved with bottle at the bottom of individual layer, this process generally needs to go down to posterity 5 ~ 6 times.
achievement:
Go down to posterity after 5 times, the natural decomposition disappearance in succeeding generations of remnant tissue's block, obtain not containing the Pure-culture cell organizing relic, passage surviving rate significantly rises, substantially confluent monolayer can be grown up to, and can in 1:2 ratio enlarged culturing, the serum proportion mark simultaneously needed for cell declines (Fig. 2: a & b) to some extent.
example 3: the routine passage cultivation of European eel liver cell and the foundation of clone:
reagent:
L-15(is purchased from Hyclone); Glycerine, methyl alcohol, Glacial acetic acid, Giemsa pulvis (purchased from the raw work in Shanghai); NaCl, Na2HPO
4, KCl, KH2PO
4, NaHCO
3, trypsin Trypsin) (purchased from Sigma); Foetal calf serum (Fetal bovine serum, FBS) (purchased from Gibco)
instrument:
Bechtop (Air Tech); Inverted fluorescence microscope (Nikon); Biochemical cultivation case (rich fast); Ultrapure water machine (Millipore)
Liquid nitrogen container (Locator), whizzer (Beckman)
consumptive material:
50mL Tissue Culture Flask (BD Falcon), 250mL Tissue Culture Flask (Corning); 15mL centrifuge tube (BD Falcon); Slide glass; 5mL elbow glass dropper; Normal glass decolouring ware
step:
1) routine passage cultivates complete culture solution preparation: preparing based on L-15 substratum, is the foetal calf serum of 10% containing final concentration, does not contain the routine passage cultivation complete culture solution of microbiotic and other nutritional supplements.
2) routine passage is cultivated: cell carries out routine passage cultivation to it after stablizing formation individual layer, with microscopy, Continuous Observation is carried out to culturing cell, go down to posterity at once when uniform slight stacking phenomenon appears in cell, with a small amount of 0.25% pancreatin rinse cellular layer, discard, add enough pancreatin (3mL/50mL Tissue Culture Flask) peptic cell again 30 seconds to 1 minute, treat that upper strata cellular contraction becomes bowlder and discards pancreatin, add the complete culture solution as described in step 1), softly blow and beat supreme confluent monolayer cells with connector bend dropping tube to come off and make suspension, whole cell suspension is moved into new bottle, cultivate 4 ~ 6 hours for 27 DEG C, whole complete culture solution is changed after the cell overwhelming majority is adherent, be placed in 27 DEG C of constant temperature culture, former bottle containing residue bottom cell adds equivalent complete culture solution to be continued to cultivate, and changes nutrient solution once completely weekly.
3) the frozen and recovery of cell: after cell enters routine passage cultivation stage, carry out liquid nitrogen freezing preservation to it, frozen storing liquid consists of 70% L-15,20% foetal calf serum and 10%DMSO in batches; Get the cell being in logarithmic phase, according to step 2) after described digestion method obtains cell suspension, centrifugal 3 minutes of 1500rpm, with frozen storing liquid re-suspended cell after supernatant discarded, by every pipe 5 × 10
5~ 1 × 10
6cell count is sub-packed in cell cryopreservation tube, is placed in the program temperature reduction box of interior Sheng Virahol, moves into-70 DEG C of refrigerators, move into liquid nitrogen and preserve for a long time after 12 hours 4 DEG C of coolings after 30 minutes to 1 hour; During recovery cell, to thaw rapidly sample with 37 DEG C of water-baths, mix with the complete culture solution described in step 1) after sucking-off cell suspension and be inoculated in culturing bottle, after 12 hours, change training liquid, continue to cultivate after removing non-viable cells and DMSO composition.
4) when cell, cultured continuously was more than 18 months in vitro, and passage number is more than after 50 generations, selected the cell of logarithmic phase, to carry out chromosomal pattern analysis after the process of colchicine method.
achievement:
Along with the increase of passage number, the growth of European eel liver cell is tending towards vigorous, and interval of going down to posterity progressively is shortened (Fig. 2: c ~ f), but possesses its characteristic (Fig. 3) that survival time is long in vitro all the time; Be 4 ~ 6 days to the cycle stability that goes down to posterity during 50th ~ 60 generation, logarithmic phase cell doubling time is 36.04 hours (Fig. 4); Preserve the cell recovery of month in liquid nitrogen after, its adherent rate can reach more than 80%, after changing liquid, surviving rate is more than 70%, external form is fibroblast-like cells, form stable homogeneous (Fig. 5), now, Establishment of Cell Line success, called after EL, dyed size analysis experiment, proves that it is the diploid cell strain (Fig. 6) meeting 2n=38.
example 4: carry out studying as the application method of aquatic animal virus host cells to self-built European eel liver cell system EL
reagent:
L-15(is purchased from Hyclone), foetal calf serum is purchased from Gibco), infect the freezing sample (this Laboratories Accession) of common eel gonad cell (EO) of common eel simplexvirus (HVA-II).
instrument:
Bechtop (Air Tech); Inverted fluorescence microscope (Nikon); Biochemical cultivation case (rich fast); Liquid nitrogen container (Locator)
consumptive material:
250mL Tissue Culture Flask (Corning); 5mL elbow glass dropper.
step:
1) according to described Secondary Culture method above with 5 × 10
5eL cell is inoculated in 250mL Tissue Culture Flask by cell count, within 36 ~ 48 hours, can grow up to fresh individual layer.
2) perfect medium is replaced by maintenance substratum, 27 DEG C of constant-temperature incubations 6 hours.
3) get common eel gonad cell (EO) the freezing sample infecting common eel simplexvirus (HVA-II), fully blow and beat mixing with connector bend dropping tube after freeze thawing 3 times for subsequent use.
4) above-mentioned steps 3 is got) the middle viral sample liquid prepared, be inoculated in above-mentioned steps 2 with the consumption of 300 μ L/ bottles) the middle experiment EL cell prepared, and not connect malicious bottle in contrast.
5) with 24 hours for interval docking poison after EL cell carry out microscopic examination, and and compared with control cells compare,
achievement:
In connecing poison after 72 hours, observe and connect number of dead cells in malicious sample and increase, 168 hours later cell death peak, and under light microscopic, the shrinkage of visible cell big area takes off wall, and having a large amount of cell debris to be dispersed in culturing bottle, 240 hours later cell mortality ratio reach 95% (Fig. 7: a); And control sample Growth of Cells is good, closely adherent, cell density has significantly increases phenomenon, only has cell debris (Fig. 7: b) that a small amount of eubolism produces in substratum.
Claims (6)
1. a strain European eel liver cell system EL, this cell lies in and is deposited in China typical culture collection center on December 16th, 2014, and preservation is registered on the books and is numbered CCTCC NO:C2014239.
2. a construction process for European eel liver cell system as claimed in claim 1, is characterized in that: the step of described construction process is as follows:
1) examination of European eel is front prepares: the black young eel of the healthy European eel young is supported 1 ~ 2 week temporarily in indoor in clean seawater, and period is feeding bait not;
2) acquisition of European eel liver organization: ready for step 1) young eel is placed in the capable frost anesthesia of trash ice, to fish body to physical stimulation stress behavior disappear; After removing mucus with 2% tincture of iodine cotton balls abundant wiping fish surface, again with 75% cotton ball soaked in alcohol wiping fish surface 2 ~ 3 times, cut off body wall by side, take out liver organization, be placed in 0 DEG C of precooling and containing penicillin and the dual anti-sterilizing of Streptomycin sulphate without calcium magnesium D-Hanks balanced salt solution, clean 5 ~ 6 times;
3) original cuiture: by step 2) the European eel liver organization that obtains shreds into 0.5 mm
3~ 1mm
3the tissue block of size, with step 2) described sterilizing is without the rinsing of calcium magnesium D-Hanks balanced salt solution; Soak culturing bottle bottom surface with the L-15 substratum not containing serum, the tissue block after rinsing is moved in 25mL culturing bottle, evenly distributed with the spacing of 0.3cm ~ 0.5cm; Be inverted body after 20 DEG C of airtight cultivations make tissue block adherent in 6 ~ 8 hours, in culturing bottle, add primary and early stage Secondary Culture complete culture solution, slowly right body, make nutrient solution invade tissues block, under 20 DEG C of constant temperature, start original cuiture;
4) early stage Secondary Culture: collect well-grown primary culture and carry out early stage Secondary Culture, the tissue block discarding nutrient solution before going down to posterity and come off, with 0.25% pancreatin rinse cellular layer, discard, add 0.25% trypsin digestion cell again, pancreatin is discarded when cell is and shrinks and be separated situation, add primary and early stage Secondary Culture complete culture solution and stop digestion, come off to the dispersion of biological cells and tissues block major part with connector bend dropping tube piping and druming culture, collecting cell and suspensions of tissues, centrifugal 3 minutes of 1000rpm, the resuspended precipitation of complete culture solution is used after removing supernatant, at 27 DEG C, enriched cultivation is carried out with the ratio of 1:1-2:1 according to biological cells and tissues number of blocks, repeat above step until confirm that culture consecutive numbers is for pollution-free sign, after going down to posterity cell can be formed smoothly and to be paved with bottle at the bottom of individual layer,
5) routine passage is cultivated: cell carries out routine passage cultivation to it after stablizing formation individual layer, with microscopy, Continuous Observation is carried out to culturing cell, go down to posterity at once when uniform slight stacking phenomenon appears in cell, with 0.25% pancreatin rinse cellular layer, discard, add 0.25% trypsin digestion cell again 30 seconds to 1 minute, treat that upper strata cellular contraction becomes bowlder and discards pancreatin, add routine passage cultivation complete culture solution, softly blow and beat supreme confluent monolayer cells with connector bend dropping tube to come off and make cell suspension, whole cell suspension is moved into new bottle, routine passage cultivation is carried out under 27 DEG C of constant temperature,
6) the frozen and recovery of cell: after cell enters routine passage cultivation stage, carry out liquid nitrogen freezing preservation to it, frozen storing liquid consists of 70%L-15,20% foetal calf serum and 10%DMSO in batches; During recovery cell, to thaw rapidly sample with 37 DEG C of water-baths, mix with routine passage cultivation complete culture solution after sucking-off cell suspension and be inoculated in culturing bottle, after 12 hours, change training liquid, continue to cultivate after removing non-viable cells and DMSO composition.
3. the construction process of European eel liver cell system as claimed in claim 2, it is characterized in that: step 3) and 4) described primary and to go down to posterity with complete culture solution be in early days based on L-15 substratum, be the foetal calf serum of 15% containing final concentration, final concentration is the penicillin of 200U/mL, and final concentration is the complete culture solution of the Streptomycin sulphate of 200 μ g/mL.
4. the construction process of European eel liver cell system as claimed in claim 2, it is characterized in that: described in step 5), routine passage cultivation complete culture solution is based on L-15 substratum, be the foetal calf serum of 10% containing final concentration, not containing the complete culture solution of microbiotic and other nutritional supplements.
5. European eel liver cell system EL as claimed in claim 1 is as the application of aquatic animal virus host cells, it is characterized in that: the EL cell in vegetative period of taking the logarithm, inoculates common eel simplexvirus HVA-II under maintenance media environment.
6. apply as claimed in claim 5, it is characterized in that: described maintenance substratum, based on L-15 substratum, comprises the foetal calf serum that final concentration is 2%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510013504.1A CN104531608A (en) | 2015-01-12 | 2015-01-12 | Anguilla anguilla liver cell line and construction method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510013504.1A CN104531608A (en) | 2015-01-12 | 2015-01-12 | Anguilla anguilla liver cell line and construction method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104531608A true CN104531608A (en) | 2015-04-22 |
Family
ID=52847243
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510013504.1A Pending CN104531608A (en) | 2015-01-12 | 2015-01-12 | Anguilla anguilla liver cell line and construction method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104531608A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108795851A (en) * | 2017-04-27 | 2018-11-13 | 大连医科大学 | The method for building up of people's salivary adenoid cystic carcinoma carcinoma-associated fibroblasts system CAF-SA a kind of and its application |
| CN109207422A (en) * | 2018-09-26 | 2019-01-15 | 福建省农业科学院生物技术研究所 | A kind of European eel kidney cell system EK and its application |
| CN109414494A (en) * | 2016-02-02 | 2019-03-01 | 基准动物健康有限公司 | The method for generating virus |
| CN110577930A (en) * | 2019-09-30 | 2019-12-17 | 重庆赛托斯创生物科技发展有限公司 | Multi-connected-tube adipose-derived stem cell extraction method |
| CN113957037A (en) * | 2021-11-22 | 2022-01-21 | 海南大学 | Bactria camelina branchia cell line, construction method and application method thereof |
-
2015
- 2015-01-12 CN CN201510013504.1A patent/CN104531608A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109414494A (en) * | 2016-02-02 | 2019-03-01 | 基准动物健康有限公司 | The method for generating virus |
| CN108795851A (en) * | 2017-04-27 | 2018-11-13 | 大连医科大学 | The method for building up of people's salivary adenoid cystic carcinoma carcinoma-associated fibroblasts system CAF-SA a kind of and its application |
| CN108795851B (en) * | 2017-04-27 | 2022-03-18 | 大连医科大学 | Establishment method and application of human salivary adenoid cystic carcinoma related fibroblast cell line CAF-SA |
| CN109207422A (en) * | 2018-09-26 | 2019-01-15 | 福建省农业科学院生物技术研究所 | A kind of European eel kidney cell system EK and its application |
| CN109207422B (en) * | 2018-09-26 | 2020-08-21 | 福建省农业科学院生物技术研究所 | European eel kidney cell line EK and application thereof |
| CN110577930A (en) * | 2019-09-30 | 2019-12-17 | 重庆赛托斯创生物科技发展有限公司 | Multi-connected-tube adipose-derived stem cell extraction method |
| CN113957037A (en) * | 2021-11-22 | 2022-01-21 | 海南大学 | Bactria camelina branchia cell line, construction method and application method thereof |
| CN113957037B (en) * | 2021-11-22 | 2023-04-28 | 海南大学 | Perch gill cell line, construction and application method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104531608A (en) | Anguilla anguilla liver cell line and construction method and application thereof | |
| CN101979518B (en) | Method for preparing pseudorabies virus | |
| CN102399743A (en) | Cell line of pterygiophore tissue of cryprinus carpiod and construction method | |
| CN105543164A (en) | Primary isolated culture method for dairy cow mammary epithelial cells | |
| CN113249308B (en) | Perch arterial ball cell line and application and culture method thereof | |
| CN104152403B (en) | A kind of method that establishing goose embryonic epithelium cell line and the goose embryonic epithelium cell line of foundation | |
| CN102424813A (en) | Simple extraction method of high-purity mouse skeletal muscle satellite cells | |
| CN104726401A (en) | Method for improving success rate of umbilical cord blood mesenchymal stem cell culture by using placental mesenchymal stem cells | |
| CN104027798A (en) | Method for culturing and producing PVC 2 antigen through whole suspension cells | |
| CN109030131A (en) | A kind of refined dragonfish method of chromosome preparation | |
| CN110499280B (en) | In-vitro construction method of Acipenser parvum thecal cell line and reagent used by same | |
| CN109207422B (en) | European eel kidney cell line EK and application thereof | |
| CN110305844B (en) | Crucian spinal cord tissue cell line and application thereof | |
| CN101451121B (en) | Construction method of Epinephelus fuscoguttatus heart cell line | |
| CN107858322B (en) | Method for establishing primary hippocampal cell culture system | |
| CN114107181B (en) | Sturgeon embryo cell line, culture medium and preparation method thereof | |
| CN103484425B (en) | Pseudosciaena crocea head kidney cell line and construction method thereof | |
| CN102120983B (en) | Separation and culture method of epidermal stem cells of Beijing ducks | |
| CN110295137B (en) | Channa argus kidney cell line and construction method and application thereof | |
| CN103725644B (en) | Cherry valley duck embryo epithelial cell line and method for building up thereof | |
| CN101012450B (en) | Mount Wuzhi Pig ear rim tissue fibroblast series and culturing method thereof | |
| CN102604887B (en) | In vitro culture method for hucho taimen body cells | |
| CN108795854A (en) | A kind of hair follicle stem cells storing liquid and its preparation method and application | |
| CN114600805B (en) | Breeding method of 'Shibei No. 1' of rapid-growing triploid portuguese oyster | |
| Schwenk | Callus formation from mechanically isolated soybean cotyledonary cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20150422 |