CN103232969A - Prawn cell culture medium - Google Patents
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Abstract
The invention relates to a novel prawn cell culture medium formula. A culture medium prepared according to the formula can be used for in vitro culture of prawn cell. By utilizing the prawn cell culture medium designed by the invention, in vitro survival and growth of the prawn cell can be well maintained, cell adherence is tight, and the appearance is stretched. When the prawn cell culture medium is used for primary culture of a tissue block of a prawn lymphoid organ, the lymphoid organ can be started to migrate out from the tissue block in 3 hours and forms a continuity cell monolayer in 1-2 days. The virus sensitivity of the cultured prawn cell is not damaged by the culture medium.
Description
Technical field
The invention belongs to zooblast, tissue culture technique field, be specifically related to a kind of novel prawn cell culture medium.
Background technology
The frequent outburst of prawn virus disease is puzzlement shrimp culture industry and a difficult problem needing to be resolved hurrily always, and prawn immortality clone is to separate and pathogenesis, development of effective diagnostic reagent and anti-control techniques that purifying prawn ' s virus, research are viral and favourable instrument and the research means of producing virus vaccines efficiently etc.Although experts and scholars cultivate and build to fasten and carried out a large amount of explorations and unremitting effort in the prawn cells in vitro both at home and abroad, the cell cultures of prawn still is in the former foster level of being commissioned to train at present, and the clone of immortality is not successfully set up always.And find effective prawn cell culture medium, be that realization prawn cell is successfully built the key that is.
At present, the prawn cell cultures does not have special business-like substratum.Most of investigator utilizes the commercialization artificial medium of Mammals and insect to be basic medium, adds serum and other nutritional factor simultaneously and carries out the cell cultures work of prawn.Leibovitz(L-15 for example), substratum such as M199, MEM, TC199, Grace substratum, TC-100, LHM, CMPLT and RPMI-1640 all once was used to the cell culture studies of prawn.Wherein with the L-15 substratum, especially the culture effect of 2 * L-15 substratum is best, is most widely used.Purushothaman etc. (1998) are commissioned to train brown prawn (Penaeus indicus) and tigar prawn (P. monodon) former and find that under the condition of interpolation 10% calf serum, the culture effect of L-15 substratum (1 *) is better than MEM in supporting.It is basic medium that Goswami etc. (2009) adopt L-15 substratum (1 *), add 20% foetal calf serum (FBS), 1% shrimp serum, 0.1% glucose and 0.5%NaCl simultaneously, carried out the heart tissue piece of Macrobrachium rosenbergii (Macrobrachium rosenbergii) former be commissioned to train foster, observe moving out of myocardial cell after 2 weeks, and finally obtain the primary cultured cell individual layer.It is basic medium that Nadala etc. (1993) adopt 2 * L-15 substratum, add 20%FBS, 8% shrimp muscle extracting solution and 20 ng/mL Urogastrons (EGF) simultaneously, carried out successfully that the Lymphoid tissue piece of the blue prawn (P.stylirostris) in South America and Penaeus vannamei (P.vannamei) is former is commissioned to train fosterly, and after 1 week, obtained the primary cultured cell individual layer.But added other basic medium of foetal calf serum, shrimp muscle extracting solution and EGF somatomedin such as M199, RPMI1640 and Grace insect substratum equally and then do not obtained satisfied culture effect.Ellender etc. (1992) adopt 2 * L-15 substratum (adding 20%FBS) successfully to cultivate the hemocyte of Penaeus vannamei.It is basic medium that Hu etc. (2008) adopt 2 * L-15 substratum, adds 20%FBS, 5 g/L NaCl, 0.75 g/L NaHCO simultaneously
3, 1% glucose and 20 μ g/mL Prostatropins (bFGF) have also successfully been carried out former being commissioned to train of Lymphoid tissue piece of Chinese prawn (P. chinensis) and have been supported and the immortality Study on Transformation.
Trophic component, pH value and the osmotic pressure of investigator by analyzing and understand the prawn hemolymph is also arranged, simulate the shrimp hemolymph then and design the prawn cell culture medium.For example, it is respectively the prawn cell culture medium of basic medium with M199 and 0.2 * L-15 that Shimizu etc. (2001) have designed 2 according to the trophic component of the blue prawn hemolymph in South America, what be used for the prawn ovary tissue formerly is commissioned to train fosterly, and compares with the culture effect of 2 * L-15 substratum (adding 20%FBS).Find that the culture effect of newly-designed 2 substratum is better than 2 * L-15 substratum.Recently, Jayesh etc. (2012) have also designed a new prawn cell culture medium according to the hemolymph trophic component of tigar prawn, and find that its culture effect is better than 2 * L-15 and Grace insect cell substratum.
Generally speaking, the prescription of the prawn cell culture medium that different investigators adopt mostly is different, and its culture effect is not repeated by other investigator basically yet, and the success ratio of acquisition prawn primary cultured cell individual layer is not high.To the greatest extent the possessor reports that resulting prawn cultured cell in vitro can be at external survival some months, but a small amount of prawn cell often of keeping, rather than healthy continuity cell monolayer, thereby it is little to build the help that is for the prawn cell.Therefore, be necessary to provide a kind of more effective prawn cell culture fluid.
Summary of the invention
The purpose of this invention is to provide a kind of novel prawn cell culture media formulations, the substratum of formulated can be used for the cultivation of prawn cells in vitro accordingly, and can keep survival and the growth of vitro culture prawn cell better, thereby remedies the deficiencies in the prior art.
The preservation liquid of prawn cell culture medium of the present invention, be to be basic medium with 1.5 * L-15 substratum, and add the component of following final concentration: volume ratio is 10% prawn muscle extracting solution, 5 g/L NaCl, 2 g/L glucose, 1g/L NaHCO
3, 10 μ g/mL taurines, 10 μ g/mL proline(Pro), 100 IU/mL benzylpenicillin sodiums and 100 μ g/mL Vetstreps; PH is 7.3-7.5.
Wherein the preparation method of prawn muscle extracting solution is as follows: with prawn muscle with 2.4% NaCl solution as damping fluid homogenate after, in 60 ℃ of water-baths 1 hour, transfer to then in the centrifuge tube, low-temperature centrifugation is collected supernatant, preparation is finished in the membrane filtration degerming.
Wherein the quality volume percent of prawn muscle and 2.4% NaCl solution is 1:3;
Described low-temperature centrifugation is 4 ℃, centrifugal 1.5 hours of 9000 rpm;
The working fluid of prawn cell culture medium of the present invention, be also to be added with the following component of final concentration in preserving liquid: volume ratio is 15% foetal calf serum (FBS), 20ng/mL Prostatropin (bFGF) and 20 ng/mL Urogastrons (EGF), substratum osmotic pressure is 621 ± 20 mOsm/kg, and the pH value is 7.3-7.5.
The compound method that novel prawn cell culture medium involved in the present invention is preserved liquid is as follows: dissolving 20.55 g L-15 substratum, 5 g NaCl, 2 g glucose, 1 g NaHCO in 1 liter of ultrapure water
3, 0.01 g taurine, 0.01 g proline(Pro), 100 mL shrimp muscle extracting solutions, 1.0 * 10
5IU benzylpenicillin sodium and 100 mg Vetstreps are regulated the pH value to 7.3-7.5; Under aseptic condition, finish the preparation of preserving liquid after with the substratum filtration sterilization with 0.22 μ m filter membrane.
The designed prawn cell culture medium of application the present invention is carried out base and is enclosed shrimp (Metapenaeus ensis, claim the cutter volume new prawn again) Lymphoid tissue piece primary cell culture, (1) the former cell of supporting in the tissue block of being commissioned to train of prawn is moved out fast, start moving out of cell in tissue block inoculation back in 2-3 hour; (2) obtain prawn primary cultured cell individual layer fast, can form the cell monolayer of 70%-80% degree of converging in tissue block inoculation back in 16-24 hour; (3) can make the former foster tissue block of being commissioned to train be repeated inoculation and utilization; (4) do not destroy the viral susceptibility of the prawn cell of cultivating.
Description of drawings
Fig. 1: the culture effect that the base of the prawn cell culture medium that the present invention is designed and Shimizu prawn cell culture medium and 2 * L-15 substratum encloses shrimp Lymphoid tissue piece primary cell culture compares;
Wherein: 1-1,1-2 and 1-3 represent the growth conditions with Shimizu prawn culture medium culturing prawn Lymphoid tissue primary cell growth 6 hours, 24 hours and 36 hours respectively;
2-1,2-2 and 2-3 represent the growth conditions with 2 * L-15 culture medium culturing prawn Lymphoid tissue piece primary cell growth 6 hours, 24 hours and 36 hours respectively;
3-1,3-2 and 3-3 represent the growth conditions with the designed prawn cell culture medium of the present invention cultivate the growth of prawn Lymphoid tissue piece primary cell 6 hours, 24 hours and 36 hours respectively;
Fig. 2: the Lymphoid tissue primary cell that the designed prawn cell culture medium substratum of the present invention encloses shrimp can be survived and be surpassed 25 days cytological map;
Fig. 3: base encloses the recycling of shrimp Lymphoid tissue piece; Prawn Lymphoid tissue piece is former is commissioned to train to support after 3 days and suspends again, and the cell growth when being inoculated into back adherent growth on the new culture plate 24 hours (A) and 4 days (B) is schemed;
Fig. 4: base encloses shrimp Lymphoid tissue primary cultured cell and inoculates 200 μ L white spot virus (WSSV) (titre is 4 * 10
6/ μ L) the light microscopic photo of back cytopathic effect,
Wherein C0, C1, C2 and C3 are respectively prawn Lymphoid tissue primary cell individual layer inoculation PBS(contrast) cell growth light microscopic photo when back 0 day, 1 day, 2 days and 5 days;
It is 4 * 10 that V0, V1, V2 and V3 are respectively prawn Lymphoid tissue primary cell individual layer virus inoculation titre
6WSSV virus liquid after microgram when 0 day, 1 day, 2 days and 5 days.
Embodiment
Prawn cell culture medium of the present invention has carried out long term studies in the proportioning of components selection, concentration, makes to give full play to the interworking effect between each component, thereby has drawn the present invention.
The present invention has used prawn muscle extracting solution, its concrete preparation method is as follows: get prawn alive, behind the cotton ball soaked in alcohol sterilization body surface, under the aseptic condition, remove head, tail and intestines and crust, get muscle and put into 500 mL volume large beakers, weigh, organize according to 1g then: the ratio of 3 mL damping fluids adds 2.4% NaCl solution, and with electric homogenizer muscle is broken into pulpous state.In 60 ℃ of water-baths 1 hour, during rock the bottle mixing for several times.Transfer to then in the 50 mL centrifuge tubes, 4 ℃, centrifugal 1.5 hours of 9000 rpm.Collect supernatant, successively use 0.45 μ m filter membrane and 0.22 μ m membrane filtration degerming ,-20 ℃ store for future use.
Prepare 20 μ g/mL bFGF mother liquors.Concrete preparation method is as follows: get 1.5 mL sterilization centrifuge tube, take by weighing 20 μ g bFGF powder, add 1 mL PBS damping fluid (prescription: dissolving 3.0 g Na in every liter of ultrapure water then
2HPO
412H
2O, 0.2 g KH
2PO
4, 8.0 g NaCl and 0.2 g KCl, pH 7.2), put upside down the mixing dissolving after, with 0.22 μ m pin type filter filtration sterilization ,-20 ℃ store for future use.During use, per 100 mL prawn substratum are preserved liquid and are added this bFGF mother liquor of 100 μ L (20 μ g/mL).
Prepare 20 μ g/mL EGF mother liquors.Concrete preparation method is as follows: get 1.5 mL sterilization centrifuge tube, take by weighing 20 μ g EGF powder, add 1 mL PBS damping fluid (it is the same fill a prescription) then, after putting upside down mixing and dissolving, with 0.22 μ m pin type filter filtration sterilization ,-20 ℃ store for future use.During use, per 100 mL prawn substratum are preserved liquid and are added this EGF mother liquor of 100 μ L (20 μ g/mL).
Prepare novel prawn cell culture medium involved in the present invention and preserve liquid.Concrete compound method is as follows: dissolving 20.55 g L-15 substratum, 5 g NaCl, 2 g glucose, 1 g NaHCO in 1 liter of ultrapure water
3, 0.01 g taurine, 0.01 g proline(Pro), 100 mL shrimp muscle extracting solutions, 1.0 * 10
5IU benzylpenicillin sodium and 100 mg Vetstreps are regulated the pH value to 7.3-7.5.Under aseptic condition, with 0.22 μ m filter membrane with the substratum filtration sterilization after, be sub-packed in 100 mL serum bottles, 85 mL/ bottles, 4 ℃ preserve or-20 ℃ frozen.
A kind of compound method of prawn cell culture medium working fluid of the present invention is as follows: get 85 mL prawn cell culture mediums and preserve liquid, in Bechtop, add 15 mL FBS, 100 μ L, 20 μ g/mL bFGF mother liquors and 100 μ L, 20 μ g/mL EGF mother liquors, mixing, 4 ℃ of preservations are standby.
Use the designed prawn cell culture medium of the present invention and carried out the Lymphoid tissue piece primary cell culture that base encloses shrimp (Metapenaeus ensis), analyze its culture effect, and compare with the culture effect of Shimizu prawn substratum and 2 * L-15 substratum.
The result shows that the cultivation of prawn cell culture medium of the present invention has good effect (Fig. 1), and the time that cell is moved out from tissue block (can begin to move out) the earliest in tissue block inoculation back in 2-3 hour; The cell growth is the fastest, and cell monolayer reaches 70%-80% degree of converging required time the shortest (being about tissue block inoculation back 16-24 hour); Cell attachment is tight, and state is good, and the growth life-span reaches 28 ± 3 days (Fig. 2).Shimizu prawn substratum and 2 * L-15 substratum then needed respectively 72-96 hour and 48-60 hour ability forms the cell monolayer of 70%-80% degree of converging, and cell survival is much shorter also, is respectively 10 ± 4 days and 15 ± 3 days.
Source and the prescription of Shimizu prawn substratum: Shimizu etc. (2001) are according to the designed prawn cell culture medium of trophic component of the blue prawn hemolymph in South America, and compound method is as follows: dissolving 2.74 g L-15 substratum, 16 g NaCl, 1 g glucose, 0.45 g KCl, 0.014 g ZnCl in 1 liter of ultrapure water
2, 0.85 g MgCl
2, 1.22 g CaCl
2, 30 mL PBS, 0.04 g Methionin, 0.1 g glutamine, 0.08 g taurine, 0.08 g proline(Pro), 150 mL FBS, 1.0 * 10
5IU benzylpenicillin sodium and 100 mg Vetstreps, 20 μ g bFGF and 20 μ g EGF, pH value 7.3-7.5, osmotic pressure are 697 ± 20 mOsm/kg.
The prescription of 2 * L-15 substratum: dissolving 27.4 g L-15 substratum, 5 g NaCl, 1 g glucose, 1 g NaHCO in 1 liter of ultrapure water
3, 100 mL shrimp muscle extracting solutions, 150 mL FBS, 20 μ g bFGF, 20 μ g EGF, 1.0 * 10
5IU benzylpenicillin sodium and 100 mg Vetstreps, pH value 7.3-7.5, osmotic pressure are 747 ± 20 mOsm/kg.
Use the result also to show, prawn substratum of the present invention can support prawn Lymphoid tissue piece to repeat growth and the survival of adherent back cell, has improved the utilization ratio of prawn Lymphoid tissue piece, thereby can obtain more prawn primary cultured cell (Fig. 3) quickly.
In addition, the former foster lymphocyte of being commissioned to train of the prawn that grows in the prawn substratum of the present invention still keeps the susceptibility to baculovirus of prawn (WSSV).Found that, the former foster lymphocyte of being commissioned to train of the prawn of the prawn culture medium culturing that the present invention is designed has susceptibility to WSSV, can see tangible cytopathic effect on the 4th day behind the virus inoculation, thereby the prawn cell of cultivating can be used for this viral correlative study (Fig. 4).
Claims (6)
1. the preservation liquid of a prawn cell culture medium, described preservation liquid is to be basic medium with 1.5 * L-15 substratum, and adds the component of following final concentration: volume ratio is 10% prawn muscle extracting solution, 5 g/L NaCl, 2 g/L glucose, 1g/L NaHCO
3, 10 μ g/mL taurines, 10 μ g/mL proline(Pro), 100 IU/mL benzylpenicillin sodiums and 100 μ g/mL Vetstreps; PH is 7.3-7.5.
2. preservation liquid as claimed in claim 1 is characterized in that described prawn muscle extracting solution, be with prawn muscle with 2.4% NaCl solution as damping fluid homogenate after, in 60 ℃ of water-baths 1 hour, transfer to then in the centrifuge tube, low-temperature centrifugation is collected supernatant, and preparation is finished in the membrane filtration degerming.
3. preservation liquid as claimed in claim 2 is characterized in that the quality volume percent of described prawn muscle and 2.4% NaCl solution is 1:3.
4. preservation liquid as claimed in claim 2 is characterized in that described low-temperature centrifugation is 4 ℃, centrifugal 1.5 hours of 9000 rpm.
5. the working fluid of a prawn cell culture medium, it is characterized in that described working fluid is that to be added with final concentration in the described preservation liquid of claim 1 be 15% foetal calf serum, 20 ng/mL Prostatropins and 20 ng/mL Urogastrons.
6. the compound method of the described preservation liquid of claim 1 is as follows: dissolving 20.55 g L-15 substratum, 5 g NaCl, 2 g glucose, 1 g NaHCO in 1 liter of ultrapure water
3, 0.01 g taurine, 0.01 g proline(Pro), 100 mL shrimp muscle extracting solutions, 1.0 * 10
5IU benzylpenicillin sodium and 100 mg Vetstreps are regulated the pH value to 7.3-7.5; Under aseptic condition, finish the preparation of preserving liquid after with the substratum filtration sterilization with 0.22 μ m filter membrane.
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Cited By (6)
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| CN105648024A (en) * | 2016-01-29 | 2016-06-08 | 中国海洋大学 | Rapid screening method of compound Chinese-herbal-medicine immunopotentiator for prawns |
| CN105754928A (en) * | 2016-05-16 | 2016-07-13 | 中国海洋大学 | Technology for dissociating and culturing prawn embryonic cells |
| CN109609435A (en) * | 2018-12-19 | 2019-04-12 | 上海海洋大学 | A kind of culture medium and its cultural method of Eriocheir sinensis haemocyte |
| US20210009943A1 (en) * | 2019-07-10 | 2021-01-14 | Ocean University Of China | Method for continuous culture of shrimp cells |
| CN113025557A (en) * | 2021-04-07 | 2021-06-25 | 华南师范大学 | Improved L-15 culture medium and application thereof in culture of prawn muscle cells |
| CN115354014A (en) * | 2022-09-16 | 2022-11-18 | 中国海洋大学 | Culture medium for long-term culture and continuous passage of prawn cells |
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| CN107858322B (en) * | 2017-10-31 | 2020-10-09 | 中国科学院南海海洋研究所 | Method for establishing primary hippocampal cell culture system |
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| CN105648024A (en) * | 2016-01-29 | 2016-06-08 | 中国海洋大学 | Rapid screening method of compound Chinese-herbal-medicine immunopotentiator for prawns |
| CN105754928A (en) * | 2016-05-16 | 2016-07-13 | 中国海洋大学 | Technology for dissociating and culturing prawn embryonic cells |
| CN109609435A (en) * | 2018-12-19 | 2019-04-12 | 上海海洋大学 | A kind of culture medium and its cultural method of Eriocheir sinensis haemocyte |
| CN109609435B (en) * | 2018-12-19 | 2022-09-02 | 上海海洋大学 | Culture medium and culture method for eriocheir sinensis blood cells |
| US20210009943A1 (en) * | 2019-07-10 | 2021-01-14 | Ocean University Of China | Method for continuous culture of shrimp cells |
| US11873509B2 (en) * | 2019-07-10 | 2024-01-16 | Ocean University Of China | Method for continuous culture of shrimp cells |
| CN113025557A (en) * | 2021-04-07 | 2021-06-25 | 华南师范大学 | Improved L-15 culture medium and application thereof in culture of prawn muscle cells |
| CN113025557B (en) * | 2021-04-07 | 2021-12-14 | 华南师范大学 | Improved L-15 culture medium and application thereof in culture of prawn muscle cells |
| CN115354014A (en) * | 2022-09-16 | 2022-11-18 | 中国海洋大学 | Culture medium for long-term culture and continuous passage of prawn cells |
| CN115354014B (en) * | 2022-09-16 | 2023-09-26 | 中国海洋大学 | Culture medium for long-term culture and continuous passage of prawn cells |
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