Method for rapid enrichment and separation of gonadal somatic cells of leopard coral grouper

Abstract

The present application relates to the technical field of aquatic animal cell culture, and particularly relates to a rapid enrichment and separation method of Plectrypops leopardus gonadal somatic cells. The method comprises the following steps: selecting healthy Plectrypops leopardus for anesthesia; taking gonadal tissue, and after aseptic treatment, cutting, centrifugal washing and filtration, inoculating in a culture medium for primary culture to obtain gonadal cells migrated from the tissue block, and after trypsin-EDTA digestion and centrifugal washing, using 1xPBS containing polysorbate for treatment; after blocking, antibody incubation and washing, verifying the labeling effect by a fluorescence microscope to realize specific enrichment of gonadal somatic cells; after filtration and purification of the enriched fluorescent labeled cells, placing the cells under a fluorescence microscope, accurately picking fluorescent positive cells under a fluorescence field, and transferring the cells to a culture dish containing complete culture medium, inoculating the separated fluorescent positive cells in optimized complete culture medium for purification culture, so that the cells adhere to the wall and realize stable subculture.

Description

Technical Field

[0001] This invention relates to the field of aquatic animal cell culture technology, and in particular to a method for rapid enrichment and separation of leopard-gill spiny perch sex gland cells. Background Technology

[0002] In the reproductive system of fish, the gonads are composed of both germ cells and somatic cells. While somatic cells themselves do not form gametes, they play an irreplaceable role in regulating gametogenesis. They establish a microenvironment conducive to the proliferation, differentiation, and maturation of germ cells by providing structural support, nutrient supply, secreting signaling molecules, and regulating hormone levels. In the fish ovary, follicle cells and theca cells are two types of somatic cells that play a crucial regulatory role in oocyte development and maturation. The oocyte is surrounded by one or more layers of follicle cells, with an outer layer composed of theca cells, together forming the basic structural unit of the follicle. Follicle cells are located around the oocyte and can form tight intercellular junctions with the oocyte through cytoplasmic processes, directly mediating the transmission of substances and signals. They not only provide the nutrients required for oocyte growth but also secrete various growth factors and gamete development signaling molecules. These signals promote oocyte growth and the initiation of meiosis by regulating the transcriptional activity and cell cycle of the oocyte. Located in the outer layer of the follicle, the tunica vaginalis (SEV) cells are derived from ovarian stromal cells and are mainly responsible for the synthesis of steroid hormones. Through hormone-signal interactions, SEV cells provide key regulation for oocyte growth, meiosis initiation, and maturation.

[0003] In fish testes, Sertoli cells and Leydig cells are two key somatic cell types that regulate gametogenesis and maintain testicular microenvironment homeostasis. Sertoli cells, located within the seminiferous tubules, directly interact with spermatogonial stem cells (SSCs) and developing spermatocytes and spermatids, playing a crucial role in supporting and regulating spermatogenesis. Sertoli cells secrete various growth factors, regulating the self-renewal and differentiation balance of SSCs. Leydig cells, located in the interseminal tubule space, are the main steroid-producing cells in the testes, primarily responsible for synthesizing and secreting androgens such as testosterone and 11-ketotestosterone (11-KT), which play important regulatory roles in spermatogenesis.

[0004] Leopard sea bass, commonly known as the Eastern Star Grouper, is a type of grouper and is a hermaphroditic fish that matures through sexual dimorphism. It is a dominant economic fish species in coastal areas of my country, with its annual output value steadily increasing. However, the leopard sea bass aquaculture industry faces numerous problems, such as: genetic resource degradation, miniaturization of parent fish, and poor reproductive development and low reproductive efficiency in indoor parent breeding. The root cause of these problems is inextricably linked to its reproductive characteristics. According to literature reports, after 12 months of age, the gonads of leopard sea bass consistently contain two types of reproductive cells, and simultaneous maturation of the testes and ovaries is clearly observed in mature parent fish. The coexistence of both sex reproductive cells is highly likely the fundamental reason for its low reproductive efficiency. In recent years, researchers have attempted to establish an in vitro culture system for fish gonadal cells to simulate the gonadal microenvironment and explore its effects on oogonial stem cells (OSCs) and spermatogonial stem cells (SSCs). In vitro cultured follicular cells and Cetori cells continuously secrete various growth factors, which can promote the proliferation and differentiation of OSCs and SSCs, respectively. Furthermore, steroid hormones (such as estrogen and 11-ketotestosterone) secreted by tunica vaginalis cells and Lesity cells also play important roles in regulating the epigenetic state of stem cells and inducing gamete meiosis. Through co-culturing with somatic cells, researchers can not only maintain the long-term in vitro survival of germline stem cells but also induce their differentiation into functional gametes under controlled conditions, thus providing an experimental basis for in vitro gamete formation and the construction of xenogeneic reproductive systems in fish.

[0005] To investigate the interaction mechanism between gonadal cells and germline stem cells in hermaphroditic leopard-gill sea bass, it is essential to first enrich and isolate gonadal cells. Current research primarily relies on differential adhesion, Percoll density gradient centrifugation, or tissue block migration for initial separation, but these methods suffer from low purity and reproducibility. Rapid enrichment and sorting of cells using reliable cell markers such as antibodies has always been the optimal method. However, due to the dual limitations of antibody specificity and cell fragility, the biggest factor currently restricting progress in non-model fish research is the difficulty in maintaining high-precision screening of target cells while ensuring cell viability. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a method for the rapid enrichment and separation of leopard-gill spiny perch glandular cells.

[0007] The present invention aims to provide a method for rapid enrichment and isolation of sex gland cells of the leopard-gill spiny perch, specifically including the following steps:

[0008] S1. Select healthy leopard-gill spiny perch for anesthesia; take gonadal tissue, aseptically process, cut, centrifuge, wash and filter, and then inoculate it into culture medium for primary culture to obtain gonadal cells that migrate from the tissue block;

[0009] S2. After trypsin-EDTA digestion, centrifugation and washing, the emigrating gonadal cells were treated with 1×PBS containing polysorbate; after blocking, antibody incubation and washing, the labeling effect was verified by fluorescence microscopy to achieve specific enrichment of gonadal cells;

[0010] S3. After filtering and purifying the enriched fluorescently labeled cells, place them under a fluorescence microscope and use a single-cell micropicking instrument to accurately pick fluorescent positive cells under the fluorescence field of view, and transfer them to a culture dish containing complete culture medium to complete the rapid separation of gonadal cells.

[0011] S4. The isolated fluorescent positive cells were seeded into an optimized complete culture medium for purification culture, allowing the cells to adhere and grow stably through passage.

[0012] Preferably, in step S1, eugenol is used to anesthetize the leopard-gill spiny perch; aseptic processing and cutting include: obtaining gonadal tissue under aseptic conditions in a sterile laminar flow hood; washing the gonadal tissue three times with a pre-cooled 1×PBS solution containing 1% penicillin-streptomycin-gentamicin to remove surface blood vessels and membranes covering the gonadal tissue, and cutting the gonadal tissue into small tissue blocks.

[0013] Preferably, in step S1, the centrifugal washing is performed by centrifuging at 800-1000 rpm for 4-6 min and discarding the supernatant; preheated 1×HBSS is added to the precipitate and mixed by inverting 3-4 times, allowed to stand for 30 seconds, and then centrifuged at 800-1000 rpm for 4-6 min and discarding the supernatant.

[0014] The filtration was performed by filtering the gonadal cell suspension using a 100μm cell filter, and the gonadal cell suspension was obtained by resuspending the cells in complete culture medium after centrifugation and washing.

[0015] The primary culture was carried out in a CO2 incubator.

[0016] Preferably, step S2 specifically includes the following sub-steps:

[0017] S21. After digesting the gonadal cells obtained in step S1 with trypsin-EDTA, centrifuge at 800~1000 rpm for 4~6 min, discard the supernatant, resuspend the cells with 1×PBS, and filter through a 40μm cell filter to obtain the cell resuspension.

[0018] S22. Take the cell resuspended solution, add 1×PBS containing polysorbate, incubate for 3~10 min, then centrifuge and discard the supernatant;

[0019] S23. Blocking and Antibody Incubation: First, resuspend the cells in 5% skim milk powder, incubate on ice for 0.5-1.5 h, centrifuge, and discard the supernatant; then add specific primary antibody diluted in 2% skim milk powder to the cells, incubate on ice for 0.5-1.5 h, centrifuge, and wash; subsequently, resuspend the cells in 5% goat serum for 1 h and block, then discard the supernatant; add fluorescently labeled secondary antibody diluted in 2% goat serum, incubate on ice in the dark for 0.5-1.5 h, centrifuge, and wash to obtain fluorescently labeled cells;

[0020] S24. Observe the fluorescently labeled cells under a fluorescence microscope to confirm that the cells exhibit specific green fluorescence, thus completing the enrichment of gonadal cells.

[0021] Preferably, the concentration of trypsin-EDTA in step S21 is 0.2~0.3%; the concentration of polysorbate in the 1×PBS containing polysorbate in step S22 is 1‰, and the incubation time is 5min.

[0022] Preferably, in step S23, the ratio of 2% skim milk powder to specific primary antibody is 1000:1, and the specific primary antibody is Gsdf primary antibody; the ratio of 2% goat serum to fluorescently labeled secondary antibody is 1000:1, and the fluorescently labeled secondary antibody is Alexa FluorPlus 488 fluorescent secondary antibody; the incubation time on ice is 1 hour.

[0023] Preferably, in step S3, a 40 μm cell filter is used for filtration and purification; 100 to 300 cells are placed in each culture dish.

[0024] Preferably, the optimized complete culture medium in step S4 includes: D-glucose DMEM basal medium, fetal bovine serum, β-mercaptoethanol, and cytokines.

[0025] Preferred, optimized complete culture media consist of: 1 g / L D-glucose DMEM basal medium, 10% fetal bovine serum, 0.1% β-mercaptoethanol, and 10 ng / mL cytokines.

[0026] Compared with the prior art, the present invention can achieve the following beneficial effects:

[0027] (1) The process of separating and culturing high-purity gonadal cells from gonadal tissue is complete, time-saving, and can quickly obtain target cells;

[0028] (2) Fluorescent labeling based on gonadal cell-specific markers, combined with single-cell picking, eliminates interference from germ cells and other cells, resulting in high cell purity;

[0029] (3) The entire process uses mechanical processing, flexible labeling, and gentle picking, which causes little damage to cells and results in high cell adhesion and strong proliferation capacity after separation;

[0030] (4) By optimizing the culture medium and culture conditions, long-term stable passage of gonadal cells of the leopard gill spiny perch was achieved, filling the gap in the in vitro culture system of gonadal cells of this fish species.

[0031] (5) The conditions are clear and the steps are standardized. It is not limited by complex equipment and is easy to apply on a large scale and carry out subsequent experiments.

[0032] In summary, the isolation method of this invention provides an extremely useful tool for the genetic breeding of leopard-gill spiny perch cells and for exploring the interaction mechanism between gonadal cells and reproductive stem cells in hermaphroditic leopard-gill spiny perch. Attached Figure Description

[0033] Figure 1 This is a suspension of leopard-gill spiny perch gonad cells and the culture status of primary cells according to an embodiment of the present invention; A is a cell suspension obtained by mechanical treatment of leopard-gill spiny perch gonad cells, and B is an optical micrograph of leopard-gill spiny perch gonad tissue blocks after 3 days of culture.

[0034] Figure 2 This figure shows the localization of the leopard-gill spiny perch antibody Gsdf in the gonads according to an embodiment of the present invention; AC in the figure are immunofluorescence staining images of leopard-gill spiny perch ovarian tissue, and DF is immunofluorescence staining image of leopard-gill spiny perch testis tissue; where A and D are DAPI staining, showing the cell nuclei of all cells; B and E are immunostaining using anti-Gsdf antibody; C and F are merged images.

[0035] Figure 3 These are images of leopard gill spiny perch gonad cell suspension before and after Gsdf antibody labeling according to embodiments of the present invention; in the figures, A represents the image under bright field (BF), B represents the image under the green fluorescence channel, and C represents the merged image.

[0036] Figure 4 The figures are flow cytometry scatter plots and immunofluorescence micrographs of quantitatively sorted gonadal cells according to embodiments of the present invention; A and B in the figures are flow cytometry scatter plots, and CE are immunofluorescence micrographs.

[0037] Figure 5 The figure shows the selected cell culture status according to the embodiments of the present invention; the figure is a bright field image of the cells, P represents the cell culture generation, and P1, P2, P3, P5, P6 and P8 represent the first generation, second generation, third generation, fifth generation, sixth generation and eighth generation, respectively. Detailed Implementation

[0038] In the following description, embodiments of the invention will be described with reference to the accompanying drawings. In the description below, the same modules are denoted by the same reference numerals. Where the same reference numerals are used, their names and functions are also the same. Therefore, their detailed description will not be repeated.

[0039] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not constitute a limitation thereof.

[0040] This invention provides a method for the rapid enrichment and isolation of leopard-gill spiny perch sex gland cells. The method employs flexible fluorescent labeling, single-cell mechanical selection, and optimized cell culture conditions to achieve rapid in vitro enrichment, isolation, and culture of leopard-gill spiny perch somatic cells. Specifically, it includes the following steps:

[0041] S1. Primary culture of gonadal cells: Healthy leopard gill spiny perch aged 2-4 months and weighing approximately 150g were harvested for gonadal tissue after anesthesia with eugenol. Under sterile conditions, the fresh gonads were washed three times with pre-cooled 1×PBS (5-10ml) containing 1% penicillin-streptomycin-gentamicin (mixed antibiotics). After removing blood vessels and the capsule, the cells were cut into pieces approximately 1mm thick. 3 The tissue blocks were cut into small pieces, centrifuged, washed, and resuspended. The filtered cell digestion solution was collected. The cell digestion solution was resuspended in 1×HBSS pre-cooled at 4°C, centrifuged horizontally at 100g for 3-5 minutes, the supernatant was discarded, and the cells were resuspended in complete culture medium. The cells were then seeded in complete culture medium and cultured in primary culture at 28°C with CO2 for about 3 days to obtain gonadal cells that migrated from the tissue blocks.

[0042] In a specific embodiment, the gonadal tissue was placed in a sterile laminar flow hood to achieve sterility; the entire tissue block was placed in a 1.5ml centrifuge tube containing complete culture medium and cut into small pieces with scissors; the specific procedures for centrifugation, washing, and resuspending were as follows: centrifuge at 800-1000 rpm for 4-6 min and discard the supernatant; add preheated 1×HBSS to the pellet and mix by inverting 3-4 times, let stand for 30 seconds, centrifuge at 800-1000 rpm for 4-6 min and discard the supernatant; 1×HBSS contains 1×PBS and 0.04% BSA; after repeating the above procedures of resuspending with 1×HBSS, centrifuging and discarding the supernatant, the cells were resuspended in complete culture medium.

[0043] S2. Immunofluorescence labeling and enrichment of gonadal cells: The emigrating cells obtained in step S1 were digested with 0.25% trypsin-EDTA, centrifuged and washed, and then treated with 1×PBS containing polysorbate; then blocked with skim milk powder, incubated with Gsdf primary antibody, and incubated with Alexa Fluor Plus 488 fluorescent secondary antibody to complete the fluorescent labeling and obtain fluorescently labeled gonadal cells;

[0044] In a specific embodiment, the centrifugation and washing procedure is as follows: centrifuge at 800-1000 rpm for 4-6 minutes, discard the supernatant; resuspend the cells in PBS, filter through a 40 μm cell filter, and add 50-100 μL of the resuspended cells to each new centrifuge tube (approximately 10 μL). 5 ~10 8 Cells were collected; 1 ml of 1×PBS containing 1‰ polysorbate was added to each tube, and the cells were incubated for 5 minutes, followed by centrifugation at 800-1000 rpm for 4-6 minutes, and the supernatant was discarded; the cells were resuspended in 5% skim milk powder, incubated on ice for 1 hour, and centrifuged at 800-1000 rpm for 4-6 minutes, and the supernatant was discarded; Gsdf antibody was diluted with 2% skim milk powder at a ratio of 1000:1, mixed well, and added to the cells; the cell suspension was incubated on ice for 1 hour, centrifuged and the supernatant was discarded, the cells were resuspended in 1×PBS, and centrifuged again and the supernatant was discarded; the cells were resuspended in 5% goat serum powder, incubated on ice for 1 hour, and the supernatant was discarded; Alexa Fluor Plus 488 fluorescent secondary antibody was diluted with 2% goat serum at a ratio of 1000:1, and incubated on ice for 1 hour; the cells were resuspended in 1×PBS, centrifuged and the supernatant was discarded; 10 μL of the sample was placed under a fluorescence microscope to observe the fluorescent labeling.

[0045] S3. Isolation of gonadal cells: Fluorescently labeled cells were filtered through a 40μm cell filter and placed in a 6-well plate containing complete culture medium. Fluorescently labeled cells were picked under fluorescent conditions using a single-cell micropicking instrument. The picked cells were transferred to a culture dish and observed under a fluorescence microscope to see if the selected cells were fluorescently labeled.

[0046] In a specific embodiment, a single-cell micro-picking instrument from Boyi Biotechnology was used. The glass capillary tube used in the instrument was set to have an outer diameter of 1.65 mm, an inner diameter of 1.0 mm, and a length of 63 mm. The picking pressure value of the instrument was 2 Pa. 100 to 300 picked cells were placed in each culture dish.

[0047] S4. Purification and culture of gonadal cells: The selected fluorescent positive gonadal cells were cultured in an optimized complete culture medium to obtain leopard-gill spiny perch gonadal cells that adhered to the wall, had a long spindle or fusiform morphology, and could be continuously passaged.

[0048] In a specific embodiment, the main components of the culture medium are: 1 g / L D-glucose DMEM basal medium, 10% fetal bovine serum, 0.1% β-mercaptoethanol, and 10 ng / mL cytokines (bFGF, EGF).

[0049] Example 1

[0050] This embodiment provides a method for rapid enrichment and isolation of sex gland cells from the leopard-gill spiny perch, specifically including the following steps:

[0051] S1. Primary culture of gonadal cells, including the following sub-steps:

[0052] S11. Gonadal Tissue Acquisition and Aseptic Processing: Healthy leopard gill spiny perch aged 2-4 months and weighing approximately 150g were anesthetized with eugenol and their gonadal tissue was aseptically obtained in a sterile laminar flow hood. The fresh gonads were washed three times with pre-cooled 1×PBS (5-10ml) containing 1% penicillin-streptomycin-gentamicin to remove surface blood vessels and the membranes covering the gonadal tissue. The gonadal tissue was then cut into pieces approximately 1mm in size. 3 The organizational blocks;

[0053] S12. Preparation of cell suspension: Place all the cut tissue pieces into a 1.5 ml centrifuge tube containing complete culture medium and further cut them with scissors; centrifuge at 800-1000 rpm for 4-6 min and discard the supernatant; add preheated 1×HBSS to the pellet and mix by inverting 3-4 times, let stand for 30 seconds, centrifuge at 800-1000 rpm for 4-6 min and discard the supernatant; 1×HBSS contains 1×PBS and 0.04% BSA; repeat the above steps of resuspending with 1×HBSS, centrifuging and discarding the supernatant, and then resuspend the cells in complete culture medium to obtain gonadal cell suspension;

[0054] S13. Filtration and Seeding Culture: Filter the above cell suspension through a 100μm cell filter and collect the filtered cell digestion solution; resuspend the cell digestion solution in 1×HBSS pre-cooled at 4℃, centrifuge horizontally at 100g for 3-5min, discard the supernatant, and resuspend the cells in complete culture medium; seed the cell suspension into culture flasks, add complete culture medium to make up the amount, and place in an incubator containing CO2 (5% concentration) at 28℃ for primary culture;

[0055] S14. Primary Cell Migration and Observation: After approximately 3 days of primary culture, cells were observed to migrate out of the gonadal tissue block, forming an adherent cell layer, thus obtaining the gonadal cells that had migrated from the tissue block. Results are as follows... Figure 1 As shown.

[0056] Figure 1 Image A is a micrograph of a suspension of gonadal cells from the leopard-gill spiny perch, showing that the cells are in a dispersed state. Figure 1 Image B is a micrograph of primary cells after 3 days of culture, showing that the gonadal cells that migrated from the tissue block have formed a typical adherent growth morphology, with the cells being long spindle-shaped or fusiform, and cell clusters formed by cell aggregation can be seen in some areas.

[0057] S2. Immunofluorescence labeling and enrichment of gonadal cells, including:

[0058] S21. Cell digestion and collection: The emigrating cells obtained in step S1 were digested with 0.25% trypsin-EDTA, centrifuged at 800-1000 rpm for 4-6 min, and the supernatant was discarded; the cells were resuspended in 1×PBS to obtain a cell resuspension.

[0059] S22. Cell pretreatment: Take 50-100 μl of the above cell resuspending solution (containing approximately 10... 5 ~10 8 (100 cells), filtered through a 40μm cell filter and added to a new centrifuge tube; 1 ml of 1×PBS containing 1‰ polysorbate was added to each tube and incubated for 5 minutes; then centrifuged at 800~1000 rpm for 4~6 minutes, and the supernatant was discarded to complete the cell pretreatment;

[0060] S23. Blocking and antibody incubation; including:

[0061] Blocking: Resuspend the pretreated cells in 5% skim milk powder and incubate on ice for 1 hour; centrifuge at 800-1000 rpm for 4-6 minutes and discard the supernatant;

[0062] Primary antibody incubation: Dilute Gsdf primary antibody with 2% skim milk powder at a volume ratio of 1000:1, mix well and add to cells; place the cell suspension on ice and incubate for 1 hour; after incubation, centrifuge at 800-1000 rpm for 4-6 min and discard the supernatant; resuspend cells with 1×PBS, centrifuge again and discard the supernatant;

[0063] Secondary antibody incubation: Resuspend cells in pre-prepared 5% goat serum and incubate on ice for 1 hour, then discard the supernatant; dilute Alexa Fluor Plus 488 fluorescent secondary antibody with 2% goat serum at a volume ratio of 1000:1 and add it to the cells; incubate on ice in the dark for 1 hour; after incubation, resuspend cells in 1×PBS, centrifuge at 800-1000 rpm for 4-6 min, and discard the supernatant;

[0064] S24. Labeling effect verification: To verify the specificity of Gsdf antibody, immunofluorescence staining was first performed on the gonad tissue sections of the leopard-spined perch. Figure 2 The results showed that the Gsdf protein was specifically expressed in somatic cells of the ovary and testis. Figure 2As shown in AC, in ovarian tissue, DAPI staining clearly marked the nuclei of all cells, and the green fluorescence signal of Gsdf protein was specifically distributed in somatic cells surrounding ovarian follicles, while no obvious signal was observed in oocytes; Figure 2 As shown in the DF diagram, the green fluorescence signal of the Gsdf protein is specifically expressed in testicular support cells, but no obvious fluorescence is observed in germ cells. These tissue-level localization results indicate that Gsdf can serve as a specific molecular marker for the gonadal cells of the leopard-gill spiny perch.

[0065] Based on this, the primary cultured gonadal cells were immunofluorescently labeled. 10 μl of the labeled cell sample was taken and observed under a fluorescence microscope. The cells were confirmed to show specific green fluorescence, thus completing the fluorescence labeling and obtaining fluorescently labeled gonadal cells.

[0066] S3. Isolation of gonadal cells, including:

[0067] S31. Cell filtration and preparation: Resuspend the fluorescently labeled cells obtained in step S2 in 1×PBS, filter through a 40μm cell filter to remove cell clumps; place the filtered cell suspension in a 6-well plate containing complete culture medium for later use;

[0068] S32. Single-cell microscopic picking: Fluorescent positive cells were picked using Boyi Biotechnology's single-cell microscopic picking instrument under fluorescent conditions; the instrument settings were: glass capillary outer diameter 1.65mm, inner diameter 1.0mm, length 63mm, and picking pressure value 2Pa.

[0069] S33. Cell Transfer and Verification: The selected fluorescently positive cells were transferred to culture dishes pre-filled with complete culture medium, with 100-300 cells per dish. Observation under a fluorescence microscope confirmed that all selected cells were fluorescently positive, thus completing the isolation of gonadal cells. Results are as follows: Figure 3 , Figure 4 As shown.

[0070] like Figure 3 As shown in Figure A, under bright field, the cells appear to be round or nearly round and are in good condition. Figure 3 As shown in Figure B, some cells exhibit clear, specific green fluorescence under the green fluorescence channel; Figure 3 Image C is a merged image, further confirming the correspondence between the green fluorescence signal and cell location. These results demonstrate that the Gsdf antibody maintains good specificity and labeling efficiency in cell suspension.

[0071] like Figure 4As shown, a large number of unlabeled contaminating cells (visible under bright field) were present in the cell suspension before sorting by the single-cell instrument, while the cells collected after sorting were all Gsdf-positive fluorescent cells with intact cell morphology and good viability, proving that the method of the present invention can efficiently and accurately separate target glandular cells.

[0072] S4. Purification and culture of gonadal cells: The selected fluorescently positive cells were added to a pre-optimized culture medium. On the second day of culture, gonadal cells were observed to adhere to the culture medium and exhibit a typical elongated spindle or fusiform shape, and the cells had been successfully cultured for more than 7 generations. Results are as follows... Figure 5 As shown in the figure. These results demonstrate the suitability of this culture medium for the gonadal cells of the leopard-gill spiny perch.

[0073] In this step, the main components of the culture medium are: 1 g / L D-glucose DMEM basal medium, 10% fetal bovine serum, 0.1% β-mercaptoethanol, and 10 ng / mL cytokines (bFGF, EGF).

[0074] The key technical points and advantages of this invention are as follows: A mechanical digestion method is used to prepare a suspension of gonadal cells from the leopard-gill spiny perch, eliminating the need for excessive enzymatic digestion and maximizing the preservation of cell viability and integrity. Polysorbate is used to enhance cell membrane permeability, and combined with immunofluorescence labeling using gonadal cell-specific antibodies, achieving efficient and specific enrichment of target somatic cells. Single-cell micropicking technology is employed, allowing for direct picking of positive cells under fluorescence visualization. This gentle operation minimizes cell damage and enables precise acquisition of individual gonadal cells. Combined with optimized complete culture medium and culture conditions, the isolated gonadal cells can rapidly adhere, proliferate normally, and achieve stable passage culture.

[0075] It should be understood that the various forms of processes shown above can be used to reorder, add, or delete steps. For example, the steps described in this invention disclosure can be executed in parallel, sequentially, or in different orders, as long as the desired result of the technical solution disclosed in this invention can be achieved, and this is not limited herein.

[0076] The specific embodiments described above do not constitute a limitation on the scope of protection of this invention. Those skilled in the art should understand that various modifications, combinations, sub-combinations, and substitutions can be made according to design requirements and other factors. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this invention should be included within the scope of protection of this invention.

Claims

1. A method for rapid enrichment and separation of sex gland cells from the leopard-gill spiny perch, characterized in that: Specifically, the steps include the following: S1. Select healthy leopard gill spiny perch aged 2-4 months and anesthetize them with eugenol; take gonadal tissue, and after aseptic treatment, mince, centrifugation, washing and filtering, inoculate it into culture medium for primary culture to obtain gonadal cells that migrate from the tissue block; S2. After digestion with 0.2-0.3% trypsin-EDTA, centrifugation and washing, the emigrating gonadal cells were treated with 1×PBS containing 1‰ polysorbate. After blocking, antibody incubation, and washing, the labeling effect was verified by fluorescence microscopy to achieve specific enrichment of gonadal cells. Specifically, the blocking, antibody incubation, and washing process included: resuspending cells in 5% skim milk powder, incubating on ice for 0.5–1.5 h, centrifuging, and discarding the supernatant; adding specific primary antibody diluted in 2% skim milk powder to the cells, incubating on ice for 0.5–1.5 h, centrifuging, and washing; subsequently, resuspending cells in 5% goat serum for 1 h, blocking, and discarding the supernatant; adding fluorescently labeled secondary antibody diluted in 2% goat serum, incubating on ice in the dark for 0.5–1.5 h, centrifuging, and washing to obtain fluorescently labeled cells; the ratio of 2% skim milk powder to specific primary antibody was 1000:1, and the specific primary antibody was Gsdf primary antibody; the ratio of 2% goat serum to fluorescently labeled secondary antibody was 1000:1, and the fluorescently labeled secondary antibody was Alexa Fluor Plus 488 fluorescent secondary antibody. S3. After filtering and purifying the enriched fluorescently labeled cells, place them under a fluorescence microscope and use a single-cell micropicking instrument to accurately pick fluorescent positive cells under the fluorescence field of view, and transfer them to a culture dish containing complete culture medium to complete the rapid separation of gonadal cells. S4. The isolated fluorescent positive cells were seeded into an optimized complete culture medium for purification and culture, allowing the cells to adhere and grow stably through passage. The optimized complete culture medium included: 1 g / L D-glucose DMEM basal medium, 10% fetal bovine serum, 0.1% β-mercaptoethanol, and 10 ng / mL cytokines; the cytokines were bFGF and EGF.

2. The method for rapid enrichment and separation of leopard-gill spiny perch sex gland cells according to claim 1, characterized in that: In step S1, eugenol is used to anesthetize the leopard-gill spiny perch; aseptic processing and cutting include: obtaining gonadal tissue under aseptic conditions in a sterile laminar flow hood; washing the gonadal tissue three times with a pre-cooled 1×PBS solution containing 1% penicillin-streptomycin-gentamicin to remove surface blood vessels and the membrane covering the gonadal tissue, and cutting the gonadal tissue into small tissue blocks.

3. The method for rapid enrichment and separation of leopard-gill spiny perch sex gland cells according to claim 1, characterized in that: In step S1, the centrifugal washing is performed by centrifuging at 800-1000 rpm for 4-6 min and discarding the supernatant; preheated 1×HBSS is added to the precipitate and mixed by inverting 3-4 times, allowed to stand for 30 seconds, and then centrifuged at 800-1000 rpm for 4-6 min and discarding the supernatant. The filtration was performed by filtering the gonadal cell suspension using a 100μm cell filter, and the gonadal cell suspension was obtained by resuspending the cells in complete culture medium after centrifugation and washing. The primary culture was carried out in a CO2 incubator.

4. The method for rapid enrichment and separation of leopard-gill spiny perch sex gland cells according to claim 1, characterized in that: Step S2 specifically includes the following sub-steps: S21. After digesting the gonadal cells obtained in step S1 with trypsin-EDTA, centrifuge at 800~1000 rpm for 4~6 min, discard the supernatant, resuspend the cells with 1×PBS, and filter through a 40μm cell filter to obtain the cell resuspension. S22. Take the cell resuspension, add 1×PBS containing 1‰ polysorbate, incubate for 3~10 min, then centrifuge and discard the supernatant; S23. Blocking and Antibody Incubation: First, resuspend the cells in 5% skim milk powder, incubate on ice for 0.5-1.5 h, centrifuge, and discard the supernatant; then add specific primary antibody diluted in 2% skim milk powder to the cells, incubate on ice for 0.5-1.5 h, centrifuge, and wash; subsequently, resuspend the cells in 5% goat serum for 1 h and block, then discard the supernatant; add fluorescently labeled secondary antibody diluted in 2% goat serum, incubate on ice in the dark for 0.5-1.5 h, centrifuge, and wash to obtain fluorescently labeled cells; S24. Observe the fluorescently labeled cells under a fluorescence microscope to confirm that the cells exhibit specific green fluorescence, thus completing the enrichment of gonadal cells.

5. The method for rapid enrichment and separation of leopard-gill spiny perch sex gland cells according to claim 4, characterized in that: In step S21, the concentration of trypsin-EDTA is 0.25%; in step S22, the incubation time of the cell resuspension is 5 min.

6. The method for rapid enrichment and separation of leopard-gill spiny perch sex gland cells according to claim 4, characterized in that: In step S23, the incubation time on ice is 1 hour.

7. The method for rapid enrichment and separation of leopard-gill spiny perch sex gland cells according to claim 1, characterized in that: In step S3, a 40μm cell filter is used for filtration and purification; 100-300 cells are placed in each culture dish.

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