CN105349484B - A kind of construction method of rhinoceros horn gold thread Barb heart cell system - Google Patents
A kind of construction method of rhinoceros horn gold thread Barb heart cell system Download PDFInfo
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Abstract
The present invention relates to a kind of construction methods of rhinoceros horn gold thread Barb heart cell system, and this method comprises the following steps: 1) acquisition of rhinoceros horn gold thread Barb heart tissue: being combined using penicillin and alcohol and carried out disinfection to rhinoceros horn gold thread Barb；2) originally culture: using glutamine, penicillin, streptomysin, streptomycin sulphate, anphotericin is contained, the M199 culture solution for the 280-330mOsm/l that osmotic pressure is is cultivated；3) secondary culture: when reaching for 10 generation, cell culture fluid changes basic culture solution into, and rhinoceros horn gold thread Barb heart cell system is successfully established.Construction method of the invention rhinoceros horn gold thread Barb heart cell system form obtained is into fiber-like, continuous passage and biological characteristic research can be directly applied to, not only it is able to satisfy the needs to this rareness species Germ-plasma resources protection and theoretical research of rhinoceros horn gold thread Barb, simultaneously it is also the first heart cell system of Chinese Cave Fish, lays a good foundation for the cave adaptation Journal of Sex Research of cellular level.
Description
Technical field
The present invention relates to a kind of methods for establishing cell line using rhinoceros horn gold thread Barb heart tissue, belong to fresh-water aquatic organisms
The technical field of cell culture and Ultra-cryofreezing preservation.
Background technique
Rhinoceros horn gold thread Barb Sinocyclocheilus rhinocerous (Regan, 1904) is under the jurisdiction of Cypriniformes
(Cypriniformes) Cyprinidae (Cyprinidae) gold thread Barb belongs to (Sinocyclocheilus), is the hole delivered for 1994
Cave novel species belongs to Nanpanjiang River water system, grinds to it currently, distributed point known to this kind is the subterranean stream in belt township, county, Luoping of Yunnan
Study carefully the confirmation and its Phylogenetic Relationships of the description, classification position that are also detected in its characteristic of division.
It because rhinoceros horn gold thread Barb population quantity is smaller, and is not easy to obtain, therefore, how to protect and save this rare cave fish
The germ plasm resource of class becomes urgent problem to be solved now.The pool is supported under environment, and rhinoceros horn gold thread Barb can't realize the normal of sexual gland
Reach maturity, realizing further artificial propagation, there is also certain difficulties, therefore, by save its sperm, ovum, embryo,
Individual etc. is infeasible come the method for saving its germ plasm resource, so inventor has turned one's attention to the body cell of rhinoceros horn gold thread Barb
Culture.
Rhinoceros horn gold thread Barb moves in groundwater environment and shows a series of compliance characteristics, for example eyes move back
Change, the reduction of scale quantity, sensory system it is highly developed etc., but it again unlike peace water gold thread Barb, certain features are degenerated
Very thoroughly, it is a transitional type of the Cave Fish to cave adaptation, therefore is indispensable in cave adaptation Journal of Sex Research
Research object, and the success of cell culture is to explore rhinoceros horn gold thread Barb on a cellular level to the adaptability machine of groundwater environment
System is laid a good foundation.
Fish cell culture arises from the 1960s, being developed so far and more than 280 strain cell lines, Chinese fish has had been established
Cell culture only established more than 50 strain cell lines after more than 30 years, established the species deficiency Chinese Fishes sum of cell line
1.5% (Chinese Fishes are more than 3500 kinds), it can be seen that, the building speed of cell line is slowly that attention rate is inadequate, joins
With worker be less likely to be one of its reason；Secondly, being easier in fish cell culture compared to mammaliancellculture
It polluting, the rate that leads to the failure rises, and it is unqualified that this phenomenon is attributed to technology mostly in existing patent or article, and
Dislike the quality problems for focusing on fish body itself less, in rhinoceros horn gold thread Barb cell culture practice, the animation of fish body also direct shadow
The success or failure of cell line building are rung；In addition, because of the limitation of rhinoceros horn gold thread Barb outdoor habitatss, to the understanding of its life habit compared with
Few, this also increases the difficulty of rhinoceros horn gold thread Barb cell culture to a certain extent.Therefore, rhinoceros horn gold thread Barb heart cell system
Success, which constructs, to be built upon on the basis of the feeding ecological habit understanding in wild to the rhinoceros horn gold thread Barb for a long time and pool, meanwhile, rhinoceros horn gold thread
Barb is the first Cave Fish for realizing heart cell culture of China, this cell line is also the first heart cell of Chinese Cave Fish
System.By literature search, it has no and identical report of the invention.
Summary of the invention
The object of the present invention is to provide a kind of construction method of rhinoceros horn gold thread Barb heart cell system easy to operation, with
Meet the needs to rhinoceros horn gold thread Barb cave adaptation Journal of Sex Research, quick karyotyping and Germ-plasma resources protection.
Specifically, according to an aspect of the invention, there is provided a kind of construction method of rhinoceros horn gold thread Barb heart cell system,
The construction method includes the following steps: the acquisition of 1) rhinoceros horn gold thread Barb heart: first using the penicillin soaking disinfection rhinoceros horn of 50IU/l
Gold thread Barb fish body 20-30 minutes, then rhinoceros horn gold thread Barb is anaesthetized with the MS-222 of 80-120ppm；Again with 75% alcohol wipe fish body
Afterwards, its heart is taken, PBS medicining liquid dipping heart tissue is added；It is after impregnating 15-30 minutes, heart tissue is clear with PBS thimerosal
It washes；2) originally culture: it will be cut into tissue fritter by the heart tissue 1) obtained, and is inoculated in 25cm2Tissue Culture Flask
In, it is inverted overnight in 16-20 DEG C of incubator, next day adds originally culture liquid, starts primary training in 16-20 DEG C of incubator
It supports, half amount replacement culture solution, the 6-8 days cells start to migrate from tissue block every three days, grow up to cell monolayer within 30-40 days；3)
Secondary culture: primary cardiac cell starts to pass on after being paved with bottom of bottle 80%, the originally culture liquid in culture bottle is sucked out, first to contain
Cell has been hanged in the trypsin digestion passage of the trypsase of 0.1-0.2%, and it is anti-to neutralize pancreatin that secondary culture liquid 1-2ml is added
It answers, passage is passed by 1:1 for the first time, is hereafter passed on by 1:2, continues to cultivate in 16-20 DEG C of incubator；Passage one in every 4-6 days
Secondary, when reaching for 10 generation, cell culture fluid changes basic culture solution into, and rhinoceros horn gold thread Barb heart cell system is successfully established.
Further, the PBS thimerosal be containing concentration be 100-300IU/ml penicillin, concentration be 100-300 μ
The amphotericin B that the streptomycin sulphate and concentration that the streptomysin of g/ml, concentration are 20-40 μ g/ml are 20-40 μ g/ml.
Further, the basic culture solution is containing the glutamine that concentration is 300-400 μ g/ml and to account for total volume
10-15% fetal calf serum M199 culture solution.
Further, the originally culture liquid is containing the glutamine that concentration is 600-800 μ g/ml, accounts for total volume
The fetal calf serum of the 15-25%, penicillin that concentration is 100-200IU/ml, the streptomysin that concentration is 100-300 μ g/ml, dense
The M199 culture solution for the amphotericin B that the streptomycin sulphate and concentration that degree is 20-40 μ g/ml are 20-40 μ g/ml.
Further, the secondary culture liquid is containing the glutamine that concentration is 500-600 μ g/ml, accounts for total volume
10-20% fetal calf serum, the kanamycin sulfate that concentration is 20-40 μ g/ml and the both sexes that concentration is 20-40 μ g/ml
The M199 culture solution of mycin B.
Further, the pH value of the originally culture liquid, the secondary culture liquid and the basic culture solution is 7.0-
7.2。
Further, the osmotic pressure of the originally culture liquid, the secondary culture liquid and the basic culture solution is 280-
330mOsm/l。
Further, the construction method further includes the steps that following cell cryopreservation and recovery: 1) prepare cells frozen storing liquid:
Frozen stock solution includes M199 culture solution, fetal calf serum and DMSO, is mixed by the volume ratio of 4:5:1, matching while using；2) cell cryopreservation:
The cell of logarithmic growth phase, after the digestion of above-mentioned pancreatin, cell suspension 800-1200rpm is centrifuged 6-10 minutes, discards supernatant
Liquid；The cells frozen storing liquid of preparation is added into cell precipitation, is resuspended, making the concentration of cell is about 1 × 106/ml, and 1ml is thin
Born of the same parents' suspension is transferred in 1.8ml cryopreservation tube, and cryopreservation tube is placed in program temperature reduction box, -80 DEG C placement 24-48 hours, then put
Enter in liquid nitrogen and saves for a long time；3) cell recovery: above-mentioned cryopreservation tube is removed from liquid nitrogen, and is put into 37 DEG C of water-baths and is quickly rocked
To thawing；Then in aseptic operating platform, defrosting cell is transferred in 15ml centrifuge tube, and equivalent basic culture solution is added,
800-1000rpm is centrifuged 6-8 minutes, removes supernatant；Cell is resuspended with basic culture solution, and is transferred in Tissue Culture Flask, 16-
It is cultivated in 20 DEG C of incubators, cell grows up to single layer within 6-9 days.
Percentage used in each step of the invention is percent by volume.
Remarkable result of the invention is:
1. construction method of the invention is easy to operation, and construction method has repeatability, and the cell line of building is steady
It is qualitative good.
2. relative to the blood Short-term Culture for karyotyping, using the heart tissue of construction method acquisition of the invention
The cell just migrated out in block is active, and cell concentration is big, can be used for chromosome analysis.
3. compared with fin ray cell line, with tissue block in the heart cell system originally culture of construction method acquisition of the invention
Pollution rate is lower, to improve success rate.
4. rhinoceros horn gold thread Barb heart cell system's form that the present invention constructs is that continuous passage and can directly answer at fiber-like
For biological characteristic research, the needs to rhinoceros horn gold thread Barb Germ-plasma resources protection and theoretical research and application are met.
5. being the first heart of Chinese Cave Fish with the rhinoceros horn gold thread Barb heart cell that construction method of the invention obtains
Cell line provides good material to the adaptability of cave environment for research Cave Fish, has established cave adaptation Journal of Sex Research
Basis.
Detailed description of the invention
Fig. 1 is the figure for showing influence of the different disinfection way to rhinoceros horn gold thread Barb heart tissue block cell migration.
Fig. 2 is the figure for showing the influence that different culture solutions grow rhinoceros horn gold thread Barb heart cell.
Specific embodiment
In from detailed description below, aforementioned aspect of the present invention and other aspects of the present invention be will be apparent.
Embodiment 1
1. preparing PBS thimerosal and cell culture fluid
PBS thimerosal: antibiotic being added into PBS, makes the concentration 100IU/ml of penicillin, and streptomysin concentration is 100 μ
G/ml, the concentration of streptomycin sulphate are 40 μ g/ml, and amphotericin B concentration is 40 μ g/ml.
Basic culture solution: glutamine and fetal calf serum are added into M199 culture solution, makes the concentration 300 of glutamine
μ g/ml, fetal calf serum of the total volume 15%.
Originally culture liquid: glutamine, fetal calf serum and antibiotic are added into M199 culture solution, makes the dense of glutamine
Degree is 600 μ g/ml, and fetal calf serum of the total volume 25%, the concentration of penicillin is 100IU/ml, and streptomysin concentration is 100 μ g/
Ml, the concentration of streptomycin sulphate are 40 μ g/ml, and amphotericin B concentration is 40 μ g/ml.
Secondary culture liquid: glutamine, fetal calf serum and antibiotic are added into M199 culture solution, makes the dense of glutamine
Degree is 500 μ g/ml, and the concentration of fetal calf serum of the total volume 20%, streptomycin sulphate is 20 μ g/ml, and amphotericin B concentration is
20μg/ml。
The pH value for adjusting above-mentioned culture solution is 7.0, osmotic pressure 280mOsm/l, is placed in 4 DEG C of refrigerators and saves backup.
Wherein, different from mycillin, streptomycin sulphate is a kind of aminoglycoside antibiotics, to many Grain-negative bars
Bacterium has stronger antibacterial action, this antimicrobial spectrum is similar to gentamicin, and in the cell culture of rhinoceros horn gold thread Barb, celebrating is big by contrast
The influence of mycin and streptomycin sulphate to cell, discovery rhinoceros horn gold thread Barb heart cell is more sensitive to streptomycin sulphate (being shown in Table 1),
So selecting streptomycin sulphate thimerosal and culture solution is added in the present embodiment.In addition, it is similar to the dual anti-antimicrobial spectrum of green chain,
Kanamycin sulfate mainly acts on Gram-positive and negative bacteria, but rhinoceros horn gold thread Barb heart cell is to kanamycin sulfate
Sensibility it is higher, therefore, in the secondary culture of rhinoceros horn gold thread Barb heart cell of the invention, using kanamycin sulfate generation
Replace the green chain in originally culture dual anti-.
The influence that 1 gentamicin of table and streptomycin sulphate move out to rhinoceros horn gold thread Barb tissue block cell
In addition, since rhinoceros horn gold thread Barb is Cave Fish, immunity is lower, therefore, in the cell culture of rhinoceros horn gold thread Barb
Each stage culture solution in joined glutamine, with improve its immunity reduce tissue block pollution rate.
2. originally culture
Penicillin soaking disinfection rhinoceros horn gold thread Barb fish body 20 minutes for first using 50IU/l, then anaesthetized with the MS-222 of 80ppm
Rhinoceros horn gold thread Barb.Again with 75% alcohol wipe fish body after, be placed in superclean bench, take its heart with sterile disscting instrument, set
In 12 orifice plates, PBS medicining liquid dipping heart tissue is added.After impregnating 30 minutes, heart tissue is cleaned 5 with PBS thimerosal
It is secondary, it is cut into tissue fritter, and be inoculated in 25cm2In Tissue Culture Flask, it is inverted overnight in 16 DEG C of incubators, next day adds again
Enter originally culture liquid 3ml, start originally culture in 16 DEG C of incubators, every three days half amount replacement culture solution, cell starts within the 9th day
It is migrated from tissue block, grows up to cell monolayer within 40 days.
Wherein, since rhinoceros horn gold thread Barb is lower to the tolerance that green chain is dual anti-, if using high dual anti-disinfection treatment, rhinoceros horn
Gold thread Barb heart cell tissue block can not move out cell.Therefore, the penicillin soaking disinfection of 50IU/L is used only in the present embodiment.
3. secondary culture
Primary cardiac cell starts to pass on after being paved with bottom of bottle 80%.The specific method is as follows for secondary culture: culture bottle is first sucked out
In originally culture liquid, be added 0.1% trypsin-EDTA solutions 2ml, digest 3 minutes, after cell rounding, passage is added
Culture solution 2ml gently blows and beats bottom of bottle, attached cell is made to fall off to neutralize pancreatin reaction, and passage is passed by 1:1 for the first time, hereafter presses 1:2
It is passed on, continues to cultivate in 16 DEG C of incubators；Passage in every 6 days is primary, and when reaching for 10 generation, cell culture fluid changes basis into
Culture solution, at this point, Establishment of Cell Line success.Currently, cell reached for 30 generations.
4. cell cryopreservation and recovery
(a) prepare cell cryopreservation and protect liquid: frozen stock solution includes M199 culture solution, fetal calf serum and DMSO, by the body of 4:5:1
Product is than mixing, matching while using.
(b) cell cryopreservation: the cell of logarithmic growth phase, after the digestion of above-mentioned pancreatin, cell suspension 800rpm centrifugation 10
Minute, discard supernatant.The cells frozen storing liquid of preparation is added into cell precipitation, is resuspended, making the concentration of cell is about 1 × 106
1ml cell suspension is transferred in 1.8ml cryopreservation tube, cryopreservation tube is placed in program temperature reduction box by a/ml, and -80 DEG C of placements 24 are small
When, it is then placed in liquid nitrogen and saves for a long time.
(c) cell recovery: above-mentioned cryopreservation tube is removed from liquid nitrogen, and is put into 37 DEG C of water-baths and is quickly rocked to thawing.
Then in aseptic operating platform, defrosting cell is transferred in 15ml centrifuge tube, and is added equivalent basic culture solution, 800rpm from
The heart 8 minutes, remove supernatant.Cell is resuspended with basic culture solution, and is transferred in Tissue Culture Flask, is cultivated in 16 DEG C of incubators, 9
Its cell grows up to single layer.
Embodiment 2
1. preparing PBS thimerosal and cell culture fluid
PBS thimerosal: antibiotic being added into PBS, makes the concentration 300IU/ml of penicillin, and streptomysin concentration is 300 μ
G/ml, the concentration of streptomycin sulphate are 20 μ g/ml, and amphotericin B concentration is 20 μ g/ml.
Basic culture solution: glutamine and fetal calf serum are added into M199 culture solution, makes the concentration 400 of glutamine
μ g/ml, fetal calf serum of the total volume 10%.
Originally culture liquid: glutamine, fetal calf serum and antibiotic are added into M199 culture solution, makes the dense of glutamine
Degree is 800 μ g/ml, and fetal calf serum of the total volume 15%, the concentration of penicillin is 200IU/ml, and streptomysin concentration is 30 μ g/
Ml, the concentration of streptomycin sulphate are 20 μ g/ml, and amphotericin B concentration is 20 μ g/ml.
Secondary culture liquid: glutamine, fetal calf serum and antibiotic are added into M199 culture solution, makes the dense of glutamine
Degree is 600 μ g/ml, and the concentration of fetal calf serum of the total volume 10%, streptomycin sulphate is 40 μ g/ml, and amphotericin B concentration is
40μg/ml。
The pH value for adjusting above-mentioned culture solution is 7.0, osmotic pressure 300mOsm/l, is placed in 4 DEG C of refrigerators and saves backup.
2. originally culture
Penicillin soaking disinfection rhinoceros horn gold thread Barb fish body 30 minutes for first using 50IU/l, then anaesthetized with the MS-222 of 120ppm
Rhinoceros horn gold thread Barb.Again with 75% alcohol wipe fish body after, be placed in superclean bench, take its heart with sterile disscting instrument, set
In 12 orifice plates, PBS medicining liquid dipping heart tissue is added.After impregnating 15 minutes, heart tissue is cleaned 3 with PBS thimerosal
It is secondary, it is cut into tissue fritter, and be inoculated in 25cm2In Tissue Culture Flask, it is inverted overnight in 20 DEG C of incubators, next day adds again
Enter originally culture liquid 5ml, start originally culture in 20 DEG C of incubators, every three days half amount replacement culture solution, cell starts within the 6th day
It is migrated from tissue block, grows up to cell monolayer within 30 days.
3. secondary culture
Primary cardiac cell starts to pass on after being paved with bottom of bottle 80%.The specific method is as follows for secondary culture: culture bottle is first sucked out
In originally culture liquid, be added 0.2% trypsin-EDTA solutions 1ml, digest 2 minutes, after cell rounding, passage is added
Culture solution 1.5ml gently blows and beats bottom of bottle, attached cell is made to fall off to neutralize pancreatin reaction, and passage is passed by 1:1 for the first time, is hereafter pressed
1:2 is passed on, and continues to cultivate in 20 DEG C of incubators；Passage in every 4 days is primary, and when reaching for 10 generation, cell culture fluid is changed into
Basic culture solution, at this point, Establishment of Cell Line success.Currently, cell reached for 30 generations.
4. cell cryopreservation and recovery
(a) prepare cell cryopreservation and protect liquid: frozen stock solution includes M199 culture solution, fetal calf serum and DMSO, by the body of 4:5:1
Product is than mixing, matching while using.
(b) cell cryopreservation: the cell of logarithmic growth phase, after the digestion of above-mentioned pancreatin, cell suspension 1200rpm centrifugation 6
Minute, discard supernatant.The cells frozen storing liquid of preparation is added into cell precipitation, is resuspended, making the concentration of cell is about 1 × 106
1ml cell suspension is transferred in 1.8ml cryopreservation tube, cryopreservation tube is placed in program temperature reduction box by a/ml, and -80 DEG C of placements 48 are small
When, it is then placed in liquid nitrogen and saves for a long time.
(c) cell recovery: above-mentioned cryopreservation tube is removed from liquid nitrogen, and is put into 37 DEG C of water-baths and is quickly rocked to thawing.
Then in aseptic operating platform, defrosting cell is transferred in 15ml centrifuge tube, and equivalent basic culture solution, 1000rpm is added
Centrifugation 6 minutes removes supernatant.Cell is resuspended with basic culture solution, and is transferred in Tissue Culture Flask, is trained in 20 DEG C of incubators
It supports, cell grows up to single layer within 6 days.
Embodiment 3
1. preparing PBS thimerosal and cell culture fluid
PBS thimerosal: antibiotic being added into PBS, makes the concentration 300IU/ml of penicillin, and streptomysin concentration is 300 μ
G/ml, the concentration of streptomycin sulphate are 20 μ g/ml, and amphotericin B concentration is 20 μ g/ml.
Basic culture solution: glutamine and fetal calf serum are added into M199 culture solution, makes the concentration 400 of glutamine
μ g/ml, fetal calf serum of the total volume 10%.
Originally culture liquid: glutamine, fetal calf serum and antibiotic are added into M199 culture solution, makes the dense of glutamine
Degree is 800 μ g/ml, and fetal calf serum of the total volume 15%, the concentration of penicillin is 200IU/ml, and streptomysin concentration is 30 μ g/
Ml, the concentration of streptomycin sulphate are 20 μ g/ml, and amphotericin B concentration is 20 μ g/ml.
Secondary culture liquid: glutamine, fetal calf serum and antibiotic are added into M199 culture solution, makes the dense of glutamine
Degree is 600 μ g/ml, and the concentration of fetal calf serum of the total volume 10%, streptomycin sulphate is 40 μ g/ml, and amphotericin B concentration is
40μg/ml。
The pH value for adjusting above-mentioned culture solution is 7.0, osmotic pressure 300mOsm/l, is placed in 4 DEG C of refrigerators and saves backup.
2. originally culture
Penicillin soaking disinfection rhinoceros horn gold thread Barb fish body 25 minutes for first using 50IU/l, then anaesthetized with the MS-222 of 100ppm
Rhinoceros horn gold thread Barb.Again with 75% alcohol wipe fish body after, be placed in superclean bench, take its heart with sterile disscting instrument, set
In 12 orifice plates, PBS medicining liquid dipping heart tissue is added.It impregnates after twenty minutes, heart tissue is cleaned 4 with PBS thimerosal
It is secondary, it is cut into tissue fritter, and be inoculated in 25cm2In Tissue Culture Flask, it is inverted overnight in 18 DEG C of incubators, next day adds again
Enter originally culture liquid 4ml, start originally culture in 18 DEG C of incubators, every three days half amount replacement culture solution, cell starts within the 8th day
It is migrated from tissue block, grows up to cell monolayer within 35 days.
3. secondary culture
Primary cardiac cell starts to pass on after being paved with bottom of bottle 80%.The specific method is as follows for secondary culture, and culture bottle is first sucked out
In originally culture liquid, be added 0.15% trypsin-EDTA solutions 1.5ml, digest 2 minutes, after cell rounding, be added and pass
Nutrient solution of being commissioned to train 1.5ml gently blows and beats bottom of bottle, attached cell is made to fall off, passage is passed by 1:1 for the first time, hereafter to neutralize pancreatin reaction
It is passed on by 1:2, continues to cultivate in 18 DEG C of incubators；Passage in every 5 days is primary, and when reaching for 10 generation, cell culture fluid is changed
At basic culture solution, at this point, Establishment of Cell Line success.Currently, cell reached for 30 generations.
4. cell cryopreservation and recovery
(a) prepare cell cryopreservation and protect liquid: frozen stock solution includes M199 culture solution, fetal calf serum and DMSO, by the body of 4:5:1
Product is than mixing, matching while using.
(b) cell cryopreservation: the cell of logarithmic growth phase, after the digestion of above-mentioned pancreatin, cell suspension 1000rpm centrifugation 8
Minute, discard supernatant.The cells frozen storing liquid of preparation is added into cell precipitation, is resuspended, making the concentration of cell is about 1 × 106
1ml cell suspension is transferred in 1.8ml cryopreservation tube, cryopreservation tube is placed in program temperature reduction box by a/ml, and -80 DEG C of placements 36 are small
When, it is then placed in liquid nitrogen and saves for a long time.
(c) cell recovery: above-mentioned cryopreservation tube is removed from liquid nitrogen, and is put into 37 DEG C of water-baths and is quickly rocked to thawing.
Then in aseptic operating platform, defrosting cell is transferred in 15ml centrifuge tube, and is added equivalent basic culture solution, 800rpm from
The heart 8 minutes, remove supernatant.Cell is resuspended with basic culture solution, and is transferred in Tissue Culture Flask, is cultivated in 18 DEG C of incubators, 8
Its cell grows up to single layer.
Embodiment 4
The fish body of the rhinoceros horn gold thread Barb of embodiment 3 is compared using different sterilization methods.
The most common potassium permanganate soaking disinfection is respectively adopted in rhinoceros horn gold thread Barb in embodiment 3, methylene blue impregnates
Disinfection, alcohol directly sterilizes and the mode of penicillin alcohol combined disinfection, other steps are identical as the step in embodiment 3.Knot
Fruit shows (referring to Fig. 1), and during entire originally culture, potassium permanganate and methylene blue soaking disinfection all show lower
Tissue block cell migration rate, and with the increase of cultivated days, certain tissue blocks for migrating out cell can fall off without weighing again
It is newly adherent to migrate out cell；At the initial stage of originally culture, alcohol, which directly sterilizes, shows higher histocyte mobility, but with
The extension of incubation time, occur tissue block pollution the case where, cause tissue block to fall off, can not save；Penicillin impregnate and
Under alcohol disinfecting synergy, tissue block shows higher cell migration rate, and with the increase of incubation time, migrates out thin
The tissue block of born of the same parents is more and more, and the tissue block to fall off is less.
Therefore, for rhinoceros horn gold thread Barb, potassium permanganate and methylene blue soaking disinfection, though it can guarantee that tissue block is not sent out
Raw pollution, but tissue block is more toxic, cell is not easy to move out, and alcohol directly sterilizes and tends to occur pollute, and culture can not
Continue, the mode of penicillin and alcohol combined disinfection is more suitable for.
Embodiment 5
The base fluid of the cell culture fluid of embodiment 3 is compared using different culture solutions.
By the culture solution in embodiment 3 be substituted for fish cell culture commonly several culture solution DMEM, DMEM (high sugar),
DMEM/F12, L-15, M199, M1640 compare the influence (ginseng that these different culture solutions grow rhinoceros horn gold thread Barb heart cell
See Fig. 2), the results show that the growth rate of cell is different, and adherent rate is also different under the conditions of different culture solutions.Cell culture one day
Afterwards, the attached cell of M199 and L-15 is more, and the cell that DMEM, DMEM (high sugar), DMEM/F12 and M1640 culture solution are adherent
It is less；With the increase of incubation time, the cell quantity of M199 and L-15 are gradually increased, and the cell quantity of M199 is higher than L-15；
The cell quantity of M199 is about twice of DMEM, DMEM (high sugar) and DMEM/F12 cell quantity；M1640 the most culture solution when,
Though cell quantity is increased slightly, reduced levels are maintained always.
Therefore, for rhinoceros horn gold thread Barb heart cell, the effect of M199 is best, followed by L-15, other four kinds trainings
Nutrient solution is not appropriate for cell growth.
Those skilled in the art is it should be understood that although for illustrative purposes, this document describes tools of the invention
Body embodiment, but it can be carry out various modifications without departing from the spirit and scope of the present invention.Therefore, of the invention specific
Embodiments and examples should not be considered as limiting the scope of the invention.The present invention is limited only by the appended claims.This Shen
Please in quote all documents be fully incorporated herein by reference.
Claims (2)
1. a kind of construction method of rhinoceros horn gold thread Barb heart cell system, the construction method include the following steps:
1) acquisition of rhinoceros horn gold thread Barb heart: penicillin soaking disinfection rhinoceros horn gold thread Barb fish body 20-30 minutes for first using 50IU/l,
Rhinoceros horn gold thread Barb is anaesthetized with the MS-222 of 80-120ppm again；Again with 75% alcohol wipe fish body after, take its heart, PBS be added and disappears
Venom impregnates heart tissue；After impregnating 15-30 minutes, heart tissue is cleaned with PBS thimerosal；
2) originally culture: it will be cut into tissue fritter by the heart tissue 1) obtained, and is inoculated in 25cm2Tissue Culture Flask
In, it is inverted overnight in 16-20 DEG C of incubator, next day adds originally culture liquid, starts primary training in 16-20 DEG C of incubator
It supports, half amount replacement culture solution, the 6-8 days cells start to migrate from tissue block every three days, grow up to cell monolayer within 30-40 days；
3) secondary culture: primary cardiac cell starts to pass on after being paved with bottom of bottle 80%, and the originally culture liquid in culture bottle is first sucked out,
Cell has been hanged with the trypsin digestion passage of the trypsase containing 0.1-0.2%, secondary culture liquid 1-2ml is added to neutralize pancreatin
Reaction, passage is passed by 1:1 for the first time, is hereafter passed on by 1:2, continues to cultivate in 16-20 DEG C of incubator；It passes within every 4-6 days
Once, when reaching for 10 generation, cell culture fluid changes basic culture solution into, and rhinoceros horn gold thread Barb heart cell system is successfully established,
Wherein the PBS thimerosal be containing concentration be 100-300IU/ml penicillin, the chain that concentration is 100-300 μ g/ml
The PBS thimerosal for the amphotericin B that the streptomycin sulphate and concentration that mycin, concentration are 20-40 μ g/ml are 20-40 μ g/ml,
Wherein the basic culture solution is the glutamine and 10-15% of the total volume for being 300-400 μ g/ml containing concentration
Fetal calf serum M199 culture solution,
Wherein the originally culture liquid is containing glutamine that concentration is 600-800 μ g/ml, 15-25% of the total volume
Fetal calf serum, concentration be 100-200IU/ml penicillin, concentration be the streptomysin of 100-300 μ g/ml, concentration is 20-40 μ
The streptomycin sulphate and concentration of g/ml is the M199 culture solution of the amphotericin B of 20-40 μ g/ml,
Wherein the secondary culture liquid is containing glutamine that concentration is 500-600 μ g/ml, 10-20% of the total volume
Fetal calf serum, the kanamycin sulfate that concentration is 20-40 μ g/ml and the amphotericin B that concentration is 20-40 μ g/ml
M199 culture solution,
Wherein the pH value of the originally culture liquid, the secondary culture liquid and the basic culture solution is 7.0-7.2,
Wherein the osmotic pressure of the originally culture liquid, the secondary culture liquid and the basic culture solution is 280-330mOsm/l.
2. construction method as described in claim 1, which further includes the steps that following cell cryopreservation and recovery:
1) prepare cells frozen storing liquid: frozen stock solution includes M199 culture solution, fetal calf serum and DMSO, is mixed by the volume ratio of 4:5:1,
Matching while using；
2) cell cryopreservation: the cell of logarithmic growth phase, after the digestion of above-mentioned pancreatin, cell suspension 800-1200rpm is centrifuged 6-
10 minutes, discard supernatant；The cells frozen storing liquid of preparation is added into cell precipitation, is resuspended, makes the concentration 1 × 10 of cell6
1ml cell suspension is transferred in 1.8ml cryopreservation tube, cryopreservation tube is placed in program temperature reduction box by a/ml, -80 DEG C of placement 24-
It 48 hours, is then placed in liquid nitrogen and saves for a long time；
3) cell recovery: above-mentioned cryopreservation tube is removed from liquid nitrogen, and is put into 37 DEG C of water-baths and is quickly rocked to thawing；Then exist
In aseptic operating platform, defrosting cell is transferred in 15ml centrifuge tube, and is added equivalent basic culture solution, 800-1000rpm from
The heart 6-8 minutes, remove supernatant；Cell is resuspended with basic culture solution, and is transferred in Tissue Culture Flask, in 16-20 DEG C of incubator
Culture, cell grows up to single layer within 6-9 days.
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| CN103305456A (en) * | 2013-07-11 | 2013-09-18 | 中国科学院昆明动物研究所 | Construction method and ultralow temperature freezing and storing method of sinocyclocheilus grahami saccus olfactorius cell line |
| CN103992981A (en) * | 2013-12-19 | 2014-08-20 | 集美大学 | Larimichthys crocea liver cell line and establishment method thereof |
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| CN103305456A (en) * | 2013-07-11 | 2013-09-18 | 中国科学院昆明动物研究所 | Construction method and ultralow temperature freezing and storing method of sinocyclocheilus grahami saccus olfactorius cell line |
| CN103992981A (en) * | 2013-12-19 | 2014-08-20 | 集美大学 | Larimichthys crocea liver cell line and establishment method thereof |
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| Title |
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| "鱼类细胞培养技术研究进展";艾庆辉 等;《渔业科学进展》;20120630;第33卷(第3期);122-128 * |
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