CN106508891B - A kind of construction method of Dali schizothoracin kidney cell system - Google Patents

A kind of construction method of Dali schizothoracin kidney cell system Download PDF

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CN106508891B
CN106508891B CN201610976570.3A CN201610976570A CN106508891B CN 106508891 B CN106508891 B CN 106508891B CN 201610976570 A CN201610976570 A CN 201610976570A CN 106508891 B CN106508891 B CN 106508891B
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schizothoracin
dali
concentration
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王晓爱
潘晓赋
杨君兴
范伟
刘倩
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Kunming Institute of Zoology of CAS
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Abstract

The present invention relates to a kind of construction methods of Dali schizothoracin kidney cell system, and this method comprises the following steps: 1) acquisition of Dali schizothoracin renal tissue: being combined using potassium permanganate and alcohol and carried out disinfection to Dali schizothoracin；2) originally culture: using Porcine HGF, penicillin, streptomysin, amphotericin B is contained, L-15 the or DMEM/F12 culture solution for the 280-300mOsm/L that osmotic pressure is is cultivated；3) secondary culture: when reaching for 10 generation, cell culture fluid changes basic culture solution into, and the Dali schizothoracin kidney cell system is successfully established.Construction method of the invention Dali schizothoracin kidney 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 Dali schizothoracin, simultaneously it is also the first cell line of Dali schizothoracin, lays a good foundation for the altitude acclimatization Journal of Sex Research of Dali schizothoracin cellular level.

Description

A kind of construction method of Dali schizothoracin kidney cell system
Technical field
The present invention relates to a kind of construction methods using Dali schizothoracin kidney cell system, belong to the training of fresh-water aquatic organisms cell Support the technical field with Ultra-cryofreezing preservation.
Background technique
Dali schizothoracin (Schizothorax taliensis) is under the jurisdiction of Cyprinidae Schizothoracinae Schizothorax, and original is Pu'er tea The economic fish of extra large specialty accounts for Erhai catches 30% in history.1970s are due to Erhai water level decreasing, tens of rivers Ditch, fish hole are dry, and Dali schizothoracin loses spawning ground, and the introducing of denizen, and the fish-egg for Dali schizothoracin of largely eating adds It, cruel fish, which is excessively captured, makes Dali schizothoracin quantity fall sharply.From Kunming Institute of Zoology, Chinese Academy of Sciences's fishes snd library acquisition and recording It sees, after last time in 1973 is collected, without discovery Dali schizothoracin, unanimously thinks big in academia within 43 years Schizothoracin is managed to become extinct from Erhai Lake in Dali basin.It is listed within Dali schizothoracin 1989 national II grades of protection animal, " Chinese in imminent danger Animal Red Data Book (fish) " in be listed in threatened level, for protect this rareness species local government formulated " Erhai manage item Example ".Dali schizothoracin lives in Lentic environment at the middle and upper levels, and food is based on planktonic organism, and when oviposition requires flowing water environment, often Year 4-5 mating period month, parent population are traced back oviposition at the river or lakebed groundwater seep waterborne for swimming over to grit bottom riverbed.Ovum need to flow Hatch in water.
For the survival state for protecting and understanding Dali schizothoracin, since last century the nineties, Chinese Academy of Sciences Kunming Institute of Botany repeatedly forms a team to Erhai Lake in Dali, the river Xi Erhe, Mi Ju and other places to carry out to investigate, until in May, 2016 just finds greatly Manage the trace of schizothoracin.But it because Dali schizothoracin population quantity is less, and is not easy to obtain, therefore, how to protect and save this The germ plasm resource of Species of Rare Fish from Qingdao becomes urgent problem to be solved now.The pool is supported under environment, because the cultivation time is too short, Dali schizothoracin It can't realize artificial propagation, therefore, the method for saving its germ plasm resource by saving its sperm, ovum, embryo, individual etc. It is infeasible, so inventor has turned one's attention to the Somatic Cell Culture of Dali schizothoracin.
48% is fish in known vertebrate, and fish cell is applied to more pupil's objects and cures as histocyte model Subject is learned, is huge resource treasure-house.Fish cell culture arises from 1962, is developed so far and more than 280 strain cell lines have had been established, The fish cell culture of China derived from more than 40 kinds of fish, establishes cell after vertical more than 70 strain of fish cell system of building together in more than 30 years 1.5% (Chinese Fishes are more than 3500 kinds) of the species deficiency Chinese Fishes sum of system.It can be seen that the building speed of cell line Be it is slowly, attention rate is not enough, the worker that participates in is less likely to be one of its reason；Secondly, compared to mammal Cell culture is easier to pollute in fish cell culture, and the rate that leads to the failure rises, and mostly will in existing patent or article This phenomenon is attributed to that technology is unqualified, and dislikes the quality problems for focusing on fish body itself less, and in Dali, schizothoracin cell culture is real In trampling, the animation of fish body also directly affects the success or failure of cell line building；In addition, because of the office of Dali schizothoracin outdoor habitatss Sex-limited, less to the understanding of its life habit, this also increases the difficulty of Dali schizothoracin cell culture to a certain extent.Through Literature search 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 Dali schizothoracin kidney cell system easy to operation, with Meet the needs to Dali schizothoracin Germ-plasma resources protection and altitude acclimatization Journal of Sex Research.
Specifically, the present invention provides a kind of construction method of Dali schizothoracin kidney cell system, which includes such as Lower step:
1) acquisition of Dali schizothoracin kidney: the potassium permanganate soaking disinfection Dali schizothoracin fish body of 10-20mg/L is first used 10-20 minutes, then Dali schizothoracin fish body is anaesthetized with the MS-222 of 80-100ppm；Again with 75% alcohol wipe fish body after, take it The renal tissue of PBS thimerosal cleaning Dali schizothoracin fish body is added in kidney；
2) originally culture: the renal tissue obtained by step 1) is cut into tissue fritter, and it is thin to be inoculated in 25cm2 In born of the same parents' culture bottle, it is inverted overnight in 18-20 DEG C of incubator, next day adds originally culture liquid, opens in 18-20 DEG C of incubator Dynamic originally culture, half amount replacement culture solution, the 10-12 days cells start to migrate from tissue block every three days, grow up within 30-40 days thin Born of the same parents' single layer, then primary kidney cell；
3) secondary culture: primary kidney cell starts to pass on after being paved with bottom of bottle 80%, and the primary training in culture bottle is first sucked out Nutrient solution has hanged cell with the trypsin digestion passage of the trypsase-EDTA 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 18-20 DEG C of incubator；Often Passage in 5-7 days is primary, and when reaching for 10 generation, cell culture fluid changes basic culture solution, the Dali schizothoracin kidney cell system into It is successfully established.
Further, the PBS thimerosal be containing concentration be containing concentration be 100-300IU/mL penicillin, concentration The mixed solution for the amphotericin B that streptomysin and concentration for 100-300 μ g/mL are 20-40 μ g/mL.
Further, the basic culture solution is the bFGF and 10- of the total volume for being 6-10ng/mL containing concentration The mixed solution of the L-15 or DMEM/F12 culture solution of 15% fetal calf serum.
Further, the originally culture liquid is bFGF, the 15- of the total volume for being 6-10ng/mL containing concentration 20% fetal calf serum, the penicillin that concentration is 200-300IU/mL, the streptomysin and concentration that concentration is 200-300 μ g/mL For the mixed solution of the L-15 or DMEM/F12 culture solution of the amphotericin B of 20-40 μ g/mL.
Further, the secondary culture liquid is bFGF, the 15- of the total volume for being 6-10ng/mL containing concentration 20% fetal calf serum, the penicillin that concentration is 100-200IU/mL, the streptomysin and concentration that concentration is 100-200 μ g/mL For L-15 the or DMEM/F12 culture solution of the amphotericin B of 15-20 μ g/mL.
Further, the pH value of the originally culture liquid, secondary culture liquid and basic culture solution is 7.0-7.2.
Further, the osmotic pressure of the originally culture liquid, secondary culture liquid and basic culture solution is 280- 300mOsm/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 L-15 or DMEM/F12 culture solution, fetal calf serum and DMSO, is mixed by the volume ratio of 3:6:1, 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, making the concentration of cell is about 1 × 106A/mL, 1mL cell suspension is transferred in 1.8mL cryopreservation tube, cryopreservation tube is placed in program temperature reduction box, -80 DEG C placement 24-48 hours, It 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 30 DEG C of water-baths fast Speed is rocked to thawing；Then in aseptic operating platform, defrosting cell is transferred in 15mL centrifuge tube, and the training of equivalent basis is added Nutrient solution, 800-1000rpm are centrifuged 6-8 minutes, remove supernatant；Cell is resuspended with basic culture solution, and is transferred to Tissue Culture Flask In, it is cultivated in 18-20 DEG C of incubator, cell grows up to single layer within 8-10 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 renal 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 other cell lines, with tissue block in the kidney cell system originally culture of construction method acquisition of the invention Pollution rate is lower, to improve success rate.
4. the Dali schizothoracin kidney cell system form that the present invention constructs is that continuous passage and can directly answer at fiber-like For biological characteristic research, the needs to Dali schizothoracin Germ-plasma resources protection and theoretical research and application are met.
5. it is the first cell line of Dali schizothoracin with the Dali schizothoracin kidney cell that construction method of the invention obtains, Good material is provided to the adaptability of altitude environment for research plateau fish, has established the basis of altitude acclimatization Journal of Sex Research.
Detailed description of the invention
Fig. 1 is to show that renal tissue in the schizothoracin originally culture of Dali migrates out the figure of the process of cell.
Fig. 2 is the figure for showing kidney cell single layer in the schizothoracin originally culture of Dali.
Fig. 3 is the figure for showing influence of the different disinfection way to Dali schizothoracin renal tissue block cell migration.
Fig. 4 is the figure for showing the influence that different culture solutions grow Dali schizothoracin kidney 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 300IU/mL of penicillin, and streptomysin concentration is 300 μ G/mL, amphotericin B concentration are 20 μ g/mL.
Basic culture solution: bFGF and fetal calf serum are added into L-15 culture solution, makes the concentration 10ng/mL of bFGF, tire Cow's serum of the total volume 20%.
Originally culture liquid: bFGF, fetal calf serum and antibiotic are added into L-15 culture solution, makes the concentration of bFGF 10ng/mL, fetal calf serum of the total volume 20%, the concentration of penicillin are 250IU/mL, and streptomysin concentration is 250 μ g/mL, two Property amphotericin B concentration be 40 μ g/mL.
Secondary culture liquid: bFGF, fetal calf serum and antibiotic are added into L-15 culture solution, makes the concentration of bFGF 10ng/mL, fetal calf serum of the total volume 20%, amphotericin B concentration are 20 μ g/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
First use the potassium permanganate soaking disinfection Dali of 20mg/l schizothoracin fish body 10 minutes, then numb with the MS-222 of 80ppm Liquor-saturated Dali schizothoracin.Again with 75% alcohol wipe fish body after, be placed in superclean bench, take its kidney with sterile disscting instrument, It is placed in 12 orifice plates, is cleaned renal tissue 5 times with PBS thimerosal, be cut into tissue fritter, and be inoculated in 25cm2Cell training It supports in bottle, is inverted overnight in 18 DEG C of incubators, next day adds originally culture liquid 3mL, starts in 18 DEG C of incubators primary Culture, half amount replacement culture solution, the 11st day cell start to migrate (Fig. 1) from tissue block every three days, grow up to cell monolayer within 40 days (Fig. 2).
3. secondary culture
Primary kidney 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 0.15% 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 18 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.
4. cell cryopreservation and recovery
(a) prepare cell cryopreservation and protect liquid: frozen stock solution includes L-15 culture solution, fetal calf serum and DMSO, by the body of 3:6:1 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 30 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, 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 250IU/mL of penicillin, and streptomysin concentration is 250 μ G/mL, amphotericin B concentration are 30 μ g/mL.
Basic culture solution: bFGF and fetal calf serum are added into DMEM/F12 culture solution, makes the concentration 10ng/ of bFGF ML, fetal calf serum of the total volume 15%.
Originally culture liquid: bFGF, fetal calf serum and antibiotic are added into DMEM/F12 culture solution, makes the concentration of bFGF 10ng/mL, fetal calf serum of the total volume 15%, the concentration of penicillin are 200IU/mL, and streptomysin concentration is 200 μ g/mL, two Property amphotericin B concentration be 30 μ g/mL.
Secondary culture liquid: bFGF, fetal calf serum and antibiotic are added into DMEM/F12 culture solution, makes the concentration of bFGF 10ng/mL, fetal calf serum of the total volume 15%, amphotericin B concentration are 20 μ g/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
First use the potassium permanganate soaking disinfection Dali of 10mg/l schizothoracin fish body 20 minutes, then numb with the MS-222 of 100ppm Liquor-saturated Dali schizothoracin.Again with 75% alcohol wipe fish body after, be placed in superclean bench, take its kidney with sterile disscting instrument, It is placed in 12 orifice plates, renal tissue is cleaned 3 times with PBS thimerosal, is cut into tissue fritter, and be inoculated in 25cm2Cell It in culture bottle, is inverted overnight in 20 DEG C of incubators, next day adds originally culture liquid 5mL, starts in 20 DEG C of incubators former It is commissioned to train feeding, every three days half amount replacement culture solution, cell starts to migrate from tissue block within the 10th day, grows up to cell monolayer within 35 days.
3. secondary culture
Primary kidney 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 5 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.
4. cell cryopreservation and recovery
(a) prepare cell cryopreservation and protect liquid: frozen stock solution includes DMEM/F12 culture solution, fetal calf serum and DMSO, by 3:6:1 Volume ratio 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 30 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 200IU/mL of penicillin, and streptomysin concentration is 200 μ G/mL, amphotericin B concentration are 40 μ g/mL.
Basic culture solution: bFGF and fetal calf serum are added into L-15 culture solution, makes the 400 μ g/mL of concentration of bFGF, tire Cow's serum of the total volume 10%.
Originally culture liquid: bFGF, fetal calf serum and antibiotic are added into L-15 culture solution, makes the concentration 8ng/ of bFGF ML, fetal calf serum of the total volume 15%, the concentration of penicillin are 200IU/mL, and streptomysin concentration is 200 μ g/mL, and both sexes are mould Plain B concentration is 20 μ g/mL.
Secondary culture liquid: bFGF, fetal calf serum and antibiotic are added into L-15 culture solution, makes the concentration 8ng/ of bFGF ML, fetal calf serum of the total volume 10%, amphotericin B concentration are 30 μ g/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
It first uses the penicillin soaking disinfection Dali of 15mg/l schizothoracin fish body 15 minutes, then is anaesthetized with the MS-222 of 100ppm Dali schizothoracin.Again with 75% alcohol wipe fish body after, be placed in superclean bench, take its kidney with sterile disscting instrument, set In 12 orifice plates, renal tissue is cleaned 4 times with PBS thimerosal, is cut into tissue fritter, and be inoculated in 25cm2Cell training It supports in bottle, is inverted overnight in 18 DEG C of incubators, next day adds originally culture liquid 4mL, starts in 18 DEG C of incubators primary Culture, half amount replaces culture solution every three days, and cell starts to migrate from tissue block within the 11st day, grows up to cell monolayer within 35 days.
3. secondary culture
Primary kidney 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.
4. cell cryopreservation and recovery
(a) prepare cell cryopreservation and protect liquid: frozen stock solution includes L-15 culture solution, fetal calf serum and DMSO, by the body of 3:6:1 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 Dali schizothoracin of embodiment 3 is compared using different sterilization methods.
The most common potassium permanganate soaking disinfection is respectively adopted in Dali schizothoracin in embodiment 3, methylene blue impregnates Disinfection, alcohol directly sterilize, methylene blue+alcohol combined disinfection and potassium permanganate+alcohol combined disinfection mode, other steps It is identical as the step in embodiment 3.As the result is shown (referring to Fig. 1), during entire originally culture, methylene blue soaking disinfection Lower tissue block cell migration rate is all shown with methylene blue+alcohol combined disinfection, and with the increase of cultivated days, certain The tissue block for migrating out cell a bit, which can fall off, migrates out cell without again adherent again；At the initial stage of originally culture, permanganic acid Potassium soaking disinfection and alcohol, which directly sterilize, shows higher histocyte mobility, but with the extension of incubation time, occurs The case where tissue block pollution, causes tissue block to fall off, can not save；Under potassium permanganate+alcohol synergy, tissue block performance Higher cell migration rate out, and with the increase of incubation time, the tissue block for migrating out cell is more and more, the tissue to fall off Block is less.
Therefore, for the schizothoracin of Dali, methylene blue is more toxic, no matter methylene blue soaking disinfection or Asia Methyl blue+alcohol combined disinfection, though can guarantee that tissue block does not pollute, larger to the damage of tissue block, cell is not easy to move Out, and alcohol directly sterilizes and potassium permanganate soaking disinfection tends to occur pollute, culture can not continue, potassium permanganate+alcohol connection The mode for closing 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 Dali schizothoracin kidney 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 1 day Afterwards, the attached cell of L-15 and DMEM/F12 is more, and the cell that DMEM, DMEM (high sugar), M199 and M1640 culture solution are adherent It is less；With the increase of incubation time, the cell quantity of L-15 and DMEM/F12 are gradually increased, and the cell quantity of L-15 is higher than DMEM/F12；The cell quantity of L-15 is about twice of DMEM, DMEM (high sugar) and M199 cell quantity；M1640 is culture solution When, though cell quantity is increased slightly, reduced levels are maintained always.
Therefore, for the schizothoracin kidney cell of Dali, the effect of L-15 is best, followed by DMEM/F12, other four Kind culture 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 (1)

1. a kind of construction method of Dali schizothoracin kidney cell system, it is characterised in that construction method includes the following steps:
1) acquisition of Dali schizothoracin kidney: the potassium permanganate soaking disinfection Dali schizothoracin fish body 10-20 of 10-20mg/L is first used Minute, then Dali schizothoracin fish body is anaesthetized with the MS-222 of 80-100ppm；Again with 75% alcohol wipe fish body after, take its kidney, The renal tissue of PBS thimerosal cleaning Dali schizothoracin fish body is added；
The PBS thimerosal be containing concentration be 100-300IU/mL penicillin, the streptomysin that concentration is 100-300 μ g/mL And concentration is the mixed solution of the amphotericin B of 20-40 μ g/mL；
2) originally culture: the renal tissue that step 1) obtains is cut into tissue fritter, and is inoculated in 25cm2Tissue Culture Flask In, it is inverted overnight in 18-20 DEG C of incubator, next day adds originally culture liquid, starts primary training in 18-20 DEG C of incubator It supports, half amount replacement culture solution, the 10-12 days cells start to migrate from tissue block every three days, grow up to cell monolayer within 30-40 days, Then primary kidney cell；
3) secondary culture: primary kidney 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-EDTA containing 0.1-0.2%, secondary culture liquid 1-2mL is added in It is reacted with pancreatin, passage is passed by 1:1 for the first time, is hereafter passed on by 1:2, continues to cultivate in 18-20 DEG C of incubator；Every 5-7 Its passage is primary, and when reaching for 10 generation, cell culture fluid changes basic culture solution into, and the Dali schizothoracin kidney cell system establishes Success；
The basic culture solution is the fetal calf serum of the bFGF and 10-15% of the total volume that are 6-10ng/mL containing concentration L-15 or DMEM/F12 culture solution mixed solution；
The originally culture liquid be containing concentration be 6-10ng/mL bFGF, 15-20% of the total volume fetal calf serum, Penicillin, the streptomysin that concentration is 200-300 μ g/mL and the concentration that concentration is 200-300IU/mL are the two of 20-40 μ g/mL The mixed solution of the L-15 or DMEM/F12 culture solution of property mycin B；
The secondary culture liquid be containing concentration be 6-10ng/mL bFGF, 15-20% of the total volume fetal calf serum, Penicillin, the streptomysin that concentration is 100-200 μ g/mL and the concentration that concentration is 100-200IU/mL are the two of 15-20 μ g/mL The mixed solution of the L-15 or DMEM/F12 culture solution of property mycin B；
The pH value of the originally culture liquid, the secondary culture liquid and the basic culture solution is 7.0-7.2；
The osmotic pressure of the originally culture liquid, the secondary culture liquid and the basic culture solution is 280-300mOsm/L；
4) cell cryopreservation and the step of recovery:
A) prepare cells frozen storing liquid: frozen stock solution includes L-15 or DMEM/F12 culture solution, fetal calf serum and DMSO, by 3:6:1's Volume ratio mixing, matching while using；
B) 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, make the concentration of cell be about 1 × 1061mL 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 placements It 24-48 hours, 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 30 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 18-20 DEG C of incubator Culture, cell grows up to single layer within 8-10 days.
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CN103409365A (en) * 2013-09-05 2013-11-27 中国科学院昆明动物研究所 Method for establishing schizothorax wangchiachii heart cell line and performing ultralow temperature cryopreservation on schizothorax wangchiachii heart cell line
CN103436490A (en) * 2013-09-05 2013-12-11 中国科学院昆明动物研究所 Construction and ultralow temperature freezing preservation method of fin cell line of schizothorax grahami
CN104974977A (en) * 2015-04-30 2015-10-14 肇庆大华农生物药品有限公司 Epinephelus lanceolatus kidney tissue cell line and construction method thereof

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CN103409365A (en) * 2013-09-05 2013-11-27 中国科学院昆明动物研究所 Method for establishing schizothorax wangchiachii heart cell line and performing ultralow temperature cryopreservation on schizothorax wangchiachii heart cell line
CN103436490A (en) * 2013-09-05 2013-12-11 中国科学院昆明动物研究所 Construction and ultralow temperature freezing preservation method of fin cell line of schizothorax grahami
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