A turbot spleen tissue cell line, its preparation method and application

Abstract

This invention provides a turbot spleen tissue cell line, with the accession number CCTCCNO:C2022229. The turbot spleen tissue cell line provided by this invention can be continuously passaged, and experimental results show that it remains stable after 65 passages, providing a large number of stable turbot spleen cells for research on immune function genes, etc. The provided cell line has excellent characteristics; it consists of fibroblasts, with spindle-shaped or irregularly triangular cells, several irregular protrusions extending outward from the cell body, and the cells are closely arranged. It can be passaged every 3-4 days, with high recovery efficiency, and can be used for plasmid transfection, siRNA interference, and various other molecular biology experiments.

Description

Technical Field

[0001] This invention belongs to the field of fish cell culture technology, specifically relating to a turbot spleen tissue cell line, its preparation method, and its application. Background Technology

[0002] Turbot (Scophthalmus maximus) is a species of fish belonging to the family Scophthalmidae and the genus Scophthalmus, and is an important economic fish species in my country. However, in recent years, turbot farming has been frequently disrupted by bacterial diseases, particularly Aeromonas salmonicida, Edwardsiella tarda, and Vibrio anguillarum, which have severely hampered the development of turbot farming. Therefore, research on the immune system and disease mechanisms of turbot is urgently needed. The spleen tissue of fish is an important central immune tissue, containing a large number of T cells, B cells, and other immune cells, playing a crucial role in resisting pathogen invasion. Therefore, isolating turbot spleen tissue cells and culturing cell lines will contribute to research on the pathogenic mechanisms of turbot pathogens and the function of turbot immune-related genes.

[0003] Cell culture technology is a crucial technique in biological research. It primarily refers to the method of extracting tissues or cells from an organism and allowing them to survive, grow, and reproduce under appropriate in vitro conditions, maintaining their structure and function. The cell lines are derived from various fish tissues, including embryos, brains, eyes, swim bladders, bones, muscles, epidermis, fins, visceral tissues, and tumors. Compared to directly using individual fish for experiments, using fish cell lines offers advantages such as ease of operation, freedom from site and seasonal constraints, and high reproducibility. The applications of in vitro cell culture encompass multiple disciplines, including fish immunology, virology, molecular genetics, pharmacology, toxicology, cell engineering breeding, and germplasm preservation. Bacterial diseases are among the most serious threats to aquatic organisms, severely impacting the development of aquaculture in my country. With the development of cell culture technology, the role of fish cell culture in fish disease and immunology research has become increasingly prominent. Although primary fish cell culture technology is currently a relatively mature technique, obtaining stable, passaged cell lines suitable for experimental model construction still presents significant challenges and opportunities for chance. Summary of the Invention

[0004] The purpose of this invention is to provide a turbot spleen tissue cell line, its preparation method, and its application. The screened and preserved cell line is stable in passage and has a high survival rate. This provides a research basis for the study of turbot spleen in bacterial disease resistance and makes up for the shortcomings of existing technologies in the study of fish spleen immunity.

[0005] The turbot spleen cells SMSP provided by this invention were deposited on August 24, 2022, at the China Center for Type Culture Collection of Wuhan University, with accession number CCTCC NO:C2022229, and deposit address: Wuhan, China.

[0006] The turbot spleen cells SMSP provided by this invention were obtained by screening after constructing spleen tissue from juvenile turbot. The method for establishing the SMSP includes the following steps:

[0007] 1) Obtaining turbot spleen tissue:

[0008] Spleen tissue was taken from 6-month-old turbot juveniles, washed with PBS, and transferred into DMEM basal medium containing penicillin, streptomycin, gentamicin and amphotericin B;

[0009] 2) Primary culture:

[0010] Turbot spleen tissue was immersed in 75% ethanol for 1 minute, then in DMEM basal medium containing penicillin, streptomycin, gentamicin, and amphotericin B for 2 hours, followed by rinsing with PBS. Finally, the turbot spleen tissue was minced to 1 mm. 3 Prepare tissue blocks of various sizes. Transfer the tissue blocks to T25 culture flasks, aspirate the culture medium, leaving only a small amount to keep them moist, and place the culture flasks at 24°C for 2 hours to allow the tissue blocks to adhere to the bottom of the flasks. Add complete culture medium and incubate, finally placing the flasks in a 24°C incubator.

[0011] 3) Subculture: When the primary culture reaches about 80% coverage of the bottom plate of the culture flask, the culture medium is aspirated, and excess serum is removed by washing with PBS. Then, the adherent cells are digested with 0.25% trypsin and subcultured to obtain turbot spleen tissue cell line.

[0012] In a preferred embodiment of the present invention, the complete culture medium is a DMEM+Hepes-based medium comprising 15% fetal bovine serum (FBS), 5‰ β-mercaptoethanol, 1× non-essential amino acids (Gibco, 11140050), 500 U / mL penicillin, 500 μg / mL streptomycin, 1.25 μg / mL amphotericin B, and 250 μg / mL gentamicin.

[0013] In another aspect, the present invention provides a method for culturing SMSP cell lines, using DMEM medium and administering 10% fetal bovine serum at an ambient temperature of 24°C to maintain rapid cell growth.

[0014] The turbot spleen tissue cell line provided by this invention is used for the study of immune function genes and pathogenic mechanisms of pathogens. Under stimulation by LPS and poly(I:C), the expression of immune-related genes TNF-β, IL-1β, IL-12A, IL-12B, and IL-10 all increased.

[0015] The turbot spleen cell line provided by this invention can be continuously passaged. Experimental results show that it remains stable after 65 passages, providing a large number of stable turbot spleen cells for research on immune function genes, etc. The provided cell line has excellent characteristics; it is a fibroblast, with spindle-shaped or irregularly triangular cells, several irregular protrusions extending outward from the cell body, and the cells are closely arranged. It can be passaged every 3-4 days, with high recovery efficiency, and can be used for plasmid transfection, siRNA interference, and various other molecular biology experiments. Attached Figure Description

[0016] Figure 1 Microscopic image (10×) of primary cultured turbot spleen cell line on day 12.

[0017] Figure 2 Microscopic image (10×) of turbot spleen cell line cultured 24 hours after resuscitation.

[0018] Figure 3 Chromosome analysis and karyotype distribution of turbot spleen cell lines

[0019] Figure 4 Image showing cell growth of turbot spleen cell lines cultured in different concentrations of fetal bovine serum for 5 days.

[0020] Figure 5 Images showing cell growth of turbot spleen cell lines after 5 days of culture at different temperatures.

[0021] Figure 6 Images showing cell growth of turbot spleen cell lines after 5 days of culture in different types of basal media.

[0022] Figure 7 Microscopic images of cell lines transfected with GFP-siRNA 48 hours later.

[0023] Figure 8 Microscopic images of cells transfected with pEGFP-N1 48 hours later.

[0024] Figure 9 Expression of immune-related genes in SMSP after 2h, 6h, 12h and 24h of stimulation with LPS(A) and poly(I:C)(B). Detailed Implementation

[0025] The complete culture medium used in this invention comprises DMEM+Hepes basal medium, fetal bovine serum (FBS), β-mercaptoethanol, non-essential amino acids, penicillin, streptomycin, amphotericin B, and gentamicin. DMEM medium, FBS, and non-essential amino acids provide essential nutrients for cell growth; Hepes provides a stable growth environment; β-mercaptoethanol stimulates cell proliferation and enhances cell viability; penicillin, streptomycin, amphotericin B, and gentamicin broaden the antibacterial spectrum, effectively inhibiting bacterial growth and preventing contamination, especially in primary cultures.

[0026] The technical solution of the present invention will be further described and illustrated below with reference to the accompanying drawings and specific embodiments.

[0027] Example 1: Construction and screening of cell lines

[0028] The turbot used to construct the cell line came from Haiyang Yellow Sea Fisheries Co., Ltd. in Yantai, Shandong Province. Before sampling, the turbot were cultured under conditions of 18℃, salinity of 28-30‰, neutral pH, and sufficient dissolved oxygen.

[0029] The basic culture medium used in this example consists of the following components: 13.5g DMEM powder (Invitrogen, 12800-058) + 9.528g Hepes powder + 1L triple-distilled water, stored at 4°C.

[0030] The complete culture medium consisted of the following: 7.5 mL fetal bovine serum (FBS, Gibco) + 0.5 mL 100× penicillin-streptomycin solution, amphotericin B at a final concentration of 1.25 μg / mL and gentamicin at a final concentration of 250 μg / mL, 50 μL β-mercaptoethanol, 0.5 mL 100× non-essential amino acid solution, and 40 mL basal culture medium, stored at 4℃.

[0031] The components and preparation of the cell cryopreservation solution are as follows: 1 mL DMSO (purchased from Invitrogen) + 2 mL fetal bovine serum + 7 mL complete culture medium, prepared fresh for each use.

[0032] The steps of establishing the turbot spleen cell line of this invention are as follows:

[0033] 1) Place 6-month-old turbot juveniles (approximately 10cm long and 25g in a bucket, then add 2-3 drops of eugenol until the fish stop swimming, lie on their side at the bottom of the bucket, and their gill covers stop opening and closing. Wipe the surface of the fish with a cotton ball soaked in 75% alcohol, and separate the tissue using sterile surgical scissors. Place the extracted spleen tissue into a 15mL centrifuge tube containing penicillin-streptomycin PBS solution, and then immediately transfer it to a clean bench for further processing.

[0034] 2) In a clean bench, sterilize with UV light and collect spleen tissue. Add PBS to 3 wells and DMEM containing 4 antibiotics (penicillin, streptomycin, gentamicin, and amphotericin B) to 2 wells of a 6-well plate. Pass the tissue sequentially through PBS, PBS, PBS, DMEM containing antibiotics (1 h), and DMEM containing antibiotics (0.5-1 h). Transfer the tissue block to a 6 cm petri dish and cut it into small pieces with scissors or a scalpel. Cut the small tissue pieces (1 mm) into smaller pieces. 3 Transfer the tissue to a T25 culture flask, arrange the tissue, aspirate excess culture medium, and incubate at 24°C. Place the flask horizontally for 2 hours, then vertically for 0.5 hours. Add complete culture medium and continue incubation. Figure 1 As shown, after primary culture of turbot spleen cells, new cells migrated from adherent cells and tissue blocks around day 12, and small clusters of cells appeared around day 15.

[0035] 3) When the primary cells cover approximately 80% of the bottom of the culture flask, they can be passaged. Aspirate the culture medium and wash with PBS to remove excess serum. Then, digest the adherent cells with 0.25% trypsin for about 3 minutes without shaking the flask. Aspirate the trypsin, add complete culture medium, and gently pipette to suspend the spleen cells. Passage at a 1:2 ratio. After passage, incubate at 24°C. Passage every 3-4 days thereafter until after 50 passages. The cells are stable during culture and have a rapid passage rate, making them suitable as experimental cells.

[0036] The turbot spleen tissue cell line obtained through final screening was named SMSP.

[0037] Example 2: Cryopreservation and thawing of turbot spleen cells (SMSP)

[0038] 1) Cryopreservation: Take one T75 culture flask containing turbot spleen cell line in logarithmic growth phase, digest with 0.25% trypsin, and collect the cells by centrifugation. Cryopreservation solution at 5 × 10⁻⁶ cells / day. 5 Resuspend the cells in cryopreservation solution, gently pipetting the cell pellet to ensure even dispersion in the solution. Aliquot the cell suspension into cryovials, 1.5-1.8 mL per tube. Follow the slow freezing principle for cell cryopreservation: incubate at 4°C for 10 min, -20°C for 30 min, and -80°C for 1 day, then transfer to liquid nitrogen for storage.

[0039] 2) Thawing: Cell thawing follows the rapid thawing principle. Remove the cryovials from liquid nitrogen and quickly place them in a 37°C water bath. Gently shake the cryovials to ensure rapid and uniform thawing; the thawing time should not exceed 1 minute. Centrifuge the thawed cell suspension at 1,200 rpm for 5 minutes to remove DMSO. Resuspend the centrifuged cells in 5 mL of complete culture medium. Transfer the cell suspension to a T25 cell culture flask and incubate at 24°C.

[0040] like Figure 2 The turbot spleen cell line achieved an adhesion rate of approximately 80% within 24 hours after thawing, and its morphology was similar to that of the cells before cryopreservation, indicating that it was in good condition.

[0041] Example 3: Chromosomal karyotype analysis of SMSP cells from turbot spleen

[0042] 1) Chromosomal karyotype analysis of SMSP cells was performed at passage 12. Spleen cells of turbot in the logarithmic growth phase were cultured for 12 hours with colchicine at a final concentration of 10 μg / mL, followed by trypsin digestion to obtain cell suspension.

[0043] 2) Centrifuge the collected cell suspension at 1,200 rpm for 10 min, and gently aspirate the supernatant. Add 2 mL of 1×PBS to a 15 mL centrifuge tube to resuspend the cells, and then add 10 mL of ice water to induce hypotonic swelling. Transfer the cells to a T75 culture flask and incubate at room temperature for 10 min. Slowly add 1 mL of fixative (glacial acetic acid: methanol = 1:3). Transfer the cells to a 15 mL centrifuge tube, centrifuge at 1200 rpm for 7 min, carefully remove the supernatant, slowly add 5 mL of fixative, and gently pipette to resuspend the cells. Incubate at room temperature for 10 min, and centrifuge at 1200 rpm for 7 min. Repeat the above steps 3 times, adding 0.5 mL of fixative to resuspend the cells. Drop the cell suspension from a height onto a pre-chilled glass slide, allow it to air dry, and then stain with 1× Giemsa at room temperature for 10 min. Gently rinse the slide surface with enzyme-free sterile water, dry, and mount with neutral resin.

[0044] The number and karyotype of chromosomes are fundamental to cytogenetics and are crucial indicators for identifying species and sex. In cell culture, chromosomes are essential for determining cell origin and whether transformation has occurred during culture. Figure 3 As shown, chromosome analysis of the turbot spleen cell line revealed a near-normal karyotype; 58% of the observed mitotic cells had 44 chromosomes; and the chromosome number ranged from haploid to tetraploid (22-88 chromosomes). In summary, this demonstrates that the cell line obtained in this invention has the same chromosome number as individual turbot.

[0045] Example 4: Screening for the optimal fetal bovine serum concentration of SMSP in turbot spleen cells

[0046] Prepare complete culture media with FBS concentrations of 2%, 5%, 10%, 15%, and 20%. Digest 53 generations of SMSP cells, adjust cell concentration, and distribute 5 × 10⁶ cells per well. 4 SMSP cells were seeded into 48-well plates, with 15 wells for each cell type and each serum concentration. Cells were incubated at 24°C. Every 24 hours, cells from each serum concentration were digested in 3 wells to collect cells, for a total of 5 days. Cell counts were performed using a cell counter to calculate the total number of cells per well. Growth curves were plotted with culture time on the x-axis and cell count on the y-axis under different basal culture conditions to determine the optimal serum concentration for cell growth. The results are shown below. Figure 4 As shown, SMSP cells grew fastest at a serum concentration of 20%, followed by 15%. The growth rates at 5% and 10% serum concentrations were essentially the same, and cells could also grow at a serum concentration of 2%. Therefore, SMSP cells can grow at lower serum concentrations; 5% and 10% serum concentrations are sufficient for stable SMSP cell growth.

[0047] Example 5: Screening for the optimal growth temperature of turbot spleen cell SMSP

[0048] 53 generations of SMSP cells were used to determine the optimal growth temperature. SMSP cells were spaced at 5 × 10⁶ cells per well. 4 Cells were seeded into six 48-well plates, with 15 wells seeded in each plate. The plates were incubated at 28°C for 3 hours. After cell attachment, the plates were transferred to incubators at 16°C, 20°C, 24°C, 28°C, and 32°C. Cells were then collected by digesting three wells every 24 hours for a total of five days. The collected cells were counted using a cell counter to calculate the total number of cells per well. A growth curve was plotted with culture time on the x-axis and cell count on the y-axis to determine the optimal growth temperature for SMSP cells. The results are shown below. Figure 5 As shown, SMSP cells grow rapidly at 20℃, 24℃, and 28℃, with the fastest growth at 24℃. Growth is slow at 16℃ and inhibited at 32℃. Therefore, the optimal culture temperature for SMSP is 24℃.

[0049] Example 6: Screening for the optimal basal culture medium for turbot spleen cell SMSP

[0050] Prepare complete culture media with DMEM, DMEM:F12, M-199, RPMI-1640 and L15 as basal media, respectively. Digest 53 generations of SMSP cells, adjust cell concentration, and divide into groups of 5 × 10⁶ cells per well. 4SMSP cells were seeded into 48-well plates, with 15 wells for each cell type and each basal medium. Cells were incubated at 24°C. Subsequently, every 24 hours, 3 wells of each cell type were digested to collect cells for a total of 5 days. Cell counts were performed using a cell counter to calculate the total number of cells per well. Growth curves were plotted with culture time on the x-axis and cell count on the y-axis under different basal medium conditions to determine the optimal basal medium for cell growth. The results are shown below. Figure 6 As shown, SMSP cells grow rapidly in DMEM and L15 media, and also grow rapidly in DMEM:F12 media. Growth is slower in 1640 basal medium, and almost nonexistent in M199 basal medium. Therefore, the optimal basal medium for SMSP cell growth is DMEM or L15.

[0051] Example 7: Detection of siRNA transfection effect of turbot spleen cells SMSP

[0052] One day in advance, turbot spleen cells were seeded in 24-well plates (3 wells), ideally with cells in the logarithmic growth phase and a cell density of approximately 60% at transfection. Using Takara's Xfect Transfection Reagent kit, 25 pmol of GFP-siRNA was first added to 30 μL of Xfect Reaction Buffer and mixed well. After standing for 5 seconds, 2.5 μL of Xfect RNA Polymer was added and mixed well, then incubated at room temperature for 10 minutes. 0.2 mL of culture medium was aspirated from each well, leaving 0.3 mL per well. The transfection reagent was then added to the 24-well plate. After 6 hours of transfection, the medium was replaced with 0.5 mL of fresh medium and cultured for another 48 hours. 48 hours after transfection, the cell transfection status was observed under an inverted fluorescence microscope and photographed.

[0053] RNA interference (RNAi) technology inhibits transcription and translation to prevent the expression of specific genes, becoming an important tool for verifying gene function and targeting drugs in the post-genomic era. 48 hours after transfection, microscopic examination under a fluorescence microscope reveals... Figure 7 Green fluorescence (GFP) was observed in the samples. The experimental results indicate that the established turbot spleen tissue cell line is suitable for small RNA interference experiments.

[0054] Example 8: Detection of plasmid transfection effect of turbot spleen cells SMSP

[0055] One day in advance, turbot spleen cells were seeded in 24-well plates (3 wells), ideally with cells in the logarithmic growth phase and a cell density of approximately 60% at transfection. Using Takara's Xfect Transfection Reagent kit, 1 μg of pEGFP-N1 was first added to 30 μL of Xfect Reaction Buffer and mixed well. After standing for 5 seconds, 0.3 μL of Xfect Polymer was added and mixed well, then incubated at room temperature for 10 minutes. 0.2 mL of culture medium was aspirated from each well, leaving 0.3 mL per well. The transfection reagent was then added to the 24-well plate. After 6 hours of transfection, the medium was replaced with 0.5 mL of fresh medium and cultured for another 48 hours. 48 hours after transfection, cell transfection was observed under an inverted fluorescence microscope and photographed. The results are as follows: Figure 8 As shown, reported green fluorescence (EGFP) was observed. The experimental results indicate that the established turbot spleen tissue cell line is suitable for plasmid transfection experiments.

[0056] Example 9: Detection of expression of immune-related genes in turbot spleen cells SMSP after LPS and poly(I:C) stimulation

[0057] After stimulation with LPS and poly(I:C), the expression of pro-inflammatory cytokines TNF-β and immune cell modulators IL-1β, IL-12A, IL-12B, and IL-10 in SMSPs was detected. Figure 9 As shown in Figure A, all detected genes were highly expressed in SMSP cells after LPS stimulation. The expression of most of these genes was upregulated with increasing LPS stimulation time. The expression of these genes after poly(I:C) stimulation was essentially the same as after LPS stimulation, and their expression was also upregulated after poly(I:C) stimulation. Figure 9 B). The above results indicate that SMSP can serve as an in vitro research model for turbot to study the pathogenic mechanisms of pathogens.

[0058] The above description is only a preferred embodiment of the present invention, and therefore cannot be used to limit the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the present invention and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A turbot spleen tissue cell line, characterized in that, The turbot spleen tissue cell line described herein has the accession number CCTCC NO:C2022229.

2. The application of the turbot spleen tissue cell line described in claim 1 in the study of immune function genes or pathogenic mechanisms of pathogens.

3. The application as described in claim 2, characterized in that, The immune-related genes mentioned are TNF-β, IL-7, IL-1β, IL-12A, IL-12B or IL-10.

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