CN105602887A - Culture medium and method for culturing chlamys farreri trochophore cell lines - Google Patents
Culture medium and method for culturing chlamys farreri trochophore cell lines Download PDFInfo
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- CN105602887A CN105602887A CN201510801587.0A CN201510801587A CN105602887A CN 105602887 A CN105602887 A CN 105602887A CN 201510801587 A CN201510801587 A CN 201510801587A CN 105602887 A CN105602887 A CN 105602887A
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Abstract
The invention discloses a culture medium and method for culturing chlamys farreri trochophore cell lines. The culture medium comprises the following components: 13.7 g/l of an L-15 base culture medium, 20.2 g/l of NaCl, 0.54 g/l of KCl, 0.60 g/l of CaCl2, 1 g/l of MgSO4, 3.9 g/l of MgCl2 and fetal bovine serum with the final concentration of 5%, 2 mmol/l of glutamine, 1% of a chlamys farreri yolk extract liquid, 1% of chlamys farreri serum, 100 IU/ml of penicillin, and 100 [mu]g/ml of streptomycin, wherein the pH is 7.2-7.4. The culture method comprises that the culture medium is used for performing single cell primary culture of trochophores, to realize early passage and serial subcultivation. The culture medium can effectively increase cell survival, and makes cells continuously proliferate; and the method has guiding significance on establishment of other marine shellfish cell lines, and has great application value.
Description
Technical field
The invention belongs to the cell culture technology field of seashells, be specifically related to a kind of comb of cultivatingCulture medium and the method for hole scallop trochophore clone.
Background technology
It is one of important means in bioengineering and common method that cell is cultivated, extensive useIn chromosome analysis, cellular metabolism, gene expression, immunity, carcinogenesis mechanism, Virus culture,The research fields such as separation, qualification and drug screening. Shellfish organizes training from 20th century 60In latter stage in age, reported have tens kinds of oyster, mussel, clams etc. Domestic fromStart to carry out the research in this field the eighties in 20th century. Whether successfully cell cultivates keyCan create adapt circumstance and make cell in vitro can continue propagation, also do not have at present a kind of forThe cell culture medium of seashells, in order to make better shellfish Growth of Cells, many researchers coupleThis has carried out the pilot studys such as a large amount of interpolation factors. But because seashells cell is cultivated manyImitate mammaliancellculture flow process, culture medium is also the base at mammalian cell cultureOn plinth, make change, and requirement and the mammalian cell of seashells cell to nutrition is poor to some extentDifferent, make seashells Growth of Cells and division lack suitable trophic factors. Therefore, althoughPass through the effort of nearly half a century, still do not set up the immortality clone of seashells,Its cell is cultivated and is rested in former culture and finite cell lines level more. Many researchers wishSet up seashells clone by adding factor optimizing culture medium.
In addition, the cell category of in vitro culture is also most important, and common cell is cultivated kind to be hadAdult tissue's cell, as gill cell, outer embrane cell, haemocyte, heart cell, neural thinBorn of the same parents etc., however adult tissue's cell division growth is limited in one's ability in vitro, therefore also nonidealCell is cultivated kind source. Grow early stage embryo or larva cell and conventionally there is the larger silk that hasDivision potential, and cell many places are in low level of differentiation or undifferentiated stem cell state, thisOne characteristic makes embryo or larva cell become the best experiment material that cell is cultivated. Seashells isImportant monoid in marine organisms, its embryo is outer owing to there being the coated difficult acquisition of film unicellular, because ofThis, larva cell is the cell derived that seashells has in vitro culture prospect. Chlamys farreri(Chlamysfarreri) be under the jurisdiction of Mollusca (Mollusca), lamellibranchiata(Lamellibranchia), pearl shell order (Pterioida), Pectenidae (Pectinidae), fanShellfish belongs to (Pecten), is the economic shellfish that only originates in China's north Coast, is northern China weightThe breed variety of wanting.
Summary of the invention
The object of the present invention is to provide a kind of for cultivating Chlamys farreri trochophore cloneCulture medium and method, be intended to solve prior art and cannot realize asking that shellfish passage cultivatesTopic.
The present invention is specifically achieved through the following technical solutions:
Cultivate a culture medium for Chlamys farreri trochophore clone, described culture medium comprisesThe L-15 basal medium of following component: 13.7g/l, 20.2g/lNaCl, 0.54g/lKCl,0.60g/lCaCl2、1g/lMgSO4、3.9g/lMgCl2And the final concentration hyclone that is 5%,The glutamine of 2mmol/L, 1% Chlamys farreri Yolk extraet, 1% Chlamys farreri bloodClearly, the penicillin of 100IU/ml and the streptomysin of 100 μ g/ml, pH is 7.2-7.4.
Chlamys farreri serum of the present invention is prepared by the following method: in closed shell flesh, extractHaemolymph of Chlamys farreri, is placed in 4 DEG C of hold over night of sterile chamber, next day by supernatant withThe centrifugal 10min of 2000g, collects after supernatant respectively micro-through 0.45 μ m and 0.22 μ m apertureHole filter filtration sterilization, is placed in-20 DEG C of preservations after packing.
Chlamys farreri Yolk extraet of the present invention is prepared by the following method: dissect ripeThe Chlamys farreri ovary of phase, the seawater that is placed in sterilizing cleans 3 times, ovary is shredded with scissorsTo chyle shape; Get being organized in 15ml centrifuge tube of 1ml chyle shape, add 10ml withoutCalcium ions and magnesium ions phosphate buffer; Ultrasonication 3min on ice, power 200W; After endLeave standstill 5min, then 4 DEG C, 8000g, centrifugal 1h; Collect supernatant through 0.45 μ m, 0.22After μ m micro porous filtration degerming, divide and install in 1.5ml centrifuge tube, every pipe 1ml, sealer, frozenFor subsequent use.
The described phosphate buffer without calcium ions and magnesium ions configures by the following method: 0.2g/lKCl、0.2g/lKH2PO4、30g/l、2.16g/lNa2HPO4·7H2The aqueous solution of O, usesUltra-pure water is as solvent, and autoclaving after adjusting pH to 7.2-7.4, is placed in 4 DEG C of preservations.
Another object of the present invention is to provide a kind of Chlamys farreri trochophore clone of cultivatingMethod, utilize described culture medium to carry out the unicellular former culture of trochophore, realize early stageThe cultivation of going down to posterity and go down to posterity, specifically comprises the following steps:
1) single celled obtaining
Get the Chlamys farreri of gonad maturity, the discharge of induction gamete, artificial insemination are also cultivated load wheelLarva, adds the phosphate buffer without calcium ions and magnesium ions after centrifugal concentrating, obtain load wheel to dissociateLarva unicellular;
2) former culture
By unicellular being inoculated in culture dish obtaining, add the above-mentioned culture medium of 1ml, startFormer culture;
3) cultivation of going down to posterity
After the cell of former culture grows clone, picking clone, realizes and going down to posterity in early days, waits to go down to posterityWhen the cell paving bottle rate of cultivating reaches 80%, use the method for machinery piping and druming with the ratio of 1:2Go down to posterity, complete the cultivation of going down to posterity of Chlamys farreri trochophore cultured cell in vitro, successfully buildVertical Chlamys farreri trochophore clone.
Beneficial effect of the present invention is: the Chlamys farreri load wheel that 1) adopts the inventive method to buildLarva clone can be carried out continuous passage, has gone down to posterity at present more than 60 generations. Cellular morphology is nearCircle, nucleocytoplasmic ratio is large, and growth curve is normal, can steady and continuous go down to posterity, freezing preservation. CanTo be applied to pathogenesis, cellular elements breeding, the function of seashells cultivation pathogenic microorganismThe research field such as gene, transgenosis. 2) the present invention has developed one and has been applicable to seashells loadThe culture medium that wheel larva cell is cultivated, determine the hyclone that adds low concentration and scallop serum withYolk extraet, the survival that can effectively improve cell, can make cell continue propagation, and shouldMethod has directive significance to the foundation of other seashells clone, and using value is very big.
Brief description of the drawings
Fig. 1 is the former culture of the Chlamys farreri trochophore cell of the present invention microscope of the 2nd dayPhoto;
Fig. 2 is the microphotograph of Chlamys farreri trochophore cell clone of the present invention;
Fig. 3 is the microphotograph of Chlamys farreri of the present invention the 10th generation trochophore cell;
Fig. 4 is the microphotograph of Chlamys farreri of the present invention the 50th generation trochophore cell;
Fig. 5 is the aobvious of Chlamys farreri of the present invention the 10th generation trochophore cell Giemsa dyeingMicro mirror photo;
Fig. 6 is the growth curve of Chlamys farreri load the 45th generation wheel larva cell of the present invention;
Fig. 7 is the ITS of Chlamys farreri of the present invention the 26th generation trochophore cell and gill tissue(18S and 28SrRNA the Internal Transcribed Spacer) pcr amplified fragment.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is described further, and the following stated is only to thisThe preferred embodiment of invention, not does other forms of restriction to the present invention, any familiarProfessional and technical personnel may utilize the technology contents of above-mentioned announcement to be changed to equal changeThe equivalent embodiment changing. Every the present invention program's content that do not depart from, real according to technology of the present inventionAny simple modification or equivalent variations that confrontation following examples are done, all drop on guarantor of the present inventionProtect in scope.
The structure of embodiment 1 Chlamys farreri trochophore clone
1) configuration culture medium
The preparation of Chlamys farreri serum: extract haemolymph of Chlamys farreri in closed shell flesh, be placed in nothing4 DEG C of hold over night in bacterium container, next day by supernatant with the centrifugal 10min of 2000g, in collectionMillipore filter filtration sterilization through 0.45 μ m and 0.22 μ m aperture respectively after clear. Packing postpositionIn-20 DEG C of preservations.
The preparation of Chlamys farreri Yolk extraet: dissect the Chlamys farreri ovary in maturity period, be placed inIn the seawater of sterilizing, clean 3 times, ovary is shredded to chyle shape with scissors. Get 1ml chyleBeing organized in 15ml centrifuge tube of shape, adds 10ml without calcium ions and magnesium ions phosphate buffer;Ultrasonication 3min (super 15s stops 10s) on ice, power 200W. After finishing, leave standstill 5min,4 DEG C again, 8000g, centrifugal 1h; Collect supernatant through 0.45 μ m, 0.22 μ m micro porous filtrationAfter degerming, divide and install in 1.5ml centrifuge tube, every pipe 1ml, sealer, frozen for subsequent use.
Configuration without the phosphate buffer of calcium ions and magnesium ions: 0.2g/lKCl, 0.2g/lKH2PO4、 30g/l、2.16g/lNa2HPO4·7H2The aqueous solution of O, uses the conduct of Millipore ultra-pure waterSolvent, autoclaving after adjusting pH to 7.2-7.4, is placed in 4 DEG C of preservations.
The preparation of clone nutrient solution: the L-15 basal medium by 13.7g/l adds respectively 20.2g/lNaCl、0.54g/lKCl、0.60g/lCaCl2、1g/lMgSO4、3.9g/lMgCl2And5% hyclone, the glutamine of 2mmol/L, 1% Chlamys farreri Yolk extraet,The strepto-of 1% Chlamys farreri serum, the penicillin of 100IU/ml and 100 μ g/ml is made,PH is 7.2-7.4.
2) cultivation of Chlamys farreri trochophore clone
A) single celled obtaining
Get the Chlamys farreri of gonad maturity, discharge, manually award according to conventional method induction gameteEssence is also cultivated trochophore. Centrifugal concentrating adds without the phosphate buffer of calcium ions and magnesium ions and dissociatesObtain the unicellular of trochophore.
B) former culture
By above-mentioned unicellular being inoculated in culture dish, add 1ml complete culture solution, start formerCulture, observe every day, changes in time complete culture solution, former culture according to pollution condition etc.Two days time, observe unicellular, its form as shown in Figure 1.
C) cultivation of going down to posterity
As shown in Figure 2, picking clone after the cell of former culture grows clone, realizes early stageGo down to posterity, when the cell of a culture to be passed paving bottle rate reaches 80%, the method that uses machinery piping and druming withThe ratio of 1:2 goes down to posterity, and successfully sets up Chlamys farreri trochophore clone.
The signature analysis of embodiment 2 Chlamys farreri trochophore clones
1) form is described
The 10th generation of Chlamys farreri trochophore cell and 50 generation microphotograph as Fig. 3 and Fig. 4Shown in, cellular morphology is subcircular, simultaneously Chlamys farreri trochophore cell the 10th generation GiemsaDyeing microphotograph as shown in Figure 5, shows that nucleocytoplasmic ratio is large, and cell proliferation is vigorous, and part hasCloning cluster.
2) growth curve
Get propagation stable, about 6d go down to posterity once 45 generation trochophore logarithmic phase cell, adjustWhole cell density is 5 × 104/ ml, is inoculated in 24 orifice plates and cultivates with the amount in 1ml/ hole.Every 24h take out 3 holes count, carry out continuously 10d, taking incubation time (d) asAbscissa, taking cell concentration as ordinate (cell/ml), carries out the drafting of growth curve, rootAccording to formula T=t × lg2/lg (Nt/N0) calculate its population doubling time (T), wherein N0For initialThe cell number of inoculation, Nt is the cell number of cultivating after the t time.
As shown in Figure 6, this cell line growth curve is normal, calculates population doubling time for resultFor T=75.6h.
3) frozen and recovery
Get the 30th generation exponential phase Chlamys farreri trochophore cell, by 106CellThe centrifugal 10min of 1500r, with frozen protection liquid (the 90%L15 complete medium and 10% of 1.5mlDMSO) resuspended, be placed in program freezing storing box (Mr.Cryo1℃FreezingContainers) carry out gradient cooling-80 DEG C of refrigerator overnight, finally at liquid nitrogenMedium-term and long-term preservation.
Take out the cryopreservation tube in liquid nitrogen, be placed in rapidly 37 DEG C of water-baths, make frozen protection liquid moltenSeparate, the centrifugal 5min of 1500r, abandons protection liquid, adds 1.5ml fresh culture re-suspended cell,The cell that takes a morsel carries out Trypan Blue, and all the other cells are trained in 23 DEG C of biochemical cultivation casesSupport, after 12h, change culture medium.
Trypan Blue counting demonstration, approximately more than 90% cell survives. Cell is through 2After d adapts to, recover multiplication capacity, start to have adherent clone to occur, after 6d, normally passGeneration.
4) Molecular Identification of cell kind
Extract Chlamys farreri trochophore the 26th generation cell and Gill of Chlamys farreri genomeDNA is template, according to the conservative 18S of scallop and 28SrRNA primers, justTo primer sequence: 5 ' GTTTCTGTAGGTGAACCTG-3 '; Reverse primer sequence:5 ' CTCGTCTGATCTGAGGTCGGA-3 ', the special ITS (18S of amplification Chlamys farreriWith 28SrRNA the Internal Transcribed Spacer) fragment. Pcr amplification condition: 94 DEG C of 5min,94 DEG C of 30s, 54 DEG C of 30s, 72 DEG C of 1min, 30 circulations, 72 DEG C of 10min. Get 1.5 μ lPCR product separates by 1.5% agarose electrophoresis, EB dyeing, and ultraviolet detects and takes pictures.
As shown in Figure 7, Chlamys farreri trochophore the 26th generation cell and Gill of Chlamys farreriAll amplification obtains object band (741bp) and illustrates that the cell of in vitro culture comes from Chlamys farreriAnd in incubation, this ITS zone length does not change in vitro.
Claims (6)
1. a culture medium of cultivating Chlamys farreri trochophore clone, is characterized in that,Described culture medium comprises L-15 basal medium, the 20.2g/l of following component: 13.7g/lNaCl、0.54g/lKCl、0.60g/lCaCl2、1g/lMgSO4、3.9g/lMgCl2And eventuallyConcentration is that 5% hyclone, the glutamine of 2mmol/L, 1% Chlamys farreri yolk are carriedGet liquid, 1% Chlamys farreri serum, the penicillin of 100IU/ml and the strepto-of 100 μ g/mlElement, pH is 7.2-7.4.
2. a kind of Chlamys farreri trochophore clone of cultivating according to claim 1Culture medium, is characterized in that, described Chlamys farreri serum is prepared by the following method: in closingIn shell flesh, extract haemolymph of Chlamys farreri, be placed in 4 DEG C of hold over night of sterile chamber, next day willSupernatant is with the centrifugal 10min of 2000g, collects after supernatant respectively through 0.45 μ m and 0.22 μ mThe millipore filter filtration sterilization in aperture, is placed in-20 DEG C of preservations after packing.
3. a kind of Chlamys farreri trochophore clone of cultivating according to claim 1Culture medium, is characterized in that, described Chlamys farreri Yolk extraet is prepared by the following method:The Chlamys farreri ovary of dissecting the maturity period, the seawater that is placed in sterilizing cleans 3 times, with scissors generalOvary shreds to chyle shape; Get being organized in 15ml centrifuge tube of 1ml chyle shape, add10ml is without calcium ions and magnesium ions phosphate buffer; Ultrasonication 3min on ice, power 200W;After finishing, leave standstill 5min, then 4 DEG C, 8000g, centrifugal 1h; Collect supernatant through 0.45 μ m,After 0.22 μ m micro porous filtration degerming, divide and install in 1.5ml centrifuge tube, every pipe 1ml, sealer,Frozen for subsequent use.
4. a kind of Chlamys farreri trochophore clone of cultivating according to claim 3Culture medium, is characterized in that, the described phosphate buffer without calcium ions and magnesium ions passes through with belowMethod configuration: 0.2g/lKCl, 0.2g/lKH2PO4、30g/l、2.16g/lNa2HPO4·7H2OThe aqueous solution, use ultra-pure water as solvent, regulate autoclaving after pH to 7.2-7.4, putIn 4 DEG C of preservations.
5. a method of cultivating Chlamys farreri trochophore clone, is characterized in that, profitCarry out the unicellular former culture of trochophore with culture medium described in claim 1, realize early stageThe cultivation of going down to posterity and go down to posterity.
6. a kind of Chlamys farreri trochophore clone of cultivating according to claim 5Method, is characterized in that,
1) single celled obtaining
Get the Chlamys farreri of gonad maturity, the discharge of induction gamete, artificial insemination are also cultivated load wheelLarva, centrifugal concentrating adds without the phosphate buffer of calcium ions and magnesium ions and dissociates and obtain trochophoreUnicellular;
2) former culture
By unicellular being inoculated in culture dish obtaining, add training described in 1ml claim 1Support base, start former culture;
3) cultivation of going down to posterity
After the cell of former culture grows clone, picking clone, realizes and going down to posterity in early days, waits to go down to posterityWhen the cell paving bottle rate of cultivating reaches 80%, use the method for machinery piping and druming with the ratio of 1:2Go down to posterity, complete the cultivation of going down to posterity of Chlamys farreri trochophore cultured cell in vitro, successfully buildVertical Chlamys farreri trochophore clone.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114134099A (en) * | 2021-11-29 | 2022-03-04 | 北部湾大学 | Balanced salt solution of blood cells of marine invertebrates |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH1156345A (en) * | 1997-08-19 | 1999-03-02 | Agency Of Ind Science & Technol | Utilization of marine waste to culture medium |
| CN102965331A (en) * | 2012-11-16 | 2013-03-13 | 中国海洋大学 | Method for culturing chlamys farreri trochophore cell line |
-
2015
- 2015-11-19 CN CN201510801587.0A patent/CN105602887A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH1156345A (en) * | 1997-08-19 | 1999-03-02 | Agency Of Ind Science & Technol | Utilization of marine waste to culture medium |
| CN102965331A (en) * | 2012-11-16 | 2013-03-13 | 中国海洋大学 | Method for culturing chlamys farreri trochophore cell line |
Non-Patent Citations (2)
| Title |
|---|
| YANG HONG-SHENG ET AL.,: ""IMPACT OF STARVATION ON SURVIVAL , MEAT CONDITION"", 《CHINESE JOURNAL OF OCEANOLOGY AND LIMNOLOGY》 * |
| 晏萌: ""栉孔扇贝（Chlamys farreri）细胞的体外培养体系建立和特征分析"", 《中国博士学位论文全文数据库农业科技辑》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114134099A (en) * | 2021-11-29 | 2022-03-04 | 北部湾大学 | Balanced salt solution of blood cells of marine invertebrates |
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