CN108004197B - Primary culture method of cuttlefish embryonic cells and special reagent - Google Patents
Primary culture method of cuttlefish embryonic cells and special reagent Download PDFInfo
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Abstract
The invention discloses a primary culture method of cuttlefish embryonic cells and a special reagent. The primary culture special reagent specifically comprises the following components: culture medium solution, methylcellulose, D-glucose, calf serum, cuttlefish serum, and double antibiotics. The beneficial effects are that: the primary culture method has the advantages that the number of cells obtained by separation is large, the disinfection is thorough, the damage to embryos during separation can be reduced, the adherent growth quantity of the embryo cells is large, the success rate of culture is high, and the survival time is long; the special reagent can stimulate cells to secrete more growth factors, accelerate the division of embryonic cells and shorten the cell culture time.
Description
Technical Field
The invention relates to the technical field of aquaculture, in particular to a primary culture method of cuttlefish embryonic cells and a special reagent.
Background
Sepiella maindroni (Sepiella maindroni) belongs to Mollusca, Cephalopoda, Dibranchia, Sepioidea, Sepiidae and Sepiella. Sepiella maindroni is a marine product with high protein, low fat and comprehensive nutrition. Its economic value is high, and its edible portion is 92% of total body. It has white meat quality, rich nutrients, high quality protein and rich trace elements, and delicious taste, and may be eaten frequently to delay intelligence, delay senility, raise hemopoiesis and immunity and prevent cardiac vascular diseases, anemia and tumor. However, with the damage to young cuttlefishes caused by the enclosing of spawning groups and the netting operation, the fishing strength is increased, so that the cuttlefish groups cannot be effectively supplemented, and in addition, the pollution and the damage to the natural environment cause the rapid decline of Sepiella maindroni resources from the end of the 80 s in the 20 th century, and the fishing news is difficult to form again. Sepiella maindroni is an important cephalopod species which is developed in large-scale breeding and breeding only in recent years. However, the totally artificial breeding technology of the cuttlefish has many problems, such as: the survival rate of the initially hatched young black is lower in the process of converting endogenous nutrition into exogenous nutrition; the newly hatched young black silkworms need living bait, and the young and adult are lack of artificial bait; under intensive culture conditions, the regulation and control of culture environment and the like, so the economic cost for culturing the cuttlefish is still higher at present.
With the rapid development of life science, cell engineering has been increasingly emphasized in the field of bioengineering technology. In a sense, genetic engineering is the core of modern biotechnology, while cellular engineering is the fundamental and common platform of modern biotechnology. Cell culture is an indispensable research means for basic research on cytology, verification of cell structure and function, and research on cell engineering. Since the research on marine organism cell culture starts late and the difference between marine organism cell culture conditions and terrestrial organisms is large due to the huge difference of living environments, the marine organism cell culture technology is not mature in many varieties at present. However, the cell culture technology of the cephalopod cuttlefish under in vitro conditions has not been overcome, and thus the cell culture-based related research is seriously affected, and therefore, a stable and effective cell culture method is urgently developed.
Disclosure of Invention
One of the purposes of the invention is to provide a primary culture method of cuttlefish embryonic cells, which has the advantages of more separated cells, thorough disinfection, reduction of damage to embryos during separation, large adherent growth amount of the embryonic cells, high culture success rate and long survival time.
The second purpose of the invention is to provide a special primary culture reagent for cuttlefish embryonic cells, which can accelerate the division of embryonic cells, shorten the cell culture time, stimulate the cells to secrete more growth factors, make the embryonic cells perform better during the initial cell adherence period, and can quickly start to migrate from the adherent tissue block to continue to grow.
Aiming at the problems mentioned in the technology, the invention adopts the technical scheme that:
a primary culture method of cuttlefish embryonic cells comprises collecting fertilized eggs, obtaining embryonic tissues and cells, and performing primary culture of the embryonic cells, and specifically comprises the following steps:
collecting fertilized eggs: selecting healthy cuttlefish parents, respectively temporarily culturing in a cement pond according to a male-female ratio of 1:1, inflating and shading, putting a cuttlefish egg laying base into the pond to collect fertilized eggs, culturing under the conditions that the seawater temperature is 16-20 ℃, the salinity is 29-31, the pH is 7.0-8.0 and the illuminance is 4.1-13.1lx, taking out the fertilized eggs from the beginning of egg laying to 50-70min after egg laying, placing the fertilized eggs in a glass water tank for incubation, culturing for 2-3d under the conditions that the seawater temperature is 22-24 ℃, the salinity is 29-31 and the pH is 7.8-8.6, continuously oxygenating for standby, wherein early embryo yolk occupies most of embryos, most of yolk and cells in the centrifugal tube are centrifugally collected after separation, embryo cells are difficult to obtain by separation, and the embryos of 2-3d fertilized embryos are easy to separate primary cells, the number of the separated cells is also large, and the success rate is higher;
obtaining embryonic tissues and cells: sterilizing fertilized eggs, tearing the egg membrane by using tweezers to release embryos and yolk, transferring the embryos to a culture dish in a super clean workbench, shearing and crushing, centrifuging for 3-5min at the speed of 1000-, the damage to embryonic cells is reduced, tissue blocks with proper and uniform sizes are obtained, an egg membrane can be removed immediately, the influence of the egg membrane on the growth of the cells is reduced, the wall-adhering effect of the cells is improved, and the sterilization is thorough, so that the embryonic cells cannot be infected with germs, and the success rate of the culture of the embryonic cells is further improved;
primary culture of embryonic cells: suspending embryo tissues and cell precipitates by using a primary culture special reagent, transferring the suspended embryo tissues and cell precipitates into a culture plate, treating 30-40 fertilized eggs at each hole, using the special reagent in an amount of 100-, there will be a large number of adherent cells and the cells will grow better and survive longer with continued addition of medium.
Preferably, the disinfection method comprises the following steps: sequentially soaking in 70-80% ethanol solution for 10-20min, washing with 7.0-8.0 sterile PBS solution for 4-6 times, soaking in 0.62-0.72% antibiotic and 0.28-0.38% ofloxacin sterile water for 20-40min, rinsing with 70-80% ethanol solution for 5-10s, and washing with 7.0-8.0 sterile PBS solution for 4-6 times. The ofloxacin contains 1.8-2.5% of dextro-ofloxacin, the ofloxacin can prevent bacterial DNA from synthesizing and copying to cause bacterial death by inhibiting the activity of bacterial DNA gyrase, the broad-spectrum antibacterial effect is achieved, the influence of bacteria on fertilized eggs can be avoided, the gain effect can be achieved by compounding with double antibiotics, the disinfection effect is greatly improved, the condition that cells are polluted can be effectively avoided, the ofloxacin containing the dextro-ofloxacin can also penetrate through micropores on an egg membrane to inactivate carbonylation and sulfylation sticky substances, meanwhile, the inner layer of the egg membrane can be disintegrated, the stickiness of the egg membrane and embryos is weakened, the egg membrane is easier to separate, the damage to the embryos during separation is reduced, and the preparation is made for improving the success rate of embryo cell culture.
A special reagent for primary culture of cuttlefish embryonic cells specifically comprises the following components: culture medium solution, methylcellulose, D-glucose, calf serum, cuttlefish serum, and double antibiotics. The special reagent can simulate the nutrient environment of the growth of embryonic cells in vivo, can meet the requirements of the embryonic cells on various methods such as nutrient components, growth promotion factors, hormones, osmotic pressure, pH and the like, promotes the growth and proliferation of the embryonic cells, and can provide adherence and expansion factors for the embryonic cells, so that a large number of cells migrate out of adherent embryonic tissue blocks and spread all around, few single cells grow independently, and can rapidly migrate out of the adherent tissue blocks and continue to grow.
A special reagent for primary culture of cuttlefish embryonic cells is prepared by the following steps:
step 1: equal volumes of 770-790mM NaCl solution, 14-15mM KCl solution, 100-110mM MgCl2Solution and 20-25mM CaCl2Mixing the solutions uniformly, and filtering with 0.22 μm filter membrane for sterilization to obtain culture medium solution for use, wherein the culture medium solution can maintain osmotic pressure of embryo cells, adjust pH value, and supply energy and inorganic ion components required by cell survival;
step 2: adding methylcellulose into the culture medium solution according to the weight-volume ratio of 1:75-85 (mg/mL), and stirring at 2-5 deg.C for 3-4h to obtain solution A;
and step 3: dissolving D-glucose in a culture medium solution according to a weight-to-volume ratio of 1:7.5-8.5 (mg/mL) to obtain a solution B, adding the solution B into the solution A according to a volume ratio of 1:5, then adding calf serum, cuttlefish serum and antibiotics to ensure that the final concentration of the calf serum is 3-5%, the final concentration of the cuttlefish serum is 0.6-0.8% and the final concentration of the antibiotics is 0.8-1.2mg/100mL, and uniformly mixing to obtain the special reagent.
Compared with the prior art, the invention has the advantages that: 1) the primary culture method has the advantages that the number of cells obtained by separation is large, the disinfection is thorough, the damage to embryos during separation can be reduced, the adherent growth quantity of the embryo cells is large, the success rate of culture is high, and the survival time is long; 2) the culture method can lead the disinfectant to penetrate through micropores on the egg membrane while disinfecting, so that the carbonylation and sulfylation sticky substances are inactivated, and meanwhile, the inner layer of the egg membrane can also be disintegrated, the stickiness of the egg membrane and the embryo is weakened, the egg membrane is easier to separate, the damage to the embryo during separation is reduced, and the preparation is made for improving the success rate of embryo cell culture; 3) the special reagent for primary culture can simulate the nutrient environment for the growth of embryonic cells in vivo, meet the requirements of the embryonic cells on various methods such as nutrient components, growth promoting factors, hormones, osmotic pressure, pH and the like, promote the growth and proliferation of the embryonic cells, and simultaneously provide adherence and expansion factors for the embryonic cells, so that a large number of cells migrate out of an adherence embryo tissue block and spread to the periphery.
Detailed Description
The scheme of the invention is further illustrated by the following examples:
example 1:
a primary culture method of cuttlefish embryonic cells comprises collecting fertilized eggs, obtaining embryonic tissues and cells, and performing primary culture of the embryonic cells, and specifically comprises the following steps:
1) collecting fertilized eggs: selecting healthy cuttlefish parents, respectively temporarily culturing in a cement pond according to a male-female ratio of 1:1, inflating and shading, putting a cuttlefish spawning base into the pond to collect fertilized eggs, culturing under the conditions of seawater temperature of 16 ℃, salinity of 31, pH of 7.0 and illuminance of 13.1lx, taking out the fertilized eggs from the beginning of spawning to 50min after spawning, placing the fertilized eggs into a glass water tank for incubation, culturing for 2d under the conditions of seawater temperature of 24 ℃, salinity of 29 and pH of 8.6, and continuously oxygenating for later use;
2) obtaining embryonic tissues and cells: disinfecting fertilized eggs, tearing an egg membrane by using forceps, releasing embryos and yolk, transferring the embryos to a culture dish in a super clean workbench, shearing, centrifuging for 3min at 1200r/min to obtain embryo tissue precipitates, adding 3 mu g/mL trypsin solution into the precipitates to suspend the embryo tissues, digesting for 5min at 30 ℃, adding primary culture special reagent with the same volume as the trypsin solution, stopping digestion of trypsin, centrifuging for 3min at 1200r/min, removing supernatant, and obtaining digested embryo tissues and cell precipitates, wherein the whole process adopts aseptic operation;
3) primary culture of embryonic cells: suspending embryonic tissues and cell sediments by using a primary culture special reagent, transferring the suspended embryonic tissues and cell sediments into a culture plate, treating 30 fertilized eggs in each hole, using 100 mu l of the special reagent, wrapping the fertilized eggs by using a Parafilm sealing membrane, then culturing the fertilized eggs in a constant-temperature incubator at 30 ℃, replacing a fresh special reagent after the tissues and the cells are attached to the wall on the 2 nd day, replacing the special reagent every 5d, obtaining a cuttlefish cell population after 40d, and performing aseptic operation in the whole process.
The sterilization method in the step 1 comprises the following steps: sequentially soaking with 70% ethanol solution for 20min, washing with 7.0 pH sterile PBS solution for 6 times, soaking with 0.62% antibiotic and 0.38% ofloxacin sterile water for 20min, washing with 70% ethanol solution for 10s, and washing with 7.0 pH sterile PBS solution for 6 times. The ofloxacin contains 1.8 percent of dextro-ofloxacin.
A special reagent for primary culture of cuttlefish embryonic cells specifically comprises the following components: culture medium solution, methylcellulose, D-glucose, calf serum, cuttlefish serum, and double antibiotics.
A special reagent for primary culture of cuttlefish embryonic cells is prepared by the following steps:
step 1: equal volumes of 770mM NaCl solution, 15mM KCl solution, 100mM MgCl2Solution and 25mM CaCl2Mixing the solutions, and filtering with 0.22 μm filter membrane for sterilization to obtain culture medium solution;
step 2: adding methylcellulose into the culture medium solution according to the weight-to-volume ratio of 1:75 (mg/mL), and stirring at 5 ℃ for 3h to obtain solution A for later use;
and step 3: dissolving D-glucose in a culture medium solution according to a weight-to-volume ratio of 1:7.5 (mg/mL) to obtain a solution B, adding the solution B into the solution A according to a volume ratio of 1:5, then adding calf serum, cuttlefish serum and antibiotics so that the final concentration of the calf serum is 5%, the final concentration of the cuttlefish serum is 0.6% and the final concentration of the antibiotics is 1.2mg/100mL, and uniformly mixing to obtain the special reagent.
Example 2:
a primary culture method of cuttlefish embryonic cells comprises collecting fertilized eggs, obtaining embryonic tissues and cells, and performing primary culture of the embryonic cells, and specifically comprises the following steps:
1) collecting fertilized eggs: selecting healthy cuttlefish parents, respectively temporarily culturing in a cement pond according to a male-female ratio of 1:1, inflating and shading, putting a cuttlefish spawning base into the pond to collect fertilized eggs, culturing under the conditions of seawater temperature of 20 ℃, salinity of 29, pH of 8.0 and illuminance of 4.1 lx, taking out the fertilized eggs from the beginning of spawning to 70min after spawning, placing the fertilized eggs into a glass water tank for incubation, culturing for 3d under the conditions of seawater temperature of 22 ℃, salinity of 31 and pH of 7.8, and continuously oxygenating for later use;
2) obtaining embryonic tissues and cells: disinfecting fertilized eggs, tearing an egg membrane by using forceps, releasing embryos and yolk, transferring the embryos to a culture dish in a super clean workbench, shearing, centrifuging for 5min at 1000r/min to obtain embryo tissue precipitates, adding 2 mu g/mL trypsin solution into the precipitates to suspend the embryo tissues, digesting for 3min at 40 ℃, adding a primary culture special reagent with the same volume as the trypsin solution, stopping digestion of trypsin, centrifuging for 5min at 1000r/min, removing supernatant, and obtaining digested embryo tissues and cell precipitates, wherein the whole process adopts aseptic operation;
3) primary culture of embryonic cells: suspending embryonic tissues and cell sediments by using a primary culture special reagent, transferring the suspended embryonic tissues and cell sediments into a culture plate, treating 40 fertilized eggs in each hole, using 150 mu l of the special reagent, wrapping the fertilized eggs by using a Parafilm sealing membrane, then culturing in a constant-temperature incubator at 20 ℃, replacing a fresh special reagent after the tissues and the cells are attached to the wall on day 2, replacing the special reagent every 8 days, obtaining a cuttlefish cell population after 20 days, and performing aseptic operation in the whole process.
The sterilization method in the step 1 comprises the following steps: sequentially soaking in 80% ethanol solution for 10min, washing with sterile PBS solution with pH of 8.0 for 4 times, soaking in sterile water containing 0.72% antibiotic and 0.28% ofloxacin for 40min, washing with 80% ethanol solution for 5s, and washing with sterile PBS solution with pH of 8.0 for 4 times. The ofloxacin contains 2.5 percent of dextro-ofloxacin.
A special reagent for primary culture of cuttlefish embryonic cells specifically comprises the following components: culture medium solution, methylcellulose, D-glucose, calf serum, cuttlefish serum, and double antibiotics.
A special reagent for primary culture of cuttlefish embryonic cells is prepared by the following steps:
step 1: equal volumes of 790mM NaCl solution, 14mM KCl solution, 110mM MgCl2Solution and 20mM CaCl2Mixing the solutions, and filtering with 0.22 μm filter membrane for sterilization to obtain culture medium solution;
step 2: adding methylcellulose into the culture medium solution according to the weight-to-volume ratio of 1:85 (mg/mL), and stirring at 2 ℃ for 4h to obtain solution A for later use;
and step 3: dissolving D-glucose in a culture medium solution according to a weight-to-volume ratio of 1:8.5 (mg/mL) to obtain a solution B, adding the solution B into the solution A according to a volume ratio of 1:5, then adding calf serum, cuttlefish serum and antibiotics so that the final concentration of the calf serum is 3%, the final concentration of the cuttlefish serum is 0.8% and the final concentration of the antibiotics is 0.8mg/100mL, and uniformly mixing to obtain the special reagent.
Example 3:
a primary culture method of cuttlefish embryonic cells comprises collecting fertilized eggs, obtaining embryonic tissues and cells, and performing primary culture of the embryonic cells, and specifically comprises the following steps:
1) collecting fertilized eggs: selecting healthy cuttlefish parents, respectively temporarily culturing in a cement pond according to a male-female ratio of 1:1, inflating and shading, putting a cuttlefish spawning base into the pond to collect fertilized eggs, culturing under the conditions of seawater temperature of 18 ℃, salinity of 30, pH of 7.5 and illuminance of 8.5 lx, taking out the fertilized eggs from the beginning of spawning to 60min after spawning, placing the fertilized eggs into a glass water tank for incubation, culturing for 2.5 days under the conditions of seawater temperature of 23 ℃, salinity of 30 and pH of 8.2, and continuously oxygenating for later use;
2) obtaining embryonic tissues and cells: sequentially soaking 210 fertilized eggs in 75% ethanol solution for 15min, washing with 7.5 pH sterile PBS solution for 5 times, soaking in sterile water containing 0.67% double antibiotics and 0.33% ofloxacin for 30min, rinsing with 75% ethanol solution for 8s, washing with 7.5 pH sterile PBS solution for 5 times, tearing the egg membrane with forceps, releasing embryo and yolk, transferring the embryo into a culture dish in a clean bench, cutting, centrifuging at 1100r/min for 4min to obtain embryo tissue precipitate, adding 2.5 μ g/mL trypsin solution into the precipitate to suspend the embryo tissue, digesting at 35 deg.C for 4min, adding primary culture special reagent with the same volume as the trypsin solution, terminating the trypsin digestion, centrifuging at 1100r/min for 4min, removing supernatant to obtain digested embryo tissue and cell precipitate, and performing sterile operation in the whole process, the ofloxacin contains 2.2 percent of dextro-ofloxacin;
3) primary culture of embryonic cells: suspending embryonic tissues and cell sediments by using a primary culture special reagent, transferring the suspended embryonic tissues and cell sediments into a culture plate, treating 35 fertilized eggs at each hole, using 120 mu l of the special reagent, wrapping the fertilized eggs by using a Parafilm sealing membrane, culturing the fertilized eggs in a constant-temperature incubator at 25 ℃, replacing a fresh special reagent after the tissues and the cells adhere to the wall on the 2 nd day, observing 80 cell clusters in each field of view under a 10-time objective lens after 3 days, replacing the special reagent once every 6d, observing a large number of adherent cells under the 10-time objective lens field of view after 30d, wherein the cell clusters overlap more, namely cuttlefish cell groups, and performing aseptic operation in the whole process.
A special reagent for primary culture of cuttlefish embryonic cells specifically comprises the following components: culture medium solution, methylcellulose, D-glucose, calf serum, cuttlefish serum, and double antibiotics.
A special reagent for primary culture of cuttlefish embryonic cells is prepared by the following steps:
step 1: equal volumes of 780mM NaCl, 14.2mM KCl, 108mM MgCl2And 21mM CaCl2Mixing, filtering with 0.22 μm filter membrane for sterilization to obtain culture medium solution;
step 2: adding 5mg of methylcellulose into 400mL of culture medium solution, and stirring at 4 ℃ for 3.5h to obtain solution A for later use;
and step 3: dissolving 10mg of D-glucose in 80mL of culture medium solution to obtain solution B, adding the solution B into the solution A, then adding calf serum, cuttlefish serum and antibiotics to ensure that the final concentration of the calf serum is 4%, the final concentration of the cuttlefish serum is 0.7% and the final concentration of the antibiotics is 1.0mg/100mL, and uniformly mixing to obtain the special reagent.
Example 4:
a primary culture method of cuttlefish embryonic cells comprises collecting fertilized eggs, obtaining embryonic tissues and cells, and performing primary culture of the embryonic cells, and specifically comprises the following steps:
1) collecting fertilized eggs: selecting healthy cuttlefish parents, respectively temporarily culturing in a cement pond according to a male-female ratio of 1:1, inflating and shading, putting a cuttlefish spawning base into the pond to collect fertilized eggs, culturing under the conditions of seawater temperature of 18 ℃, salinity of 30, pH of 7.5 and illuminance of 8.5 lx, taking out the fertilized eggs from the beginning of spawning to 60min after spawning, placing the fertilized eggs into a glass water tank for incubation, culturing for 2.5 days under the conditions of seawater temperature of 23 ℃, salinity of 30 and pH of 8.2, and continuously oxygenating for later use;
2) obtaining embryonic tissues and cells: sequentially soaking 210 fertilized eggs in 75% ethanol solution for 15min, washing with 7.5 pH sterile PBS solution for 5 times, soaking in sterile water containing 0.67% double antibiotics and 0.33% ofloxacin for 30min, rinsing with 75% ethanol solution for 8s, washing with 7.5 pH sterile PBS solution for 5 times, tearing the egg membrane with forceps, releasing embryo and yolk, transferring the embryo into a culture dish in a clean bench, cutting, centrifuging at 1100r/min for 4min to obtain embryo tissue precipitate, adding 2.5 μ g/mL trypsin solution into the precipitate to suspend the embryo tissue, digesting at 35 deg.C for 4min, adding primary culture special reagent with the same volume as the trypsin solution, terminating the trypsin digestion, centrifuging at 1100r/min for 4min, removing supernatant to obtain digested embryo tissue and cell precipitate, and performing sterile operation in the whole process, the ofloxacin contains 2.2 percent of dextro-ofloxacin;
3) primary culture of embryonic cells: suspending embryonic tissues and cell sediments by using a primary culture special reagent, transferring the suspended embryonic tissues and cell sediments into a culture plate, treating 35 fertilized eggs at each hole, using 120 mu l of the special reagent, wrapping the fertilized eggs by using a Parafilm sealing membrane, culturing the fertilized eggs in a constant-temperature incubator at 25 ℃, replacing a fresh special reagent after the tissues and the cells adhere to the wall on the 2 nd day, observing 84 cell clusters in each field of view under a 10-time objective lens after 3 days, replacing the special reagent once every 6d, observing a large number of adherent cells under the 10-time objective lens field of view after 30d, wherein the cell clusters overlap more, namely cuttlefish cell groups, and the whole process adopts aseptic operation.
A special reagent for primary culture of cuttlefish embryonic cells specifically comprises the following components: culture medium solution, methylcellulose, D-glucose, calf serum, cuttlefish serum, and double antibiotics.
A special reagent for primary culture of cuttlefish embryonic cells is prepared by the following steps:
step 1: equal volumes of 780mM NaCl, 14.2mM KCl, 108mM MgCl2And 21mM CaCl2Mixing, filtering with 0.22 μm filter membrane for sterilization to obtain culture medium solution;
step 2: adding 5mg of methylcellulose into 400mL of culture medium solution, and stirring at 4 ℃ for 3.5h to obtain solution A for later use;
and step 3: dissolving 10mg of D-glucose in 80mL of culture medium solution to obtain solution B, adding B into the solution A, then adding calf serum, cuttlefish serum, ancitabine and double antibiotics to ensure that the final concentration of the calf serum is 4%, the final concentration of the cuttlefish serum is 0.7%, the final concentration of the ancitabine is 0.24mg/100mL and the final concentration of the double antibiotics is 1.0mg/100mL, and uniformly mixing to obtain the special reagent.
Example 5:
a primary culture method of cuttlefish embryonic cells comprises collecting fertilized eggs, obtaining embryonic tissues and cells, and performing primary culture of the embryonic cells, and specifically comprises the following steps:
1) collecting fertilized eggs: selecting healthy cuttlefish parents, respectively temporarily culturing in a cement pond according to a male-female ratio of 1:1, inflating and shading, putting a cuttlefish spawning base into the pond to collect fertilized eggs, culturing under the conditions of seawater temperature of 18 ℃, salinity of 30, pH of 7.5 and illuminance of 8.5 lx, taking out the fertilized eggs from the beginning of spawning to 60min after spawning, placing the fertilized eggs into a glass water tank for incubation, culturing for 2.5 days under the conditions of seawater temperature of 23 ℃, salinity of 30 and pH of 8.2, and continuously oxygenating for later use;
2) obtaining embryonic tissues and cells: sequentially soaking 210 fertilized eggs in 75% ethanol solution for 15min, washing with 7.5 pH sterile PBS solution for 5 times, soaking in sterile water containing 0.67% double antibiotics and 0.33% ofloxacin for 30min, rinsing with 75% ethanol solution for 8s, washing with 7.5 pH sterile PBS solution for 5 times, tearing the egg membrane with forceps, releasing embryo and yolk, transferring the embryo into a culture dish in a clean bench, cutting, centrifuging at 1100r/min for 4min to obtain embryo tissue precipitate, adding 2.5 μ g/mL trypsin solution into the precipitate to suspend embryo tissue, digesting at 35 deg.C for 4min, adding common culture reagent with the same volume as trypsin solution, terminating trypsin digestion, centrifuging at 1100r/min for 4min, removing supernatant to obtain digested embryo tissue and cell precipitate, and performing sterile operation in the whole process, the ofloxacin contains 2.2 percent of dextro-ofloxacin;
3) primary culture of embryonic cells: suspending embryonic tissues and cell sediments by using a common culture reagent, transferring the suspended embryonic tissues and cell sediments into a culture plate, treating 35 fertilized eggs at each hole, wherein the using amount of the common culture reagent is 120 mu l, wrapping the fertilized eggs by using a Parafilm sealing membrane, culturing the fertilized eggs in a constant-temperature incubator at 25 ℃, changing a fresh common culture reagent after the tissues and the cells adhere to the wall on day 2, observing 48 cell clusters in each visual field under a 10-time objective lens after 3 days, changing the special common culture reagent once every 6d, and observing a large number of adherent cells under the 10-time objective lens visual field after 30d, but the overlapping condition of the cell clusters is less, which shows that the culture effect of the special reagent used in the embodiment 3 is far better than that of the common culture reagent.
Conventional operations in the operation steps of the present invention are well known to those skilled in the art and will not be described herein.
The embodiments described above are intended to illustrate the technical solutions of the present invention in detail, and it should be understood that the above-mentioned embodiments are only specific embodiments of the present invention, and are not intended to limit the present invention, and any modification, supplement or similar substitution made within the scope of the principles of the present invention should be included in the protection scope of the present invention.
Claims (4)
1. A primary culture method of cuttlefish embryonic cells comprises collecting fertilized eggs, obtaining embryonic tissues and cells, and primary culture of the embryonic cells, and is characterized in that:
the fertilized egg collecting step comprises: selecting healthy cuttlefish parents, respectively temporarily culturing in a water tank according to a male-female ratio of 1:1, inflating and shading, putting a cuttlefish egg laying base into the tank to collect fertilized eggs, culturing under the conditions that the seawater temperature is 16-20 ℃, the salinity is 29-31, the pH is 7.0-8.0 and the illuminance is 4.1-13.1lx, taking out the fertilized eggs from the beginning of egg laying to 50-70min after egg laying, placing the fertilized eggs into a glass water tank for incubation, culturing for 2-3d under the conditions that the seawater temperature is 22-24 ℃, the salinity is 29-31 and the pH is 7.8-8.6, and continuously oxygenating for later use;
the step of obtaining embryo tissues and cells comprises the following steps: disinfecting fertilized eggs, tearing an egg membrane by using tweezers, releasing embryos and yolk, transferring the embryos to a culture dish in a super clean workbench, shearing and crushing, centrifuging for 3-5min at the speed of 1000-;
the primary culture special reagent consists of the following components: culture medium solution, methylcellulose, D-glucose, calf serum, cuttlefish serum and double antibiotics, and specifically comprises the following components: 770-790mM NaCl solution, 14-15mM KCl solution, 100-110mM MgCl solution with equal volume2Solution and 20-25mM CaCl2Mixing the solutions, and filtering with 0.22 μm filter membrane for sterilization to obtain culture medium solution; adding methylcellulose into the culture medium solution according to the weight-volume ratio of 1mg:75-85mL, and uniformly stirring to obtain a solution A; dissolving D-glucose in the culture medium solution according to the weight-volume ratio of 1mg:7.5-8.5mL to obtain a solution B; then adding the solution B into the solution A according to the volume ratio of 1:5,finally adding other components to make the final concentration of calf serum be 3-5%, the final concentration of cuttlefish serum be 0.6-0.8% and the final concentration of double antibiotics be 0.8-1.2mg/100ml, and mixing well to obtain the final product;
the method for disinfecting the fertilized eggs in the step of obtaining the embryo tissues and the cells comprises the following steps: sequentially soaking in 70-80% ethanol solution for 10-20min, washing with 7.0-8.0 sterile PBS solution for 4-6 times, soaking in 0.62-0.72% antibiotic and 0.28-0.38% ofloxacin sterile water for 20-40min, rinsing with 70-80% ethanol solution for 5-10s, and washing with 7.0-8.0 sterile PBS solution for 4-6 times;
the ofloxacin contains 1.8-2.5% of dextro-ofloxacin;
the adding amount of the trypsin is 2-3 mug/mL, the digestion temperature is 30-40 ℃, and the time is 3-5 min.
2. The primary culture method of cuttlefish embryo cells according to claim 1, which comprises: the primary culture steps of the embryonic cells are as follows: suspending embryonic tissue and cell sediment by using a primary culture special reagent, transferring the suspended embryonic tissue and cell sediment into a culture plate, wrapping the culture plate by using a Parafilm sealing film, culturing the culture plate in a constant-temperature incubator at the temperature of 20-30 ℃, replacing the special reagent after the tissue and the cell are attached to the wall on day 2, replacing the special reagent once every 5-8 days according to the growth condition of the cell, and obtaining the cuttlefish cell population after 20-40 days.
3. The primary culture method of cuttlefish embryo cells as claimed in claim 2, which comprises: 30-40 fertilized eggs are processed in each hole in the primary culture step of the embryo cells, and the dosage of the special reagent is 100-.
4. A special reagent for primary culture of cuttlefish embryonic cells is characterized in that: the proprietary reagent consisted of the following components: culture medium solution, methylcellulose, D-glucose, calf serum, cuttlefish serum, and double antibiotics;
the preparation method of the special reagent comprises the following steps:
1) an equal volume of 770-790mM NaCl solution14-15mM KCl solution, 100-110mM MgCl2Solution and 20-25mM CaCl2Mixing the solutions, and filtering with 0.22 μm filter membrane for sterilization to obtain culture medium solution;
2) adding methylcellulose into the culture medium solution according to the weight-volume ratio of 1mg:75-85mL, and stirring at 2-5 ℃ for 3-4h to obtain solution A for later use;
3) dissolving D-glucose into the culture medium solution according to the weight-volume ratio of 1mg:7.5-8.5mL to obtain a solution B, adding the solution B into the solution A according to the volume ratio of 1:5, then adding calf serum, cuttlefish serum and antibiotics so that the final concentration of the calf serum is 3-5%, the final concentration of the cuttlefish serum is 0.6-0.8% and the final concentration of the antibiotics is 0.8-1.2mg/100mL, and uniformly mixing to obtain the special reagent.
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