Separation and in-vitro culture method of spermatogonial stem cells of grass carp

Abstract

The invention belongs to the technical field of in-vitro culture of germline stem cells, and particularly relates to a separation and in-vitro culture method of spermatogonial stem cells of grass carp. The method comprises the following steps: by taking male grass carps as donors, anesthetizing, killing, cleaning, disinfecting, dissecting and separating gonad tissues, crushing into fragments by adopting a mode of combining magnetic bead crushing with shearing, digesting with trypsin, filtering and centrifuging to obtain high-purity spermatogonial stem cells, and performing in-vitro culture on the spermatogonial stem cells by adopting a culture solution. By optimizing the age of the male grass carp, an enzyme digestion process and an in-vitro culture system and adopting a one-step enzyme digestion method, the digestion time is shortened, the operation process is simplified, and the industrial problems that the spermatogonial stem cells of the grass carp are low in separation efficiency and culture is easy to differentiate are solved. According to the invention, a separation, purification and primary culture technical system of the spermatogonial stem cells of the grass carp is systematically established for the first time, and important cell materials and technical supports are provided for breeding technologies such as transplantation of germline stem cells of the grass carp, gene editing and the like.

Description

Technical Field

[0001] This invention belongs to the field of reproductive stem cell in vitro culture technology, specifically relating to a method for isolating and culturing grass carp spermatogonial stem cells in vitro. Background Technology

[0002] Grass carp, as the highest-yielding economic fish species in freshwater aquaculture, has long relied on the domestication of wild species, leading to a decline in genetic diversity and reduced resilience, among other genetic degradation issues. Traditional breeding cycles, reaching up to 20 years, are insufficient to meet industry demands. Fish reproductive stem cell transplantation technology can overcome the limitations of a long sexual maturation cycle, and combined with surrogacy techniques, significantly shorten the time required for selecting superior breeds. Spermatoglyphics, as reproductive stem cells with self-renewal and multi-directional differentiation potential, can be cultured in vitro to establish stable cell lines, providing a sustainable source of donor cells for reproductive cell transplantation. Furthermore, establishing in vitro culture systems allows for in-depth research into the molecular mechanisms of spermatogonial stem cell proliferation and differentiation, providing a theoretical basis for optimizing transplantation parameters (such as cell concentration and timing). Ultimately, this technology can not only address the grass carp germplasm resource crisis but also provide a technological paradigm for the protection of other endangered fish species, promoting the sustainable development of aquaculture.

[0003] Compared to mammals, research on spermatogonial stem cells in fish started later. Currently, spermatogonial stem cell isolation and culture techniques have been successfully established in fish such as medaka, zebrafish, and rainbow trout. However, research on spermatogonial stem cells in economically important fish still faces many challenges, including low cell isolation purity, imperfect long-term in vitro culture systems, and difficulty in maintaining stem cell characteristics. These problems restrict the application of spermatogonial stem cells in aquaculture breeding. Furthermore, there are few research reports on grass carp spermatogonial stem cells, and grass carp-specific isolation and culture conditions are lacking.

[0004] Currently, there is no method for isolating and culturing spermatogonial stem cells specifically for grass carp. Firstly, existing isolation and culture conditions largely reference other species, such as mammals and carp, and have not been optimized for grass carp, resulting in low efficiency. Secondly, current techniques use donor fish of random ages, and the characteristics of spermatogonial stem cells at different developmental stages vary significantly, leading to unstable cell quality and proliferation capacity. Furthermore, current techniques often employ combined enzyme digestion methods, which are time-consuming and more prone to cell damage.

[0005] Therefore, there is an urgent need to solve the industry problem of low isolation efficiency and easy differentiation of grass carp spermatogonial stem cells. Summary of the Invention

[0006] To overcome the shortcomings of the existing technology, this invention provides a method for isolating and culturing grass carp spermatogonial stem cells in vitro. This invention systematically compared the proportion of type A spermatogonial stem cells and cell proliferation activity in spermatogonial stem cells from donor fish of different ages, ensuring optimal cell source quality. Secondly, this invention develops a one-step enzymatic digestion system, shortening digestion time, thereby simplifying the operation process and reducing the risk of contamination. Finally, this invention specifically optimizes the culture system for grass carp. Ultimately, this makes the isolation and culture of grass carp spermatogonial stem cells more efficient, solving the industry problem of low isolation efficiency and easy differentiation of grass carp spermatogonial stem cells.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This invention provides a method for isolating and culturing grass carp spermatogonial stem cells in vitro, comprising the following steps: (1) Anesthetize the male grass carp, kill it, wash its body surface, disinfect it, and remove its gonads; (2) The dissected gonads were rinsed in sterile phosphate buffer and then broken into small pieces by a combination of magnetic bead breaking and shearing. (3) The fragments were digested with trypsin solution, filtered, and centrifuged to obtain isolated spermatogonial stem cells, which were then cultured in vitro in culture medium. The culture medium contains basal culture medium, fetal bovine serum, grass carp serum, epidermal growth factor, and basic fibroblast growth factor.

[0008] Furthermore, in step (1), the male grass carp is a 1-4 year old male grass carp.

[0009] Furthermore, in step (1), the male grass carp is a 2-year-old male grass carp.

[0010] Furthermore, in step (2), the volume of the fragment is <1 mm. 3 .

[0011] Furthermore, in step (3), the working concentration of trypsin in the trypsin solution is 0.25%.

[0012] Furthermore, in step (3), the digestion temperature is 37 °C and the digestion time is 6-7 min.

[0013] Furthermore, the basal culture medium is any one of L-15, M199, and DMEM-F12.

[0014] Furthermore, the fetal bovine serum is Clark fetal bovine serum.

[0015] Furthermore, the concentration of fetal bovine serum in the culture medium is 7-15%.

[0016] Furthermore, the concentration of fetal bovine serum in the culture medium is 10%.

[0017] Further, in step (3), the conditions for the in vitro primary culture are: culture at a CO2 concentration of 5%, saturated humidity and constant temperature of 28 °C, with the culture medium being changed every 3 days.

[0018] Compared with the prior art, the beneficial effects of the present invention are: This invention provides a method for isolating and culturing grass carp spermatogonial stem cells in vitro. Using male grass carp as donors, the fish are anesthetized, euthanized, cleaned, disinfected, and then dissected to separate the gonadal tissue. The tissue is broken into small pieces using a combination of magnetic bead fragmentation and shearing, digested with trypsin, filtered, and centrifuged to obtain high-purity spermatogonial stem cells. These cells are then cultured in vitro using a culture medium. This invention optimizes the age of the male grass carp, the enzyme digestion process, and the in vitro culture system, employing a one-step enzyme digestion method that shortens the digestion time. This simplifies the operation process and solves the industry-wide problems of low isolation efficiency and easy differentiation of grass carp spermatogonial stem cells. This invention is the first to systematically establish a technical system for the isolation, purification, and primary culture of grass carp spermatogonial stem cells, providing important cellular materials and technical support for grass carp reproductive stem cell transplantation, gene editing, and other breeding technologies. Attached Figure Description

[0019] Figure 1 The cell viability of grass carp donors of different ages in Examples 1-4 (n=12) is shown, where different letters indicate significant differences (p<0.05).

[0020] Figure 2 The images show the morphology of cells cultured in vitro for different days in Example 2, with the arrows pointing to spermatogonial stem cells.

[0021] Figure 3 The figure shows the effect of different fetal bovine serum sources on grass carp spermatogonial stem cells in Example 2, Comparative Example 5, and Comparative Example 6 (n=12). The Sijiqing group was defined as the control, and the arrows point to spermatogonial stem cells. Different letters indicate significant differences (p<0.05).

[0022] Figure 4 The graph shows the effect of different serum concentrations on grass carp spermatogonial stem cells in Examples 2, 7, and 8 (n=12). 7% is defined as the control, arrows indicate spermatogonial stem cells, and different letters indicate significant differences (p<0.05).

[0023] Figure 5 The image shows a comparison of the effects of different basal culture media on grass carp spermatogonial stem cells in Examples 2, 5, and 6 (n=12). L-15 is defined as the control, and the arrow points to the spermatogonial stem cells.

[0024] Figure 6 The image shows a comparison of the effects of different cytokines on grass carp spermatogonial stem cells in Example 2, Comparative Example 7, and Comparative Example 8 (n=12). In this image, EGF+bFGF is defined as the control, and different letters indicate significant differences (p<0.05). Detailed Implementation

[0025] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0026] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0027] Example 1 One-year-old male grass carp (November 19th month) were selected as donors from a grass carp hatchery in Zhaoqing City, Guangdong Province. First, the fish were anesthetized with 0.01% 2-phenoxyethanol (Sigma-Aldrich), then euthanized by cutting off the gill arches, and their bodies were washed clean under running water. Next, the entire body surface of the fish was disinfected with 75% alcohol, and the gonads were removed using sterile scissors and forceps. The removed gonads were rinsed 3-4 times in sterile PBS (pH=7.4), placed in sterile centrifuge tubes, and fragmented into particles <1 mm using a combination of magnetic bead fragmentation and shearing. 3Small fragments were then digested with 0.25% trypsin preheated to 37 °C for 7 min, followed by termination of digestion with 5 times the volume of trypsin in PBS (pH=7.4), centrifuged at 800 g for 5 min, and then rinsed once with sterile PBS (pH=7.4). Cell suspension was filtered through a 100-mesh nylon filter to remove cell debris. Centrifuged at 800 g for 5 min, and the supernatant was discarded. Cells that settled at the bottom of the tube were identified as grass carp spermatogonial stem cells. These cells were cultured in a medium containing 0.02 g NaHCO3, 0.1 mmol / L β-mercaptoethanol (Sigma-Aldrich), and 1% grass carp serum. The grass carp serum was prepared as follows: Healthy, disease-free grass carp (approximately 2 kg) were selected. The fish were first anesthetized with 0.01% 2-phenoxyethanol (Sigma-Aldrich), then their gill arches were cut to euthanize them. Their bodies were washed under running water. Next, the entire body surface of the fish was disinfected with 75% alcohol. Blood was drawn from the tail vein into a sterile centrifuge tube, which was then sealed and placed at 4 ℃. After approximately 4-6 hours, the tube was centrifuged at 800 g for 5 min, and the supernatant was collected. The supernatant was placed in a sterile centrifuge tube, sealed with sealing film, and then incubated in a 56 ℃ water bath for 30 minutes. The cells were prepared by resuspending the cells in L-15 medium containing grass carp serum, 2 mmol / L glutamine (Sigma-Aldrich), 1 mmol / L non-essential amino acids (Sigma-Aldrich), 2‰ penicillin and streptomycin, 10% Clark FBS, 10 ng / mL EGF and 10 ng / mL bFGF. The cells were then cultured under conditions of 5% CO2 concentration, saturated humidity and constant temperature of 28 °C, with the culture medium replaced with fresh medium every 3 days.

[0028] Example 2 The only difference between Example 2 and Example 1 is that a 2-year-old male grass carp is used instead of a 1-year-old male grass carp in Example 1, while the rest is the same as in Example 1.

[0029] After grass carp spermatogonial stem cells were inoculated into the culture medium, cell morphology was observed daily, such as... Figure 2 As shown, one day after inoculation, most spermatogonial stem cells were still attached to the surface of supporting cells, and adhesion was completed on the second day. From the third day onwards, spermatogonial stem cells began to proliferate slowly. Subsequently, as the cells continued to proliferate, the number of spermatogonia began to increase.

[0030] Example 3 The only difference between Example 3 and Example 1 is that a 3-year-old male grass carp is used instead of a 1-year-old male grass carp in Example 1, while the rest is the same as in Example 1.

[0031] Example 4 The only difference between Example 4 and Example 1 is that a 4-year-old male grass carp is used instead of a 1-year-old male grass carp in Example 1, while the rest is the same as in Example 1.

[0032] Example 5 The only difference between Example 5 and Example 2 is that M199 is used instead of L-15 in Example 2 as the basal culture medium, while the rest is the same as in Example 2.

[0033] Example 6 The only difference between Example 6 and Example 2 is that DMEM-F12 (high sugar type) (Sigma-Aldrich) was used instead of L-15 in Example 2 as the basal culture medium. The rest is the same as in Example 2.

[0034] Example 7 The only difference between Example 7 and Example 2 is that the concentration of Clark FBS is changed to 7%, while the rest remains the same as Example 2.

[0035] Example 8 The only difference between Example 8 and Example 2 is that the concentration of Clark FBS is changed to 15%, while the rest remains the same as Example 2.

[0036] Comparative Example 1 The only difference between Comparative Example 1 and Example 2 is that the digestive enzyme used is 0.1% type I collagenase instead of 0.25% trypsin in Example 2, and the digestion time is 12 min. The rest is the same as Example 2.

[0037] Comparative Example 2 The only difference between Comparative Example 2 and Example 2 is that the digestive enzyme used is 0.1% type II collagenase instead of 0.25% trypsin in Example 2, and the digestion time is 18 min. The rest is the same as Example 2.

[0038] Comparative Example 3 The only difference between Comparative Example 3 and Example 2 is that the digestive enzyme used is 0.1% type III collagenase instead of 0.25% trypsin in Example 2, and the digestion time is 15 min. The rest is the same as Example 2.

[0039] Comparative Example 4 The only difference between Comparative Example 4 and Example 2 is that the digestive enzyme used is 0.1% type IV collagenase instead of 0.25% trypsin in Example 2, and the digestion time is 15 min. The rest is the same as Example 2.

[0040] Comparative Example 5 The only difference between Comparative Example 5 and Example 2 is that Hyclone fetal bovine serum was used instead of Clark fetal bovine serum in Example 2, while the rest remained the same as Example 2.

[0041] Comparative Example 6 The only difference between Comparative Example 6 and Example 2 is that the fetal bovine serum used is domestic Sijiqing fetal bovine serum instead of Clark fetal bovine serum in Example 2, and the rest is the same as Example 2.

[0042] Comparative Example 7 The only difference between Comparative Example 7 and Example 2 is that the growth factor in the culture medium is 20 ng / mL bFGF instead of 10 ng / mL EGF and 10 ng / mL bFGF in Example 2, and the rest is the same as in Example 2.

[0043] Comparative Example 8 The only difference between Comparative Example 8 and Example 2 is that the growth factor in the culture medium is 20 ng / mL EGF instead of 10 ng / mL EGF and 10 ng / mL bFGF in Example 2, and the rest is the same as Example 2.

[0044] Test Example 1 Cell viability was assessed using a CCK-8 assay kit at 3, 6, and 9 days after spermatogonial stem cell seeding in Examples 1, 2, 3, and 4, respectively. The results are as follows: Figure 1 As shown, there was no significant difference in cell viability among grass carp of different ages 3 days after inoculation. However, on days 6 and 9 of culture, the cell viability of 2-year-old grass carp was significantly higher than that of 3- and 4-year-old grass carp, while the cell viability of 1-year-old grass carp was the lowest.

[0045] Test Example 2 After the digestion steps in Examples 2, 1, 2, 3, and 4 were completed, a small amount of cell suspension was taken and stained with 0.4% trypan blue (at a volume ratio of cell suspension / trypan blue = 9 / 1). The total number of cells and the number of viable cells were calculated using a hemocytometer. Furthermore, the cell suspension was seeded into cell culture plates, and cell adhesion was observed every 12 hours. The results are shown in Table 1 (n=6). Different letters indicate significant differences (p<0.05). Adhesion time was defined as the time required for more than 20% of cells to adhere. It can be seen that digestion with 0.25% trypsin resulted in the shortest digestion time, and the total cell number was significantly higher than that with 0.1% type I and type II collagenase. Regarding viable cell rate, 0.1% type II collagenase had the lowest rate, while the other groups showed no significant differences. In addition, there were no significant differences in adhesion time among the groups.

[0046] Table 1 enzymes Digestion time (min) Total number of cells (cells) viable cell rate (%) Time spent adhering to the wall (h) 0.25% trypsin 7 32.55±7.87a 85.34±3.41a 32±6.20 0.1% Type I collagenase 12 21.54±5.98b 89.91±5.10a 30±6.57 0.1% Type II collagenase 18 17.44±8.71bc 73.32±4.34b 34±4.90 0.1% Type III Collagenase 15 25.44±7.91ab 82.19±3.70ab 32±6.20 0.1% Type IV collagenase 15 28.13±9.83ab 86.55±2.91a 30±6.57 Test Example 3 Cell morphology was observed on day 6 post-inoculation for Examples 2, 5, and 6, and cell viability was assessed using a CCK-8 assay kit. Results are as follows: Figure 3 As shown, the Clark serum group exhibited the most significant growth advantage in stem cells, and its cell viability was significantly higher than that of the other two serum groups.

[0047] Test Example 4 Cell morphology was observed on day 6 post-inoculation for Examples 2, 7, and 8, and cell viability was assessed using a CCK-8 assay kit. Results are as follows: Figure 4 As shown in the figure, the 10% group showed the best cell growth advantage, followed by the 15% and 7% groups.

[0048] Test Example 5 Cell morphology was observed on day 6 post-inoculation for Examples 2, 5, and 6, and cell viability was assessed using a CCK-8 assay kit. Results are as follows: Figure 5 As shown, there is no significant difference among the three groups.

[0049] Test Example 6 Cell morphology was observed on day 6 after inoculation of Examples 2, 7, and 8, and cell viability was detected using a CCK-8 assay kit. Results are as follows: Figure 6 As shown, the combined use of 10 ng / mL EGF and 10 ng / mL bFGF exhibits a greater growth advantage than using them alone, indicating that EGF and bFGF have a synergistic promoting effect on the in vitro culture of grass carp spermatogonial stem cells.

[0050] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. A method for isolating and culturing grass carp spermatogonial stem cells in vitro, characterized in that, Includes the following steps: (1) Anesthetize the male grass carp, kill it, wash its body surface, disinfect it, and remove its gonads; (2) The dissected gonads were rinsed in sterile phosphate buffer and then broken into small pieces by a combination of magnetic bead breaking and shearing. (3) The fragments were digested with trypsin solution, filtered, and centrifuged to obtain isolated spermatogonial stem cells, which were then cultured in vitro in culture medium. The culture medium contains basal culture medium, fetal bovine serum, grass carp serum, epidermal growth factor, and basic fibroblast growth factor.

2. The method for isolating and culturing grass carp spermatogonial stem cells according to claim 1, characterized in that, In step (1), the male grass carp is a 1-4 year old male grass carp.

3. The method for isolating and culturing grass carp spermatogonial stem cells according to claim 2, characterized in that, In step (1), the male grass carp is a 2-year-old male grass carp.

4. The method for isolating and culturing grass carp spermatogonial stem cells according to claim 1, characterized in that, In step (2), the volume of the fragment is <1 mm. 3 .

5. The method for isolating and culturing grass carp spermatogonial stem cells according to claim 1, characterized in that, In step (3), the working concentration of trypsin in the trypsin solution is 0.25%.

6. The method for isolating and culturing grass carp spermatogonial stem cells according to claim 1, characterized in that, In step (3), the digestion temperature is 37 °C and the digestion time is 6-7 min.

7. The method for isolating and culturing grass carp spermatogonial stem cells according to claim 1, characterized in that, The basal culture medium is any one of L-15, M199, and DMEM-F12.

8. The method for isolating and culturing grass carp spermatogonial stem cells according to claim 1, characterized in that, The fetal bovine serum in question is Clark fetal bovine serum.

9. The method for isolating and culturing grass carp spermatogonial stem cells according to claim 1, characterized in that, The concentration of fetal bovine serum in the culture medium is 7-15%.

10. A method for isolating and culturing grass carp spermatogonial stem cells according to claim 9, characterized in that, The concentration of fetal bovine serum in the culture medium is 10%.

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