CN112920987A - Separation and primary culture method of vascular sac cells of lateolabrax japonicus - Google Patents
Separation and primary culture method of vascular sac cells of lateolabrax japonicus Download PDFInfo
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- CN112920987A CN112920987A CN202110158231.5A CN202110158231A CN112920987A CN 112920987 A CN112920987 A CN 112920987A CN 202110158231 A CN202110158231 A CN 202110158231A CN 112920987 A CN112920987 A CN 112920987A
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Abstract
The invention discloses a method for separating vascular sac cells of Perch and performing primary culture, which comprises the steps of taking complete vascular sac tissues, rinsing with a buffer solution and a culture medium, cutting the vascular sac tissues into not less than 3 blocks, and placing the vascular sac tissue blocks in a pore plate for static culture under optimized culture conditions. The vascular sac separation method of the lateolabrax japonicus adopted by the invention can reduce cell loss and cell morphological damage in the operation and rinsing processes; the primary culture method adopted by the invention can allow a large amount of hemangiocyst cells to adhere to the wall, and the passage effect is good.
Description
Technical Field
The invention relates to a fish cell culture method, in particular to a separation and primary culture method of vascular sac cells of Perch.
Technical Field
Lateolabrax major (Lateolabrax), Osteichthyes, Actinopterygii, Perciformes, Percoidei, Serratidae (Serranidae), Lateolabrax (Lateolabrax). The lateolabrax japonicus is widely distributed in China, Korea and Japan coastal areas, has the characteristics of wide temperature adaptation range, strong salt adaptability, delicious meat quality, high economic benefit and the like, and is an important aquaculture variety in China.
Perch belongs to short-day seasonal breeding fish, and the egg laying period in the north is 10 months to 12 months, and the south is 11 months to 1 month in the next year. The photoperiod reaction can appear in the seasonally propagated fish and the thyroxine can participate in the regulation and control of the seasonal reproduction, because the lateolabrax japonicus breeds the winter wintery, a series of problems (such as low survival rate of larval fish, higher bait cultivation cost and the like) caused by low temperature become the main restriction factors of the scale production of lateolabrax japonicus offspring seeds, the gonads of the lateolabrax japonicus can be induced to mature in advance or after the growth by regulating the photoperiod length, so the spawning propagation in different seasons can be realized by regulating and controlling the ecological environment conditions, and the method is an important problem to be solved in the artificial propagation of the lateolabrax. Researches show that the vascular sac is an organ for regulating the fish photoperiod, and primary cells have similar characteristics with in vivo cells and can truly reflect the activity and the function of the in vivo cells. The fish cells can provide great convenience for researches on fish virus separation, an antiviral mechanism, vaccine research and development, a transgenic technology, a chimeric tissue engineering and the like, and the requirements on resources such as facility manpower and the like are low compared with those of a living body experiment, the repeatability is good, and the research period is short. At present, no data related to vascular sac separation and primary culture of the lateolabrax japonicus exists at home and abroad, so that a standard and reproducible method for vascular sac cell separation and primary culture of the lateolabrax japonicus is very necessary to be established, and an experimental platform is provided for seasonal propagation research of the lateolabrax japonicus.
Disclosure of Invention
The invention aims to provide a method for separating vascular sac cells of lateolabrax japonicus and performing primary culture.
In order to achieve the purpose, the invention adopts the technical scheme that: a process for separating the vascular sac cells of Japanese sea bass includes such steps as washing the vascular sac tissue with buffer solution and culture medium, cutting into at least 3 blocks, and laying the vascular sac tissue blocks in perforated plate for static culture.
In the invention, the complete vascular sac tissue is cut into 3-4 blocks, if the number of the vascular sac tissue blocks is less than 3, the cell amount climbed out from the section is too small, which directly results in poor cell adherence condition, if the number of the vascular sac tissue blocks is more than 5, the vascular sac is only 3-4 mm, due to excessive cutting, the tissue is dispersed, in the process of dripping the culture medium, a large number of cells are suspended in the culture medium, while the suspension cells of the vascular sac hardly adhere to the wall, and the suspended cells have low activity and can not grow.
In the present invention, the medium used in rinsing and culturing was L-15 complete medium comprising 300-500U/mL penicillin, 300-500U/mL streptomycin and 10-30% FBS. Preferably, the L-15 complete medium comprises 350-450U/mL penicillin, 350-450U/mL streptomycin and 15-25% FBS.
According to the method, the vascular sac tissue block is spread on a pore plate soaked by serum, the adherence of cells can be assisted by the serum soaking, then the vascular sac tissue block is placed in an incubator at 28 ℃ and is inversely cultured for 1.5-2 hours, a small amount of L-15 complete culture medium is dripped right above the tissue block, the method can assist the tissue block in spreading, the vascular sac tissue block is prevented from floating, the adherence of the cells can be separated out, the culture medium is replenished to 500 microliters after the overnight culture is carried out for 12 hours, the culture medium is cultured under normal atmospheric pressure, and the growth spreading condition of the vascular sac tissue block is observed by using a microscope. A large number of adherent cells can be observed after 4 days of culture, and subculture can be carried out after one week.
In the present invention, the buffer used in the rinsing was a double-resistant Duchenne phosphate buffer (double-resistant DPBS) containing 400U/mL of penicillin and 400U/mL of streptomycin. The cultures used in the rinse also included a dual-resistant L-15 medium containing 400U/mL penicillin and 400U/mL streptomycin.
The invention has the advantages that:
1. the vascular sac separation method of the lateolabrax japonicus can reduce cell loss and cell morphological damage in the operation and rinsing processes.
2. The vascular sac culture method of the lateolabrax japonicus adopted by the invention can ensure that a large number of vascular sac cells adhere to the wall, and the passage effect is good.
3. The invention refers to and improves the existing sea water fish cell method, searches for an in vitro culture method suitable for the vascular sac of the lateolabrax japonicus, and provides an experimental platform for the research of functional genes of the lateolabrax japonicus.
4. The method provided by the invention is not limited to lateolabrax japonicus and can be popularized to the separation and culture of the vascular sac cells of other fishes.
Drawings
FIG. 1 shows the complete medium screening results.
FIG. 2 shows the hypothalamus and the vascular sac tissue of a lateolabrax japonicus.
FIG. 3 is a photomicrograph of vascular sacs of lateolabrax japonicus according to the present invention after 24 hours of primary culture.
FIG. 4 is a photomicrograph of the vascular sac primary culture of lateolabrax japonicus of the present invention at day 4.
FIG. 5 is a photomicrograph of the vascular sac primary culture of lateolabrax japonicus of the present invention at day 7.
FIG. 6 is a microphotograph of vascular sacs of lateolabrax japonicus after passage of culture according to the present invention.
FIG. 7 is a comparison of the number of vascular sac sections of weever of the present invention versus the effect of cell migration.
Detailed Description
The technical solution of the present invention will be further illustrated and described below with reference to the accompanying drawings by means of specific embodiments.
DPBS basic (1X) buffer, L-15 basal medium, penicillin streptomycin bis-antibody were purchased from GIBCO, USA, in the following examples; fetal bovine serum FBS was purchased from GIBCO and pancreatic digest from Biosharp.
1. Preparing solution
Double-antibody DPBS: penicillin and streptomycin double antibody were added to the DPBS to 400U/mL.
Double-resistant L-15 medium: penicillin streptomycin double antibody is added into L-15 basic culture medium to 400U/mL.
L-15 complete Medium 1: penicillin streptomycin double antibody is added to 400U/mL in L-15 basic medium, and 20% FBS is added at the same time.
L-15 complete Medium 2: penicillin streptomycin double antibody is added to 400U/mL in L-15 basic medium, and 10% FBS is added at the same time.
L-15 complete Medium 3: penicillin streptomycin double antibody is added to 400U/mL in L-15 basic medium, and 5% FBS is added at the same time.
2. Aseptic blood taking sac
Selecting Lateolabrax japonicus with healthy constitution, no damage on the surface and weight of 0.5kg-1kg, cutting off the top of Lateolabrax japonicus under sterile environment with sterile scissors, and gently inverting the brain tissue with sterile forceps to observe white round pituitary tissue, red vascular sac tissue and a pair of crescent shaped hypothalamus tissues (figure 2). Carefully taking out the complete vascular sac tissue of the lateolabrax japonicus, putting the vascular sac tissue into a culture dish which is prepared in advance and is added with the double-antibody DPBS for rinsing for 2 times, transferring the vascular sac tissue into the culture dish which is added with the double-antibody L-15 culture medium for rinsing for 2 times by using a rubber tube or a forceps after primary cleaning, and putting the tissue into the culture dish which is added with the L-15 complete culture medium containing serum for rinsing twice.
3. Vascular sac tissue mass culture
3.1 cutting the vascular sac tissue into 3-4 pieces in a culture dish by using a sterile blade, uniformly placing the cut vascular sac tissue on a 24-hole plate soaked by serum, placing the cut vascular sac tissue on an incubator at 28 ℃, carrying out inverted culture for 1.5-2 hours, then slightly dropwise adding a small amount of L-15 complete culture medium for overnight culture, avoiding the tissue pieces from floating up to the greatest extent, supplementing the culture medium to 500 microliters after 12 hours, carrying out culture under normal atmospheric pressure, and observing the growth and spreading conditions by using a microscope. After a typical 24-hour incubation, a large number of precipitated cells were observed at the edge of the cell mass (FIG. 2), and after 2-3 days the medium was changed and the tissue mass was gently blown off, cultured and observed. As shown in fig. 1, the cells cultured in the L-15 complete medium 1 were deposited and spread over the culture well on the first day, a large number of adherent cells were observed to spread over the culture well and a small number of suspended cells were observed on the fourth day (fig. 1, fig. 3 to 4), and about 7 days, the cells adhered to the whole 24-well plate by 90% or more (fig. 5), and were subcultured with stable growth and passable once every two days (fig. 1). The cells cultured in the L-15 complete medium 2 were found to have cell-separated cells around the tissue mass but small cell numbers far away on the first day, and a small number of adherent cells were found on the fourth day (FIG. 1). In the cells cultured in the L-15 complete medium 3, the cell analysis around the tissue mass was small on the first day, and almost no adherent cells were observed on the fourth day (FIG. 1), and since the amount of adherent cells was small, the growth was inhibited after the passage, and the subculture was impossible.
3.2 cutting the vascular sac tissue into 2 or 5 pieces in a culture dish by using a sterile blade, uniformly placing the cut vascular sac tissue on a 24-hole plate soaked by serum, placing the cut vascular sac tissue on an incubator at 28 ℃, carrying out inverted culture for 1.5-2 hours, then slightly dropwise adding a small amount of L-15 complete culture medium for 1 overnight culture, avoiding the tissue pieces from floating up to the greatest extent, supplementing the culture medium to 500 microliters after 12 hours, carrying out culture under normal atmospheric pressure, and observing the growth and spreading conditions by using a microscope. As shown in FIG. 7, the vascular sac tissue was cut into 2 pieces, and the amount of cells that climbed out of the cut surface was too small, which directly resulted in poor cell adhesion. And 5 pieces of the hemangiocyst are cut, the hemangiocyst is only 3-4 mm, the cutting is excessive, the tissue is scattered, a large amount of cells are suspended in the culture in the process of dripping the culture medium, the suspension cells of the hemangiocyst are hardly attached to the wall, and the suspended cells have low activity and cannot grow.
4. Passage of vascular sac
The medium in the 24-well plate was aspirated, 300. mu.l of double-resistant Duchenne phosphate buffer (double-resistant DPBS) was added, the plate was gently shaken, the cells were rinsed, the double-resistant DPBS was aspirated, 300. mu.l of pancreatin digestive enzyme solution was added, digestion was carried out for 3-5min, and 1 ml of complete medium was added to stop digestion. The liquid was centrifuged at 1000rpm for 10 minutes, the supernatant was discarded, the white cell pellet was resuspended by adding complete medium, and the pellet was dispensed to a new plate for culture. After 2 days the planking reached 80% (fig. 6). Subsequent passages can be performed as appropriate.
Finally, it should also be noted that the above example is only one of several embodiments of the present invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
Claims (9)
1. A method for separating vascular sac cells of lateolabrax japonicus and primary culture is characterized in that complete vascular sac tissues are taken, rinsed by buffer solution and culture medium, cut into not less than 3 pieces, and then placed in a pore plate for standing culture.
2. The method for isolation and primary culture of vascular sac cells of lateolabrax japonicus as claimed in claim 1, wherein the whole vascular sac tissue is cut into 3-4 pieces.
3. The method for isolating and primary culturing vascular sac cells of lateolabrax japonicus as claimed in claim 1 or 2, wherein the culture medium used in the rinsing and culturing is L-15 complete culture medium comprising 300-500U/mL penicillin, 300-500U/mL streptomycin and 10-30% FBS.
4. The method for isolating and primary culturing vascular sac cells of lateolabrax japonicus as claimed in claim 3, wherein the L-15 complete medium comprises 350-450U/mL penicillin, 350-450U/mL streptomycin and 15-25% FBS.
5. The method for separating and primary culturing vascular sac cells of lateolabrax japonicus according to claim 3 or 4, wherein the culturing is carried out by spreading the vascular sac tissue blocks on a serum-infiltrated pore plate, then placing the vascular sac tissue blocks in an incubator at 28 ℃ for inverted culturing for 1.5-2 hours, then dripping a small amount of L-15 complete culture medium on the vascular sac tissue blocks, culturing for 12 hours overnight, then supplementing the culture medium to 500 microliters, and culturing under normal atmospheric pressure.
6. The method for isolating and primary culturing vascular sac cells of lateolabrax japonicus as claimed in claim 5, wherein the buffer used in the rinsing is a double-resistant DPBS comprising 400U/mL penicillin and 400U/mL streptomycin.
7. The method for isolating and primary culturing vascular sac cells of lateolabrax japonicus as claimed in claim 5, wherein the culture used in said rinsing further comprises a double-resistant L-15 medium containing 400U/mL penicillin and 400U/mL streptomycin.
8. The method for isolating and primary culturing vascular sac cells of lateolabrax japonicus as claimed in claim 1, 2, 3 or 4, wherein the buffer used in rinsing is a bis-Duchenne phosphate buffer containing 400U/mL penicillin and 400U/mL streptomycin.
9. The method for isolating and primary culturing vascular sac cells of lateolabrax japonicus as claimed in claim 1, 2, 3 or 4, wherein the culture medium used in rinsing further comprises a double-resistant L-15 medium containing 400U/mL penicillin and 400U/mL streptomycin.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1737130A (en) * | 2005-07-12 | 2006-02-22 | 中国水产科学研究院黄海水产研究所 | Lateolabrax japonicus embryo stem cell and cultural method |
| CN102634481A (en) * | 2012-04-23 | 2012-08-15 | 上海海洋大学 | In vitro culture method for scatophagus argus kidney cells |
| CN111411073A (en) * | 2020-03-19 | 2020-07-14 | 华南农业大学 | Construction method of sea bass fry cell line |
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2021
- 2021-02-04 CN CN202110158231.5A patent/CN112920987A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1737130A (en) * | 2005-07-12 | 2006-02-22 | 中国水产科学研究院黄海水产研究所 | Lateolabrax japonicus embryo stem cell and cultural method |
| CN102634481A (en) * | 2012-04-23 | 2012-08-15 | 上海海洋大学 | In vitro culture method for scatophagus argus kidney cells |
| CN111411073A (en) * | 2020-03-19 | 2020-07-14 | 华南农业大学 | Construction method of sea bass fry cell line |
Non-Patent Citations (3)
| Title |
|---|
| YUSUKE NAKANE, ET AL.: ""The saccus vasculosus of fish is a sensor of seasonal changes in day length"", 《NAT COMMUN》 * |
| 周怡 等: ""草鱼脑细胞原代培养"", 《渔业研究》 * |
| 袁满: ""Dynorphin 在光周期诱导花鲈生殖发育中的分子机制"", 《上海海洋大学 硕士学位论文》 * |
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