CN1766092A - Fish embryo cell separates and cultural method - Google Patents
Fish embryo cell separates and cultural method Download PDFInfo
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Abstract
A kind of fish embryo cell separates and cultural method, and its schedule of operation is: the collection of fish embryo; Embryo's (blastaea-primitive gut later stage) cellular segregation; The former generation of embryonic cell and the cultivation of going down to posterity; The evaluation of embryonic cell feature comprises the evaluation of morphology, multiplication characteristic, karyotype.70% alcohol-pickled sterilization is adopted in embryo's sterilization, method of Tearing is adopted in the separation of cell from the embryo, and the separation of embryonic cell is all carried out in perfect medium with cultivation, and cell cultures is carried out in common incubator, culture temperature is 24 ℃, goes down to posterity 2-3 time weekly.The seawater fish embryonic cell of cultivating with this method has that little, the rounded or polygon of cell, growth are stable, propagation fast, features such as diploid cell ratio height.The technology of the present invention is simple to operate, and is easy to use, can be applied to the separation and the cultivation of nearly all fish embryo cell.
Description
Technical field:
The invention belongs to the culture technique of aquatic living things technology Mesichthyes cell, is under external artificial culture condition, is a kind of technological method that the material acquisition has the embryo cell line of unlimited multiplication capacity with the fish embryo cell.
Background technology:
Fish embryo cell is the cell with unlimited multiplication capacity that separation and Culture goes out from fish blastula stage to primitive gut later stage embryo.Before the present invention makes, yet there are no relevant marine fish embryonic cell both at home and abroad and separate and the special report of cultivating.Aspect the cell cultures of embryonic origin, mainly concentrate in the world in the research of small-sized pattern fish-zebra fish and blue or green Medaka embryonic cell cultivation: Collodi etc. (2004) have carried out separating and culture studies of zebrafish embryo cell, what they adopted is trophocyte's culture method, promptly cultivated the rainbow trout splenocyte (RTS34st) of individual layer earlier, to from the embryo, place rainbow trout individual layer splenocyte to cultivate by isolated cell then, after treating that the zebrafish embryo cell colony grows up to, with the pasteur glass pipette colony is transferred in the test tube, be digested to individual cells, form suspension, cell suspension places on the individual layer rainbow trout splenocyte again and carries out succeeding transfer culture; (1984) such as Lannan CN have set up the Oncorhynchi embryo cell line, and the embryonic cell separation method complicated operation that they adopt is not easy successfully; Cell cultivation process is more loaded down with trivial details, and the cell speed of growth is slower; A large amount of culturing cell difficulty are big, are unfavorable for doing a large amount of experiments with cell.
Summary of the invention:
The objective of the invention is to set up fish embryo cell and separate and the simple and easy method of cultivating, make cell keep good growth conditions and typical embryonic cell feature thereof, for seawater fish cell germplasm storehouse and functional gene research provide cell base.
The present invention realizes by following technology contents: comprising: the separation of embryo's collection, embryonic cell (blastula stage-primitive gut later stage), the cultivation of embryonic cell and go down to posterity, the embryonic cell characterized---morphology evaluation, growth and multiplication characteristic are identified, ploidy detects.
Embryo's collection: the insemination ovum 50ml of seawater fish (lefteye flounder, turbot etc.) is placed in the 500ml graduated cylinder of filling seawater, and after 10 minutes, the ovum of normal fertilization floats over the upper strata of water, and dead ovum sinks to the bottom.Collect natant embryo, stand-by.
The separation of embryonic cell (blastula stage-primitive gut later stage): gather fish embryo, be cultured to the blastaea later stage at normal temperatures after microscopy is qualified or primitive gut used during the later stage.With embryo's grouping, every group of 50-80 embryo embathes in 1 * PBS solution repeatedly, moves on to then on the Bechtop, soaks several seconds in 70% alcohol, sterilizes fast, uses 1 * PBS (the pH value is 7.4) solution to clean then three times.The embryo is drawn in the aseptic culture dish, blots PBS, add an amount of perfect medium, divest egg membrane with the tip tweezers, isolate cell mass, therefrom isolate embryonic cell at microscopically, be used for former be commissioned to train foster.
The cultivation of embryonic cell and going down to posterity:
With cell inoculation embryonic stage monolayer culture in the 24 hole tissue culturing plates of collecting, add perfect medium 1.0ml, place 24 ℃ of incubators to cultivate. cultivate after 2-3 days, change substratum, cover with culture hole in cell, ratio in 1: 2 goes down to posterity, and changes a subculture in every 2-3 days, and 1 week went down to posterity 2-3 time.The perfect medium prescription is the DMEM basic medium, add 4.5g/ml glucose, 20m mol/L Hepes (pH7.4), 100U/ml penicillin, 100U/ml Streptomycin sulphate, 2m mol/L L-glutamic acid, 1m mol/L Sodium.alpha.-ketopropionate, 1mmol/L non-essential amino acid, 15% foetal calf serum, the 5ng/ml fibroblast growth factor, 27.5 μ M mercaptoethanols.After forming stable embryo cell line, cell keeps growth and rate of propagation faster, keeps the form of embryonic cell.
The embryonic cell characterized:
Morphology is identified: 100 * and 400 * phase microscope under the profile and the cell size of observation of cell, photomicrography record cellular form, it is circular that embryonic cell mostly is, polygon and Polygons, have big nucleus, one or two kernel, the diameter of cell are about 15-30 μ m.
Growth and multiplication characteristic are identified: after embryonic cell is cultivated through repeatedly going down to posterity and set up clone, still keep growing fast and multiplication characteristic, cell doubling time is 24-30 hour.
Ploidy detects: the embryonic cell that is passaged to 15-20 generation is carried out chromosome analysis, and microscopically is observed chromosome morphology, the ratio of statistics diploid cell, and the normal diploid cell proportion is generally more than 60%.
The present invention and prior art contrast are characterized in:
The present invention has set up the seawater fish embryonic cell and has separated and cultural method, be characterized in that the embryonic cell lock out operation is simple, it is convenient to cultivate, it is wide to need not specific cellular segregation liquid, culture medium prescription and cultural method practicality, can be applied to nearly all fish.
Description of drawings:
Fig. 1: morphology qualification result figure, A: turbot 16 generation cell B: turbot 35 generation cell.
Fig. 2: differing temps is to the figure that influences of turbot embryonic cell growth,
Fig. 3: turbot embryonic cell chromosome morphology figure,
Fig. 4: turbot embryonic cell chromosome number distribution plan,
Fig. 5: turbot diploid embryos fetus cells karyogram.
Embodiment:
The present invention is in the research of carrying out fishes in bothid and true plaice embryonic cells cultivations such as lefteye flounder and turbot, the separation of foundation, the method for cultivating fish embryo cell.
Cultivation with the turbot embryonic cell is that example is described in detail method content of the present invention below:
Its method content comprises: the separation of embryo's collection, embryonic cell (blastula stage-primitive gut later stage), the cultivation of embryonic cell and go down to posterity, the embryonic cell characterized---morphology evaluation, growth and multiplication characteristic are identified, ploidy detects.
Embryo's collection: collect turbot insemination ovum 50ml, be placed in the 500ml graduated cylinder of filling seawater, leave standstill 10 minutes after, the ovum of normal fertilization floats over the upper strata of water, dead ovum sinks to the bottom, the ovum that guarantees normal fertilization has hundreds of piece, uses in order to experiment.Collect natant fetal tissues, transfer in other incubation vessels and cultivate, stand-by.
The separation of embryonic cell: gather turbot embryo soon after the insemination, microscopically select the spilting of an egg normal, grow neat zygote, be cultured to gastrul stage at normal temperatures, each former be commissioned to train to support need embryo 50-80 piece.At first the embryo is placed in 1 * PBS solution and embathes repeatedly, move on to then on the Bechtop, in 70% alcohol, soak several seconds, sterilize fast, use 1 * PBS (the pH value is 7.4) solution to clean then three times.The embryo is drawn in the aseptic culture dish, blots PBS, add an amount of perfect medium, divest egg membrane with tip tweezers (through high-temperature sterilization), isolate cell mass, the egg membrane of not wanting is transferred in the other container at microscopically.Blow and beat cell mass gently with the rifle head, form individual cells or less agglomerate, the sucking-off substratum, the centrifuge tube of packing into is centrifugal 5 minutes with 800 rev/mins, remove supernatant (containing lecithality and oily ball), add perfect medium the cell in the centrifuge tube is blown and beaten, make single cell suspension.
The former generation of turbot embryonic cell and the cultivation of going down to posterity:
In 24 orifice plates, every hole adds the 1ml perfect medium with the above embryonic cell suspension inoculation that obtains, and the incubator temperature is made as 24 ℃.The perfect medium prescription is: DMEM is a basic medium, add 4.5g/ml glucose, 20m mol/L Hepes (pH7.4), 100U/ml penicillin, the 100U/ml Streptomycin sulphate, 2m mol/L L-glutamic acid, 1m mol/L Sodium.alpha.-ketopropionate, 1m mol/L non-essential amino acid, 15% foetal calf serum, the 5ng/ml fibroblast growth factor, 27.5 μ M mercaptoethanols, 0.1% embryo's extract.Often examine under a microscope the cell attachment situation, after 2-3 days, when treating that cell covers with, go down to posterity with 1: 2 ratio, changed a subculture in every 2-3 days, 1 week went down to posterity 2-3 time.At present, the turbot embryonic cell was cultivated more than 300 day, went down to posterity generation more than 100.
The evaluation of turbot embryonic cell:
Morphology is identified: 100 * or 400 * phase microscope under can be observed the form of turbot embryonic cell, embryonic cell is generally less, and rounded, Polygons or polygon have big nucleus, one or two kernel, the diameter of cell is about 15-25um.This also is the important morphological feature of embryonic cell.In the culturing process in embryonic cell early stage, the inoblast proportion is more, and in the culturing process that continues to go down to posterity, inoblast reduces gradually, and circular, Polygons or polygon occupy main ratio, as shown in Figure 1.A is turbot 16 a generation cell, and the inoblast proportion is more; B is turbot 35 a generation cell, and circle, Polygons or polygon occupy main ratio.
Growth and multiplication characteristic: turbot embryonic cell (TEC) growth is rapid, and rate of propagation is fast, and is general, can go down to posterity once in 2-3 days.Under extra-low density (10
3Cells/ml) be inoculated in the culturing bottle, can form the mono-clonal colony by a cell in 10-15 days.Cell doubling time is 24-30 hour.Fast growth, propagation also are one of embryonic cell and other somatic important difference fast.Experiment shows that turbot embryonic cell growth temperature range is wider, and the speed of growth is the fastest in the time of 24 ℃, as shown in Figure 2.
Ploidy detects: the turbot embryonic cell grows to logarithmic phase, adding colchicine in substratum, to make final concentration be 0.5 μ g/ml, harvested cell after 4 hours, the hypotonic processing of 75mmol/L KCl solution 25 minutes, 3:1 methyl alcohol, Glacial acetic acid fixed cell 3 times, each 15 minutes, cold sheet, dry air, 5%Giemsa dyeing 20 minutes.Microscopically is observed chromosome morphology (referring to Fig. 3), and counting chromosome number (referring to Fig. 4) is made caryogram (referring to Fig. 5).Choose 100 metacinesis phases, the ratio of counting diploid cell, statistics shows that the ratio with 44 chromosomal diploid cells is more than 70%, karyotype formulas is 2n=4m+12st+28t, and is identical with turbot somatic chromosome number and caryogram.In general, the diploid cell ratio (more than 65%) in the embryonic cell of vitro culture is than diploid cell ratio (less than 50%) height in the common aspect cell of cultivating.
Claims (1)
1. a fish embryo cell separates and cultural method, it is characterized in that its schedule of operation is: the separation of fish embryo cell; The former generation of embryonic cell and the cultivation of going down to posterity; The evaluation of embryonic cell: comprise that morphology evaluation, growth and multiplication characteristic are identified and ploidy detects;
Embryonic cell the blastula stage-separation in primitive gut later stage: gather fish embryo, be cultured to the blastaea later stage at normal temperatures after microscopy is qualified or primitive gut used during the later stage; Earlier the embryo is placed in 1 * PBS solution and embathes repeatedly, soaking disinfection in 70% alcohol uses 1 * PBS (the pH value is 7.4) solution to clean at last three times then; The embryo is put in a small amount of perfect medium, divests egg membrane with the tip tweezers under anatomical lens, isolate cell mass, therefrom isolate embryonic cell, centrifugal back is resuspended with perfect medium, makes cell suspension;
The former generation of embryonic cell and the cultivation of going down to posterity:
The embryonic cell for preparing is inoculated in the 24 hole tissue culturing plates cultivates, add perfect medium 1.0ml, place 24 ℃ of incubators to cultivate. cultivate after 2-3 days, change substratum, cover with culture hole in cell, go down to posterity, changed a subculture in every 2-3 days in 1: 2 ratio; The perfect medium prescription is the DMEM basic medium, add 4.5g/ml glucose, 20m mol/L HepespH 7.4,100U/ml penicillin, 100U/ml Streptomycin sulphate, 2mmol/L L-glutamic acid, the 1mmol/L Sodium.alpha.-ketopropionate, 1mmol/L non-essential amino acid, 15% foetal calf serum, the 5ng/ml fibroblast growth factor, 27.5 μ M mercaptoethanols.After forming stable embryo cell line, cell keeps growth and rate of propagation faster, keeps the form of embryonic cell;
The evaluation of embryonic cell:
Morphology is identified: 100 * and 400 * phase microscope under the profile and the cell size of observation of cell, it is circular that embryonic cell mostly is, polygon and Polygons have big nucleus, one or two kernel, the diameter of cell are about 15-30 μ m;
Growth and multiplication characteristic are identified: after embryonic cell is cultivated through repeatedly going down to posterity and set up clone, still keep growth and multiplication characteristic fast, generally went down to posterity once in 2-3 days, cell doubling time is 24-30 hour;
Ploidy detects: the turbot embryonic cell grows to logarithmic phase, adding colchicine in substratum, to make final concentration be 0.5 μ g/ml, harvested cell after 4 hours, the hypotonic processing of 75mmol/L KCl solution 25 minutes, 3: 1 methyl alcohol, Glacial acetic acid fixed cell 3 times, each 15 minutes, cold sheet, dry air, 5%Giemsa dyeing 20 minutes.Microscopically is observed chromosome morphology, the ratio of statistics diploid cell, and the normal diploid cell proportion is generally more than 60%.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101126077B (en) * | 2007-08-31 | 2010-07-14 | 中国水产科学研究院黑龙江水产研究所 | Blood serum substituting agent and blood serum free rainbow trout embryo cell line culture medium |
| CN101825533A (en) * | 2010-06-01 | 2010-09-08 | 苏州大学 | Giemsa staining method of mouse macrophage cells |
| CN103060259A (en) * | 2012-12-06 | 2013-04-24 | 沙珍霞 | Building method of fish head and kidney tissue derived cell lines |
| CN109652365A (en) * | 2019-02-01 | 2019-04-19 | 南京大学 | A kind of preparation method of zebrafish embryo single cell suspension |
| CN110055213A (en) * | 2019-04-23 | 2019-07-26 | 中国海洋大学 | A kind of separation method of dwarf clam egg membrane |
| CN110551682A (en) * | 2018-06-01 | 2019-12-10 | 深圳华大生命科学研究院 | Kits and methods for dissociation of animal embryos |
| CN114107181A (en) * | 2021-11-19 | 2022-03-01 | 中国水产科学研究院黄海水产研究所 | Sturgeon embryonic cell line, culture medium and preparation method thereof |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101720697B (en) * | 2009-12-09 | 2011-07-27 | 湖南师范大学 | Cultivation method of gynogenesis megalobrama amblycephala |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN85102491A (en) * | 1985-04-01 | 1986-09-24 | 中国科学院水生生物研究所 | Technique of fish cell clone culture |
| WO2003051109A1 (en) * | 2001-12-13 | 2003-06-26 | Purdue Research Foundation | Cell culture system and methods of use |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101126077B (en) * | 2007-08-31 | 2010-07-14 | 中国水产科学研究院黑龙江水产研究所 | Blood serum substituting agent and blood serum free rainbow trout embryo cell line culture medium |
| CN101825533A (en) * | 2010-06-01 | 2010-09-08 | 苏州大学 | Giemsa staining method of mouse macrophage cells |
| CN103060259A (en) * | 2012-12-06 | 2013-04-24 | 沙珍霞 | Building method of fish head and kidney tissue derived cell lines |
| CN110551682A (en) * | 2018-06-01 | 2019-12-10 | 深圳华大生命科学研究院 | Kits and methods for dissociation of animal embryos |
| CN109652365A (en) * | 2019-02-01 | 2019-04-19 | 南京大学 | A kind of preparation method of zebrafish embryo single cell suspension |
| CN110055213A (en) * | 2019-04-23 | 2019-07-26 | 中国海洋大学 | A kind of separation method of dwarf clam egg membrane |
| CN114107181A (en) * | 2021-11-19 | 2022-03-01 | 中国水产科学研究院黄海水产研究所 | Sturgeon embryonic cell line, culture medium and preparation method thereof |
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