CN111876370B - Abalone cell culture medium and abalone cell culture method - Google Patents

Abstract

The invention relates to an abalone cell culture medium and an abalone cell culture method, wherein the abalone cell culture medium comprises the following components: l-15 basal medium and mixed serum, wherein the mixed serum comprises: fetal calf serum accounting for 15% of the volume of the abalone cell culture medium, and inactivated abalone serum accounting for 5% of the volume of the abalone cell culture medium; the basic culture medium is added with the following components: penicillin streptomycin mixed liquor 100 x, gentamicin, amphotericin B, NaCl, KCl and CaCl₂、MgSO₄、MgCl₂. According to the invention, polylysine coating improvement is carried out on the surface of the 6-hole cell culture plate, a complete culture medium formula for primary culture of the Haliotis discus hannai blood cells is adjusted, and the Haliotis discus hannai blood cells with strong adherence capability, stable state and good activity can be rapidly obtained in a short time. Besides, the application can also provide abalone cells for researches such as nutrition, immunity, environmental toxicity, gene function analysis and the like.

Description

Abalone cell culture medium and abalone cell culture method

technical field

The invention belongs to the technical field of animal cell culture, and in particular relates to an abalone cell culture medium and an abalone cell culture method.

Background technique

Wrinkled disc abalone (Haliotis discus hannai Ino) is one of the important cultured shellfish in my country. In recent years, the abalone breeding industry has developed rapidly, but it is also faced with illness and death caused by high temperature in summer and pathogens. Regarding the research on the nutritional requirements of the wrinkled abalone, the demand for amino acids, fatty acids and other nutrients and the research on physiological metabolism still need to be further studied.

The slow growth rate and long breeding period of abalone limit the exploration efficiency of in vivo experiments, and the in vivo experiments are affected by many environmental factors, which cannot accurately reflect the deep molecular mechanism. Therefore, in addition to the traditional in vivo research methods, it is urgent to establish a suitable model to provide a convenient condition for short-term experiments. Cell-level studies have a short experimental period, strong tissue specificity, and stable conditions. Marine invertebrates lack specific immunity, and blood cells play an important innate immune function. As an efficient and stable in vitro experimental model, blood cell culture is suitable for the study of cell responses to environmental toxicants and nutrient metabolism. Therefore, the cell culture of abalone is helpful to explore the high temperature stress of abalone and the pathogenic mechanism of pathogenic organisms, provide an experimental model for pathogen identification and disease prevention, and also contribute to the research on precise nutrition of abalone and facilitate the perception of nutrients. , metabolism and other mechanisms, as well as gene knockdown, overexpression and other experiments.

The culture of abalone blood cells mainly adopts the method of direct blood drawing and adherence. However, the experiment found that the current culture method of abalone blood cells has poor cell viability. After 2 days of culture, the blood cells gradually fall off, and all fall off and no longer grow on the 3rd day. The cultured blood cells cannot meet the experimental needs.

SUMMARY OF THE INVENTION

In view of this, the purpose of the present invention is to overcome the deficiencies of the prior art, and to provide a culture medium for abalone cells and a method for culturing abalone cells, so as to solve the problem that the cultured abalone blood cells in the prior art are difficult to meet experimental requirements.

In order to achieve the above purpose, the present invention adopts the following technical scheme: an abalone cell culture medium, the abalone cell culture medium components include: L-15 basal medium and mixed serum, and the mixed serum includes: accounting for the abalone cells 15% fetal bovine serum by volume of the medium, and inactivated abalone serum that accounts for 5% of the volume of the abalone cell culture medium;

Each 100ml of the basal medium was added with the following components at final concentrations: 1ml penicillin-streptomycin mixture 100×, 20mg gentamicin, 100μg amphotericin B, 2.02g NaCl, 0.05g KCl, 0.06g CaCl ₂ , 0.1 g MgSO ₄ , 0.18 g MgCl ₂ .

Further, the preparation method of described inactivated abalone serum comprises:

The blood was collected from the blood sinuses of the gastro-foot of the wrinkled abalone;

Centrifuge the removed blood and take the supernatant;

The supernatant was placed in a 56°C water bath for 30min to inactivate;

The inactivated supernatant was filtered and sterilized with a filter with a diameter of 0.22 μm to obtain inactivated abalone serum.

Further, after the preparation of the abalone cell culture medium is completed, a filter with a filtration diameter of 0.22 μm is used for filtration and sterilization for use.

The embodiments of the present application provide a method for culturing abalone cells, which includes: selecting healthy individuals of the wrinkled abalone, using a brush to remove body surface attachments, and using a circulating water system with an ultraviolet germicidal lamp to temporarily cultivate the wrinkled abalone to be tested for 24 hours ;

Take out the wrinkled abalone to be tested, wipe the body surface of the wrinkled abalone with 75% alcohol to remove the mucus on the surface of the gastropod, move it into the ultra-clean workbench, and use a sterile scalpel to cut the wrinkled abalone muscle for blood. The blood taken out is immediately put into the heparin sodium blood collection tube;

The blood was mixed with the abalone cell culture medium and shaken, and then inoculated into a polylysine-coated six-well cell culture plate, sealed with a membrane, and placed in an incubator, and the abalone cell culture medium was replaced every 3 days.

Further, it also includes:

Blood cells were seeded in a 6-well cell culture plate for 24 h, and then the blood cells were modeled by ROS in abalone cell culture medium containing H ₂ O ₂ , and incubated with DCFH-DA working solution.

Further, the ROS modeling method includes:

The blood cells were inoculated into a 6-well cell culture plate at a density of 10 6 per well, placed in an incubator, and after 24 h, the abalone cell culture medium was sucked out, washed with PBS, and then added with a concentration of 50 mmol/L H ₂ O ₂ abalone. cell culture medium;

After 1 h, slowly rinse three times with PBS, add working solution containing 50 μmol/L DCFH-DA to each well, and incubate in an incubator for 20 min;

After the incubation, the working solution was sucked out, rinsed three times slowly with PBS, and 2 ml of abalone cell culture medium was added to each well;

Cell fluorescence was observed using a fluorescence microscope at an excitation wavelength of 490 nm.

Further, the preparation method of the culture plate coating includes:

Choose a six-well cell culture plate;

Pipette 400 μl of 0.01% poly-lysine into the wells, infiltrate the bottom of the plate evenly, suck out the poly-lysine after 30 minutes, rinse the wells three times with ultrapure water, cover the plate and place it in an incubator for later use .

Further, the temperature in the incubator is 17°C-25°C.

Further, the temperature in the incubator is 22°C.

Further, the water in the circulating water system is seawater, and the concentration added to the seawater is 0.5mg/L povidone-iodine;

The blood and abalone cell culture medium were mixed in a volume ratio of 1:3.

The present invention adopts the above technical solutions, and the beneficial effects that can be achieved include:

The invention provides an abalone cell culture medium and an abalone cell culture method. The invention adjusts the complete culture medium formula for the primary culture of abalone blood cells in the wrinkled disc, and further improves the surface of the 6-well cell culture plate by coating with polylysine. The abalone cells can quickly obtain the wrinkled disc abalone blood cells with strong adherence ability, stable state and good activity in a short time. In addition, the technical solution of the present application can be used for studies on nutrition, immunity, environmental toxicity, gene function analysis, and the like.

Description of drawings

In order to illustrate the embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings that need to be used in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only These are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained according to these drawings without creative efforts.

Fig. 1 is the blood cell state diagram of the wrinkled disc abalone that adheres to the wall for 2h of the present invention;

Fig. 2 is the blood cell state diagram of the wrinkled disc abalone attached to the wall for 24h of the present invention;

Fig. 3 is the blood cell state diagram of the wrinkled disc abalone after adhering to the wall for 9d of the present invention;

Fig. 4 is a graph showing the relationship between culture time and relative activity of blood cells of abalone in the present invention;

Fig. 5 is a comparison diagram of the effect of different culture media on the state of blood cells of abalone;

Fig. 6 is a blood cell fluorescence image after ROS modeling of the present invention.

Detailed ways

In order to make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be described in detail below. Obviously, the described embodiments are only some, but not all, embodiments of the present invention. Based on the embodiments of the present invention, all other implementations obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present invention.

A specific abalone cell culture medium and abalone cell culture method provided in the embodiments of the present application are described below with reference to the accompanying drawings.

In the abalone cell culture medium provided in the examples of the present application, the components of the abalone cell culture medium include: L-15 basal medium and mixed serum, and the mixed serum includes: 15% of the volume of the abalone cell culture medium. bovine serum, inactivated abalone serum accounting for 5% of the volume of the abalone cell culture medium;

Each 100ml of the basal medium was added with the following components at final concentrations: 1ml penicillin-streptomycin mixture 100×, 20mg gentamicin, 100μg amphotericin B, 2.02g NaCl, 0.05g KCl, 0.06g CaCl ₂ , 0.1 g MgSO ₄ , 0.18 g MgCl ₂ .

The present application adjusts the complete medium formula for primary culture of abalone hemocytes, and can quickly obtain hemocytes with strong adherence ability, stable state and good activity in a short period of time.

Preferably, the preparation method of the inactivated abalone serum comprises:

The blood was collected from the blood sinuses of the gastro-foot of the wrinkled abalone;

Centrifuge the removed blood and take the supernatant;

The supernatant was placed in a 56°C water bath for 30min to inactivate;

The inactivated supernatant was filtered and sterilized with a filter with a diameter of 0.22 μm to obtain inactivated abalone serum.

Preferably, after the abalone cell culture medium is configured, a filter with a diameter of 0.22 μm is used for filtration and sterilization for later use.

The application provides an abalone cell culture method, comprising:

Select healthy individuals of wrinkled abalone, use a brush to remove body surface attachments, and use a circulating water system with an ultraviolet germicidal lamp to temporarily raise the wrinkled abalone to be tested for 24 hours;

Take out the wrinkled abalone to be tested, wipe the body surface of the wrinkled abalone with 75% alcohol to remove the mucus on the surface of the gastropod, move it into the ultra-clean workbench, and use a sterile scalpel to cut the wrinkled abalone muscle for blood. The blood taken out is immediately put into the heparin sodium blood collection tube;

The blood was mixed with the abalone cell culture medium and shaken, and then inoculated into a polylysine-coated six-well cell culture plate, sealed with a membrane, and placed in an incubator, and the abalone cell culture medium was replaced every 3 days.

Preferably, the temperature in the incubator is 17°C-25°C.

Preferably, the temperature in the incubator is 22°C

In some embodiments, the water in the circulating water system is seawater, and the seawater is added with a concentration of 0.5 mg/L povidone-iodine;

The blood and abalone cell culture medium were mixed in a volume ratio of 1:3.

Specifically, the main reagents used in the experiment in this application include: L15 medium, fetal bovine serum (Biological Industries), penicillin, streptomycin, gentamicin, amphotericin, polylysine solution, 3-( 4,5-Dimethylthiazole-2)-2,5-diphenyltetrazolium bromide (MTT) kit, sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, magnesium chloride, collagen type II Enzyme, H2O2, ₂ ',7' _- dichlorofluorescein diacetate (DCFH-DA), 75% alcohol, heparin sodium vacuum blood collection tube.

Experimental materials include: 0.22μm filter, syringe, T25 petri dish, 6-well cell culture plate, elbow forceps, surgical scissors, scalpel, 100μm cell sieve, Pasteur pipette.

The experimental materials were sterilized before the experiment, and the experimental consumables such as forceps, scissors, scalpels used for dissection were sterilized in an autoclave at 121 °C for 30 min.

The specific steps of primary culture of blood cells are as follows:

Selection of experimental abalone: Selection of healthy wrinkled abalone.

Temporary cultivation of experimental abalone: the surface of the abalone was washed with a brush, and the experimental abalone was temporarily cultivated for 24 hours in a circulating water system with an ultraviolet germicidal lamp, and 0.5 mg/L povidone-iodine was added to the seawater.

Obtainment of blood cells: The experimental abalone was taken out, and the body surface was scrubbed three times with 75% alcohol, and the mucus on the surface of the gastropod was wiped off. The scrubbed abalone was moved to the ultra-clean bench, and the abalone foot muscle was cut with a sterile scalpel to collect blood and immediately add it to the heparin sodium blood collection tube. The blood and complete medium were mixed at a ratio of 1:3, and then inoculated into a polylysine-coated six-well cell culture plate, sealed with a parafilm, and placed in a biochemical incubator at 22 °C. The medium was changed once a day with complete medium.

The experimental results of the abalone cell culture method used in this application: 2 hours after the initiation of primary culture of abalone blood cells in the wrinkled disc, some cells began to adhere to the wall, and the cells in the dense blood cells were radial, as shown in Figure 1; 24 hours after the initiation of primary culture, abalone After the blood cells were attached to the bottom of the plate, the refraction of the cells was good, and the cell confluence rate was about 90%, as shown in Figure 2; 9 days after the primary culture was started, the cells adhered well, the shape stretched, the blood cells converged into a monolayer, and the confluence rate About 100%, as shown in Figure 3; the cell viability was detected by MTT colorimetry, and its viability was not significantly different from that after 24h of primary culture, as shown in Figure 4.

In the present application, a comparative experiment was carried out on the influence of different media on the culture state of the blood cells of abalone, and the results are shown in Table 1.

Table 1

Among them, the L-15 medium on the blood cell culture state of abalone in the wrinkled disc is shown in Figure 5A;

Figure 5B shows the culture state of blood cells of abalone with salinity-adjusted L-15 medium;

Figure 5C shows the culturing state of blood cells of abalone with salinity-adjusted L-15 medium + abalone serum.

In this experiment, the basal medium formula was optimized, and it was found that adding penicillin-streptomycin mixture, gentamicin, amphotericin B, NaCl, KCl, CaCl ₂ , MgSO ₄ and MgCl ₂ to L-15 medium could reduce the Chances of bacterial contamination during blood cell culture while keeping blood cells in good shape.

It can be seen from the above experimental results that by optimizing the formula of the abalone cell culture medium in the present application, it is found that adding abalone serum to the salinity-adjusted L-15 medium can prolong the adherence time of abalone blood cells and maintain the proliferation activity of blood cells.

Preferably, the method for making the culture plate coating includes:

Choose a six-well cell culture plate;

Pipette 400 μl of 0.01% poly-lysine into the wells, infiltrate the bottom of the plate evenly, suck out the poly-lysine after 30 minutes, rinse the wells three times with ultrapure water, cover the plate and place it in an incubator for later use .

Specifically, in this experiment, the surface of the six-well cell culture plate was coated, and it was found that the coated culture plate can shorten the adherence time of abalone blood cells by 3-4 hours, and at the same time accelerate the confluence of adherent blood cells into a monolayer at the bottom of the plate.

Preferably, the abalone cell culture method provided by this application also includes:

Blood cells were seeded in a 6-well cell culture plate for 24 h, and then the blood cells were modeled by ROS in abalone cell culture medium containing H ₂ O ₂ , and incubated with DCFH-DA working solution.

In this application, a fluorescent probe (2', 7'-dichlorofluorescein yellow diacetate, referred to as DCFH-DA) was used to observe the fluorescence emitted by the reactive oxygen species in the blood cells oxidized by the non-fluorescent DCFH to generate the DCF. The constructed blood cell model of wrinkled disc in vitro can be used for oxidative stress modeling of abalone.

Preferably, the ROS modeling method includes:

The blood cells were inoculated into a 6-well cell culture plate at a density of 10 6 per well, placed in an incubator, and after 24 h, the abalone cell culture medium was sucked out, washed with PBS, and then added with a concentration of 50 mmol/L H ₂ O ₂ abalone. cell culture medium;

After 1 h, slowly rinse three times with PBS, add working solution containing 50 μmol/L DCFH-DA to each well, and incubate in an incubator for 20 min;

After the incubation, the working solution was sucked out, rinsed three times slowly with PBS, and 2 ml of abalone cell culture medium was added to each well;

Cell fluorescence was observed using a fluorescence microscope at an excitation wavelength of 490 nm.

The specific modeling and testing steps include:

Abalone blood cells were seeded into a 6-well cell culture plate at a density of 106/well, cultured in a biochemical incubator at 22 °C, and after 24 h, the complete medium was aspirated, rinsed with PBS, and added with 50 mmol/LH ₂ O ₂ . culture medium;

After 1 h, slowly rinse three times with PBS, add a working solution containing 50 μmol/L DCFH-DA to each well, and incubate in a 22 °C incubator for 20 min;

After the incubation, aspirate the working solution, rinse slowly with PBS three times, and add 2 ml of complete medium to each well;

Cell fluorescence was observed using a fluorescence microscope at an excitation wavelength of 490 nm.

Specifically, 24 hours after inoculating blood cells in a 6-well plate, H ₂ O ₂ was used for ROS modeling of blood cells, and DCFH-DA working solution was added to incubate. The cells after ROS modeling were observed using a fluorescence microscope at an excitation wavelength of 490 nm. The green fluorescence emitted proves that the in vitro cultured model of corrugated disc blood cells can be used for the related research on the immunity and anti-oxidation of abalone.

As shown in Figure 6, the experimental results show that after the blood cells are modeled in H ₂ O ₂ , the reactive oxygen species in the blood cells can be observed under the Echo Revolve fluorescence microscope to oxidize the non-fluorescent DCFH to the fluorescent DCF. The constructed in vitro cultured model of blood cells in wrinkled disc can be used for oxidative stress modeling of abalone, and further applied to related researches on immunity and anti-oxidation.

To sum up, the present invention provides an abalone cell culture medium and abalone cell culture method. The components of the abalone cell culture medium include: L-15 basal medium and mixed serum, and the mixed serum includes: accounting for 15% of the volume of the abalone cell culture medium The following components were added to the basal medium: penicillin-streptomycin mixture 100×, gentamicin, amphotericin B, NaCl, KCl , CaCl ₂ , MgSO ₄ , MgCl ₂ . The invention improves the polylysine coating on the surface of the 6-well cell culture plate, adjusts the complete medium formula of the primary culture of the blood cells of abalone, and can quickly obtain strong adherence ability, stable state and activity in a short period of time. Well wrinkled disc abalone blood cells. In addition, this application can also provide abalone cells for research on nutrition, immunity, environmental toxicity, and gene function analysis.

The above are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention. should be included within the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims (5)

1. A culture method of abalone cells is characterized in that,

selecting healthy Haliotis discus hannai individuals, removing body surface attachments by using a brush, and temporarily culturing the Haliotis discus hannai to be tested for 24h by using a circulating water system with an ultraviolet sterilizing lamp;

taking out the Haliotis discus hannai to be tested, wiping the body surface of the Haliotis discus hannai with 75% alcohol to remove mucus on the surface of the gastropoda, transferring into a superclean workbench, cutting open the foot muscle of the Haliotis discus hannai with a sterile scalpel to take blood, and immediately putting the taken blood into a heparin sodium blood collection tube;

mixing the blood and the abalone cell culture medium according to the volume ratio of 1: 3, uniformly mixing the blood and the abalone cell culture medium, then inoculating the mixture into a poly-lysine coated six-hole cell culture plate, sealing the culture plate by adopting a membrane, then placing the culture plate into an incubator, and replacing the abalone cell culture medium every 3 days to obtain blood cells;

further comprising:

after the blood cells were seeded on 6-well cell culture plates for 24 hours, the plates containing H were used₂O₂Performing ROS modeling on blood cells by using the abalone cell culture medium, and adding DCFH-DA working solution for incubation;

the ROS molding method comprises the following steps:

mixing blood cells at 10 deg.C⁶Inoculating the density of each well into a 6-well cell culture plate, placing in an incubator, sucking out the abalone cell culture medium after 24H, washing with PBS, and adding H with the concentration of 50mmol/L₂O₂The culture medium of abalone cells;

slowly washing with PBS for 3 times after 1h, adding working solution containing 50 mu mol/L DCFH-DA into each hole, and incubating for 20min in an incubator;

after the incubation is finished, sucking out the working solution, slowly washing the working solution for 3 times by using PBS, and adding 2ml of abalone cell culture medium into each hole;

observing cell fluorescence at an excitation wavelength of 490nm using a fluorescence microscope;

the manufacturing method of the polylysine coated six-hole cell culture plate comprises the following steps:

selecting a six-hole cell culture plate;

sucking 400 mul of 0.01% polylysine, dripping into the holes, uniformly infiltrating the bottom of the plate, sucking out the polylysine after 30min, flushing the holes for 3 times by adopting ultrapure water, covering the plate, and placing the plate in an incubator for later use;

wherein the abalone cell culture medium comprises the following components: l-15 basal medium and a serum cocktail comprising: fetal calf serum accounting for 15% of the volume of the abalone cell culture medium, and inactivated abalone serum accounting for 5% of the volume of the abalone cell culture medium;

the preparation method of the inactivated abalone serum comprises the following steps:

blood is taken from the blood sinuses of the gastropoda of haliotis discus hannai;

centrifuging the blood and collecting supernatant;

inactivating the supernatant in a 56 ℃ water bath for 30 min;

filtering and sterilizing the inactivated clear liquid by adopting a filter with the filtering diameter of 0.22 mu m to obtain inactivated abalone serum;

the following final concentrations of components were added per 100ml of the basal medium: 1ml penicillin streptomycin mixed liquor 100X, 20mg gentamicin, 100. mu.g amphotericin B, 2.02g NaCl, 0.05g KCl, 0.06g CaCl₂、0.1g MgSO₄、0.18g MgCl₂。

2. The abalone cell culture method according to claim 1, wherein,

and after the abalone cell culture medium is configured, filtering and sterilizing the abalone cell culture medium by using a filter with the filtering diameter of 0.22 mu m for later use.

3. The abalone cell culture method according to claim 1 or 2, wherein the temperature in the incubator is 17 ℃ -25 ℃.

4. The abalone cell culture method according to claim 3, characterized in that,

the temperature in the incubator was 22 ℃.

5. The abalone cell culture method according to claim 1, wherein,

the water in the circulating water system is seawater, and povidone iodine with the concentration of 0.5mg/L is added into the seawater.

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