CN114317419A - Method for constructing muscle cell line of Gymnocypris przewalskii - Google Patents

Abstract

The invention discloses a construction method of a muscle cell line of Gymnocypris przewalskii, which comprises primary culture, subculture, cell cryopreservation and recovery, wherein the cell culture solution of the primary culture comprises the following components: a basic culture medium, 10-30% of bovine serum, 200-400 IU/mL of penicillin, 200-400 mug/mL of streptomycin, 1-5 mmol/L of glutamine and 1-5 mmol/L of sodium pyruvate; the components of the subcultured cell culture solution comprise: a basic culture medium, 10-30% bovine serum, 50-250 IU/mL penicillin, 50-250 mug/mL streptomycin, 1-5 mmol/L glutamine and 1-5 mmol/L sodium pyruvate; the frozen stock solution for freezing the cells comprises the following components: a basic culture medium, 10-30% of bovine serum, 50-250 IU/mL of penicillin, 50-250 mug/mL of streptomycin, 5-30% of DMSO, 1-5 mmol/L of glutamine and 1-5 mmol/L of sodium pyruvate. The preparation method is simple, does not need special equipment, can be operated in a common sterile culture room, and the prepared cells have good and stable growth and can be used for researches on nutrition, immunity, environmental toxicity, gene function analysis and the like.

Description

A method for constructing Qinghai Lake naked carp muscle cell line

technical field

The invention belongs to the technical field of cell culture, in particular to a method for constructing a Qinghai Lake naked carp muscle cell line.

Background technique

Qinghai Lake naked carp (Gymnocyprisprzewalskii), commonly known as Huang fish, belongs to Cypriniformes (CyPriniformes), Cyprinidae (Cyprinidae), Schizothorax subfamily, Gymnocyprisprzewalskii, mainly distributed in Qinghai Lake and its tributaries around the lake in China, which is a plateau The economic fish in low-temperature saline-alkaline waters is the only economic fish in Qinghai Lake and one of the important economic fish in my country. It likes to inhabit beachside, slow-flowing places between large rock piles, deep pools or rock crevices. It has strong adaptability and can live in brackish water (the salt content of Qinghai Lake is 12-13‰) or fresh water. Under natural conditions, the growth of naked carp in Qinghai Lake is extremely slow, and it takes an average of 7 to 10 years to grow to 300-500 g. Since the 1960s, hundreds of thousands of acres of grasslands around Qinghai Lake have been reclaimed as farmland, and 108 rivers that flow into Qinghai Lake have been dammed and dammed, blocking breeding channels. spawning in the middle of the estuary, resulting in the death of a large number of naked carp in the estuary. Relevant information shows that the existing naked carp resources in Qinghai Lake are about 7,500 tons, which is less than 1/10 of the initial stage of development. At present, birds inhabiting Bird Island swallow nearly 1,000 tons of naked carp every year. The living environment of carp has been polluted, destroyed and overfished, resulting in a serious decline in the resources of naked carp in Qinghai Lake and serious damage to fishery resources. important challenge.

In order to protect the Qinghai Lake naked carp resources, the Qinghai Lake naked carp artificial proliferation and release technology has gradually matured in the past two decades. This technology requires the Qinghai Lake naked carp mature parents under natural conditions as the source of sex cells for artificial insemination. And larvae hatching culture, the technology needs to be carried out in a specific period of time. The artificial breeding technology for rearing naked carp in Qinghai Lake has not yet been established, and there is no relevant report on artificial breeding of naked carp in Qinghai Lake. A large number of Qinghai Lake naked carp individuals are needed, and the source of Qinghai Lake naked carp individuals can only be obtained by capturing wild individuals, which forms an urgent contradiction with the protection of the rare fish germplasm resources.

The establishment of Qinghai Lake naked carp cell line as a cell model is an important method to solve the above problems. Fish cell culture started in the 1960s, and more than 280 cell lines have been established so far. After more than 30 years of fish cell culture in China, only more than 50 cell lines have been established, and the species established for cell lines are not enough for Chinese fish. 1.5% of the total (more than 3,500 species of fish in China), it can be seen that the construction speed of cell lines is very slow, and there are certain difficulties in itself. And compared with other fish cell cultures, because the naked carp individuals in Qinghai Lake under natural conditions are usually diseased individuals, carrying a variety of parasites, they are more prone to contamination in cell culture, and the cell lethality rate is higher, resulting in an increase in the failure rate. . Therefore, there is an urgent need to establish a cell line construction method with low cell lethality and the ability to obtain a large number of muscle cells for basic research on the growth of naked carp in Qinghai Lake.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a method for constructing a Qinghai Lake naked carp muscle cell line, which can obtain a large number of muscle cells for basic research on fish growth.

In order to achieve the above object, the present invention adopts the following technical solutions:

The invention provides a method for constructing a Qinghai Lake naked carp muscle cell line, including primary culture, subculture, cell cryopreservation and recovery. 30% bovine serum, 200～400IU/mL penicillin, 200～400μg/mL streptomycin;

The components of the subcultured cell culture fluid include: basal medium, 10-30% bovine serum, 50-250 IU/mL penicillin, and 50-250 μg/mL streptomycin;

The components of the cryopreservation liquid for freezing the cells include: basal medium, 10-30% bovine serum, 50-250 IU/mL penicillin, 50-250 μg/mL streptomycin, and 5-30% DMSO.

Further, the components of the primary cultured cell culture fluid include: basal medium, 10-20% bovine serum, 200-300 IU/mL penicillin, 200-300 μg/mL streptomycin;

The components of the subcultured cell culture fluid include: basal medium, 10-20% bovine serum, 50-100 IU/mL penicillin, and 50-100 μg/mL streptomycin;

The components of the cryopreservation liquid for freezing the cells include: basal medium, 10-20% bovine serum, 50-100 IU/mL penicillin, 50-100 μg/mL streptomycin, and 10-20% DMSO;

Further, the components of the primary cultured cell culture fluid include: basal medium, 15% bovine serum, 300 IU/mL penicillin, and 300 μg/mL streptomycin;

The components of the subcultured cell culture fluid include: basal medium, 15% bovine serum, 100 IU/mL penicillin, and 100 μg/mL streptomycin;

The components of the cryopreservation solution for cryopreservation of the cells include: basal medium, 15% bovine serum, 100 IU/mL penicillin, 100 μg/mL streptomycin, and 10% DMSO.

The "%" in the present invention is the volume percentage concentration; each component in the medium is added according to the concentration, and each component is followed by a concentration unit.

Further, the basal medium in described primary culture, subculture is selected from the one in DMEM (high glucose), DMEM (low glucose), L-15, RPMI 1640 medium, preferably DMEM (high glucose) culture medium;

The basal medium in the cryopreservation solution is DMEM (high glucose) medium;

The bovine serum is selected from one of fetal bovine serum and calf serum, preferably fetal bovine serum.

The basic medium was purchased from Gibco company DMEM high glucose medium, the product number is 11960044, DMEM high glucose medium contains 4500mg/L D-glucose, does not contain L-glutamine, does not contain sodium pyruvate.

Further, the culture medium components of the primary culture, subculture, and cell cryopreservation all further include 1-5 mmol/L glutamine and 1-5 mmol/L sodium pyruvate.

Further, the components of the primary cultured cell culture fluid include: basal medium, 10-20% bovine serum, 200-300 IU/mL penicillin, 200-300 μg/mL streptomycin, 1-5 mmol/L glutathione Amino amide, 1～5mmol/L sodium pyruvate;

The components of the subcultured cell culture fluid include: basal medium, 10-20% bovine serum, 50-100 IU/mL penicillin, 50-100 μg/mL streptomycin, 1-5 mmol/L glutamine, 1-1 ~5mmol/L sodium pyruvate;

The cryopreservation liquid components of the cell cryopreservation include: basal medium, 10-20% bovine serum, 50-100 IU/mL penicillin, 50-100 μg/mL streptomycin, 10-20% DMSO, 1-5 mmol/mL L glutamine, 1～5mmol/L sodium pyruvate;

In a specific embodiment of the present invention, the components of the primary cultured cell culture fluid include: basal medium, 15% bovine serum, 300 IU/mL penicillin, 300 μg/mL streptomycin, 2.5 mmol/L glutamine Amide, 2.5mmol/L sodium pyruvate;

The components of the subcultured cell culture fluid include: basal medium, 15% bovine serum, 100 IU/mL penicillin, 100 μg/mL streptomycin, 2.5 mmol/L glutamine, 2.5 mmol/L sodium pyruvate;

The components of the cryopreserved cell cryopreservation include: basal medium, 15% bovine serum, 100IU/mL penicillin, 100μg/mL streptomycin, 10% DMSO, 2.5mmol/L glutamine, 2.5mmol/L Sodium pyruvate.

Further, the step of the primary culture includes: after washing the muscle tissue block with sterile PBS, inoculate it in a culture flask and inoculate it upside down, and add 1 ml of primary culture medium to the culture flask to keep the humidity in the culture flask and prevent the muscle tissue from drying. When the cells died, 3 ml of cell culture medium was added the next day, and the cells were cultured to grow into a cell monolayer;

The conditions for the primary culture are: inversion in a 22-26°C incubator for 24 hours, preferably overnight inversion in a 25°C incubator.

The steps of obtaining the muscle cells include: rinsing the 3-month-old larvae with sterile water, then soaking in 75% alcohol for 40-100 s, and drying the subcutaneous muscle tissue.

Further, the step of subculturing includes: the primary muscle cells cover 60% of the bottom of the bottle, suck out the primary culture medium, suspend the cells, add the cell culture medium to carry out subculture.

Further, the method for suspending cells is trypsin digestion; further, the reagents used in the trypsin digestion include 0.2-0.4% trypsin-EDTA, preferably 0.25% trypsin-EDTA;

The method of the subculture is as follows: the first passage is 1:1, and then the passage is 1:2, and the passage is once every 8 to 12 days, preferably once every 10 days;

The temperature of the subculture is 22-26°C, preferably 25°C.

Further, the step of cryopreserving the cells includes: taking the cells in the logarithmic growth phase and digesting them with trypsin, centrifuging, mixing the obtained precipitate with the cryopreservation solution, resuspending, and storing in liquid nitrogen;

The resuspended cell concentration is 1×10 ⁵ to 1×10 ⁷ cells/mL, preferably 1×10 ⁶ cells/mL.

Further, the resuscitation step includes: rapidly thawing the cryopreserved solution in a 37°C water bath, centrifuging, resuspending with a subculture medium after obtaining the precipitate, and culturing it at 25°C until it grows into a cell monolayer.

The beneficial effects of the present invention are:

(1) The muscle cells obtained by the method of the present invention have a low lethality rate, and a large number of muscle cells can be obtained, which are convenient for research on nutrition, immunity, environmental toxicity and gene function analysis of naked carp.

(2) The method of the present invention is simple and easy to implement, does not require special equipment, and can be operated in a general sterile culture room.

Description of drawings

Figure 1 is a primary culture diagram;

Figure 2 is a graph of the 20th passage subculture.

Detailed ways

The technical solutions of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, but not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present invention.

The DMEM high-glucose medium of the present invention was purchased from Gibco Company, the product number is 11960044, and the component is 4500 mg/L D-glucose; the fetal bovine serum was purchased from Shanghai Sangon Bioengineering Co., Ltd., the product number was E510008. Unless otherwise specified, the reagents used in the present invention are all commercially available.

Example 1

(1) Muscle acquisition of naked carp in Qinghai Lake

Select the naked carp larvae from Qinghai Lake that have been cultured in the pond for 3 months, soak and rinse in sterile water for 3 times for 10 to 20 minutes each time, and then soak the fish body with 75% alcohol for 1 minute and then dry it. Tissue, placed in sterile PBS, for use.

(2) Primary culture

First, 12 explants were tested with three basal media, DMEM (high glucose), Leibovitz'sL-15 and RPMI 1640, and the best basal medium was selected.

The results show that in DMEM (high glucose) and Leibovitz's L-15 medium, cells can migrate from the gill tissue and assume a fibroblast or epithelioid-like morphology. After culturing in DMEM (high glucose) supplemented with fetal bovine serum and penicillin-streptomycin solution for 25 days, the monolayer cells explanted from the explants can meet the requirements of passage for subculture. In contrast, in L-15 medium, the migration of monolayer cells around the tissue block was too slow. Therefore, during subsequent passages, DMEM (high glucose) was used as the basal medium.

In order to obtain a large number of cells after subculture for 20 generations, the present invention firstly prepares the following primary cell culture medium: DMEM high glucose medium (Gibco), 15% fetal bovine serum, 300IU/mL penicillin, 300μg/mL streptavidin element, 2.5mmol/L glutamine, 1.5mmol/L sodium pyruvate.

Cut 1 g of the obtained muscle tissue into small pieces, each small piece is 1 mm ² , rinse with sterile PBS for 3 times, 5 minutes each time, inoculate it in a 25 cm ² cell culture flask, add 1 ml of primary medium at 23 Incubate at ℃ overnight, add 3 ml of primary culture medium the next day, and replace this medium every three days. After 10 days, cells begin to migrate from the tissue block, and they can grow into a cell monolayer in 30 to 40 days. Cover the bottom of the flask by 40-60%. This treatment method can control pollution to less than 20%.

(3) Subculture

The passaged cell culture medium was prepared according to the following formula: DMEM high glucose medium (Gibco), 15% fetal bovine serum, 100 IU/mL penicillin, 100 μg/mL streptomycin, 2.5 mmol/L glutamine and 1.5 mmol/L pyruvate sodium.

After the muscle cells in the primary culture are covered with 60% of the bottom of the culture flask, the subculture is started. The steps are as follows: firstly, aspirate the primary culture medium in the culture flask, and use 0.25% trypsin-EDTA for trypsinization. The cells were generated, and 3 mL of subculture medium was added to neutralize the trypsin reaction. The first passage was 1:1, then 1:2, and the culture was continued in a 25°C incubator. The average passage was once every 10 days. The cells were in good and stable condition.

(4) Cell cryopreservation

Cell cryopreservation was prepared as follows: DMEM high glucose medium (Gibco), 15% fetal bovine serum, 100 IU/mL penicillin, 100 μg/mL streptomycin, 10% DMSO, 2.5 mmol/L glutamine, and 1.5 mmol /L sodium pyruvate.

After subculture for 20 generations, after the muscle cell line was successfully established (Figure 2), the cells in the logarithmic growth phase were taken, and after trypsinization, the cell suspension was centrifuged at 1200 rpm for 5 minutes, the supernatant was discarded, and the prepared cell pellet was added to the cell pellet. Cell cryopreservation solution, resuspend, control the cell concentration to 1×10 ⁶ ~ 1×10 ⁷ cells/mL, transfer 1 mL of cell suspension to a 1.8 mL cryopreservation tube, and place the cryogenic tube in a programmed cooling box After being placed at -80°C for 24 hours, it was placed in liquid nitrogen for long-term storage.

(5) Cell recovery

Take out the frozen cells from the liquid nitrogen, put them into a 37°C water bath and quickly shake until thawed. Centrifuge the cell suspension at 1200 rpm for 5 minutes to remove the supernatant; resuspend the cells with 1 ml of subculture medium and transfer them to a cell culture flask , 25 ℃ incubator, 8 to 10 days, the cells grow into a monolayer.

Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, and substitutions can be made in these embodiments without departing from the principle and spirit of the invention and modifications, the scope of the present invention is defined by the appended claims and their equivalents.

Claims (10)

1. A construction method of a muscle cell line of Gymnocypris przewalskii comprises primary culture, subculture, cell cryopreservation and recovery, and is characterized in that the components of a cell culture solution of the primary culture comprise: a basic culture medium, 10-30% of bovine serum, 200-400 IU/mL of penicillin and 200-400 mu g/mL of streptomycin;

the components of the subcultured cell culture solution comprise: a basic culture medium, 10-30% bovine serum, 50-250 IU/mL penicillin and 50-250 mug/mL streptomycin;

the frozen stock solution for freezing the cells comprises the following components: a basic culture medium, 10-30% bovine serum, 50-250 IU/mL penicillin, 50-250 mug/mL streptomycin and 5-30% DMSO.

2. The method of claim 1, wherein the components of the cell culture fluid of the primary culture comprise: a basic culture medium, 10-20% bovine serum, 200-300 IU/mL penicillin and 200-300 mug/mL streptomycin;

the components of the subcultured cell culture solution comprise: a basic culture medium, 10-20% bovine serum, 50-100 IU/mL penicillin and 50-100 mug/mL streptomycin;

the frozen stock solution for freezing the cells comprises the following components: a basic culture medium, 10-20% of bovine serum, 50-100 IU/mL of penicillin, 50-100 mu g/mL of streptomycin and 10-20% of DMSO;

further, the components of the cell culture solution of the primary culture include: basic culture medium, 15% bovine serum, 300IU/mL penicillin and 300 mug/mL streptomycin;

the components of the subcultured cell culture solution comprise: basic culture medium, 15% bovine serum, 100IU/mL penicillin and 100 mug/mL streptomycin;

the frozen stock solution for freezing the cells comprises the following components: basal medium, 15% bovine serum, 100IU/mL penicillin, 100. mu.g/mL streptomycin, 10% DMSO.

3. The method according to claim 1 or 2, wherein the basic medium in the primary culture and subculture is selected from one of dmem (high glucose), dmem (low glucose), L-15, RPMI 1640 medium, preferably dmem (high glucose) medium;

the basic culture medium in the frozen stock solution is DMEM (high glucose) culture medium;

the bovine serum is selected from one of fetal bovine serum and calf serum, and is preferably fetal bovine serum;

further, the DMEM (high glucose) medium was 4500mg/L D-glucose.

4. The construction method according to any one of claims 1 to 3, wherein the culture medium components of the primary culture, the subculture and the cell cryopreservation further comprise 1 to 5mmol/L glutamine and 1 to 5mmol/L sodium pyruvate.

5. The method of claim 4, wherein the components of the cell culture fluid of the primary culture comprise: a basic culture medium, 10-20% of bovine serum, 200-300 IU/mL of penicillin, 200-300 mug/mL of streptomycin, 1-5 mmol/L of glutamine and 1-5 mmol/L of sodium pyruvate;

the components of the subcultured cell culture solution comprise: a basic culture medium, 10-20% of bovine serum, 50-100 IU/mL of penicillin, 50-100 mu g/mL of streptomycin, 1-5 mmol/L of glutamine and 1-5 mmol/L of sodium pyruvate;

the frozen stock solution for freezing the cells comprises the following components: a basic culture medium, 10-20% of bovine serum, 50-100 IU/mL of penicillin, 50-100 mug/mL of streptomycin, 10-20% of DMSO, 1-5 mmol/L of glutamine and 1-5 mmol/L of sodium pyruvate;

further, the components of the cell culture solution of the primary culture include: basic culture medium, 15% bovine serum, 300IU/mL penicillin, 300 mu g/mL streptomycin, 2.5mmol/L glutamine, 2.5mmol/L sodium pyruvate;

the components of the subcultured cell culture solution comprise: basic culture medium, 15% bovine serum, 100IU/mL penicillin, 100 mug/mL streptomycin, 2.5mmol/L glutamine, 2.5mmol/L sodium pyruvate;

the frozen stock solution for freezing the cells comprises the following components: basic culture medium, 15% bovine serum, 100IU/mL penicillin, 100 mug/mL streptomycin, 10% DMSO, 2.5mmol/L glutamine, 2.5mmol/L sodium pyruvate.

6. The method of constructing according to claim 1, wherein the step of primary culture comprises: washing muscle tissue blocks with sterile PBS, inoculating to a culture bottle, performing inverted culture, adding 1ml of primary culture medium into the culture bottle to maintain the humidity in the culture bottle and prevent dry cells of the muscle tissue from dying, adding 3ml of cell culture solution the next day, and culturing to grow into a cell monolayer;

the primary culture conditions are as follows: and (3) inverting the mixture in an incubator at 22-26 ℃ for 24 hours, preferably inverting the mixture in an incubator at 25 ℃ overnight.

7. The method of construction according to claim 1, wherein the step of subculturing comprises: and (3) spreading the primary muscle cells at the bottom of the bottle by 60%, sucking out primary culture solution, suspending the cells, adding cell culture solution, and performing subculture.

8. The method of constructing according to claim 7, wherein the method of suspending cells is trypsinization; further, the reagent used in the trypsinization method comprises 0.2-0.4% of trypsin-EDTA, preferably 0.25% of trypsin-EDTA;

the subculture mode is as follows: the first passage is carried out according to the ratio of 1:1, then the passage is carried out according to the ratio of 1:2, the passage is carried out once every 8-12 days, and the passage is preferably carried out once every 10 days;

the temperature of the subculture is 22-26 ℃, and preferably 25 ℃.

9. The method of constructing according to claim 1, wherein the step of cryopreserving the cells comprises: taking cells in logarithmic phase of growth, digesting with pancreatin, centrifuging, mixing the obtained precipitate with a frozen stock solution, resuspending, and preserving with liquid nitrogen;

the cell concentration after resuspension is 1X 10⁵～1×10⁷one/mL, preferably 1X 10⁶one/mL.

10. The construction method according to claim 1, wherein the resuscitation step comprises: and (3) rapidly melting and centrifuging the frozen stock solution in water bath at 37 ℃, re-suspending the obtained precipitate by using a subculture cell culture solution, and culturing the precipitate at 25 ℃ until a cell monolayer grows.

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