CN108004197A - The primary culture method and proprietary reagent of a kind of cuttlefish embryonic cell - Google Patents
The primary culture method and proprietary reagent of a kind of cuttlefish embryonic cell Download PDFInfo
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Abstract
Primary culture method and proprietary reagent the invention discloses a kind of cuttlefish embryonic cell, primary culture method include collecting embryonated egg, obtain embryonic tissue and cell, the original cuiture of embryonic cell.The proprietary reagent of original cuiture specifically includes following components：Culture medium solution, methylcellulose, D glucose, calf serum, cuttlefish serum, twin antibiotic.Have the beneficial effect that：The separating obtained cell quantity of primary culture method of the present invention is more, and disinfection is thorough, damage when can reduce separation to embryo, and embryonic cell adherent growth amount is more, the success rate of culture is high, time-to-live length；Proprietary reagent can stimulate cell itself more to secrete growth factor, accelerate the division of embryonic cell, shorten the cell culture time.
Description
Technical field
The present invention relates to technical field of aquaculture, a kind of primary culture method more particularly, to cuttlefish embryonic cell and
Proprietary reagent.
Background technology
Sepiella maindroni（Sepiella maindroni）Belong to Mollusca Mollusca, Cephalopoda
Cephalopoda, Dibranchia Dibranchia, Sepiida Sepioidea, Sepiidae Sepiidae, sepiella maindroni de Rochebrune category
Sepiella.Sepiella maindroni is a kind of high protein, low fat, the more comprehensive marine product of nutrition.Its economic value is very
Height, its edible portion account for overall 92%.Its meat is pure white, full of nutrition, containing good protein and enriches micro member
Element, delicious flavour is often edible clever to prolong intelligence, anti-aging, improve hematopoiesis and immune function, to preventing cardiovascular disease
Disease, anaemia and tumour have certain effect.But with to spawning colony surround and seize and Swing net is to the damage of young cuttlefish
Evil, the increasing of catching intensity, prevents cuttlefish colony from obtaining effective supplement, along with the pollution and destruction of natural environment, from
Late 1980s start the drastically decline for Sepiella maindroni resource occur, and difficult formation is interrogated in fishing.Sepiella maindroni is
Just it is able to the important siphonopods species of propagation in scale and aquaculture exploitation recent years.But the full artificial breeding technology of cuttlefish
Also there are many problems, such as：The survival rate for just incubating young crow into exogenous nutrition transition process due to endogenous nutrition is relatively low；
Just incubating young crow needs living body bait, and the young and adult lack artitificial food；Under the conditions of intensive cultivation, regulation and control of breeding environment etc.,
So the financial cost of cultivation cuttlefish is still higher at present.
With the fast development of life science, in technical field of bioengineering, cell engineering is more and more taken seriously.
In a sense, genetic engineering is the core of modern biotechnology, and cell engineering be modern biotechnology basis and
Common platform.Cell culture is that the research of development Cytological Basis, cellular structures and functions verification and cellular engineering research must not
The research means that can lack.Marine organisms cell culture studies are due to starting late, while marine organisms cell culture condition is because of life
The greatest differences of environment living are also larger with terrestrial life difference, at present marine organisms cell culture technology in many types not yet
It is ripe.But the cell culture technology in relation to siphonopods squid body under the conditions of outer is not captured also, thus is seriously affected with cell culture
Based on correlative study, therefore urgent develop one kind and stablize effective cell culture processes.
The content of the invention
More it is an object of the present invention to providing a kind of separating obtained cell quantity, disinfection is thorough, reduces separation
When damage to embryo, embryonic cell adherent growth amount is more, the success rate of culture is high, the cuttlefish embryonic cell of time-to-live length
Primary culture method.
The second object of the present invention is to provide a kind of division for accelerating embryonic cell, shortens the cell culture time, can
Cell itself is stimulated more to secrete growth factor so that embryonic cell is performed better than during cell attachment in the early stage, can be quick
Migrated out since adherent tissue block, the proprietary reagent of original cuiture of the cuttlefish embryonic cell of continued growth.
The present invention is directed to the problem of being mentioned in above-mentioned technology, and the technical solution taken is：
A kind of primary culture method of cuttlefish embryonic cell, including collect embryonated egg, obtain embryonic tissue and cell, embryonic cell
Original cuiture, specifically comprise the following steps：
Collect embryonated egg：The healthy cuttlefish parent of selection, by sex ration 1:1 temporarily supports in cement pit respectively, inflates, shading, by crow
Crafty spawning base is put into pond collection embryonated egg, ocean temperature be 16-20 DEG C, salinity 29-31, pH 7.0-8.0, illuminance
To be cultivated under conditions of 4.1-13.1 lx, start to after laying eggs to take out embryonated egg in 50-70min in oviposition, be placed in bath of glass
Hatch in groove, cultivate 2-3d under conditions of ocean temperature is 22-24 DEG C, salinity 29-31, pH is 7.8-8.6, continuously fill
Oxygen, spare, body early embryo yolk occupies the overwhelming majority of embryo, and after separation, it is mostly yolk to be collected by centrifugation in centrifuge tube
With the mixture of cell, it is more difficult to separation obtains embryonic cell, and the embryo for the 2-3d that is fertilized is easier to carry out primary cell separation, point
It is also more from the cell quantity of gained, have higher success rate；
Obtain embryonic tissue and cell：Embryonated egg is sterilized, egg membrane is torn with tweezers, embryo and yolk are discharged, ultra-clean
Embryo is moved in culture dish in workbench, is shredded, 3-5min is centrifuged under 1000-1200r/min, embryonic tissue is obtained and sinks
Form sediment, the trypsin solution suspension embryonic tissue of 2-3 μ g/mL is added in precipitation, digests 3-5min at 30-40 DEG C, to remove
Cytoplasm between histocyte, makes tissue become loose, is separated into individual cells or the cell mass of smaller, adds and pancreas
The proprietary reagent of the isometric original cuiture of protein enzyme solution, terminates Trypsin Induced, 3- is centrifuged under 1000-1200r/min
5min, removes supernatant, obtains postdigestive embryonic tissue and cell precipitation, whole process uses sterile working, the processing side
Method can ensure ovum sterilize and rinse during integrality, reduce the damage to embryonic cell, obtain suitable size and
Even tissue block, can remove egg membrane immediately, reduce the influence of cell growth after egg membrane, improve the adherent effect of cell,
Sterilize at the same time thorough so that embryonic cell cannot infect germ, and then improve the success rate of embryonic cell culture；
The original cuiture of embryonic cell：With the proprietary reagent suspension embryonic tissue of original cuiture and cell precipitation, culture plate is transferred to
In, each hole handles 30-40 pieces of embryonated egg, and proprietary reagent dosage is 100-150 μ l, is wrapped with Parafilm sealed membranes, then
Cultivated in 20-30 DEG C of constant incubator, the 2nd day, after tissue and cell attachment, replace fresh proprietary reagent, and regarding thin
Intracellular growth situation, replaces once proprietary reagent every 5-8d, can obtain cuttlefish cell mass after 20-40d, whole process uses nothing
Bacterium operates, and the adherent of cell is realized by the combination of special cell surface receptor and extracellular matrix molecule, culture
Each hole gauge in plate determines embryo's quantity and the volume of culture medium, and be is ensuring that incipient cell adherent period can obtain foot
Enough anchoring factors and trophic factors for sprawling space and secreted by cell itself, just have a large amount of adherent cells, and
With constantly adding cell and also could preferably grow down with wild Oryza species, for longer periods survival is gone down.
Preferably, sterilization method is：Alcohol solution dipping 10-20min, the pH 7.0- for being successively 70-80% with concentration
8.0 sterile PBS solution is rinsed 4-6 times, the Ofloxacin of twin antibiotic and 0.28-0.38% containing 0.62-0.72% it is sterile
Water soaks 20-40min, and ethanol solution the rinsing 5-10s, pH of 70-80% are the sterile PBS solution wash rinse 4-6 of 7.0-8.0
It is secondary.Dextrorotation Ofloxacin containing 1.8-2.5% in above-mentioned Ofloxacin, Ofloxacin is by inhibiting bacteria the work of DNA gyrases
Property, prevent DNA of bacteria synthesis and duplication and cause bacterial death, there is broad-spectrum antibacterial action, can avoid bacterium to fertilization
The influence of ovum, and twin antibiotic compounding can reach gain effect, substantially increase disinfection, can effectively avoid the occurrence of thin
Born of the same parents are contaminated situation, and the Ofloxacin containing dextrorotation Ofloxacin can also pass through the micropore on egg membrane so that carbonylation and
The emplastic inactivation of sulfation, while egg membrane internal layer is disintegrated, weaken egg membrane and the viscous stickiness of embryo so that egg membrane is more
It is easily isolated, damage when reducing separation to embryo, prepares to improve the success rate of embryonic cell culture.
A kind of proprietary reagent of the original cuiture of cuttlefish embryonic cell, specifically includes following components：Culture medium solution, methyl
Cellulose, D-Glucose, calf serum, cuttlefish serum, twin antibiotic.The proprietary reagent can simulate embryonic cell and give birth in vivo
Long nutrient environment, can meet embryonic cell to all multi-methods such as nutritional ingredient, somatomedin, hormone, osmotic pressure and pH
Demand, promotes the growing multiplication of embryonic cell, while adherent and spreading factor can be provided for embryonic cell so that a large amount of cells
Migrate out from adherent embryonic tissue block and extend around and spread, few single cell independent growths, while energy
Quickly since adherent tissue block migrate out, continued growth.
A kind of proprietary reagent of the original cuiture of cuttlefish embryonic cell, its preparation method are：
Step 1：By isometric 770-790mM NaCl solutions, 14-15mM KCl solution, 100-110mM MgCl2Solution and
20-25mM CaCl2Solution is uniformly mixed, and then the membrane filtration with 0.22 μm is degerming, spare up to culture medium solution, the training
Foster based sols can maintain the osmotic pressure of embryonic cell, adjust pH value, energy and inorganic ions needed for supply cells survival into
Point；
Step 2：Volume ratio is 1 by weight:75-85（mg/mL）Methylcellulose is added in culture medium solution, at 2-5 DEG C
3-4h is stirred, obtains solution A, it is spare；
Step 3：Volume ratio is 1 by weight:7.5-8.5（mg/mL）D-Glucose is dissolved in culture medium solution and obtains B solution, then
By B solution according to volume ratio be 1:5 add in solution A, then add calf serum, cuttlefish serum and twin antibiotic so that
The final concentration of 3-5% of calf serum, the final concentration of 0.6-0.8% of cuttlefish serum, the final concentration of 0.8-1.2mg/ of twin antibiotic
100ml, is uniformly mixed, up to proprietary reagent.
Compared with prior art, the advantage of the invention is that：1）The separating obtained cell number of primary culture method of the present invention
Measure more, disinfection is thorough, damage when can reduce separation to embryo, and embryonic cell adherent growth amount is more, the success rate cultivated
High, time-to-live length；2）The cultural method can also make disinfectant through the micropore on egg membrane while disinfection so that carbonylation
Inactivated with the emplastic of sulfation, while egg membrane internal layer is disintegrated, weaken egg membrane and the viscous stickiness of embryo so that egg membrane
It is easier to separate, damage when reducing separation to embryo, prepares to improve the success rate of embryonic cell culture；3）It is of the invention former
Foster proprietary reagent of being commissioned to train can simulate the nutrient environment that embryonic cell is grown in vivo, meet embryonic cell to nutritional ingredient,
The demand of all multi-methods such as somatomedin, hormone, osmotic pressure and pH, promotes the growing multiplication of embryonic cell, while can be
Embryonic cell provides adherent and spreading factor so that a large amount of cells are migrated out from adherent embryonic tissue block and prolonged around
Spread dissipates.
Embodiment
The present invention program is described further below by embodiment：
Embodiment 1：
A kind of primary culture method of cuttlefish embryonic cell, including collect embryonated egg, obtain embryonic tissue and cell, embryonic cell
Original cuiture, specifically comprise the following steps：
1）Collect embryonated egg：The healthy cuttlefish parent of selection, by sex ration 1:1 temporarily supports in cement pit respectively, and inflation, shading will
Inkfish spawning base is put into pond collection embryonated egg, ocean temperature be 16 DEG C, salinity 31, pH 7.0, illuminance 13.1
Cultivated under conditions of lx, start to after laying eggs to take out embryonated egg in 50min in oviposition, be placed in glass flume and hatch, in sea
Coolant-temperature gage is 24 DEG C, salinity 29, pH cultivate 2d under conditions of being 8.6, continuous oxygenation, spare；
2）Obtain embryonic tissue and cell：Embryonated egg is sterilized, egg membrane is torn with tweezers, discharges embryo and yolk, super
Embryo is moved in culture dish in net workbench, is shredded, 3min is centrifuged under 1200r/min, obtains embryonic tissue precipitation, precipitation
The trypsin solution suspension embryonic tissue of 3 μ g/mL of middle addition, digests 5min at 30 DEG C, adds and trypsin solution
The proprietary reagent of isometric original cuiture, terminates Trypsin Induced, and 3min is centrifuged under 1200r/min, removes supernatant, obtains
Postdigestive embryonic tissue and cell precipitation are obtained, whole process uses sterile working；
3）The original cuiture of embryonic cell：With the proprietary reagent suspension embryonic tissue of original cuiture and cell precipitation, culture is transferred to
In plate, each hole handles 30 pieces of embryonated eggs, and proprietary reagent dosage is 100 μ l, is wrapped with Parafilm sealed membranes, then at 30 DEG C
Constant incubator in cultivate, the 2nd day, after tissue and cell attachment, replace fresh proprietary reagent, replaced every 5d once special
There is reagent, can obtain cuttlefish cell mass after 40d, whole process uses sterile working.
Sterilization method is in above-mentioned steps 1：It is successively 7.0 nothing with the alcohol solution dipping 20min, pH that concentration is 70%
Bacterium PBS solution is rinsed 6 times, soaks 20min, 70% second containing the sterile water of 0.62% twin antibiotic and 0.38% Ofloxacin
Alcoholic solution rinses 10s, the sterile PBS solution wash rinse that pH is 7.0 6 times.Contain 1.8% dextrorotation oxygen in above-mentioned Ofloxacin
Flucloxacillin.
A kind of proprietary reagent of the original cuiture of cuttlefish embryonic cell, specifically includes following components：Culture medium solution, methyl
Cellulose, D-Glucose, calf serum, cuttlefish serum, twin antibiotic.
A kind of proprietary reagent of the original cuiture of cuttlefish embryonic cell, its preparation method are：
Step 1：By isometric 770mM NaCl solutions, 15mM KCl solution, 100mM MgCl2Solution and 25mM CaCl2It is molten
Liquid is uniformly mixed, and then the membrane filtration with 0.22 μm is degerming, spare up to culture medium solution；
Step 2：Volume ratio is 1 by weight:75（mg/mL）Methylcellulose is added in culture medium solution, is stirred at 5 DEG C
3h, obtains solution A, spare；
Step 3：Volume ratio is 1 by weight:7.5（mg/mL）D-Glucose is dissolved in culture medium solution and obtains B solution, then B is molten
Liquid is 1 according to volume ratio:5 add in solution A, then add calf serum, cuttlefish serum and twin antibiotic so that small ox blood
Clear final concentration of 5%, cuttlefish serum final concentration of 0.6%, the final concentration of 1.2mg/100ml of twin antibiotic, is uniformly mixed, up to specially
There is reagent.
Embodiment 2：
A kind of primary culture method of cuttlefish embryonic cell, including collect embryonated egg, obtain embryonic tissue and cell, embryonic cell
Original cuiture, specifically comprise the following steps：
1）Collect embryonated egg：The healthy cuttlefish parent of selection, by sex ration 1:1 temporarily supports in cement pit respectively, and inflation, shading will
Inkfish spawning base is put into pond collection embryonated egg, ocean temperature be 20 DEG C, salinity 29, pH 8.0, illuminance be 4.1 lx
Under conditions of cultivate, start to after laying eggs to take out embryonated egg in 70min in oviposition, be placed in glass flume and hatch, in seawater
Temperature is 22 DEG C, salinity 31, pH cultivate 3d under conditions of being 7.8, continuous oxygenation, spare；
2）Obtain embryonic tissue and cell：Embryonated egg is sterilized, egg membrane is torn with tweezers, discharges embryo and yolk, super
Embryo is moved in culture dish in net workbench, is shredded, 5min is centrifuged under 1000r/min, obtains embryonic tissue precipitation, precipitation
The trypsin solution suspension embryonic tissue of 2 μ g/mL of middle addition, digests 3min at 40 DEG C, adds and trypsin solution
The proprietary reagent of isometric original cuiture, terminates Trypsin Induced, and 5min is centrifuged under 1000r/min, removes supernatant, obtains
Postdigestive embryonic tissue and cell precipitation are obtained, whole process uses sterile working；
3）The original cuiture of embryonic cell：With the proprietary reagent suspension embryonic tissue of original cuiture and cell precipitation, culture is transferred to
In plate, each hole handles 40 pieces of embryonated eggs, and proprietary reagent dosage is 150 μ l, is wrapped with Parafilm sealed membranes, then at 20 DEG C
Constant incubator in cultivate, the 2nd day, after tissue and cell attachment, replace fresh proprietary reagent, replaced every 8d once special
There is reagent, can obtain cuttlefish cell mass after 20d, whole process uses sterile working.
Sterilization method is in above-mentioned steps 1：It is successively 8.0 nothing with the alcohol solution dipping 10min, pH that concentration is 80%
Bacterium PBS solution is rinsed 4 times, soaks 40min, 80% second containing the sterile water of 0.72% twin antibiotic and 0.28% Ofloxacin
Alcoholic solution rinses 5s, the sterile PBS solution wash rinse that pH is 8.0 4 times.Contain 2.5% dextrorotation oxygen fluorine in above-mentioned Ofloxacin
Sha Xing.
A kind of proprietary reagent of the original cuiture of cuttlefish embryonic cell, specifically includes following components：Culture medium solution, methyl
Cellulose, D-Glucose, calf serum, cuttlefish serum, twin antibiotic.
A kind of proprietary reagent of the original cuiture of cuttlefish embryonic cell, its preparation method are：
Step 1：By isometric 790mM NaCl solutions, 14mM KCl solution, 110mM MgCl2Solution and 20mM CaCl2It is molten
Liquid is uniformly mixed, and then the membrane filtration with 0.22 μm is degerming, spare up to culture medium solution；
Step 2：Volume ratio is 1 by weight:85（mg/mL）Methylcellulose is added in culture medium solution, is stirred at 2 DEG C
4h, obtains solution A, spare；
Step 3：Volume ratio is 1 by weight:8.5（mg/mL）D-Glucose is dissolved in culture medium solution and obtains B solution, then B is molten
Liquid is 1 according to volume ratio:5 add in solution A, then add calf serum, cuttlefish serum and twin antibiotic so that small ox blood
Clear final concentration of 3%, cuttlefish serum final concentration of 0.8%, the final concentration of 0.8mg/100ml of twin antibiotic, is uniformly mixed, up to specially
There is reagent.
Embodiment 3：
A kind of primary culture method of cuttlefish embryonic cell, including collect embryonated egg, obtain embryonic tissue and cell, embryonic cell
Original cuiture, specifically comprise the following steps：
1）Collect embryonated egg：The healthy cuttlefish parent of selection, by sex ration 1:1 temporarily supports in cement pit respectively, and inflation, shading will
Inkfish spawning base is put into pond collection embryonated egg, ocean temperature be 18 DEG C, salinity 30, pH 7.5, illuminance be 8.5 lx
Under conditions of cultivate, start to after laying eggs to take out embryonated egg in 60min in oviposition, be placed in glass flume and hatch, in seawater
Temperature is 23 DEG C, salinity 30, pH cultivate 2.5d under conditions of being 8.2, continuous oxygenation, spare；
2）Obtain embryonic tissue and cell：It is with the alcohol solution dipping 15min, pH that concentration is 75% successively by 210 pieces of embryonated eggs
7.5 sterile PBS solution is rinsed 5 times, soaks 30min containing the sterile water of 0.67% twin antibiotic and 0.33% Ofloxacin,
The sterile PBS solution wash rinse that 75% ethanol solution rinsing 8s, pH is 7.5 5 times, then egg membrane is torn, discharge with tweezers
Go out embryo and yolk, move to embryo in culture dish in superclean bench, shred, 4min is centrifuged under 1100r/min, obtain
Embryonic tissue precipitates, and the trypsin solution suspension embryonic tissue of 2.5 μ g/mL is added in precipitation, 4min is digested at 35 DEG C, then
Addition and the proprietary reagent of the isometric original cuiture of trypsin solution, terminate Trypsin Induced, are centrifuged under 1100r/min
4min, removes supernatant, obtains postdigestive embryonic tissue and cell precipitation, whole process uses sterile working, above-mentioned oxygen fluorine
Sha Xingzhong contains 2.2% dextrorotation Ofloxacin；
3）The original cuiture of embryonic cell：With the proprietary reagent suspension embryonic tissue of original cuiture and cell precipitation, culture is transferred to
In plate, each hole handles 35 pieces of embryonated eggs, and proprietary reagent dosage is 120 μ l, is wrapped with Parafilm sealed membranes, then at 25 DEG C
Constant incubator in cultivate, the 2nd day, after tissue and cell attachment, fresh proprietary reagent is replaced, in 10 times of object lens after 3 days
Each visual field is averagely observed that 80 cell masses down, once proprietary reagent is replaced every 6d, in 10 times of object lens visuals field after 30d
Under visible substantial amounts of attached cell, cell mass overlapping phenomenon is more, is cuttlefish cell mass, whole process uses sterile working.
A kind of proprietary reagent of the original cuiture of cuttlefish embryonic cell, specifically includes following components：Culture medium solution, methyl
Cellulose, D-Glucose, calf serum, cuttlefish serum, twin antibiotic.
A kind of proprietary reagent of the original cuiture of cuttlefish embryonic cell, its preparation method are：
Step 1：By isometric 780mM NaCl, 14.2mM KCl, 108mM MgCl2With 21mM CaCl2It is uniformly mixed, so
Membrane filtration afterwards with 0.22 μm is degerming, spare up to culture medium solution；
Step 2：5mg methylcellulose is added in 400mL culture medium solutions, 3.5h is stirred at 4 DEG C, obtains solution A, it is spare；
Step 3：10mg D-Glucoses are dissolved in 80mL culture medium solutions and obtain B solution, then B are added in solution A, Ran Houzai
Add calf serum, cuttlefish serum and twin antibiotic so that calf serum final concentration of 4%, cuttlefish serum final concentration of 0.7%,
The final concentration of 1.0mg/100ml of twin antibiotic, is uniformly mixed, up to proprietary reagent.
Embodiment 4：
A kind of primary culture method of cuttlefish embryonic cell, including collect embryonated egg, obtain embryonic tissue and cell, embryonic cell
Original cuiture, specifically comprise the following steps：
1）Collect embryonated egg：The healthy cuttlefish parent of selection, by sex ration 1:1 temporarily supports in cement pit respectively, and inflation, shading will
Inkfish spawning base is put into pond collection embryonated egg, ocean temperature be 18 DEG C, salinity 30, pH 7.5, illuminance be 8.5 lx
Under conditions of cultivate, start to after laying eggs to take out embryonated egg in 60min in oviposition, be placed in glass flume and hatch, in seawater
Temperature is 23 DEG C, salinity 30, pH cultivate 2.5d under conditions of being 8.2, continuous oxygenation, spare；
2）Obtain embryonic tissue and cell：It is with the alcohol solution dipping 15min, pH that concentration is 75% successively by 210 pieces of embryonated eggs
7.5 sterile PBS solution is rinsed 5 times, soaks 30min containing the sterile water of 0.67% twin antibiotic and 0.33% Ofloxacin,
The sterile PBS solution wash rinse that 75% ethanol solution rinsing 8s, pH is 7.5 5 times, then egg membrane is torn, discharge with tweezers
Go out embryo and yolk, move to embryo in culture dish in superclean bench, shred, 4min is centrifuged under 1100r/min, obtain
Embryonic tissue precipitates, and the trypsin solution suspension embryonic tissue of 2.5 μ g/mL is added in precipitation, 4min is digested at 35 DEG C, then
Addition and the proprietary reagent of the isometric original cuiture of trypsin solution, terminate Trypsin Induced, are centrifuged under 1100r/min
4min, removes supernatant, obtains postdigestive embryonic tissue and cell precipitation, whole process uses sterile working, above-mentioned oxygen fluorine
Sha Xingzhong contains 2.2% dextrorotation Ofloxacin；
3）The original cuiture of embryonic cell：With the proprietary reagent suspension embryonic tissue of original cuiture and cell precipitation, culture is transferred to
In plate, each hole handles 35 pieces of embryonated eggs, and proprietary reagent dosage is 120 μ l, is wrapped with Parafilm sealed membranes, then at 25 DEG C
Constant incubator in cultivate, the 2nd day, after tissue and cell attachment, fresh proprietary reagent is replaced, in 10 times of object lens after 3 days
Each visual field is averagely observed that 84 cell masses down, once proprietary reagent is replaced every 6d, in 10 times of object lens visuals field after 30d
Under visible substantial amounts of attached cell, cell mass overlapping phenomenon is more, is cuttlefish cell mass, whole process uses sterile working.
A kind of proprietary reagent of the original cuiture of cuttlefish embryonic cell, specifically includes following components：Culture medium solution, methyl
Cellulose, D-Glucose, calf serum, cuttlefish serum, twin antibiotic.
A kind of proprietary reagent of the original cuiture of cuttlefish embryonic cell, its preparation method are：
Step 1：By isometric 780mM NaCl, 14.2mM KCl, 108mM MgCl2With 21mM CaCl2It is uniformly mixed, so
Membrane filtration afterwards with 0.22 μm is degerming, spare up to culture medium solution；
Step 2：5mg methylcellulose is added in 400mL culture medium solutions, 3.5h is stirred at 4 DEG C, obtains solution A, it is spare；
Step 3：10mg D-Glucoses are dissolved in 80mL culture medium solutions and obtain B solution, then B are added in solution A, Ran Houzai
Add calf serum, cuttlefish serum, ancitabine and twin antibiotic so that calf serum final concentration of 4%, cuttlefish serum is dense eventually
Spend for 0.7%, the final concentration of 0.24mg/100ml of ancitabine, the final concentration of 1.0mg/100ml of twin antibiotic, be uniformly mixed, i.e.,
Proprietary reagent is obtained, ancitabine can not only accelerate the division of embryonic cell, shorten the cell culture time, while can stimulate thin
Born of the same parents itself more secrete growth factor, and action time is longer, improve cell division and growth, it may have certain maintenance is thin
The effect of born of the same parents' initial configuration, improves cell algebraically.
Embodiment 5：
A kind of primary culture method of cuttlefish embryonic cell, including collect embryonated egg, obtain embryonic tissue and cell, embryonic cell
Original cuiture, specifically comprise the following steps：
1）Collect embryonated egg：The healthy cuttlefish parent of selection, by sex ration 1:1 temporarily supports in cement pit respectively, and inflation, shading will
Inkfish spawning base is put into pond collection embryonated egg, ocean temperature be 18 DEG C, salinity 30, pH 7.5, illuminance be 8.5 lx
Under conditions of cultivate, start to after laying eggs to take out embryonated egg in 60min in oviposition, be placed in glass flume and hatch, in seawater
Temperature is 23 DEG C, salinity 30, pH cultivate 2.5d under conditions of being 8.2, continuous oxygenation, spare；
2）Obtain embryonic tissue and cell：It is with the alcohol solution dipping 15min, pH that concentration is 75% successively by 210 pieces of embryonated eggs
7.5 sterile PBS solution is rinsed 5 times, soaks 30min containing the sterile water of 0.67% twin antibiotic and 0.33% Ofloxacin,
The sterile PBS solution wash rinse that 75% ethanol solution rinsing 8s, pH is 7.5 5 times, then egg membrane is torn, discharge with tweezers
Go out embryo and yolk, move to embryo in culture dish in superclean bench, shred, 4min is centrifuged under 1100r/min, obtain
Embryonic tissue precipitates, and the trypsin solution suspension embryonic tissue of 2.5 μ g/mL is added in precipitation, 4min is digested at 35 DEG C, then
The common cultivate reagent isometric with trypsin solution is added, Trypsin Induced is terminated, is centrifuged under 1100r/min
4min, removes supernatant, obtains postdigestive embryonic tissue and cell precipitation, whole process uses sterile working, above-mentioned oxygen fluorine
Sha Xingzhong contains 2.2% dextrorotation Ofloxacin；
3）The original cuiture of embryonic cell：With common cultivate reagent suspension embryonic tissue and cell precipitation, it is transferred in culture plate,
Each hole handles 35 pieces of embryonated eggs, and it is 120 μ l to commonly use cultivate reagent dosage, is wrapped with Parafilm sealed membranes, then at 25 DEG C
Constant incubator in cultivate, the 2nd day, after tissue and cell attachment, fresh common cultivate reagent is replaced, at 10 times after 3 days
Each visual field is averagely observed that 48 cell masses under object lens, is replaced every 6d and once specially commonly uses cultivate reagent, 10 after 30d
Visible substantial amounts of attached cell under times object lens visual field, but cell mass it is overlapping feelings it is less, illustrate training of the embodiment 3 with proprietary reagent
Effect is supported far better than common cultivate reagent.
Routine operation in the operating procedure of the present invention is well known to those skilled in the art, herein without repeating.
Technical scheme is described in detail in embodiment described above, it should be understood that the above is only
For the specific embodiment of the present invention, it is not intended to limit the invention, all any modifications made in the spirit of the present invention,
Supplement or similar fashion replacement etc., should all be included in the protection scope of the present invention.
Claims (9)
1. a kind of primary culture method of cuttlefish embryonic cell, including collect embryonated egg, obtain embryonic tissue and cell, embryo are thin
The original cuiture of born of the same parents, it is characterised in that：The acquisition embryonic tissue and cell step are：Embryonated egg is sterilized, with tweezers by ovum
Film is torn, and discharges embryo and yolk, embryo is moved in culture dish, shred, and is centrifuged, and is obtained embryonic tissue and is precipitated, in precipitation
Add trypsin solution suspension embryonic tissue to be digested, add the proprietary reagent of original cuiture and terminate Trypsin Induced,
Centrifugation, obtains postdigestive embryonic tissue and cell precipitation.
A kind of 2. primary culture method of cuttlefish embryonic cell according to claim 1, it is characterised in that：The acquisition embryo
The sterilization method of embryonated egg is in tire tissue and cell step：The alcohol solution dipping 10- for being successively 70-80% with concentration
The sterile PBS solution that 20min, pH are 7.0-8.0 is rinsed 4-6 times, twin antibiotic and 0.28-0.38% containing 0.62-0.72%
Ethanol solution the rinsing 5-10s, the sterile PBS that pH is 7.0-8.0 of sterile water the immersion 20-40min, 70-80% of Ofloxacin are molten
Liquid wash rinse 4-6 times.
A kind of 3. primary culture method of cuttlefish embryonic cell according to claim 2, it is characterised in that：The oxygen fluorine is husky
Dextrorotation Ofloxacin containing 1.8-2.5% in star.
A kind of 4. primary culture method of cuttlefish embryonic cell according to claim 1, it is characterised in that：The tryptose
The addition of enzyme is 2-3 μ g/mL, and digestion temperature is 30-40 DEG C, time 3-5min.
A kind of 5. primary culture method of cuttlefish embryonic cell according to claim 1, it is characterised in that：The collection
Embryonated egg step is：The healthy cuttlefish parent of selection, by sex ration 1:1 temporarily supports in pond respectively, and inflation, shading, cuttlefish is produced
Ovum base is put into pond collection embryonated egg, ocean temperature be 16-20 DEG C, salinity 29-31, pH 7.0-8.0, illuminance be
Cultivated under conditions of 4.1-13.1 lx, start to after laying eggs to take out embryonated egg in 50-70min in oviposition, be placed in glass flume
Middle hatching, 2-3d is cultivated under conditions of ocean temperature is 22-24 DEG C, salinity 29-31, pH is 7.8-8.6, continuous oxygenation,
It is spare.
A kind of 6. primary culture method of cuttlefish embryonic cell according to claim 1, it is characterised in that：The embryo is thin
The original cuiture step of born of the same parents is：With the proprietary reagent suspension embryonic tissue of original cuiture and cell precipitation, it is transferred in culture plate, uses
Parafilm sealed membranes are wrapped, and are then cultivated in 20-30 DEG C of constant incubator, the 2nd day, after tissue and cell attachment,
Proprietary reagent, and visual cell's growing state are replaced, replaces once proprietary reagent every 5-8d, it is thin to can obtain cuttlefish after 20-40d
Born of the same parents group.
A kind of 7. primary culture method of cuttlefish embryonic cell according to claim 6, it is characterised in that：The embryo is thin
Each hole handles 30-40 pieces of embryonated egg in the original cuiture step of born of the same parents, and proprietary reagent dosage is 100-150 μ l.
A kind of 8. proprietary reagent of the original cuiture of cuttlefish embryonic cell, it is characterised in that：The proprietary reagent is included with the following group
Point：Culture medium solution, methylcellulose, D-Glucose, calf serum, cuttlefish serum, twin antibiotic.
A kind of 9. proprietary reagent of the original cuiture of cuttlefish embryonic cell according to claim 8, it is characterised in that：It is described
The preparation method of proprietary reagent is：
1）By isometric 770-790mM NaCl solutions, 14-15mM KCl solution, 100-110mM MgCl2Solution and 20-
25mM CaCl2Solution is uniformly mixed, and then the membrane filtration with 0.22 μm is degerming, spare up to culture medium solution；
2）Volume ratio is 1 by weight:75-85（mg/mL）Methylcellulose is added in culture medium solution, is stirred at 2-5 DEG C
3-4h, obtains solution A, spare；
3）Volume ratio is 1 by weight:7.5-8.5（mg/mL）D-Glucose is dissolved in culture medium solution and obtains B solution, then B is molten
Liquid is 1 according to volume ratio:5 add in solution A, then add calf serum, cuttlefish serum and twin antibiotic so that small ox blood
Clear final concentration of 3-5%, the final concentration of 0.6-0.8% of cuttlefish serum, the final concentration of 0.8-1.2mg/100ml of twin antibiotic, mixing
Uniformly, up to proprietary reagent.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110055213A (en) * | 2019-04-23 | 2019-07-26 | 中国海洋大学 | A kind of separation method of dwarf clam egg membrane |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1737130A (en) * | 2005-07-12 | 2006-02-22 | 中国水产科学研究院黄海水产研究所 | Lateolabrax japonicus embryo stem cell and cultural method |
| AU2007283564A1 (en) * | 2006-08-09 | 2008-02-14 | Valneva | Method of production of transgenic avian using embryonic stem cells |
| CN104059875A (en) * | 2014-04-28 | 2014-09-24 | 浙江省淡水水产研究所 | Primary culture method of embryonic cell tissue pieces of Chinese softshell turtle |
| CN105483075A (en) * | 2015-11-19 | 2016-04-13 | 中国海洋大学 | Culture medium and method for culturing urechis unicinctus trochophore cell line |
| CN105754928A (en) * | 2016-05-16 | 2016-07-13 | 中国海洋大学 | Technology for dissociating and culturing prawn embryonic cells |
| CN106916779A (en) * | 2017-03-24 | 2017-07-04 | 厦门医学院 | A kind of sea mollusk primary cell separation and cultivate reagent box and application thereof |
-
2017
- 2017-12-14 CN CN201711339117.2A patent/CN108004197B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1737130A (en) * | 2005-07-12 | 2006-02-22 | 中国水产科学研究院黄海水产研究所 | Lateolabrax japonicus embryo stem cell and cultural method |
| AU2007283564A1 (en) * | 2006-08-09 | 2008-02-14 | Valneva | Method of production of transgenic avian using embryonic stem cells |
| CN104059875A (en) * | 2014-04-28 | 2014-09-24 | 浙江省淡水水产研究所 | Primary culture method of embryonic cell tissue pieces of Chinese softshell turtle |
| CN105483075A (en) * | 2015-11-19 | 2016-04-13 | 中国海洋大学 | Culture medium and method for culturing urechis unicinctus trochophore cell line |
| CN105754928A (en) * | 2016-05-16 | 2016-07-13 | 中国海洋大学 | Technology for dissociating and culturing prawn embryonic cells |
| CN106916779A (en) * | 2017-03-24 | 2017-07-04 | 厦门医学院 | A kind of sea mollusk primary cell separation and cultivate reagent box and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 蒋霞敏 等: "曼氏无针乌贼胚胎发育形态及发育时序的研究", 《宁波大学学报(理工版)》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110055213A (en) * | 2019-04-23 | 2019-07-26 | 中国海洋大学 | A kind of separation method of dwarf clam egg membrane |
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