CN112266894A - In-vitro culture solution for spermatogonial stem cells - Google Patents
In-vitro culture solution for spermatogonial stem cells Download PDFInfo
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- CN112266894A CN112266894A CN202011328586.6A CN202011328586A CN112266894A CN 112266894 A CN112266894 A CN 112266894A CN 202011328586 A CN202011328586 A CN 202011328586A CN 112266894 A CN112266894 A CN 112266894A
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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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- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
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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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/70—Undefined extracts
- C12N2500/80—Undefined extracts from animals
- C12N2500/84—Undefined extracts from animals from mammals
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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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/11—Epidermal growth factor [EGF]
Abstract
The invention relates to the technical field of cell culture, in particular to an in-vitro culture solution of spermatogonial stem cells, which consists of a basic culture solution, testicular tissue fluid, cistanchis, carica calycosin, Retinoic Acid (RA) and Epidermal Growth Factor (EGF), wherein the basic culture solution is based on a high-sugar DMEM/F12 cell culture medium containing L-glutamine, and fetal bovine serum with the volume of 10% and double antibody with the volume of 5% are added into the basic culture solution. The invention finds that the cistanoside and the caramel calyx-shaped flavonoid can be applied to promoting the proliferation of the spermatogonial stem cells, further provides a new culture solution for the culture of the spermatogonial stem cells, and can realize the stable and efficient proliferation of the spermatogonial stem cells.
Description
Technical Field
The invention relates to the technical field of cell culture, in particular to an in-vitro culture solution for spermatogonial stem cells.
Background
The Spermatogonial Stem Cells (SSCs) are only diploid somatic cells which can transmit genetic information to offspring in a mammal body, and research on in-vitro proliferation and induced differentiation of the cells not only can provide technical support for preservation of the genetic information of the mammal, but also can provide important theoretical basis for research on spermatogenesis of the mammal. The establishment of a rapid and efficient culture method in vitro to obtain spermatogonial stem cells that meet clinical therapeutic applications is a major problem to be solved for transplantation of spermatogonial stem cells.
Disclosure of Invention
In order to solve the problems, the invention provides an in-vitro culture solution of spermatogonial stem cells, which can realize the stable and efficient proliferation of the spermatogonial stem cells.
In order to achieve the purpose, the invention adopts the technical scheme that:
an in vitro culture solution of spermatogonial stem cells comprises basic culture solution, testis tissue fluid, cistanoside, calyx Hibisci Maflavone, Retinoic Acid (RA), and Epidermal Growth Factor (EGF), wherein the basic culture solution is based on high-sugar DMEM/F12 cell culture medium containing L-glutamine, and 10% fetal calf serum and 5% double antibody are added.
Preferably, the basic culture solution accounts for 70% of the total volume of the culture solution, the addition amount of Epidermal Growth Factor (EGF) is 20-40 ng/ml, the addition amount of Retinoic Acid (RA) is 2-7 mug/L, the addition amount of cistanchis glucoside is 100-200 mg/L, the addition amount of caramel calyx-shaped flavones is 50-100 mg/L, and the balance is testis tissue solution.
Preferably, the basic culture solution accounts for 70% of the total volume of the culture solution, the addition amount of Epidermal Growth Factor (EGF) is 20-30 ng/ml, the addition amount of Retinoic Acid (RA) is 2-6 mug/L, the addition amount of cistanoside is 120-180 mg/L, the addition amount of carica calyx flavones is 50-80 mg/L, and the balance is testis tissue solution.
Preferably, the basic culture solution accounts for 70% of the total volume of the culture solution, the addition amount of Epidermal Growth Factor (EGF) is 20-25 ng/ml, the addition amount of Retinoic Acid (RA) is 2-5 mug/L, the addition amount of cistanoside is 140-160 mg/L, the addition amount of caramel calyx-shaped flavone is 60-70 mg/L, and the balance is testis tissue solution.
The invention finds that the cistanoside and the caramel calyx-shaped flavonoid can be applied to promoting the proliferation of the spermatogonial stem cells, further provides a new culture solution for the culture of the spermatogonial stem cells, and can realize the stable and efficient proliferation of the spermatogonial stem cells.
Detailed Description
The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications can be made by persons skilled in the art without departing from the spirit of the invention. All falling within the scope of the present invention.
Example 1
An in vitro culture solution of spermatogonial stem cells comprises a basic culture solution, a testis tissue fluid, cistanoside, caramel flower pedicle macadamia flavone, Retinoic Acid (RA) and Epidermal Growth Factor (EGF), wherein the basic culture solution accounts for 70% of the total volume of the culture solution, the addition amount of the Epidermal Growth Factor (EGF) is 20-40 ng/ml, the addition amount of the Retinoic Acid (RA) is 2-7 mug/L, the addition amount of the cistanoside is 100-200 mg/L, the addition amount of the caramel flower pedicle macadamia flavone is 50-100 mg/L, and the balance is the testis tissue fluid; the basic culture solution is based on high-sugar DMEM/F12 cell culture medium containing L-glutamine, and 10% by volume of fetal bovine serum and 5% by volume of double antibody are added into the basic culture solution.
Example 2
An in vitro culture solution of spermatogonial stem cells comprises a basic culture solution, a testis tissue fluid, cistanoside, caramel flower pedicle macadamia flavone, Retinoic Acid (RA) and Epidermal Growth Factor (EGF), wherein the basic culture solution accounts for 70% of the total volume of the culture solution, the addition amount of the Epidermal Growth Factor (EGF) is 20-30 ng/ml, the addition amount of the Retinoic Acid (RA) is 2-6 mug/L, the addition amount of the cistanoside is 120-180 mg/L, the addition amount of the caramel flower pedicle macadamia flavone is 50-80 mg/L, and the balance is the testis tissue fluid; the basic culture solution is based on high-sugar DMEM/F12 cell culture medium containing L-glutamine, and 10% by volume of fetal bovine serum and 5% by volume of double antibody are added into the basic culture solution.
Example 3
An in vitro culture solution of spermatogonial stem cells comprises a basic culture solution, a testis tissue fluid, cistanoside, caramel flower pedicle macadamia flavone, Retinoic Acid (RA) and Epidermal Growth Factor (EGF), wherein the basic culture solution accounts for 70% of the total volume of the culture solution, the addition amount of the Epidermal Growth Factor (EGF) is 20-25 ng/ml, the addition amount of the Retinoic Acid (RA) is 2-5 mug/L, the addition amount of the cistanoside is 140-160 mg/L, the addition amount of the caramel flower pedicle macadamia flavone is 60-70 mg/L, and the balance is the testis tissue fluid; the basic culture solution is based on high-sugar DMEM/F12 cell culture medium containing L-glutamine, and 10% by volume of fetal bovine serum and 5% by volume of double antibody are added into the basic culture solution.
Example 4
An in vitro culture solution of spermatogonial stem cells comprises basic culture solution, testis tissue fluid, cistanoside, calyx Hibisci Sabdariffae flavone, Retinoic Acid (RA) and Epidermal Growth Factor (EGF), wherein the basic culture solution accounts for 70% of the total volume of the culture solution, the addition amount of Epidermal Growth Factor (EGF) is 20ng/ml, the addition amount of Retinoic Acid (RA) is 2 μ g/L, the addition amount of cistanoside is 140mg/L, the addition amount of calyx Hibisci Sabdariffae flavone is 60mg/L, and the balance is testis tissue fluid; the basic culture solution is based on high-sugar DMEM/F12 cell culture medium containing L-glutamine, and 10% by volume of fetal bovine serum and 5% by volume of double antibody are added into the basic culture solution.
Example 5
An in vitro culture solution of spermatogonial stem cells comprises basic culture solution, testis tissue fluid, cistanoside, calyx Hibisci Sabdariffae flavone, Retinoic Acid (RA) and Epidermal Growth Factor (EGF), wherein the basic culture solution accounts for 70% of the total volume of the culture solution, the addition amount of Epidermal Growth Factor (EGF) is 25 ng/ml, the addition amount of Retinoic Acid (RA) is 5 μ g/L, the addition amount of cistanoside is 160mg/L, the addition amount of calyx Hibisci Sabdariffae flavone is 70mg/L, and the balance is testis tissue fluid; the basic culture solution is based on high-sugar DMEM/F12 cell culture medium containing L-glutamine, and 10% by volume of fetal bovine serum and 5% by volume of double antibody are added into the basic culture solution.
Example 6
An in vitro culture solution of spermatogonial stem cells comprises basic culture solution, testis tissue fluid, cistanoside, calyx Hibisci Sabdariffae flavone, Retinoic Acid (RA) and Epidermal Growth Factor (EGF), wherein the basic culture solution accounts for 70% of the total volume of the culture solution, the addition amount of Epidermal Growth Factor (EGF) is 22 ng/ml, the addition amount of Retinoic Acid (RA) is 3 μ g/L, the addition amount of cistanoside is 150mg/L, the addition amount of calyx Hibisci Sabdariffae flavone is 65mg/L, and the balance is testis tissue fluid; the basic culture solution is based on high-sugar DMEM/F12 cell culture medium containing L-glutamine, and 10% by volume of fetal bovine serum and 5% by volume of double antibody are added into the basic culture solution.
Comparative example 1
The culture medium consists of a basic culture solution and a testis tissue solution, wherein the basic culture solution accounts for 70% of the volume of the total culture solution, the testis tissue solution accounts for 30% of the volume of the total culture solution, the basic culture solution is based on a high-sugar DMEM/F12 cell culture medium containing L-glutamine, and fetal calf serum and 5% of double antibody are added into the basic culture solution in volume.
Comparative example 2
The medium consists of a basic culture solution, a testis tissue solution and Epidermal Growth Factor (EGF), wherein the basic culture solution accounts for 70% of the volume of the total culture solution, the addition amount of the EGF is 22 ng/ml, and the balance is the testis tissue solution, wherein the basic culture solution is based on a high-sugar DMEM/F12 cell culture medium containing L-glutamine, and 10% of fetal bovine serum and 5% of double antibody by volume are added into the medium.
Comparative example 3
The medium consists of a basic culture solution, a testis tissue solution, Retinoic Acid (RA) and Epidermal Growth Factor (EGF), wherein the basic culture solution accounts for 70% of the volume of the total culture solution, the addition amount of the Epidermal Growth Factor (EGF) is 22 ng/ml, the addition amount of the Retinoic Acid (RA) is 3 mug/L, and the balance is the testis tissue solution, wherein the basic culture solution is based on a high-sugar DMEM/F12 cell culture medium containing L-glutamine, and fetal calf serum of 10% by volume and double antibody of 5% by volume are added into the basic culture solution.
Comparative example 4
The feed consists of a basic culture solution, a testis tissue solution, Retinoic Acid (RA), Epidermal Growth Factor (EGF) and caramel calyx Ma flavone, wherein the basic culture solution accounts for 70% of the volume of the total culture solution, the addition amount of the EGF is 22 ng/ml, the addition amount of the Retinoic Acid (RA) is 3 mug/L, the addition amount of the caramel calyx Ma flavone is 65mg/L, and the balance is the testis tissue solution, wherein the basic culture solution is based on a high-sugar DMEM/F12 cell culture medium containing L-glutamine, and fetal calf serum with 10% of volume and double antibody with 5% of volume are added into the basic culture solution.
Comparative example 5
The composition comprises a basic culture solution, a testis tissue solution, Retinoic Acid (RA), Epidermal Growth Factor (EGF) and cistanchis, wherein the basic culture solution accounts for 70% of the total volume of the culture solution, the addition amount of the EGF is 22 ng/ml, the addition amount of the Retinoic Acid (RA) is 3 mug/L, the addition amount of the cistanchis is 150mg/L, and the balance is the testis tissue solution, wherein the basic culture solution is based on a high-sugar DMEM/F12 cell culture medium containing L-glutamine, and fetal bovine serum with the volume of 10% and a double antibody with the volume of 5% are added into the basic culture solution.
Examples of the experiments
The culture of spermatogonial stem cells was performed using the culture solutions of example 4, example 5 and example 6 as the culture solutions of experimental group 1, experimental group 2 and experimental group 3, and the culture solutions of comparative examples 1 to 5 as the culture solutions of control group 1, control group 2, control group 3, control group 4 and control group 5.
The results show that: after the experimental group and the control group are cultured for 7 days, the cells grow rapidly, the growth forms of the cells are colony-like and grape bunch-like, the number of cell clusters is obviously increased, and the proliferation characteristics of the spermatogonial stem cells are met.
Spermatogonial stem cell activity rate: the experimental group 3 is larger than the experimental group 2, the experimental group 1 is larger than the control group 5, the control group 4 is larger than the control group 3, the control group 2 is larger than the control group 1, and the difference has significance (P < 0.05) when two groups are compared.
In conclusion, the in vitro culture solution of the invention can promote the stable and efficient proliferation of spermatogonial stem cells.
The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes or modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention. The embodiments and features of the embodiments of the present application may be combined with each other arbitrarily without conflict.
Claims (4)
1. An in vitro culture solution of spermatogonial stem cells, which is characterized in that: consists of basic culture solution, testis tissue fluid, cistanchis glycoside, cactus flower pedicle macadamia flavone, Retinoic Acid (RA) and Epidermal Growth Factor (EGF), wherein the basic culture solution is based on high-sugar DMEM/F12 cell culture medium containing L-glutamine, and fetal calf serum of 10% by volume and double antibody of 5% by volume are added into the basic culture solution.
2. An in vitro culture of spermatogonial stem cells according to claim 1, wherein: the basic culture solution accounts for 70% of the total volume of the culture solution, the addition amount of Epidermal Growth Factor (EGF) is 20-40 ng/ml, the addition amount of Retinoic Acid (RA) is 2-7 mug/L, the addition amount of cistanoside is 100-200 mg/L, the addition amount of carica calyx flavones is 50-100 mg/L, and the balance is testis tissue solution.
3. An in vitro culture of spermatogonial stem cells according to claim 1, wherein: the basic culture solution accounts for 70% of the total volume of the culture solution, the addition amount of Epidermal Growth Factor (EGF) is 20-30 ng/ml, the addition amount of Retinoic Acid (RA) is 2-6 mug/L, the addition amount of cistanoside is 120-180 mg/L, the addition amount of carica calyx flavones is 50-80 mg/L, and the balance is testis tissue solution.
4. An in vitro culture of spermatogonial stem cells according to claim 1, wherein: the basic culture solution accounts for 70% of the total volume of the culture solution, the addition amount of Epidermal Growth Factor (EGF) is 20-25 ng/ml, the addition amount of Retinoic Acid (RA) is 2-5 mug/L, the addition amount of cistanoside is 140-160 mg/L, the addition amount of carica calyx flavones is 60-70 mg/L, and the balance is testis tissue solution.
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