CN111876370B - Abalone cell culture medium and abalone cell culture method - Google Patents
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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
Technical Field
The invention belongs to the technical field of animal cell culture, and particularly relates to an abalone cell culture medium and an abalone cell culture method.
Background
Haliotis discus hannai Ino is one of the important cultured shellfish in China, and in recent years, the abalone culture industry develops rapidly, but is also faced with the disease and death caused by high temperature and pathogeny in summer. Aiming at the research aspect of the nutrient requirement of the haliotis discus hannai, the research on the required amount of nutrient substances such as amino acid, fatty acid and the like and the physiological metabolism is needed to be deep.
The slow growth speed and the long culture period of the abalones limit the exploration efficiency of in-vivo experiments, and the in-vivo experiments are influenced by a plurality of environmental factors and cannot accurately reflect deep molecular mechanisms. Therefore, in addition to the traditional in vivo research means, it is urgently needed to establish a suitable model to provide a convenient condition for short-term experiments. The experimental period of the cell level research is short, the tissue specificity is strong, and the conditions are stable. The specific immunity of the marine invertebrate is lacked, and blood cells play an important innate immune function. The blood cell culture is used as an efficient and stable in-vitro experimental model, and is suitable for researches on the reaction of cells to environmental toxicants, nutrient metabolism and the like. Therefore, the cell culture of the haliotis discus hannai is beneficial to researching the high-temperature stress of the haliotis discus hannai and the pathogenic mechanism of pathogenic organisms, provides an experimental model for pathogen identification and disease prevention, is also beneficial to researching the accurate nutrition of the haliotis discus hannai, and is convenient for researching the mechanisms of sensing, metabolism and the like of nutrients and carrying out experiments such as gene knockdown, over-expression and the like.
The culture of the abalone blood cells mainly adopts a direct blood drawing and wall pasting method. However, experiments show that the cell viability of the conventional culture mode of abalone blood cells is poor, the blood cells gradually drop after 2d culture and do not grow until 3d culture, and the cultured blood cells are difficult to meet the experimental requirements.
Disclosure of Invention
In view of the above, the present invention provides an abalone cell culture medium and an abalone cell culture method to overcome the deficiencies of the prior art, so as to solve the problem that the cultured abalone blood cells in the prior art are difficult to meet the experimental requirements.
In order to achieve the purpose, the invention adopts the following technical scheme: an abalone cell culture medium, the abalone cell culture medium components comprising: 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 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。
Further, 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 the supernatant;
inactivating the supernatant in a 56 ℃ water bath for 30 min;
and filtering and sterilizing the inactivated clear liquid by adopting a filter with the filtering diameter of 0.22 mu m to obtain the inactivated abalone serum.
Further, after the abalone cell culture medium is configured, a filter with the filtering diameter of 0.22 μm is adopted for filtering and sterilization for standby.
The embodiment of the application provides an abalone cell culture method, which comprises the following steps: 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;
and mixing the blood with the abalone cell culture medium, shaking up, inoculating the mixture into a polylysine coated six-hole cell culture plate, sealing the culture plate by adopting a membrane, placing the sealed culture plate into an incubator, and replacing the abalone cell culture medium every 3 days.
Further, the method also comprises the following steps:
after the blood cells were seeded on 6-well cell culture plates for 24 hours, H-containing cells were used2O2Performing ROS modeling on blood cells by using the abalone cell culture medium, and adding DCFH-DA working solution for incubation.
Further, the ROS molding method comprises the following steps:
inoculating blood cells into 6-well cell culture plate at a density of 106 per well, placing in incubator, sucking out abalone cell culture medium after 24H, washing with PBS, and adding H with 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;
sucking out the working solution after incubation is finished, slowly washing the working solution for 3 times by using PBS, and adding 2ml of abalone cell culture medium into each hole;
cell fluorescence was observed using a fluorescence microscope at an excitation wavelength of 490 nm.
Further, the method for manufacturing the culture plate coating comprises the following steps:
selecting a six-hole cell culture plate;
sucking 400 μ l of 0.01% polylysine, dripping into the holes, soaking the plate bottom uniformly, sucking out polylysine after 30min, washing the holes for 3 times with ultrapure water, covering the plate cover, and placing in an incubator for later use.
Further, the temperature in the incubator is 17 ℃ to 25 ℃.
Further, the temperature in the incubator was 22 ℃.
Further, the water in the circulating water system is seawater, and povidone iodine with the concentration of 0.5mg/L is added into the seawater;
the blood and the abalone cell culture medium are mixed according to the volume ratio of 1: 3.
By adopting the technical scheme, the invention can achieve the following beneficial effects:
the invention provides an abalone cell culture medium and an abalone cell culture method, the complete culture medium formula of the primary culture of abalone blood cells in the wrinkled giant disc is adjusted, and polylysine coating improvement is also carried out on the surface of a 6-hole cell culture plate, so that abalone cells can rapidly obtain the abalone blood cells in the wrinkled giant disc with strong adherence capability, stable state and good activity in a short time. In addition, the technical scheme of the application can be used for researches on nutrition, immunity, environmental toxicity, gene function analysis and the like.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a diagram showing the state of the blood cells of the Haliotis discus hannai adhered to the wall for 2h according to the present invention;
FIG. 2 is a diagram showing the state of the blood cells of the Haliotis discus hannai, adhered to the wall for 24h according to the present invention;
FIG. 3 is a diagram showing the state of the blood cells of the Haliotis discus hannai after the adherence of the blood cells to the wall 9d according to the present invention;
FIG. 4 is a graph showing the relationship between the blood cell culture time and the relative viability of the haliotis discus hannai according to the present invention;
FIG. 5 is a graph comparing the effect of different media on the status of abalone blood cells in a wrinkled disc;
FIG. 6 is a fluorescence plot of blood cells after modeling with ROS of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention.
The following describes a specific culture medium and culture method for abalone cells provided in the embodiments of the present application with reference to the drawings.
The abalone cell culture medium provided in the embodiments of this application, the abalone cell culture medium components include: 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 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。
The complete culture medium formula of the primary culture of the haliotis discus hannai blood cells is adjusted, and haliotis discus hannai blood cells with strong adherence capability, stable state and good activity can be rapidly obtained in a short time.
Preferably, 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;
and filtering and sterilizing the inactivated clear liquid by adopting a filter with the filtering diameter of 0.22 mu m to obtain the inactivated abalone serum.
Preferably, the abalone cell culture medium is prepared, and then is filtered and sterilized by a filter with the filtering diameter of 0.22 μm for standby.
The application provides an abalone cell culture method, which comprises the following steps:
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;
and mixing the blood with the abalone cell culture medium, shaking up, inoculating the mixture into a polylysine coated six-hole cell culture plate, sealing the culture plate by adopting a membrane, placing the sealed culture plate into an incubator, and replacing the abalone cell culture medium every 3 days.
Preferably, the temperature in the incubator is 17 ℃ to 25 ℃.
Preferably, the temperature in the incubator is 22 DEG C
In some embodiments, the water in the circulating water system is seawater to which povidone-iodine is added at a concentration of 0.5 mg/L;
the blood and the abalone cell culture medium are mixed according to the volume ratio of 1: 3.
In particular, the present application was adopted in experimentsThe main reagents used included: l15 culture medium, fetal bovine serum (biologicals industries), penicillin, streptomycin, gentamicin, amphotericin, polylysine solution, 3- (4, 5-dimethylthiazole-2) -2, 5-diphenyl tetrazolium bromide (MTT) kit, sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, magnesium chloride, type II collagenase, H2O22 ', 7' -dichlorofluorescein diacetate (DCFH-DA), 75% alcohol, heparin sodium vacuum blood collection tube.
The experimental materials included: 0.22 μm filter, syringe, T25 petri dish, 6-well cell culture plate, bent forceps, surgical scissors, scalpel, 100 μm cell sieve, pasteur pipette.
Sterilizing the experimental materials before experiment, and sterilizing the experimental consumables such as forceps, scissors, scalpel, etc. in autoclave at 121 deg.C for 30 min.
The blood cell primary culture comprises the following specific steps:
selection of experimental abalone: and (4) selecting healthy haliotis discus hannai.
Temporary culture of experimental abalone: the body surfaces of the abalones are washed clean by using a brush, the experimental abalones are temporarily cultured for 24 hours by using a circulating water system with an ultraviolet germicidal lamp, and 0.5mg/L povidone iodine is added into seawater.
Obtaining of blood cells: the experimental abalone is fished out, the body surface is scrubbed for three times by 75 percent alcohol, and mucus on the surfaces of the ventral legs is wiped off. The scrubbed abalone is moved into a super clean bench, and a sterile scalpel is used for scratching the foot muscle of the abalone to take blood, and the blood is immediately added into a heparin sodium blood collection tube. Mixing blood and complete culture medium at a ratio of 1: 3, gently mixing, inoculating into a polylysine coated six-hole cell culture plate, sealing with a sealing film, placing in a 22 deg.C biochemical incubator, and changing the culture medium once every three days.
The experimental results of the abalone cell culture method adopted in the application are as follows: after the primary culture of the abalone blood cells of the haliotis discus hannai is started for 2 hours, part of the cells begin to adhere to the wall, and the cells at the dense part of the blood cells are radial, which is shown in figure 1 specifically; after the primary culture is started for 24 hours, the abalone hemocytes are attached to the bottom of the plate, the refractivity of the cells is good, and the cell confluence rate is about 90 percent, which is shown in figure 2 specifically; after the primary culture is started for 9d, the cells are in a good adherent state, the shapes of the cells are extended, the blood cells are converged into a monolayer, and the confluence rate is about 100 percent, which is shown in figure 3 specifically; the cell viability was measured using MTT colorimetry and was not significantly different from that after 24h of primary culture, as shown in fig. 4.
The application makes a comparative experiment on the influence of different culture media on the culture state of the abalone blood cells in the wrinkled giant hyssop, and the result is shown in the table 1.
TABLE 1
Wherein the culture state of the L-15 culture medium on the abalone blood cells in the wrinkled giant hyssop is shown in FIG. 5A;
the state of the salinity-adjusting L-15 culture medium on the culture of the abalone blood cells in the wrinkled giant hyssop is shown in figure 5B;
the state of the culture of the blood cells of Haliotis discus hannai by the salinity-adjusting L-15 culture medium and the abalone serum is shown in FIG. 5C.
The basic culture medium formula is optimized in the experiment, and the discovery that penicillin streptomycin mixed liquor, gentamicin, amphotericin B, NaCl, KCl and CaCl are added into an L-15 culture medium2、MgSO4And MgCl2The probability of bacterial contamination in the blood cell culture process can be reduced, and the blood cells can keep good shapes.
According to the experimental results, the abalone cell culture medium has the advantages that the abalone serum is added into the salinity-adjusting L-15 culture medium to prolong the adherent time of abalone blood cells and keep the proliferation activity of the blood cells by optimizing the formula of the abalone cell culture medium.
Preferably, the method for producing the culture plate coating comprises:
selecting a six-hole cell culture plate;
sucking 400 mul of 0.01% polylysine, dripping into the holes, uniformly wetting 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.
Specifically, the surface of a six-hole cell culture plate is coated in the experiment, and the coated culture plate can shorten the adherent time of the abalone blood cells by 3-4h and accelerate the adherent blood cells to converge into a monolayer at the bottom of the plate.
Preferably, the culture method of abalone cells provided by the present application further comprises:
after the blood cells were seeded on 6-well cell culture plates for 24 hours, H-containing cells were used2O2The abalone cell culture medium carries out ROS modeling on blood cells, and DCFH-DA working solution is added for incubation.
In the application, a fluorescent probe (2 ', 7' -dichlorofluorescein diacetate, DCFH-DA for short) is used, and then the fluorescence emitted by DCF generated by DCFH in which active oxygen in blood cells oxidizes non-fluorescence is observed, so that the constructed crinkle disc blood cell in-vitro culture model can be used for the oxidative stress modeling of abalone.
Preferably, the ROS molding method comprises the following steps:
inoculating blood cells into 6-well cell culture plate at a density of 106 per well, placing in incubator, sucking out abalone cell culture medium after 24H, washing with PBS, and adding H with 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;
cell fluorescence was observed using a fluorescence microscope at an excitation wavelength of 490 nm.
The specific molding and detecting steps comprise:
inoculating Haliotis discus hannai blood cells into 6-well cell culture plate at density of 106/well, culturing in biochemical incubator at 22 deg.C, sucking out complete culture medium after 24 hr, washing with PBS, and adding 50mmol/L H solution2O2The culture medium of (1);
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 at 22 ℃;
after the incubation is finished, sucking out the working solution, slowly washing for 3 times by using PBS, and adding 2ml of complete culture medium into each hole;
cell fluorescence was observed using a fluorescence microscope at an excitation wavelength of 490 nm.
Specifically, after the blood cells of the haliotis discus hannai is inoculated on a 6-well plate for 24H, H2O2And carrying out ROS modeling on blood cells, adding DCFH-DA working solution for incubation, and observing green fluorescence emitted by the cells after the ROS modeling at the excitation wavelength of 490nm by using a fluorescence microscope, so as to prove that the constructed crinkle disc blood cell in-vitro culture model can be used for related researches on immunity and oxidation resistance of abalones.
As shown in FIG. 6, the results of the experiment showed that blood cells were in H2O2After modeling, the fluorescence emitted by DCF generated by DCFH that has no fluorescence and is oxidized by active oxygen in blood cells can be observed under an Echo Revolve fluorescence microscope, and the constructed crinkle discal blood cell in-vitro culture model can be used for oxidative stress modeling of abalone and further applied to relevant researches on immunity and antioxidation.
In summary, the present invention provides a culture medium for abalone cells and a culture method for abalone cells, wherein the culture medium for abalone cells comprises: 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.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended 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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