Culture medium and culture method for promoting in-vitro proliferation of spermatogonial stem cells
By adding N-cyclohexylethanolamine and atorvastatin to the culture medium, the metabolic activity of spermatogenetic stem cells and feeder cells was affected, and the problems of long-term proliferation of spermatogenetic stem cells and maintenance of stem cell characteristics were solved, and efficient cell proliferation was achieved and potential maintenance provides new pathways for research and treatment.
Patent Information
- Application Number
- CN202411509137.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-25
- Publication Date
- 2024-12-20
AI Technical Summary
The prior art is difficult to maintain long-term proliferation of spermatogenic stem cells in vitro, and their stem cell characteristics are gradually lost during the culture process, resulting in research difficulties.
The culture medium containing N-cyclohexylethanolamine and atorvastatin is used to reduce the competitive trophic activity of feeder cells and promote the self-renewal and proliferation of spermatogenic stem cells by affecting the metabolic activity of spermatogenic stem cells and feeder cells.
It has achieved efficient in vitro proliferation of spermatogenic stem cells, maintained their stem cell potential, and provided a new culture system, providing important scientific value for the study and treatment of male reproductive system diseases.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of stem cell culture, and in particular to a culture medium and a culture method for promoting the proliferation of spermatogonial stem cells in vitro. Background Art
[0002] Spermatogonial stem cells (SSCs) are a special type of adult stem cells in the testis and are the basis of spermatogenesis. Since SSCs are rare in the testis, the establishment of an in vitro culture system is an important prerequisite for studying and manipulating this rare cell population of SSCs, and the long-term culture of SSCs in vitro is a prerequisite for exploring the self-renewal and differentiation of SSCs. However, due to the complexity of the microenvironment in which SSCs are located and their small proportion in the entire testicular tissue, it is extremely difficult to construct a niche microenvironment or purify SSCs during in vitro culture, which has a certain impact on the research of SSCs.
[0003] Even if SSCs are successfully isolated, enriched and initially cultured, the existing culture system is difficult to maintain their long-term proliferation in vitro. During the culture process, the stem cell characteristics of SSCs will gradually be lost. As time goes by, differentiation and apoptosis play a leading role in cell events, and the final result is the stagnation of SSCs in vitro culture. Therefore, it is particularly important to study the mechanism of maintaining the stemness of SSCs and establish a culture system that supports the self-renewal of SSCs and maintains their stem cell potential. Summary of the invention
[0004] The purpose of the present invention is to provide a culture medium and a culture method for promoting the proliferation of spermatogonial stem cells in vitro, so as to solve the problems existing in the above-mentioned prior art. The culture medium of the present invention adds N-cyclohexylethanolamine and atorvastatin, which can affect the metabolic activity of spermatogonial stem cells and feeder layer cells, reduce the activity of feeder layer cells in competing for nutrients, and has a synergistic effect in promoting the self-renewal and proliferation of spermatogonial stem cells.
[0005] To achieve the above object, the present invention provides the following solutions:
[0006] The invention provides a composition for promoting the proliferation of spermatogonial stem cells in vitro, comprising N-cyclohexylethanolamine and atorvastatin.
[0007] The present invention also provides a culture medium for promoting the proliferation of spermatogonial stem cells in vitro, comprising the composition.
[0008] Preferably, in the culture medium, the mass ratio of the N-cyclohexylethanolamine to the atorvastatin is (2-6):1.
[0009] Preferably, in the culture medium, the mass ratio of the N-cyclohexylethanolamine to the atorvastatin is 3:1.
[0010] Preferably, in the culture medium, the concentration of N-cyclohexylethanolamine is 15 μg / mL, and the concentration of atorvastatin is 5 μg / mL.
[0011] Preferably, the method for preparing the culture medium comprises the following steps:
[0012] 15 mg of N-cyclohexylethanolamine and 5 mg of atorvastatin were mixed, dissolved in DMSO, the concentration of N-cyclohexylethanolamine was adjusted to 15 mg / mL, the concentration of atorvastatin was adjusted to 5 mg / mL, and sterilized by filtering with a 0.22 μm filter to obtain a mixed solution;
[0013] Preparing spermatogonial stem cell basal culture medium;
[0014] The mixed solution is added to the spermatogonial stem cell basal culture medium at a volume ratio of 1:1000 to obtain the culture medium.
[0015] Preferably, the spermatogonial stem cell basal culture medium comprises DMEM / F12 culture medium, FBS, glutamine concentrate, non-essential amino acid stock solution and β-mercaptoethanol.
[0016] Preferably, the method for preparing 100 mL of the spermatogonial stem cell basal medium comprises the following steps:
[0017] Measure 87.9 mL of DMEM / F12 medium and add it to a 200 mL container. Then add 10 mL of FBS, 1 mL of glutamine concentrate, 1 mL of non-essential amino acid stock solution, and 0.1 mL of β-mercaptoethanol.
[0018] After being fully mixed, the mixture was filtered and sterilized using a 0.22 μm filter to obtain the spermatogonial stem cell basal medium.
[0019] The present invention also provides the use of the composition or the culture medium in promoting the proliferation of spermatogonial stem cells in vitro.
[0020] The present invention also provides a culture method for promoting the proliferation of spermatogonial stem cells in vitro, comprising the step of using the culture medium to culture spermatogonial stem cells in vitro.
[0021] The present invention discloses the following technical effects:
[0022] The present invention adds N-cyclohexylethanolamine and atorvastatin to a spermatogonial stem cell basal culture medium to prepare a culture medium that promotes the in vitro proliferation of spermatogonial stem cells; experiments have shown that N-cyclohexylethanolamine and atorvastatin in a specific concentration ratio can jointly affect the metabolic activity of spermatogonial stem cells and feeder layer cells, reduce the activity of feeder layer cells in competing for nutrients, and have a synergistic effect in promoting the self-renewal and proliferation of spermatogonial stem cells. Utilizing the culture medium of the present invention, spermatogonial stem cells can be simply and efficiently amplified in large quantities, providing a new path for the in vitro culture and amplification of human spermatogonial stem cells that is different from the traditional addition of growth factors. The present invention is used to culture spermatogonial stem cells and treat male reproductive system diseases, which has very important scientific value. DETAILED DESCRIPTION
[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.
[0024] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.
[0025] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.
[0026] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.
[0027] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.
[0028] Example 1
[0029] 1. Preparation of spermatogonial stem cell basal culture medium
[0030] Preparation method of 100mL spermatogonial stem cell basal medium:
[0031] Measure 87.9 mL of DMEM / F12 medium and add it to a 200 mL container, then add 10 mL of FBS, 1 mL of glutamine concentrate (100×), 1 mL of non-essential amino acid stock solution (100×), and 0.1 mL of β-mercaptoethanol (1000×);
[0032] After being fully mixed with a magnetic stirrer, the mixture was filtered and sterilized using a 0.22 μm filter to obtain spermatogonial stem cell basal medium, which was added into a sterile plastic bottle for later use.
[0033] 2. Preparation of culture medium to promote spermatogonial stem cell proliferation in vitro
[0034] 10 mg of N-cyclohexylethanolamine and 10 mg of atorvastatin were mixed, dissolved in DMSO, the total concentration of N-cyclohexylethanolamine and atorvastatin was adjusted to 20 mg / mL, and sterilized by filtration using a 0.22 μm filter to prepare a mixed solution;
[0035] 0.1 mL of the mixed solution was added to the spermatogonial stem cell basal culture medium to obtain a culture medium for promoting the proliferation of spermatogonial stem cells in vitro.
[0036] Example 2
[0037] The only difference from Example 1 is that 13.33 mg of N-cyclohexylethanolamine and 6.67 mg of atorvastatin were mixed.
[0038] Example 3
[0039] The only difference from Example 1 is that 15 mg of N-cyclohexylethanolamine and 5 mg of atorvastatin were mixed.
[0040] Example 4
[0041] The only difference from Example 1 is that 16 mg of N-cyclohexylethanolamine and 4 mg of atorvastatin were mixed.
[0042] Example 5
[0043] The only difference from Example 1 is that 17.14 mg of N-cyclohexylethanolamine and 2.86 mg of atorvastatin were mixed.
[0044] Example 6
[0045] The only difference from Example 1 is that 18 mg of N-cyclohexylethanolamine and 2 mg of atorvastatin were mixed.
[0046] Comparative Example 1
[0047] The only difference from Example 1 is that no other drugs are added to the spermatogonial stem cell basal culture medium.
[0048] Comparative Example 2
[0049] The only difference from Example 1 is that 20 mg of N-cyclohexylethanolamine is used instead of 10 mg of N-cyclohexylethanolamine and 10 mg of atorvastatin.
[0050] Comparative Example 3
[0051] The only difference from Example 1 is that 20 mg of atorvastatin is used instead of 10 mg of N-cyclohexylethanolamine and 10 mg of atorvastatin.
[0052] Experimental Example 1
[0053] 1. Cultivation of Mouse Spermatogonial Stem Cells
[0054] The mouse spermatogonial stem cells C18-4 stored in the laboratory were taken out from liquid nitrogen and quickly thawed in a 37°C water bath;
[0055] The cell suspension in the cryopreservation tube was aspirated and added to a centrifuge tube containing DMEM / F12 medium, centrifuged at 1500 rpm for 5 min, and the supernatant was discarded;
[0056] Add an appropriate amount of DMEM / F12 medium containing 10% FBS, mix well by pipetting, and add 2×10 6 The cell suspension of 10 cells was inoculated into a 10 cm culture dish pre-coated with MEF feeder cells and placed in a 37°C cell culture incubator with 37°C and 5% CO2 for culture.
[0057] The medium was changed every 1-2 days according to the cell density. When the cell density reached 85% or more, the medium was discarded, and trypsin was added to digest the cells. After preparing a cell suspension, 2×10 6 The cell suspension of 10 cells was inoculated into a 10 cm culture dish pre-coated with MEF feeder cells, and when the cells reached the third generation, they were set aside.
[0058] 2. Experimental test
[0059] The mouse spermatogonial stem cells C18-4 prepared above were digested with trypsin, the digested cells were prepared into a cell suspension and the cell density was calculated, and then the cell suspension was inoculated into a 96-well cell culture plate, 5000 cells were inoculated in each well, and the total volume of each well was 100 μL;
[0060] The 96-well cell culture plate inoculated with cells was placed in a cell culture incubator. After culturing overnight, the culture medium was removed, and the culture medium prepared in Examples 1 to 6 and Comparative Examples 1 to 3 was added respectively. Then, the 96-well plate was placed in a cell culture incubator, and the culture medium conditions were set to 37° C. and 5% CO2. Three replicate wells were set for each treatment.
[0061] After 48 h of culture, the 96-well plate was taken out from the cell culture incubator, 10 μL of CCK-8 solution was added to each well, and the plate was returned to the cell culture incubator for incubation for 4 h. The 96-well plate was then placed in an ELISA reader with a modulated wavelength of 450 nm to detect the absorbance of each treatment. The results are recorded in Table 1.
[0062] Table 1 Absorbance value record table after different culture medium treatment
[0063]
[0064]
[0065] Note: Compared with Comparative Example 1, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001;
[0066] Compared with Comparative Example 2, # indicates P < 0.05, ## indicates P < 0.01, ### indicates P < 0.001;
[0067] According to Table 1, the absorbance of spermatogonial stem cells cultured in the culture medium of Comparative Example 2 and Comparative Example 3 was significantly improved compared with the absorbance of spermatogonial stem cells cultured in the culture medium of Comparative Example 1, indicating that adding N-cyclohexylethanolamine or atorvastatin alone to the spermatogonial stem cell culture medium can effectively improve the proliferation ability of spermatogonial stem cells;
[0068] Secondly, compared with the cells cultured in the culture medium of Comparative Example 2 or 3, the absorbance of spermatogonial stem cells cultured in the culture medium of Examples 1 to 6 was further improved, and the difference was significant, indicating that the proliferation ability of spermatogonial stem cells can be more significantly improved by the combined use of N-cyclohexylethanolamine and atorvastatin;
[0069] Furthermore, although the absorbance of spermatogonial stem cells cultured in the culture medium of Examples 1 and 6 was significantly higher than that of spermatogonial stem cells cultured in the culture medium of Control Example 2, it indicates that when N-cyclohexylethanolamine and atorvastatin are used in combination at a mass ratio of (2-6):1, the absorbance of spermatogonial stem cells increases significantly when the total addition amount is the same; especially when used in combination at a mass ratio of 3:1, the most significant synergistic effect on the proliferation of spermatogonial stem cells can be produced (i.e., Example 3).
[0070] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.
Claims
1. A composition for promoting the proliferation of spermatogonial stem cells in vitro, characterized in that: These include N-cyclohexylethanolamine and atorvastatin.
2. A culture medium for promoting the proliferation of spermatogonial stem cells in vitro, characterized in that: The invention comprises the composition according to claim 1.
3. The culture medium according to claim 2, characterized in that In the culture medium, the mass ratio of the N-cyclohexylethanolamine to the atorvastatin is (2-6):
1.
4. The culture medium according to claim 3, characterized in that In the culture medium, the mass ratio of the N-cyclohexylethanolamine to the atorvastatin is 3:
1.
5. The culture medium according to claim 4, characterized in that In the culture medium, the concentration of N-cyclohexylethanolamine is 15 μg / mL, and the concentration of atorvastatin is 5 μg / mL.
6. The culture medium according to claim 5, characterized in that The preparation method of the culture medium comprises the following steps: 15 mg of N-cyclohexylethanolamine and 5 mg of atorvastatin were mixed, dissolved in DMSO, the concentration of N-cyclohexylethanolamine was adjusted to 15 mg / mL, the concentration of atorvastatin was adjusted to 5 mg / mL, and sterilized by filtering with a 0.22 μm filter to obtain a mixed solution; Preparing spermatogonial stem cell basal culture medium; The mixed solution is added to the spermatogonial stem cell basal culture medium at a volume ratio of 1:1000 to obtain the culture medium.
7. The culture medium according to claim 6, characterized in that The spermatogonial stem cell basal culture medium comprises DMEM / F12 culture medium, FBS, glutamine concentrate, non-essential amino acid stock solution and β-mercaptoethanol.
8. The culture medium according to claim 7, characterized in that The preparation method of each 100 mL of the spermatogonial stem cell basal culture medium comprises the following steps: Measure 87.9 mL of DMEM / F12 medium and add it to a 200 mL container. Then add 10 mL of FBS, 1 mL of glutamine concentrate, 1 mL of non-essential amino acid stock solution, and 0.1 mL of β-mercaptoethanol. After being fully mixed, the mixture was filtered and sterilized using a 0.22 μm filter to obtain the spermatogonial stem cell basal medium.
9. Use of the composition according to claim 1 or the culture medium according to any one of claims 2 to 8 in promoting the proliferation of spermatogonial stem cells in vitro.
10. A method for promoting the proliferation of spermatogonial stem cells in vitro, characterized in that: The method comprises the step of using the culture medium described in any one of claims 1 to 8 to culture spermatogonial stem cells in vitro.