CN1935984A - In vitro culture method for avian stem spermatogonium - Google Patents
In vitro culture method for avian stem spermatogonium Download PDFInfo
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- CN1935984A CN1935984A CNA2006100966296A CN200610096629A CN1935984A CN 1935984 A CN1935984 A CN 1935984A CN A2006100966296 A CNA2006100966296 A CN A2006100966296A CN 200610096629 A CN200610096629 A CN 200610096629A CN 1935984 A CN1935984 A CN 1935984A
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Abstract
The invention discloses to the method used to culture bird stem spermatogonium in vitro. It includes the following steps: inoculating the bird stem spermatogonium to CEF feeder layer; adding 10% calf blood serum, 2% chicken blood serum, 2mmol/L L-glutamine, 1mmol/L sodium pyruvate, 5.5*10-15ng/ml beta-mercaptoethanol, 10 mul/ml non-essential amino acid, 15-20ng/ml hSCF, 10U/ml mLIF, 10-15ng/ml bFGF, 0.04ng/ml hIL-11, 10ng/ml IGF, 100U/ml gentamicin sulphate culture solution into DMEM high glucose culture medium. This culturing method can process vitro operation for chicken stem spermatogonium, and lay study foundation for chicken embryo stem spermatogonium further series building.
Description
Technical field
The present invention relates to transgenosis, animal cloning, fields such as preservation of rare animal and tissue and cell engineering.
Background technology
Stem spermatogonium (SSCs) is the precursor cell that sperm forms, and is a kind of multipotential stem cell with self and multidirectional differentiation potential.Along with the continuous development and the maturation of Spermatogonial Stem Cells culture technique, frozen technology and implantation technique, make the application of stem spermatogonium become possibility.And stem spermatogonium in physianthropy, set up in the protection of transgenic animal model and animals on the brink of extinction and all have very important significance.The research of stem spermatogonium is become a focus in the stem-cell research field gradually.Because the reproductive physiology structure and the anatomic characteristic of bird uniqueness make few to the research of bird tissue and cell engineering, transgenosis and animal cloning aspect.
Be transplanted in the testis of sterile mouse and observe that sperm produces and after Clonthier in 1996 was transplanted to the rat testicle sexual cell immunodeficient mouse and produces sperm, spermatogonium became a research focus gradually from the stem spermatogonium that Brister in 1994 and Zimmerman can educate mouse.People such as Li Dexue, Yin Ming studies the condition of spermatogonial stem cells into mouse vitro culture and the phenomenon of division growth.The culture condition of stem spermatogonium, authentication method and implantation technique are all in constantly renewal and perfect.
Studies show that, having on the basis of feeder layer, be basic medium with DMEM and add some additives and some somatomedin forms external survival, division growth and the clone of Mammals stem spermatogonium and has good action, but for the long-term cultivation of Embryo Gallus domesticus stem spermatogonium and the research report of building aspects such as being seldom, still do not grope the suitableeest vitro culture and the condition that goes down to posterity.
Summary of the invention
The extracorporeal culturing method that the purpose of this invention is to provide a kind of bird stem spermatogonium, vitro culture for the former stem cell of chickens' extract, provide necessary base to technology such as chickens' extract former stem cell long-term cultivation system and stem spermatogonium transplanting, transgenosiss, material preferably is provided for the differentiation of in vitro study cells whose development.
Technical scheme of the present invention is that a kind of extracorporeal culturing method of bird stem spermatogonium is characterized in that chicken embryo SSCs is inoculated on chick embryo fibroblast (CEF) feeder layer, re-uses the suitableeest nutrient solution of finding out by experiment and cultivates.Used nutrient solution is: add 10% calf serum, 2% chicken serum, 2mmol/L L-glutaminate, 1mmol/L Sodium.alpha.-ketopropionate, 5.5 * 10 in the DMEM high glucose medium
-5Mol/L beta-mercaptoethanol, 10 μ l/ml non-essential amino acid, 15-20ng/mlhSCF, 10U/ml mLIF, 10-15ng/ml bFGF, 0.04ng/ml hIL-11,10ng/ml IGF, 100U/ml gentamicin sulphate.
The present invention obtains the effective ways of the long-term vitro culture of bird stem spermatogonium, is to establish the research basis for utilizing the former stem cell of chickens' extract to carry out further building of manipulation in vitro and Embryo Gallus domesticus stem spermatogonium.Advantage is as follows:
1, easy and simple to handle, repeatable strong, can satisfy the requirement that general using SSCs carries out manipulation in vitro.
2, can carry out the cultivation of long period to SSCs external, make its a large amount of propagation, carry out the operation of cell and organizational project.
3, the SSCs that is obtained can carry out the preparation of transgenosis, animal cloning, mosaic chicken.
Description of drawings
Fig. 1 is that former generation SSCs cultivates colony * 200 that formed in 48 hours.
Fig. 2 is that former generation SSCs cultivates colony * 200 that formed in 96 hours.
Fig. 3 is that the 2nd generation SSCs cultivates colony * 200 that formed in 48 hours.
Fig. 4 is the AKP dyeing * 200 of the 2nd generation SSCs.
Fig. 5 is that the SSEA-1 dyeing of chicken SSCs identifies * 200.
Embodiment
Embodiment
Step 1: the separation of Embryo Gallus domesticus SSCs
Take out the chick of shell in one week, the aseptic testis Gallus domesticu that obtains with embathing among the PBS three times, is peelled off tunica albuginea etc. under anatomical lens.Add firmly piping and druming of an amount of PBS.At first use 10 times of collagenases (1mg/mL) digestion, at 37 ℃, 5%CO to the tissue block volume
2Act on 5～8 minutes under the concentration conditions, mesenchymal cell and some vascular components of convoluted seminiferous tubules are removed in its effect, use Unidasa (1.5mg/mL) and trypsin 0.25% afterwards) mixed solution digestion 3～5 minutes, obtain SSCs and sustenticular cell on the seminiferous epithelium, FCS with Digestive system consumption 10% stops digestion, and indigested structural constituent removes by filter with 350 order filter sieve.Filtered liquid was abandoned supernatant with 1000r/ minute centrifugal 8 minutes, made single cell suspension with nutrient solution suspension cell again, carried out trypan blue dyeing and calculated viable count, adjusted cell concn to 10
4Be inoculated into behind individual/ml to grow in the culturing bottle that the CEF feeder layer is arranged and cultivate.
Step 2: the biological characteristics after the Embryo Gallus domesticus SSCs vitro culture detects
Detect with morphology evaluation, alkaline phosphatase (AKP) dyeing, phasic specificity embryo surface antigen (SSEA-1) respectively and identify Embryo Gallus domesticus SSCs.Morphology is identified as figure
Step 3: the vitro culture of Embryo Gallus domesticus SSCs
The preparation nutrient solution:
Nutrient solution: the DMEM high glucose medium, add 10% calf serum, 2% chicken serum, 2mmol/LL-glutamine, 1mmol/L Sodium.alpha.-ketopropionate, 5.5 * 10
-5Mol/L beta-mercaptoethanol, 10 μ l/ml non-essential amino acid, 15-20ng/ml hSCF, 10U/ml mLIF, 10-15ng/ml bFGF, 0.04ng/ml hIL-11,10ng/ml IGF, 100U/ml gentamicin sulphate.
Above-mentioned DMEM (Dulbecco ' s modify Eagle ' s medium), that is: the grace Ge Shi nutrient solution of Du Beikeshi improvement.
Support and succeeding transfer culture by SSCs being carried out external former being commissioned to train, found out the better culture system of suitable SSCs at external long-term surviving, under the condition that has feeder layer to exist, in this culture system, the SSCs well-grown, adherent speed is fast, the cloning efficiency height, the SSCs in 1-4 generation has the AKP positive colony to form, and cloning efficiency is respectively 47%, 36%, 26%, 13%.Explanation is thus used the CEF feeder layer and is added vitro culture and the propagation that the nutrient solution that cytokine is not arranged is suitable for chicken SSCs most, is best cultivation body.
Claims (1)
1, a kind of extracorporeal culturing method of bird stem spermatogonium is characterized in that the bird stem spermatogonium is inoculated on the chick embryo fibroblast feeder layer, re-uses nutrient solution and cultivates; Described nutrient solution is: add 10% calf serum, 2% chicken serum, 2mmol/LL-glutamine, 1mmol/L Sodium.alpha.-ketopropionate, 5.5 * 10 in the DMEM high glucose medium
-5Mol/L beta-mercaptoethanol, 10 μ l/ml non-essential amino acid, 15-20ng/ml hSCF, 10U/ml mLIF, 10-15ng/ml bFGF, 0.04ng/ml hIL-11,10ng/ml IGF, 100U/ml gentamicin sulphate.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2006100966296A CN100455661C (en) | 2006-10-13 | 2006-10-13 | In vitro culture method for avian stem spermatogonium |
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| Application Number | Priority Date | Filing Date | Title |
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| CNB2006100966296A CN100455661C (en) | 2006-10-13 | 2006-10-13 | In vitro culture method for avian stem spermatogonium |
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| CN1935984A true CN1935984A (en) | 2007-03-28 |
| CN100455661C CN100455661C (en) | 2009-01-28 |
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| CNB2006100966296A Expired - Fee Related CN100455661C (en) | 2006-10-13 | 2006-10-13 | In vitro culture method for avian stem spermatogonium |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102031242A (en) * | 2010-11-19 | 2011-04-27 | 西北农林科技大学 | Culture solution system for enhancing reproduction rate of spermatogonial stem cells of animal and use method thereof |
| CN102382799A (en) * | 2010-08-31 | 2012-03-21 | 吴际 | In-vitro isolation and culture method of mouse spermatogonial stem cells and special culture medium thereof |
| CN102382798A (en) * | 2010-08-31 | 2012-03-21 | 吴际 | Separation method and culture in vitro method for mouse female germline stem cells |
| CN106701662A (en) * | 2016-11-28 | 2017-05-24 | 中国农业大学 | Method for in-vitro long-time stable culture of chicken embryonic stem cells |
| CN107125240A (en) * | 2016-08-01 | 2017-09-05 | 北京世纪劲得生物技术有限公司 | A kind of skin spermatogonium protection liquid and preparation method thereof |
| CN110283779A (en) * | 2019-07-17 | 2019-09-27 | 吉林省农业科学院 | A kind of isolated culture method and culture medium of chicken embryonic stem cells |
-
2006
- 2006-10-13 CN CNB2006100966296A patent/CN100455661C/en not_active Expired - Fee Related
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102382799A (en) * | 2010-08-31 | 2012-03-21 | 吴际 | In-vitro isolation and culture method of mouse spermatogonial stem cells and special culture medium thereof |
| CN102382798A (en) * | 2010-08-31 | 2012-03-21 | 吴际 | Separation method and culture in vitro method for mouse female germline stem cells |
| CN102031242A (en) * | 2010-11-19 | 2011-04-27 | 西北农林科技大学 | Culture solution system for enhancing reproduction rate of spermatogonial stem cells of animal and use method thereof |
| CN107125240A (en) * | 2016-08-01 | 2017-09-05 | 北京世纪劲得生物技术有限公司 | A kind of skin spermatogonium protection liquid and preparation method thereof |
| WO2018023828A1 (en) * | 2016-08-01 | 2018-02-08 | 北京世纪劲得生物技术有限公司 | Protective fluid for skin and spermatogonial cells and preparation method thereof |
| CN106701662A (en) * | 2016-11-28 | 2017-05-24 | 中国农业大学 | Method for in-vitro long-time stable culture of chicken embryonic stem cells |
| CN106701662B (en) * | 2016-11-28 | 2019-09-27 | 中国农业大学 | A method of external culture chicken embryonic stem cells steady in a long-term |
| CN110283779A (en) * | 2019-07-17 | 2019-09-27 | 吉林省农业科学院 | A kind of isolated culture method and culture medium of chicken embryonic stem cells |
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| CN100455661C (en) | 2009-01-28 |
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