CN106520681A - In vitro culture solution and cryopreservation solution of Ietalurus Punetaus spermatogonial stem cells, and in vitro culture and cryopreservation method - Google Patents
In vitro culture solution and cryopreservation solution of Ietalurus Punetaus spermatogonial stem cells, and in vitro culture and cryopreservation method Download PDFInfo
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- CN106520681A CN106520681A CN201610984110.5A CN201610984110A CN106520681A CN 106520681 A CN106520681 A CN 106520681A CN 201610984110 A CN201610984110 A CN 201610984110A CN 106520681 A CN106520681 A CN 106520681A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0608—Germ cells
- C12N5/061—Sperm cells, spermatogonia
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0236—Mechanical aspects
- A01N1/0242—Apparatuses, i.e. devices used in the process of preservation of living parts, such as pumps, refrigeration devices or any other devices featuring moving parts and/or temperature controlling components
- A01N1/0252—Temperature controlling refrigerating apparatus, i.e. devices used to actively control the temperature of a designated internal volume, e.g. refrigerators, freeze-drying apparatus or liquid nitrogen baths
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- C12N2500/00—Specific components of cell culture medium
- C12N2500/05—Inorganic components
- C12N2500/10—Metals; Metal chelators
- C12N2500/12—Light metals, i.e. alkali, alkaline earth, Be, Al, Mg
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- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/32—Amino acids
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/113—Acidic fibroblast growth factor (aFGF, FGF-1)
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/119—Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
Abstract
The invention relates to an in vitro culture solution and cryopreservation solution of fish spermatogonial stem cells (SSCs), and an in vitro culture and cryopreservation method, and solves the existing problems of relatively short in vitro culture time of SSCs and complex components of SSCs medium. The in vitro culture solution is prepared by taking an L-15 medium as the basis and adding HEPES, penicillin, streptomycin, NaHCO3, L-glutamic acid, fetal bovine serum, catfish serum and bFGF. The in vitro culture method includes: 1. Ietalurus Punetaus testis cell separation and SSCs enrichment; and 2. SSCs culture. The in vitro culture solution and cryopreservation solution of Ietalurus Punetaus spermatogonial stem cells provided by the invention have the characteristics of simple formula components, easy acquisition and preparation, and low price, and can realize long-term in vitro culture and cryopreservation of Ietalurus Punetaus spermatogonial stem cells.
Description
Technical field
The present invention relates to a kind of in vitro culture liquid of fish stem spermatogonium, frozen stock solution and in vitro culture, cryopreservation methods.
Background technology
Fish stem spermatogonium (spermatogonial stem cells, SSCs) is that a class is present in milter sexual gland
With self and can be divided into the germline stem cell of sperm.The in vitro culture of fish SSCs is for research animals' reproduction
Stem cell occurs, develops, differentiation and regulatory mechanism are significant.SSCs Vitro Culture Techniques combine SSCs heteroplastic transplantation skills
Art, can be used for shortening the cyclostage of Important Economic fingerling, saves endangered fish species；The SSCs of in vitro culture is also
Can be used for fish virology and toxicologic study.
Relatively more, fish SSCs is reported in the in-vitro separation of SSCs and culture in the mammals such as mouse, rat, people, pig
Research it is fewer.The most long record of incubation time in the SSCs in vitro cultures reported at present is created by Panda etc. (2011)
Make, its in vitro culture South Asia open country dace SSCs is two months.
The content of the invention
Present invention aim to address existing SSCs Time in Vitro is relatively short, and SSCs culture medium prescriptions composition is multiple
Miscellaneous problem, and a kind of in vitro culture liquid of the channel catfish stem spermatogonium for providing, frozen stock solution and in vitro culture, frozen side
Method.
The in vitro culture liquid of channel catfish stem spermatogonium of the present invention adds based on L-15 culture mediums and by per milliliter
Enter 25 μm of ol 4- HEPESs (HEPES), 100unit penicillin, 100 μ g streptomysins, 1.0 μ g NaHCO3、0.3
μ g Pidolidones, 0.2mL hyclones (FBS), 0.05mL catfish serum, the ratio of 1ng fibroblast growth factors (bFGF)
Example is made, and pH value is 7.2～7.4.
The extracorporeal culturing method of channel catfish stem spermatogonium is carried out according to the following steps：
First, the separation of channel catfish spermary cell and the enrichment of SSCs；
2nd, the culture of SSCs：The SSCs that step one is enriched with is inoculated into the Tissue Culture Plate for being covered with gelatin, big compression ring is placed in
Cultivate under border, in the incubator that saturated humidity, constant temperature are 26 DEG C；Wherein, the culture of SSCs is former from above-mentioned channel catfish essence
The in vitro culture liquid of stem cell.
The in vitro culture liquid of above-mentioned channel catfish stem spermatogonium adds 5% DMSO to make channel catfish by volume
The frozen stock solution of stem spermatogonium.
The external cryopreservation methods of channel catfish stem spermatogonium are carried out according to the following steps：
First, by the channel catfish SSCs Trypsin Induced 1min of channel catfish SSCs or amplification cultivation, terminate
Digestion moves to centrifuge tube, and 500g, centrifugation 10min abandon supernatant；Above-mentioned frozen stock solution is subsequently adding, piping and druming makes channel catfish
SSCs proceeds to cryopreservation tube after fully suspending；
2nd, cryopreservation tube is put into into cell cryopreservation box, cell cryopreservation box program is lowered the temperature automatically, rate of temperature fall is -1 DEG C/min,
Freezing storing box is placed in -80 DEG C of low temperature refrigerators to deposit again cryopreservation tube to be transferred in liquid nitrogen after 24h and is preserved, that is, complete channel catfish
Stem spermatogonium it is external frozen.
The extracorporeal culturing method of channel catfish stem spermatogonium of the present invention is capable of achieving the long-term steady of channel catfish SSCs
Determine in vitro culture, ensure stem spermatogonium multiplication capacity and it is undifferentiated under the premise of at least can cultivate 3 months.The present invention is
Important basis has been established in SSCs later stage heteroplastic transplantations and SSCs differentiation mechanism researchs.The body of channel catfish SSCs of the present invention
Outer cultural method provides donor material for SSCs heteroplastic transplantations using the amount reproduction of SSCs, it is to avoid donor fish is by excessive
Slaughter.
The present invention establishes the cryopreservation methods of in vitro culture SSCs, can be used to preserve endangered fish species heredity money
Material.
The in vitro culture liquid and frozen stock solution formula components of channel catfish stem spermatogonium of the present invention is simple, it is easy to obtain and
Prepare, it is cheap；And the long-term in vitro culture of channel catfish SSCs and frozen can be realized.
Description of the drawings
Fig. 1 is using the inventive method culture channel catfish SSCs phase contrast microscope observation figures of 92 days.
Fig. 2 is that Plzf, Thy1 and Integrin6 gene is former in the channel catfish essence of the inventive method culture 57d and 92d
Relative expression quantity column diagram in stem cell and sperm.
Specific embodiment
Technical solution of the present invention is not limited to act specific embodiment set forth below, also including between each specific embodiment
Any combination.
Specific embodiment one：The in vitro culture liquid of present embodiment channel catfish stem spermatogonium is with L-15 culture mediums
Based on and by per milliliter add 25 μm of ol 4- HEPESs (HEPES), 100unit penicillin, 100 μ g strepto-s
Element, 1.0 μ g NaHCO3, 0.3 μ g Pidolidones, 0.2mL hyclones (FBS), 0.05mL catfish serum, 1ng is into fiber finer
The ratio of the intracellular growth factor (bFGF) is made, and pH value is 7.2～7.4.
Present embodiment adjusts pH value with the NaOH solution of the HCl solution or 1mol/L of 1mol/L.
The in vitro culture liquid of the channel catfish stem spermatogonium that present embodiment is configured is with 0.22 μm of membrane filtration
It is degerming, packing, 4 DEG C of preservations.
Specific embodiment two：The extracorporeal culturing method of present embodiment channel catfish stem spermatogonium is according to the following steps
Carry out：
First, the separation of channel catfish spermary cell and the enrichment of SSCs；
2nd, the culture of SSCs：The SSCs that step one is enriched with is inoculated into the Tissue Culture Plate for being covered with gelatin, big compression ring is placed in
Cultivate under border, in the incubator that saturated humidity, constant temperature are 26 DEG C；Wherein, the culture of SSCs is from described in specific embodiment one
The in vitro culture liquid of channel catfish stem spermatogonium.
Present embodiment step one channel catfish spermary cell is separated and the enrichment of SSCs can be carried out according to the following steps：Not
It is placed on ice after reaching sexually matured two ages channel catfish (25～30cm of total length) MD-222 anesthesia and takes back laboratory.Often
, with after 70% alcohol disinfecting, abdominal cut is aseptic to win bilateral spermary for bar fish body table, is put into and (contains equipped with the dual anti-solution of 10mL
100unit/mL penicillin, 100 μ g/mL streptomysins and 1.0 μ g/mL NaHCO3Hanks ' salting liquids) 15mL sterile centrifugations
Guan Zhong.After all experimentss are dissected with fish and finished, centrifuge tube pipe outer wall moves into superclean bench after disinfecting in alcohol.In ultra-clean work
Make spermary tissue in each centrifuge tube in platform respectively to entering in a culture dish, with scalpel by other groups of adhesion on spermary
Knit.Then rinsed 3 times with the dual anti-solution of 1mL, be subsequently soaked into 2min in 0.5% bleaching waters of 5mL, taken out
Afterwards at once with Hanks ' flusheds 3 times.Spermary tissue shear is broken to into 1mm3Fritter, move into the aseptic triangle containing magneton
In bottle, 0.25% trypsase-EDTA digestive juices of 15mL are subsequently added.After 30min is incubated on ice, under 22 DEG C of magnetic agitations
Digestion 1h.Digestion is added after terminating and digestive juice equivalent or slightly more than in vitro culture liquid end of the channel catfish SSCs of digestive juice
Only digest；500g, centrifugation 10min after being filtered with the nylon leaching net in 40 μm of apertures again.Supernatant is abandoned, 2mL channel catfish is added
The in vitro culture liquid of SSCs, gently dispels the cell for being deposited in centrifugation bottom of the tube with pipettor, makes cell suspension.By cell
Suspension be gently added to Percoll density gradient liquid (70%, 45%, each 2ml of 35%Percoll be placed in from the bottom up 15ml centrifugation
Pipe) on, 4 DEG C, 800g, centrifugation 40min.Gradient centrifugation terminate after as there is 4 confluent monolayer cells, SSCs in cell size and density variation
It is primarily present the superiors.Hanks ' the salting liquids of two volumes, 500g, centrifugation 10min are added after the superiors' cell is suctioned out.Abandon
Supernatant, adds the in vitro culture liquid of 2mL channel catfish SSCs, gently dispels the cell for being deposited in centrifugation bottom of the tube, makes
Channel catfish SSCs suspensions；Both the enrichment of channel catfish stem spermatogonium had been realized.
A small amount of channel catfish SSCs suspensions are taken, with 0.4% Trypan Blue (cell suspension:Trypan blue=9: 1), blood
Cell counting count board calculates total cell number, viable count, dead cell number and living cell rate.
Present embodiment channel catfish SSCs living cell rates are 85.1%.
Experiment：
Secondary Culture is carried out to channel catfish SSCs using present embodiment method, (incubation time is to take the 6th culture
57d) identified with the channel catfish SSCs of the 11st culture (incubation time is 92d).
(1) cell observation：The 6th culture of observation of cell (incubation time is 57d) and the 11st under 400 × phase contrast microscope
The channel catfish SSCs of culture (incubation time is 92d), and while observation has just been enriched with the SSCs of acquisition.
Observation result：The SSCs cells for being just enriched with acquisition are larger, rounded.Using present embodiment method culture 57d
Size and shape, the indifference of SSCs are still all kept with the cell of 92d, in nucleus, contains " nuage " or idioplasm particle
(incubation time is as shown in Figure 1 for the channel catfish SSCs of 92d).
(2) Molecular Detection：Detect Plzf, Thy1 and Integrin6 gene in culture 57d the (the 6th by fluorescent quantitation method
Generation) and 92d (the 11st generation) channel catfish stem spermatogonium expression, while determining these three genes in channel catfish
Expression in sperm, using 18s as internal reference.
Testing result：Phase of Plzf, Thy1 and Integrin6 gene in different cells (every kind of cell selects 3 groups of tests)
It is as shown in table 1 to expression, by 2-ΔΔCtCalculate (Livak and Schmittgen 2001), as a result such as 2 institute of Fig. 2 and Biao
Show；The high expression in the channel catfish stem spermatogonium of culture 57d and 92d of Plzf, Thy1 and Integrin6 gene, and
In sperm, expression is extremely low.
Table 1
Table 2
The results show is still located using the channel catfish stem spermatogonium of present embodiment method culture 57d and 92d
In stem spermatogonium state, multiplication capacity is strong and undifferentiated.
Specific embodiment three：Difference of the present embodiment from specific embodiment two is：8 × 10 are pressed in step 26
～10 × 106The SSCs that step one is enriched with is inoculated into and is covered with the Tissue Culture Plate of 0.1% gelatin by the density of/mL.Other steps
Rapid and parameter is identical with embodiment two.
Specific embodiment four：Difference of the present embodiment from specific embodiment two is：4 in step 2 incubation
～5d changes half nutrient solution；Treat that 80～90% culture plate bottoms are paved with cell, then Secondary Culture is carried out with 1: 3 ratio.Other
Step and parameter are identical with embodiment two.
Specific embodiment five：The in vitro culture liquid of channel catfish SSCs described in specific embodiment one adds by volume
The DMSO for entering 5% makes the frozen stock solution of channel catfish stem spermatogonium.
Specific embodiment six：The external cryopreservation methods of present embodiment channel catfish stem spermatogonium are according to the following steps
Carry out：
First, by the channel catfish SSCs Trypsin Induced 1min of channel catfish SSCs or amplification cultivation, terminate
Digestion moves to centrifuge tube, and 500g, centrifugation 10min abandon supernatant；The frozen stock solution being subsequently adding described in specific embodiment five, blows
Beating makes channel catfish SSCs proceed to cryopreservation tube after fully suspending；
2nd, cryopreservation tube is put into into cell cryopreservation box, cell cryopreservation box program is lowered the temperature automatically, rate of temperature fall is -1 DEG C/min,
Freezing storing box is placed in -80 DEG C of low temperature refrigerators to deposit again cryopreservation tube to be transferred in liquid nitrogen after 24h and is preserved, that is, complete channel catfish
Stem spermatogonium it is external frozen.
Present embodiment cell cryopreservation box selects Mr.Frosty cell cryopreservation boxes.
Experiment：
The present embodiment Liquid nitrogen storage channel catfish SSCs of two weeks (6 generation of amplification cultivation) is taken out and is checked, solved
Freeze cell, then 26 DEG C of 1～2min of water-bath remove frozen stock solution.
Experimental result：Channel catfish SSCs after defrosting detects cytoactive with trypan blue, and 77.4% cell is living
Cell, and normal proliferative can pass in follow-up cultivation.
The results show present embodiment method can be used for the frozen of channel catfish SSCs, and spot can be kept to pitch
The original cytoactives of tail SSCs.
Specific embodiment seven：Difference of the present embodiment from specific embodiment six is：Step one is by amplification cultivation 6
The channel catfish SSCs Trypsin Induceds in generation.Other steps and parameter are identical with embodiment six.
Claims (6)
1. the in vitro culture liquid of channel catfish stem spermatogonium, it is characterised in that the external training of channel catfish stem spermatogonium
Nutrient solution based on L-15 culture mediums and by per milliliter add 25 μm of ol 4- HEPESs, 100unit penicillin,
100 μ g streptomysins, 1.0 μ g NaHCO3, 0.3 μ g Pidolidones, 0.2mL hyclones, 0.05mL catfish serum, 1ng is into fibre
The ratio of dimension Porcine HGF is made, and pH value is 7.2～7.4.
2. the extracorporeal culturing method of channel catfish stem spermatogonium, it is characterised in that the extracorporeal culturing method enters according to the following steps
OK：
First, the separation of channel catfish spermary cell and the enrichment of SSCs；
2nd, the culture of SSCs：The SSCs that step one is enriched with is inoculated into the Tissue Culture Plate for being covered with gelatin, atmospheric environment is placed in
Under, in the incubator that saturated humidity, constant temperature are 26 DEG C cultivate；Wherein, channel catfish described in claim 1 is selected in the culture of SSCs
The in vitro culture liquid of stem spermatogonium.
3. the extracorporeal culturing method of channel catfish stem spermatogonium according to claim 2, it is characterised in that step 2
In press 8 × 106～10 × 106The SSCs that step one is enriched with is inoculated into the density of/mL the Tissue Culture Plate for being covered with 0.1% gelatin
In.
4. the extracorporeal culturing method of channel catfish stem spermatogonium according to claim 2, it is characterised in that step 2
In incubation, 4～5d changes half nutrient solution；Treat that 80～90% culture plate bottoms are paved with cell, then passed with 1: 3 ratio
Culture.
5. the frozen stock solution of channel catfish stem spermatogonium, it is characterised in that channel catfish essence described in claim 1 is former dry thin
The in vitro culture liquid of born of the same parents adds 5% DMSO to make the frozen stock solution of channel catfish stem spermatogonium by volume.
6. external cryopreservation methods of channel catfish stem spermatogonium, it is characterised in that the external cryopreservation methods enter according to the following steps
OK：
First, by the channel catfish SSCs Trypsin Induced 1min of channel catfish SSCs or amplification cultivation, terminate digestion
Centrifuge tube is moved to, 500g, centrifugation 10min abandon supernatant；The frozen stock solution being subsequently adding described in claim 5, piping and druming pitch spot
Tail SSCs proceeds to cryopreservation tube after fully suspending；
2nd, cryopreservation tube is put into into cell cryopreservation box, cell cryopreservation box program is lowered the temperature automatically, rate of temperature fall is -1 DEG C/min, will freeze
Deposit box be placed in -80 DEG C of low temperature refrigerators deposit 24h after again by cryopreservation tube be transferred in liquid nitrogen preserve, that is, complete channel catfish essence
Former stem cell it is external frozen.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107306940A (en) * | 2017-08-08 | 2017-11-03 | 浙江省海洋水产研究所 | The spermary Cryoprotectant and the preparation method of spermatogonium of a kind of seawater fish |
| CN110684724A (en) * | 2019-09-18 | 2020-01-14 | 中山大学 | Method for producing sperms by 3D culture of odontobutis sinensis spermary cells and application |
| CN112300983A (en) * | 2020-10-28 | 2021-02-02 | 广东海大集团股份有限公司 | Method for separating fish spermatogonial stem cells by mechanical method |
| WO2023101008A1 (en) * | 2021-12-03 | 2023-06-08 | 国立大学法人九州大学 | Culture method of fish germ stem cells |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104830756A (en) * | 2015-05-07 | 2015-08-12 | 湖州师范学院 | Erythroculter ilishaeformis spermatogonia stem cell separation and culture method |
-
2016
- 2016-11-09 CN CN201610984110.5A patent/CN106520681A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104830756A (en) * | 2015-05-07 | 2015-08-12 | 湖州师范学院 | Erythroculter ilishaeformis spermatogonia stem cell separation and culture method |
Non-Patent Citations (2)
| Title |
|---|
| 王爱珍: "吉富罗非鱼精原干细胞分离与体外培养的研究", 《中国优秀硕士学位论文全文数据库-农业科技辑》 * |
| 陈秋宇等: "鱼类精原干细胞的研究进展", 《广东农业科学》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107306940A (en) * | 2017-08-08 | 2017-11-03 | 浙江省海洋水产研究所 | The spermary Cryoprotectant and the preparation method of spermatogonium of a kind of seawater fish |
| CN110684724A (en) * | 2019-09-18 | 2020-01-14 | 中山大学 | Method for producing sperms by 3D culture of odontobutis sinensis spermary cells and application |
| CN112300983A (en) * | 2020-10-28 | 2021-02-02 | 广东海大集团股份有限公司 | Method for separating fish spermatogonial stem cells by mechanical method |
| WO2023101008A1 (en) * | 2021-12-03 | 2023-06-08 | 国立大学法人九州大学 | Culture method of fish germ stem cells |
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