A red tilapia skin tissue cell line and its application

Abstract

This application relates to a red tilapia skin tissue cell line and its applications. This red tilapia skin tissue cell line is derived from red tilapia skin tissue and is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: C202619. This cell line can be stably and continuously passaged to the 88th generation under DMEM-based culture conditions containing 15% fetal bovine serum, 200 IU / mL penicillin, and 200 μg / mL streptomycin. After cryopreservation and thawing, the cell viability is above 86%, the karyotype is predominantly diploid (2n=44), and it possesses the ability to express exogenous genes, with a transfection efficiency of approximately 40%. This cell line can be used for fish pathogen isolation and culture, immunological research, and gene function analysis, providing a stable in vitro cell model for tilapia disease control and related basic research.

Description

Technical Field

[0001] This application relates to the field of fish cell line technology, and in particular to a red tilapia skin tissue cell line and its application. Background Technology

[0002] Red tilapia ( Oreochromis spp. Red tilapia is one of my country's important freshwater aquaculture fish species, possessing advantages such as rapid growth, high feed conversion rate, strong resistance to adverse conditions, and wide adaptability, thus occupying an important position in my country's freshwater aquaculture industry. However, with the continuous improvement of intensive farming practices, the frequency of bacterial and viral diseases in red tilapia has increased significantly, seriously hindering the sustainable development of the tilapia industry.

[0003] Fish cell lines are important experimental materials for studying the isolation and identification of fish viruses, pathogenic mechanisms, host immune responses, and vaccine evaluation. Currently established tilapia cell lines are mainly derived from tissues such as fins, kidneys, spleen, and brain. For example, cell lines derived from tilapia fins and kidneys have been used for the isolation and pathogenic mechanism research of pathogens such as neuronecrosis virus and streptococcus.

[0004] However, fish skin, as the body's first physical and immune barrier against pathogen invasion, plays a crucial role in mucus immunity, innate immune responses, and stress responses. Currently, there is a lack of stable, continuously passaged cell lines derived from red tilapia skin tissue. This, to some extent, limits in vitro research on the mechanisms of pathogen transdermal invasion, mucosal immunity, and skin-related immune responses.

[0005] Therefore, constructing a red tilapia skin cell line with a clear origin, stable genetic traits, long-term continuous passage culture, and suitability for standardized experimental applications is of great theoretical significance and practical value for conducting research on tilapia pathogen infection mechanisms, vaccine screening, and immune regulation. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a red tilapia skin tissue cell line and its application.

[0007] The first objective of this invention is to provide a red tilapia skin tissue cell line.

[0008] A second objective of this invention is to provide the application of the aforementioned red tilapia skin tissue cell line in the preparation of tilapia in vitro cell models.

[0009] A third objective of this invention is to provide the application of the aforementioned red tilapia skin tissue cell line in the expression of exogenous proteins.

[0010] A fourth objective of this invention is to provide the application of the aforementioned red tilapia skin tissue cell line in the construction of gene knockout cells.

[0011] The fifth objective of this invention is to provide the application of the aforementioned red tilapia skin tissue cell line in constructing a fish gene function research model.

[0012] The sixth objective of this invention is to provide the application of the aforementioned red tilapia skin tissue cell line in the isolation, culture, and immunoassay of fish pathogens.

[0013] The seventh objective of this invention is to provide the application of the aforementioned red tilapia skin tissue cell line in fish drug screening.

[0014] The eighth object of the present invention is to provide a method for cryopreserving the aforementioned red tilapia skin tissue cell line.

[0015] The ninth object of the present invention is to provide a method for culturing the aforementioned red tilapia skin tissue cell line.

[0016] To achieve the above objectives, the present invention is implemented through the following technical solution: This invention claims protection for a red tilapia skin tissue cell line, which was deposited at the China Center for Type Culture Collection on February 8, 2026, with accession number CCTCC NO: C202619.

[0017] This invention also claims protection for the following applications: The application of the red tilapia skin tissue cell line in the preparation of tilapia in vitro cell models.

[0018] The application of the red tilapia skin tissue cell line in the expression of exogenous proteins.

[0019] The application of the red tilapia skin tissue cell line in the construction of gene knockout cells.

[0020] The application of the red tilapia skin tissue cell line in constructing a fish gene function research model.

[0021] The application of the red tilapia skin tissue cell line in the isolation, culture and immunodetection of fish pathogens.

[0022] The application of the red tilapia skin tissue cell line in fish drug screening.

[0023] The present invention also claims protection for a cryopreservation method for the red tilapia skin tissue cell line, wherein a mixture containing DMSO and fetal bovine serum is used as the cryopreservation solution, and the volume ratio of DMSO to fetal bovine serum in the mixture is (8-12):(88-92).

[0024] The present invention also claims protection for the method of culturing the red tilapia skin tissue cell line, wherein the cell line is cultured in a basal medium containing 10% to 20% fetal bovine serum at 26 to 28°C and 4 to 6% CO2, wherein the basal medium is DMEM medium, MEM medium, and / or L15 medium.

[0025] Preferably, the culture is carried out in a basal medium containing 15% fetal bovine serum at 27°C and 5% CO2, wherein the basal medium is DMEM medium, MEM medium, and / or L15 medium.

[0026] Preferably, the basal culture medium is DMEM medium.

[0027] Preferably, the basal culture medium also contains penicillin and streptomycin.

[0028] Preferably, the content of the fetal bovine serum is 14-16%.

[0029] Preferably, the final concentration of the penicillin is 180–220 IU / mL.

[0030] Preferably, the final concentration of the streptomycin is 200 IU / mL.

[0031] Compared with the prior art, the present invention has the following beneficial effects: This invention discloses a cell line isolated from the skin tissue of red tilapia, deposited at the China Center for Type Culture Collection (CCTCC) under accession number CCTCC NO: C202619. This cell line can be stably and continuously passaged to passage 88 under DMEM-based conditions containing 15% fetal bovine serum, 200 IU / mL penicillin, and 200 μg / mL streptomycin. After cryopreservation and thawing, the cell viability is above 86%, the karyotype is predominantly diploid (2n=44), and it exhibits the ability to express exogenous genes, with a transfection efficiency of approximately 40%. This cell line can be used for the isolation and culture of fish pathogens, immunological research, and gene function analysis, providing a stable in vitro cell model for tilapia disease control and related basic research. Attached Figure Description

[0032] Figure 1 A shows cell morphology under a microscope; B shows the migration of primary cultured cells from the skin tissue of red tilapia; C shows the morphology of skin cells from the 10th generation red tilapia; D shows the morphology of skin cells from the 25th generation red tilapia; and D shows the morphology of skin cells from the 50th generation red tilapia.

[0033] Figure 2 This is a screening diagram of the optimal culture medium type for the growth of skin cells from the 50th generation of red tilapia in Example 2.

[0034] Figure 3 This image shows the morphology of the chromosome division phase and the chromosome karyotype analysis of skin cells from the 66th generation of red tilapia.

[0035] Figure 4 The results show the expression of the exogenous gene GFP in red tilapia skin cells transfected with GFP. Among them, A is the GFP fluorescence image of red tilapia skin cells transfected with GFP under a fluorescence microscope; B is the cell morphology image of red tilapia skin cells transfected with GFP under a bright field microscope. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0037] Example 1: Construction of Red Tilapia Skin Cell Line (1) Treatment of skin tissue of red tilapia Juvenile red tilapia (approximately 100 g) around 4 months old were anesthetized with crushed ice at low temperature. The fish were then wiped with 75% alcohol swabs and aseptically transferred to a laminar flow hood for rapid dissection. Skin tissue blocks were separated and washed 2-3 times with PBS (containing 400 U / mL penicillin-streptomycin mixture) to remove blood. The tissue blocks were then minced in DMEM basal medium, using ophthalmic scissors to cut the skin tissue into 1 mm pieces. 3 Small tissue fragments were inoculated into six-well plates soaked in gelatin, placed in an incubator at 37 degrees Celsius and inverted for 30 minutes to dry-attach the cells, and then an appropriate amount of complete cell culture medium was added and cultured at 28 degrees Celsius in a 5% CO2 incubator. The complete cell culture medium used was DMEM medium containing 20 vol% fetal bovine serum, 200 IU / mL penicillin, and 200 μg / mL streptomycin.

[0038] (2) Primary culture On day 6 of primary culture using the tissue block method, cells migrated from the tissue block (see...). Figure 1 (A) During primary culture, replace half of the old complete culture medium with fresh complete culture medium every two days. Specifically, discard half of the old medium and replace with fresh complete culture medium; wait until cell confluence reaches 80%. At 90% success rate, subculturing can be performed.

[0039] (3) Subculture Once the red tilapia skin cells have formed a monolayer (with a cell confluence of approximately 90%), remove the old culture medium using a sterile pipette, wash twice with PBS, digest the adherent cells with 0.25% trypsin containing EDTA, and observe under an inverted microscope when the cells become rounded and begin to detach. Add complete cell culture medium to stop the digestion, gently pipette to disperse and suspend the cells, and divide the cells into flasks at a 1:2 ratio, bringing the complete culture medium to 5 mL per flask. Place the flasks in an incubator at 28°C and 5% CO2 for subculturing, approximately every 3–4 days.

[0040] This cell line was derived from the skin tissue of red tilapia and was deposited at the China Center for Type Culture Collection (CCTCC) on February 8, 2026, with accession number CCTCC NO: C202619. The deposit address is Wuhan University, Wuhan, China, and the classification name is: skin cells of red tilapia (Oreochromis spp.).

[0041] Figure 1 Images B, C, and D show the skin cell morphology of red tilapia from the 10th, 25th, and 50th generations, respectively.

[0042] The red tilapia skin cell line has been continuously passaged for 12 months, and the cells can proliferate stably, having reached the 88th generation.

[0043] (4) Cell cryopreservation and thawing Collect the cell suspension after trypsin digestion, centrifuge and discard the supernatant, resuspend the cells in pre-cooled cryopreservation solution, place them in a gradient cooling box, freeze overnight at -80°C, and then transfer them to liquid nitrogen for long-term storage to complete cell cryopreservation; after removing them from liquid nitrogen, quickly place them on ice to thaw, then transfer them to a 37°C constant temperature water bath and shake until thawed, add complete cell culture medium, centrifuge and discard the culture medium and cryopreservation solution, resuspend the cells in complete cell culture medium, and evenly spread them in culture flasks for culture to complete cell revival; the viability of the revived cells is above 86%.

[0044] Example 2: Culture conditions of red tilapia skin cell line I. Experimental Methods Cells from the 50th generation of the cell line obtained in Example 1, which exhibited stable growth and good morphology, were digested with 0.25% trypsin and seeded into 96-well plates at a density of 2 × 10⁻⁶ cells / well. 4Initially, cells were seeded at a density of [number] cells / mL in each well of a culture plate. After the cells adhered and stabilized, the original culture medium was aspirated, and the cells were washed with PBS and cultured in L-15, MEM, and DMEM media, respectively. All media were supplemented with a mixture of 15% FBS and 200 U / mL penicillin / streptomycin. Cells were cultured at 28°C and 5% CO2. Cells were collected from each well of the three media daily, with three wells collected from each experimental group. The cells were digested, detached from the culture plate, and counted under a microscope using a CountStar cell counter. The experiment was repeated three times, and the growth status of the cell lines under different culture medium types was plotted. II. Experimental Results See results Figure 2 The results showed that, under the same FBS concentration and culture temperature, the red tilapia skin cells of the present invention grew best in DMEM medium.

[0045] Example 3 Chromosomal karyotype analysis of red tilapia skin cell line I. Experimental Methods Chromosome number and karyotype are fundamental to cytogenetics and are important indicators for identifying species and sex. Chromosome karyotype analysis was performed on the 77th generation cells from the cell line obtained in Example 1. The specific steps are as follows: 1. When the cells are in the logarithmic growth phase, add colchicine to a final concentration of 0.8 μg / mL and incubate at 28℃ for 6 h.

[0046] 2. Discard the old culture medium, wash with PBS, digest the cells using the standard trypsin digestion method, add the above complete culture medium to stop digestion, centrifuge at 800 g for 3 min, and collect the cells.

[0047] 3. Hypotonic treatment: Discard the supernatant, add 5 mL of 0.075 mol / L KCl solution to the cell pellet, gently disperse the pellet, and treat with hypotonic treatment in a 37℃ water bath for 40 min.

[0048] 4. Pre-fixation treatment: Add 2 mL of pre-cooled Carno fixative (methanol: glacial acetic acid = 3:1) for 5 min for pre-fixation, and then centrifuge at 800 g for 3 min.

[0049] 5. Fixation: Discard the supernatant, add 5 mL of pre-cooled Carnot fixative to the cell pellet and fix for 15 min, centrifuge at 800 g for 3 min, collect the cells, and repeat the operation twice.

[0050] 6. Slide preparation: Resuspend the cell pellet in 0.5 mL of pre-cooled Carno fixative, and then drop the cell suspension onto a slide using the cold drop method (pre-cool the slide beforehand).

[0051] 7. Staining: Stain with 10% Giemsa for 20 minutes, then rinse with water and air dry.

[0052] 8. Microscopic examination.

[0053] II. Experimental Results See results Figure 3 A total of 200 metaphase plates were counted. The metaphase plates spread with a normal diploid number. The count showed that the chromosome number ranged from 18 to 66, and more than 25% of the cells had a model value of 44 chromosomes at the 77th generation. The chromosome karyotype was predominantly diploid with 2n=44. In summary, this proves that the cell line obtained by this invention has the same chromosome number as individual red tilapia, which is consistent with the chromosome number characteristics of red tilapia.

[0054] Example 4: Transfection of red tilapia skin cell line with exogenous gene I. Experimental Methods The 40th generation of red tilapia skin cells of this invention were used at 5×10 4 Cells were initially seeded at a density of 100 cells / mL in 24-well plates and cultured in antibiotic-free DMEM medium until adherent. The plates were then incubated overnight at 28°C with 5% CO2. For transfection, the volume ratio of plasmid DNA to Lipo3000 transfection reagent was 1:2 to 1:3.

[0055] The specific steps are as follows: Take two sterile centrifuge tubes, A and B. Add Opti-MEM diluted eGFP-N3 plasmid DNA to centrifuge tube A, along with P3000 reagent, and mix gently. Add Opti-MEM diluted Lipo3000 transfection reagent to centrifuge tube B, mix gently, and incubate at room temperature for about 5 minutes. Then, slowly add the solution from centrifuge tube A to centrifuge tube B, mix gently, and incubate at room temperature for about 10–20 minutes to form a transfection complex. Add the transfection complex dropwise to each well of cells (each well pre-contains approximately 500 μL of antibiotic-free DMEM medium), and gently shake to mix. After 4–6 hours of transfection, replace with antibiotic-containing DMEM complete medium and continue culturing.

[0056] II. Experimental Results The results show that ( Figure 4 Fluorescent signals were observed 48 h after transfection, with a transfection efficiency of approximately 40%, indicating that the red tilapia skin cell line of this invention has the ability to express exogenous genes and can be used for gene function analysis and viral protein expression studies.

Claims

1. A red tilapia skin tissue cell line, characterized in that, It was deposited at the China Center for Type Culture Collection on February 8, 2026, with accession number CCTCC NO: C202619.

2. The application of the red tilapia skin tissue cell line according to claim 1 in the preparation of tilapia in vitro cell models.

3. The application of the red tilapia skin tissue cell line according to claim 1 in the expression of exogenous proteins.

4. The application of the red tilapia skin tissue cell line according to claim 1 in the construction of gene knockout cells.

5. The application of the red tilapia skin tissue cell line according to claim 1 in constructing a fish gene function research model.

6. The application of the red tilapia skin tissue cell line according to claim 1 in the isolation, culture and immunodetection of fish pathogens.

7. The application of the red tilapia skin tissue cell line according to claim 1 in fish drug screening.

8. The method for cryopreserving the red tilapia skin tissue cell line according to claim 1, characterized in that, A mixture containing DMSO and fetal bovine serum was used as the cryopreservation solution for cryopreservation, wherein the volume ratio of DMSO to fetal bovine serum in the mixture was (8-12):(88-92).

9. The method for culturing the red tilapia skin tissue cell line according to claim 1, characterized in that, Cultured in a basal medium containing 10%–20% fetal bovine serum at 26–28°C and 4–6% CO2, wherein the basal medium is DMEM medium, MEM medium, and / or L15 medium.

10. The cultivation method according to claim 9, characterized in that, The basal culture medium is DMEM medium.

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