Establishment and application of gobiocypris rarus eye cell line
By establishing the rare gudgeon eye cell line Gbr-eye, the problems of insufficient cell line development and insensitive toxicity assessment in existing technologies have been solved, enabling rapid and sensitive screening for chemical and environmental water toxicity assessment, and reducing the number of laboratory animals used.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
- Filing Date
- 2026-01-30
- Publication Date
- 2026-05-01
AI Technical Summary
The number of rare gudgeon cell lines developed is limited. Fish cornea and retinal cells are sensitive to chemicals and pollutants. Existing acute toxicity assessment methods lack effective cell models, making it difficult to achieve rapid and sensitive screening of chemical and environmental water toxicity.
A rare gudgeon eye cell line, Gbr-eye, capable of continuous passage, was established. Cells were cultured and preserved using digestion and cryopreservation techniques. A method for assessing the acute toxicity of chemicals and environmental water bodies based on this cell line was developed, and cell viability was evaluated using multi-index toxicity detection technology.
This method enables long-term passage culture and cryopreservation of rare gudgeon eye cells, providing a rapid and sensitive method for assessing the toxicity of chemicals and environmental water bodies. It can quickly screen out toxic substances, replace acute toxicity tests on fish, and reduce the number of laboratory animals used.
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Figure CN121950701A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of fish cell culture technology, specifically to the establishment and application of a rare gudgeon eye cell line. Background Technology
[0002] Rare gudgeon ( Gobiocypris rarus The rare gudgeon, belonging to the genus *Gudgeon* in the family Cyprinidae, is a small cyprinid fish endemic to my country and is the native type species established in my country's acute toxicity testing standards. As a small laboratory animal, the rare gudgeon possesses the potential to become a testing fish species for aquatic ecotoxicology in China due to its short maturation time, long breeding season, high spawning volume, and sensitivity to environmental pollutants.
[0003] To reduce the amount of experimental fish used, increase the throughput of chemical toxicity testing, and facilitate the monitoring of environmental water bodies, acute toxicity testing using fish cells has been proposed to predict acute toxicity in fish. However, to date, the number of rare gudgeon cell lines developed is limited and they have not yet been commercialized. Furthermore, the corneal and retinal cells of fish eyes are light-sensitive and structurally fragile; many chemicals and pollutants, even at low concentrations, can cause ocular surface damage and visual dysfunction, representing non-lethal but highly sensitive acute toxicity endpoints. Therefore, developing rare gudgeon eye cell lines and establishing acute toxicity screening technologies for chemicals and environmental water bodies based on rare gudgeon eye cells are urgent problems that need to be solved by those skilled in the art. Summary of the Invention
[0004] The purpose of this invention is to provide the establishment and application of a rare gudgeon eye cell line, to develop a rare gudgeon eye cell line using the eye, and to establish a method for acute toxicity assessment of chemicals and environmental water bodies based on the rare gudgeon eye cell line.
[0005] This invention is achieved through the following technical solutions:
[0006] The first objective of this invention is to provide a rare gudgeon eye cell line, Gbr-eye, which is a continuously passaged rare gudgeon eye cell line. It was deposited on January 5, 2026, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, 510070, China, with accession number GDMCC No: 67596.
[0007] The second objective of this invention is to provide a method for constructing the aforementioned rare gudgeon eye cells (Gbr-eye), comprising the following steps:
[0008] (1) Primary culture: Take the eyes of rare gudgeon, cut them into pieces, digest them, and inoculate them into primary cell culture medium to obtain primary cultured cells;
[0009] (2) Subculture: The primary cultured cells from step (1) were digested and cultured in subculture medium to obtain rare gudgeon eye cells Gbr-eye.
[0010] (3) Cell cryopreservation and thawing: During cryopreservation, the rare gudgeon eye cells Gbr-eye obtained in step (2) were digested and mixed with cell cryopreservation solution, and then stored in an ultra-low temperature environment after gradient freezing; during thawing, the cryopreserved cells were thawed and resuspended in passaged cell culture medium for culture.
[0011] Preferably, it includes the following steps:
[0012] (1) Primary culture: Rare crucian carp were starved, and their eyes were taken under sterile conditions. The tissue fragments were cut into pieces, washed with HBSS buffer containing 3% penicillin-streptomycin-amphoteric B, and then digested with 0.25% trypsin for 1-2 h. The supernatant was discarded by centrifugation, and the cells were resuspended in primary cell culture medium to obtain primary cultured cells. The primary cell culture medium was a complete culture medium containing phenol red L-15 containing 20% fetal bovine serum, 2% penicillin-streptomycin-amphoteric B (triple antibody).
[0013] (2) Subculture: The primary cultured cells from step (1) were digested with 0.25% trypsin. After the cells became round, the cells were resuspended in subculture medium and subcultured to obtain rare gudgeon eye cells Gbr-eye. The subculture medium was L-15 complete culture medium containing 10% fetal bovine serum, 1% penicillin-streptomycin-amphoteric B (triple antibiotics) and phenol red.
[0014] (3) Cell cryopreservation and thawing: When cryopreserving cells, take the rare gudgeon eye cells Gbr-eye from step (2), wash them, add 0.25% trypsin to digest them until the cells become round, centrifuge to obtain the precipitate, resuspend it in cell cryopreservation solution, perform gradient freezing, and then place it in liquid nitrogen for long-term cryopreservation; when thawing cells, thaw the cryopreserved rare gudgeon eye cells Gbr-eye, then transfer them to passaged cell culture medium, centrifuge to obtain the precipitate, and resuspend it in passaged cell culture medium for culture.
[0015] Further optimization includes the following steps:
[0016] (1) Primary culture: Healthy rare gudgeon were starved for 24 h, their tails were cut off and they were bled out. Their eyes were then collected under aseptic conditions. The eye tissue was cut into 1 mm pieces. 2Tissue fragments were mixed with HBSS buffer containing 3% penicillin-streptomycin-amphoteric B (triple antibody), and centrifuged to obtain a precipitate. The mixing and centrifugation were repeated 3 times to obtain pretreated tissue blocks. 12 mL of 0.25% trypsin was added for digestion for 1-2 h, the supernatant was discarded after centrifugation, and the cells were resuspended in primary cell culture medium to obtain a cell suspension. The suspension was transferred to a T-25 cell culture flask and cultured at 28°C until the cells reached the bottom of the culture flask, thus obtaining primary cultured cells of rare gudgeon eyes.
[0017] (2) Passage culture: Discard the primary cell culture medium, wash with HBSS buffer, add 1-2 mL of 0.25% trypsin to digest and treat the primary cultured cells in step (1) until the cells become round, add 12 mL of passage cell culture medium to resuspend the cells, and passage culture at a ratio of 1:2 to obtain rare gudgeon eye cells Gbr-eye.
[0018] (3) Cell cryopreservation and thawing: When cryopreserving cells, take the rare gudgeon eye cells Gbr-eye from step (2), wash with HBSS buffer, then add 1-2 mL of 0.25% trypsin for digestion. After the cells become round, add 5-6 mL of passaged cell culture medium to stop the digestion, centrifuge to obtain the precipitate, add cell cryopreservation medium to resuspend the cells, and transfer to cryovials; transfer the cryovials to a programmed cooling box, place them in a -80℃ environment for 24 h, and then transfer the cryovials to liquid nitrogen for long-term storage; when thawing cells, take the cryopreserved rare gudgeon eye cells Gbr-eye from liquid nitrogen, place them in a 28℃ water bath to thaw the liquid in the cryovials, quickly transfer the cell suspension in the tube to 5-6 mL of passaged cell culture medium, centrifuge to obtain the precipitate, add 5-6 mL of passaged cell culture medium to resuspend the cells, transfer to a T-25 culture flask, and culture at 28℃ for 24 h. After h, the culture medium was replaced with fresh culture medium and cultured again to obtain revived rare gudgeon eye cells Gbr-eye.
[0019] The third objective of this invention is to provide a method for assessing the acute toxicity of chemicals and environmental water based on the aforementioned rare gudgeon eye cells (Gbr-eye), comprising the following steps:
[0020] (A) Cell inoculation and culture: The above-mentioned rare gudgeon eye cells Gbr-eye were inoculated into cell culture plates and cultured until the bottom was confluent with cells;
[0021] (B) Exposure to the test substance: Prepare an exposure solution from the test chemical or environmental water sample, add it to the cell culture plate from step (A), and incubate it with the cells for a certain period of time to complete the exposure test;
[0022] (C) Multi-index toxicity detection: Take the cell culture plate from the exposure test completed in step (B), add the working solution for detecting cell viability, incubate, and then detect the corresponding fluorescence signal;
[0023] (D) Data analysis: Process the raw data obtained in step (C), plot the concentration effect curve, and calculate the half-maximum effect concentration of the test chemical or environmental water sample.
[0024] Preferably, the following steps are included:
[0025] (A) Cell seeding and culture: Rare gudgeon eye cells (Gbr-eye) with a confluence of 90-100% were obtained, digested, resuspended, and seeded at 4-6 × 10⁻⁶ cells / year. 5 The cells were seeded at a density of cells / mL in a cell culture plate and cultured at 28°C for 24-48 hours until the bottom of the cell culture plate was confluent.
[0026] (B) Exposure to the test substance: Dilute the test chemical or environmental water sample with added nutrient salt stock solution with L-15 / ex medium to prepare an exposure solution; add it to the cell culture plate of step (A) and place it at 28°C for continuous exposure for 24 h to complete the exposure test;
[0027] (C) Multi-index toxicity test: Take the cell culture plate from the exposure test completed in step (B), add the mixed working solution A and working solution B, incubate in the dark, and then use an ELISA reader to detect the solution. Discard the mixture, add working solution C, incubate in the dark, and then perform fixation and extraction operations. Detect the solution using an ELISA reader. The mixed working solution A and working solution B include resazurin, CFDA-AM and buffer solution, and the working solution C includes neutral red and buffer solution.
[0028] (D) Data analysis: Calculate cell viability based on the fluorescence signal measured in step (C), plot the concentration-effect curve, and calculate the half-maximum effective concentration of the test chemical or environmental water sample accordingly.
[0029] Further preferred, in step (B), the nutrient salt storage solution includes: (1) a storage solution A containing sodium salt, potassium salt and magnesium salt; (2) a storage solution B containing calcium salt; (3) a storage solution C containing phosphate; (4) a storage solution D containing galactose; and (5) a storage solution E containing sodium pyruvate.
[0030] Further preferably, in step (B), the L-15 / ex culture medium comprises sodium salt, potassium salt, magnesium salt, calcium salt, phosphate, galactose, and sodium pyruvate, and the solvent is water.
[0031] Further preferred, the L-15 / ex culture medium is prepared in 500 mL batches as follows: Take 30 mL of salt solution A (80 g NaCl, 4 g KCl, 0.98 g MgSO4, 0.94 g MgCl2, diluted to 600 mL with deionized water, autoclaved and stored at room temperature), 5 mL of salt solution B (1.4 g CaCl2, diluted to 100 mL with deionized water, autoclaved and stored at room temperature), 15 mL of salt solution C (1.9 g Na2HPO4, 0.6 g KH2PO4, diluted to 300 mL with deionized water, autoclaved and stored at room temperature), 5 mL of galactose solution (9 g galactose, diluted to 100 mL with deionized water, sterilized by 0.22 μm filtration, aliquoted and stored at -20℃), and 5 mL of sodium pyruvate solution (5.5 g sodium pyruvate, diluted to 100 mL with deionized water, 0.22 μm filtration). (Sterilize by filtration at μm, dispense and store at -20℃) Mix 5 mL of the solution thoroughly and add deionized water to bring the volume to 500 mL.
[0032] Further preferred, the nutrient salt stock solution is prepared as follows: Stock solution A consists of 80 g NaCl, 4 g KCl, 0.98 g MgSO4, and 0.94 g MgCl2, diluted to 300 mL with deionized water and autoclaved; Stock solution B consists of 4.2 g CaCl2, diluted to 100 mL with deionized water and autoclaved; Stock solution C consists of 1.9 g Na2HPO4 and 0.6 g KH2PO4, diluted to 100 mL with deionized water and autoclaved; Stock solution D consists of 27 g galactose, diluted to 100 mL with deionized water and sterilized by filtration; Stock solution E consists of 16.5 g sodium pyruvate, diluted to 100 mL with deionized water and sterilized by filtration.
[0033] Further preferably, in step (C), the mixed working solution A and working solution B comprise: approximately 10% resazurin solution, 0.1% CFDA-AM, and the remainder DPBS buffer; the working solution C comprises: 1-2% neutral red solution and 98-99% DPBS buffer.
[0034] Preferably, the steps include the following:
[0035] (A) Take rare gudgeon eye cells (Gbr-eye) with a confluence of 90-100%, wash with HBSS buffer, add 1-2 mL of 0.25% trypsin for digestion, and after the cells become rounded, add passaged cell culture medium to adjust the cell density to 4-6 × 10⁻⁶. 5The cell suspension to be inoculated was obtained by adding 0.5-1 mL of HBSS buffer to rinse all wells of the 24-well cell culture plate. After discarding the solution, the cell suspension to be inoculated was transferred to the 24-well cell culture plate at an inoculation rate of 500 μL / well. The plate was then incubated at 28°C for 24-48 h until the bottom of each cell culture plate was filled with rare gudgeon eye cells.
[0036] (B) Dissolve the test chemical in DMSO to obtain a concentrated solution or dilute the environmental water sample obtained by adding nutrient salt stock solution in L-15 / ex medium according to the ratio to obtain an exposure solution; take the cell culture plate with rare gudgeon eye cells at the bottom from step (A), gently add 0.5-1 mL of HBSS buffer to each well, shake the plate slightly and discard it, add 2 mL of exposure solution to each well, and then place it in an environment of 28℃ for continuous exposure for 24 h to obtain a cell culture plate that has completed the exposure test;
[0037] (C) Take the cell culture plate from step (B) after the exposure test, discard the exposure solution, add 0.5-1 mL of HBSS buffer to each well, gently shake the plate and discard it. Under sterile and light-protected conditions, add 400 μL of the mixed working solution A and working solution B to each well, incubate in the dark for 30 min, and measure the fluorescence intensity of working solution A using an excitation wavelength of 530 nm and an emission wavelength of 590 nm, and measure the fluorescence intensity of working solution B using an excitation wavelength of 493 nm and an emission wavelength of 541 nm. Discard the mixed working solution A and working solution B, add 400 μL of working solution C to each well under light-protected conditions, incubate in the dark for 60 min, discard working solution C, add 400 μL of fixative to each well, fix for 1-3 min, discard the fixative, add 400 μL of extraction buffer to each well, gently shake the plate for 10 min in a microplate reader, and then measure the fluorescence intensity using an excitation wavelength of 530 nm and an emission wavelength of 645 nm.
[0038] (D) The value J is obtained by subtracting the cell-free blank wells from the wells containing cells and the wells containing exposed solutions, and the value P is obtained by subtracting the cell-free blank wells from the control group. Cell viability at each exposure concentration is then calculated using the formula: Based on the set concentration of the test chemical or the environmental water sample and the corresponding cell viability, the concentration-effect curve of the test chemical or the environmental water sample on the rare gudgeon eye cell Gbr-eye was plotted. The half-maximal effective concentration of the test chemical or the environmental water sample on the rare gudgeon eye cell Gbr-eye was calculated using the sigmoid nonlinear fitting curve.
[0039] Further preferably, in step (C), the fixative is prepared as follows: 2.5 g of CaCl2 is dissolved in 200 mL of ultrapure water, 3.375 mL of 37% formaldehyde solution is added, and then the volume is adjusted to 500 mL with ultrapure water. The extract is prepared as follows: 5 mL of acetic acid is dissolved in 100 mL of ultrapure water, 250 mL of anhydrous ethanol is added, and then the volume is adjusted to 500 mL with ultrapure water.
[0040] Further preferred, in step (C), when discarding the exposure solution, the mixed working solution A and working solution B, working solution C, fixative, and extraction solution, the liquid is discarded into the waste liquid tank by quickly inverting the cell culture plate.
[0041] The fourth objective of this invention is to provide the use of the aforementioned rare gudgeon eye cells (Gbr-eye) in the acute toxicity assessment of chemicals and environmental water samples.
[0042] The beneficial effects of this invention are as follows:
[0043] (1) The rare gudgeon eye cells Gbr-eye of the present invention can be passaged for a long time, and can be cryopreserved and thawed at low temperature; it can be applied to the acute toxicity assessment of chemicals and water samples, and used for in vitro substitution of acute toxicity of fish, and to explore the toxic effects of chemicals and water samples.
[0044] (2) The acute toxicity assessment method for chemicals and environmental water bodies based on rare gudgeon eye cells Gbr-eye constructed in this invention has uniform cell plating, is convenient and fast, and can quickly screen out toxic test substances, providing cytotoxicity information and alternative in vitro methods for the toxicity assessment of chemicals and environmental water bodies.
[0045] A rare gudgeon eye cell, Gbr-eye, was deposited on January 5, 2026, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, 510070, China, with accession number GDMCC No:67596. Attached Figure Description
[0046] Figure 1 The image shows the morphological observation of rare gudgeon eye cells under an inverted microscope in Example 1 of this invention, where A represents the morphology of generation 0 cells and B represents the morphology of generation 3 cells.
[0047] Figure 2 Morphological observation of the 32nd generation of rare crucian carp eye cells in Example 1 of this invention under an inverted microscope (100x magnification).
[0048] Figure 3This is a schematic diagram of the cell plating and drug delivery protocol in Embodiment 2 of the present invention. In this diagram, A is a schematic diagram of the cell seeding and pipetting protocol in a 24-well plate; B is a schematic diagram of the 24-well plate layout when cells are exposed using DMSO; and C is a schematic diagram of the 24-well plate layout when cells are exposed without using DMSO.
[0049] Figure 4 This is a concentration-response curve of rare gudgeon eye cells (Gbr-eye) exposed to 3,4-dichloroaniline and a certain environmental water sample in Example 2 of the present invention. In this figure, A-3,4-DCA is the concentration-response curve of 3,4-dichloroaniline on rare gudgeon eye cells (Gbr-eye); B-environmental water sample is the concentration-response curve of an environmental water sample on rare gudgeon eye cells (Gbr-eye). Detailed Implementation
[0050] The following embodiments are further illustrations of the present invention, but not limitations thereof. Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents; and the experimental methods described are conventional methods.
[0051] Example 1: Construction of rare gudgeon eye cells Gbr-eye
[0052] The primary cell culture medium was a complete culture medium containing phenol red L-15 with 20% (v / v) fetal bovine serum and 2% (v / v) penicillin-streptomycin-amphoteric B (triple antibody); the passaged cell culture medium was a complete culture medium containing phenol red L-15 with 10% (v / v) fetal bovine serum and 1% (v / v) penicillin-streptomycin-amphoteric B (triple antibody); wherein the triple antibody included 100 U / mL penicillin, 100 μg / mL streptomycin and 0.25 μg / mL amphotericin B.
[0053] (1) Obtaining primary cultured cells of rare gudgeon eye cells
[0054] Using healthy rare gudgeon, their eyes were removed, chopped up, and digested with trypsin. The resulting suspended cells and tissue fragments were collected, inoculated into primary cell culture medium, and cultured in primary culture to obtain primary cultured cells of rare gudgeon eye cells.
[0055] The specific steps are as follows: Five healthy rare gudgeon were starved for 24 hours in advance, their tails were cut off and they were bled. Under sterile conditions, their eyes were removed and cut into 1 mm pieces. 2Tissue fragments were mixed with HBSS buffer containing 3% (v / v) penicillin-streptomycin-amphoteric B (triple antibody); the precipitate was obtained by centrifugation, and the process of mixing with HBSS buffer containing triple antibody and centrifuging to obtain the precipitate was repeated 3 times to obtain pretreated tissue blocks; 12 mL of 0.25% trypsin was added to the pretreated tissue blocks for digestion for 1-2 h, the supernatant was discarded by centrifugation, 5-6 mL of primary cell culture medium was added, and the cells were resuspended to obtain a cell suspension; the cell suspension was transferred to a T-25 cell culture flask and cultured at 28℃. After the cells reached the bottom of the culture flask, the primary cultured cells of rare gudgeon eyes were obtained.
[0056] (2) Establishment of rare gudgeon eye cell line
[0057] The primary cell culture medium of rare gudgeon eyes was discarded, washed with HBSS buffer, and digested with 1-2 mL of 0.25% trypsin. After the cells became round, 12 mL of passaged cell culture medium was added to resuspend the cells, and the cells were passaged at a ratio of 1:2 to obtain the rare gudgeon eye cell line, which was named rare gudgeon eye cell Gbr-eye.
[0058] (3) Cell cryopreservation and thawing
[0059] When performing cell cryopreservation, the rare gudgeon eye cells Gbr-eye from step (2) were washed with HBSS buffer, and 1-2 mL of 0.25% trypsin was added for digestion. After the cells became round, 5 mL of passaged cell culture medium was added to stop the digestion. The cells were then transferred to centrifuge tubes, centrifuged at 800 g for 5 min to obtain a precipitate. The cells were then resuspended in cell cryopreservation solution and transferred to cryopreservation tubes. The cryopreservation tubes were then transferred to a programmed cooling box and placed at -80℃ for 24 h. After that, the cryopreservation tubes were transferred to liquid nitrogen for long-term storage.
[0060] During cell resuscitation, the cryopreserved rare gudgeon eye cells (Gbr-eye) were removed from liquid nitrogen and quickly placed in a 28°C water bath. The cryopreservation tube containing the cells was picked up with tweezers and rapidly shaken in the water bath to thaw the liquid inside. The cell suspension in the cryopreservation tube was transferred to 5-6 mL of passaged cell culture medium, centrifuged at 800 g for 5 min to obtain a precipitate, and then 5-6 mL of passaged cell culture medium was added to resuspend the cells. The cells were then transferred to a T-25 culture flask and cultured at 28°C. After 24 h, the culture medium was replaced with fresh medium and cultured for another 24 h to obtain the revived rare gudgeon eye cells (Gbr-eye).
[0061] The aforementioned rare gudgeon eye cell, Gbr-eye, was deposited on January 5, 2026, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, 510070, China, with accession number GDMCC No:67596.
[0062] Example 2
[0063] An acute toxicity assessment method for chemicals and environmental water bodies based on rare gudgeon eye cells (Gbr-eye) includes the following steps:
[0064] (A) Inoculation of rare gudgeon eye cells Gbr-eye into 24-well plates
[0065] Take rare gudgeon eye cells (Gbr-eye) with a fusion degree of 90-100% as described in step (2) of Example 1, wash with HBSS buffer, add 1-2 mL of 0.25% trypsin for digestion, wait for the cells to become round, add an appropriate amount of the passaged cell culture medium from Example 1, and adjust the cell density to 4-6 × 10⁻⁶. 5 The cell suspension to be inoculated was obtained by measuring cells / mL.
[0066] Before inoculation, rinse all wells of the 24-well cell culture plate with 0.5-1 mL of HBSS buffer, discard the solution, and then place the cell suspension to be inoculated according to the plate-laying and drug administration diagram (). Figure 3 The inoculum was transferred to a 24-well cell culture plate at a rate of 500 μL / well and cultured at 28°C for 24 h until the bottom of the cell culture plate was filled with rare crucian carp eye cells.
[0067] (B) Exposure rare gudgeon eye cells (Gbr-eye) to the test chemical (3,4-dichloroaniline) or ambient water.
[0068] 3,4-Dichloroaniline was dissolved in DMSO to obtain a concentrated solution. The concentrated solution was diluted in L-15 / ex medium at a ratio of 1:200. Alternatively, environmental water samples with added nutrient stock solutions (for example, per 100 ml of environmental water sample: 3 mL of stock solution A, 0.333 mL of stock solution B, 1 mL of stock solution C, 0.333 mL of stock solution D, and 0.333 mL of stock solution E) were diluted in L-15 / ex medium to obtain exposure solutions. The concentrations of the test chemicals (concentration intervals ≤ 2.5) were set as follows: 100 mg / L, 50 mg / L, 25 mg / L, 12.5 mg / L, 6.25 mg / L, and 3.13 mg / L. The concentrations (volume fraction, v / v) of a test environmental water sample were set as follows: 100%, 50%, 25%, 12.5%, 6.25%, and 3.13%.
[0069] The L-15 / ex culture medium was prepared in 500 mL batches as follows: 30 mL of salt solution A (80 g NaCl, 4 g KCl, 0.98 g MgSO4, 0.94 g MgCl2, diluted to 600 mL with deionized water, autoclaved and stored at room temperature), 5 mL of salt solution B (1.4 g CaCl2, diluted to 100 mL with deionized water, autoclaved and stored at room temperature), 15 mL of salt solution C (1.9 g Na2HPO4, 0.6 g KH2PO4, diluted to 300 mL with deionized water, autoclaved and stored at room temperature), 5 mL of galactose solution (9 g galactose, diluted to 100 mL with deionized water, sterilized by 0.22 μm filtration, aliquoted and stored at -20℃), and 5 mL of sodium pyruvate solution (5.5 g sodium pyruvate, diluted to 100 mL with deionized water, 0.22 μm filtration). (Sterilize by filtration at μm, dispense and store at -20℃) Mix 5 mL of the solution thoroughly and add water to bring the volume to 500 mL.
[0070] The nutrient salt stock solution is prepared as follows: Stock solution A consists of 80 g NaCl, 4 g KCl, 0.98 g MgSO4, and 0.94 g MgCl2, diluted to 300 mL with deionized water and autoclaved; Stock solution B consists of 4.2 g CaCl2, diluted to 100 mL with deionized water and autoclaved; Stock solution C consists of 1.9 g Na2HPO4 and 0.6 g KH2PO4, diluted to 100 mL with deionized water and autoclaved; Stock solution D consists of 27 g galactose, diluted to 100 mL with deionized water and filtered for sterilization; Stock solution E consists of 16.5 g sodium pyruvate, diluted to 100 mL with deionized water and filtered for sterilization.
[0071] Take the cell culture plate from step (A) with rare gudgeon eye cells at the bottom, gently add 0.5-1 mL of HBSS buffer to each well, gently shake the plate and discard it. Then, according to the chemical plating and administration diagram (using L-15 / ex medium containing 0.5% DMSO as the control group), or the environmental water sample plating and administration diagram (using L-15 / ex medium without 0.5% DMSO as the control group), gently add 2 mL of exposure solution to each well, and then place it in a dark environment at 28℃ for 24 h of continuous exposure to obtain the cell culture plate that has completed the exposure experiment.
[0072] (C) Detection of the effects of 3,4-dichloroaniline and a certain environmental water sample on the Gbr-eye cells of rare gudgeon.
[0073] Take the cell culture plate from step (B) where the exposure test was completed, gently add 0.5-1 mL of HBSS buffer to each well, gently shake the plate and discard it. In a dark environment, add 400 μL of a mixture of working solution A and working solution B to each well and incubate in the dark for 30 min. First, measure the fluorescence intensity of working solution A (alamar Blue) using an excitation wavelength of 530 nm and an emission wavelength of 590 nm, and then measure the fluorescence intensity of working solution B (CFDA-AM) using an excitation wavelength of 493 nm and an emission wavelength of 541 nm. The working solution A and working solution B mixed in each cell culture plate include: approximately 10% resamar blue solution, 0.1% CFDA-AM, and the remainder DPBS buffer (1.2 mL resamar blue solution, 12 μL CFDA-AM, and 10.8 mL DPBS buffer).
[0074] After measuring the fluorescence intensity of working solutions A and B, discard the mixed working solutions A and B. In a dark environment, add 400 μL of working solution C to each well and incubate in the dark for 60 min. Then discard working solution C and add 400 μL of fixative to each well for 1-3 min. After discarding the fixative, add 400 μL of extraction buffer to each well and gently shake the plate in the microplate reader for 10 min. Finally, measure the fluorescence intensity of working solution C (Neutral Red) using an excitation wavelength of 530 nm and an emission wavelength of 645 nm. Working solution C comprises: 1-2% Neutral Red solution and 98-99% DPBS buffer (180 μL Neutral Red solution and 11.82 mL DPBS buffer). The fixative is prepared as follows: dissolve 2.5 g of CaCl2 in 200 mL of ultrapure water, add 3.337 mL of 37% formaldehyde solution, and then bring the volume to 500 mL with ultrapure water. The extraction buffer is prepared as follows: 5... Dissolve 100 mL of acetic acid in 100 mL of ultrapure water, add 250 mL of anhydrous ethanol, and then bring the volume up to 500 mL with ultrapure water.
[0075] (D) Data Analysis
[0076] The value J is obtained by subtracting the cell-free blank wells from the wells containing cells and the wells containing the exposed solution. The value P is obtained by subtracting the cell-free blank wells from the control group. Cell viability at each exposure concentration is then calculated using the following formula:
[0077]
[0078] Based on the set concentrations of the test chemicals and environmental water samples and the corresponding cell viability, concentration-effect curves of the test chemicals and environmental water samples on the rare gudgeon eye cells Gbr-eye were plotted. The half-maximal effective concentrations of 3,4-dichloroaniline and environmental water samples on the rare gudgeon eye cells Gbr-eye were calculated using nonlinear fitting curves.
[0079] Experimental results:
[0080] The concentration-effect curves of 3,4-dichloroaniline and a certain environmental water sample on the eye cells of rare gudgeon are shown below. Figure 4 As shown.
[0081] When the cytotoxic effects of the test chemical on the rare gudgeon eye cells Gbr-eye were quantified using working solutions A (alamar Blue), B (CFDA-AM), and C (Neutral Red), the half-maximal effective concentrations (IC50) of 3,4-dichloroaniline were 29.7 mg / L, 73.4 mg / L, and 27.3 mg / L, respectively.
[0082] When using working solutions A (alamar Blue), B (CFDA-AM), and C (Neutral Red) to quantify the cytotoxic effect of an environmental water sample on the rare gudgeon eye cells Gbr-eye, the half-maximal effective concentrations were 17.1%, 32.8%, and 30.1%, respectively.
Claims
1. A rare gudgeon eye cell, Gbr-eye, characterized by, It is a rare gudgeon eye cell line, with the accession number GDMCC No: 67596.
2. The method for constructing the rare gudgeon eye cells Gbr-eye as described in claim 1, characterized in that, Includes the following steps: (1) Primary culture: Take the eyes of rare gudgeon, cut them into pieces, digest them, and inoculate them into primary cell culture medium to obtain primary cultured cells; (2) Subculture: The primary cultured cells from step (1) were digested and cultured in subculture medium to obtain rare gudgeon eye cells Gbr-eye. (3) Cell cryopreservation and thawing: During cryopreservation, the rare gudgeon eye cells Gbr-eye obtained in step (2) were digested and mixed with cell cryopreservation solution, and then stored in an ultra-low temperature environment after gradient freezing; during thawing, the cryopreserved cells were thawed and resuspended in passaged cell culture medium for culture.
3. The method according to claim 2, characterized in that, Includes the following steps: (1) Primary culture: Rare gudgeon were starved, and their eyes were taken under sterile conditions. The eyes were cut into tissue fragments and washed with HBSS buffer containing 3% penicillin-streptomycin-amphoteric acid B. Then, they were digested with 0.25% trypsin for 1-2 h. The supernatant was discarded by centrifugation and the cells were resuspended in primary cell culture medium to obtain primary cultured cells. The primary cell culture medium was a complete culture medium containing 20% fetal bovine serum, 2% penicillin-streptomycin-amphoteric acid B and phenol red L-15. (2) Subculture: The primary cultured cells from step (1) were digested with 0.25% trypsin. After the cells became round, the cells were resuspended in subculture medium and subcultured to obtain rare gudgeon eye cells Gbr-eye. The subculture medium was L-15 complete culture medium containing 10% fetal bovine serum, 1% penicillin-streptomycin-amphoteric B, and phenol red. (3) Cell cryopreservation and thawing: When cryopreserving cells, take the rare gudgeon eye cells Gbr-eye from step (2), wash them, add 0.25% trypsin to digest them until the cells become round, centrifuge to obtain the precipitate, resuspend it in cell cryopreservation solution, perform gradient freezing, and then place it in liquid nitrogen for long-term cryopreservation; when thawing cells, thaw the cryopreserved rare gudgeon eye cells Gbr-eye, then transfer them to passaged cell culture medium, centrifuge to obtain the precipitate, and resuspend it in passaged cell culture medium for culture.
4. The method according to claim 3, characterized in that, Includes the following steps: (1) Primary culture: Healthy rare gudgeon were starved for 24 h, their tails were cut off and they were bled out. Their eyes were then collected under aseptic conditions. The eye tissue was cut into 1 mm pieces. 2 Tissue fragments were mixed with HBSS buffer containing 3% penicillin-streptomycin-amphoteric B, and centrifuged to obtain a precipitate. The mixing and centrifugation were repeated 3 times to obtain pretreated tissue blocks. 12 mL of 0.25% trypsin was added for digestion for 1-2 h, the supernatant was discarded after centrifugation, and the cells were resuspended in primary cell culture medium to obtain a cell suspension. The suspension was transferred to a T-25 cell culture flask and cultured at 28°C until the cells reached the bottom of the culture flask, thus obtaining the primary cultured cells of rare gudgeon eyes. (2) Passage culture: Discard the primary cell culture medium, wash with HBSS buffer, add 1-2 mL of 0.25% trypsin to digest and treat the primary cultured cells in step (1) until the cells become round, add 12 mL of passage cell culture medium to resuspend the cells, and passage culture at a ratio of 1:2 to obtain rare gudgeon eye cells Gbr-eye. (3) Cell cryopreservation and thawing: When cryopreserving cells, take the rare gudgeon eye cells Gbr-eye from step (2), wash them with HBSS buffer, and then add 1-2 mL of 0.25% trypsin for digestion. After the cells become round, add 5-6 mL of passaged cell culture medium to stop the digestion, centrifuge to obtain the precipitate, add cell cryopreservation medium to resuspend the cells, and transfer them to cryovials; transfer the cryovials to a programmed cooling box, place them in an environment of -80℃ for 24 h, and then transfer the cryovials to liquid nitrogen for long-term storage; when thawing cells, take the cryopreserved rare gudgeon eye cells Gbr-eye from the liquid nitrogen, place them in a 28℃ water bath to melt the liquid in the cryovials, quickly transfer the cell suspension in the tube to 5-6 mL of passaged cell culture medium, centrifuge to obtain the precipitate, add 5-6 mL of passaged cell culture medium to resuspend the cells, transfer them to a T-25 culture flask, and culture them in an environment of 28℃. After 24 h, replace with fresh culture medium and continue culturing to obtain thawed rare gudgeon eye cells.
5. A method for acute toxicity assessment of chemicals and environmental water samples based on the rare gudgeon eye cells Gbr-eye as described in claim 1, characterized in that, Includes the following steps: (A) Cell inoculation and culture: The rare gudgeon eye cells Gbr-eye described in claim 1 are inoculated into a cell culture plate and cultured until the bottom is confluent with cells; (B) Exposure to the test substance: Prepare an exposure solution from the test chemical or environmental water sample, add it to the cell culture plate from step (A), and incubate it with the cells for a certain period of time to complete the exposure test; (C) Multi-index toxicity detection: Take the cell culture plate from the exposure test completed in step (B), add the working solution for detecting cell viability, incubate, and then detect the corresponding fluorescence signal; (D) Data analysis: Process the raw data obtained in step (C), plot the concentration effect curve, and calculate the half-maximum effect concentration of the test chemical or environmental water sample.
6. The method according to claim 5, characterized in that, Includes the following steps: (A) Cell seeding and culture: Rare gudgeon eye cells (Gbr-eye) with a confluence of 90-100% were obtained, digested, resuspended, and seeded at 4-6 × 10⁻⁶ cells / year. 5 The cells were seeded at a density of cells / mL in a cell culture plate and cultured at 28°C for 24-48 hours until the bottom of the cell culture plate was confluent. (B) Exposure to the test substance: Dilute the test chemical or environmental water sample with added nutrient salt stock solution with L-15 / ex medium to prepare an exposure solution; add it to the cell culture plate of step (A) and place it at 28°C for continuous exposure for 24 h to complete the exposure test; (C) Multi-index toxicity test: Take the cell culture plate from the exposure test completed in step (B), add the mixed working solution A and working solution B, incubate in the dark, and then use an ELISA reader to detect the solution. Discard the mixture, add working solution C, incubate in the dark, and then perform fixation and extraction operations. Detect the solution using an ELISA reader. The mixed working solution A and working solution B include resazurin, CFDA-AM and buffer solution, and the working solution C includes neutral red and buffer solution. (D) Data analysis: Calculate cell viability based on the fluorescence signal measured in step (C), plot the concentration-effect curve, and calculate the half-maximum effective concentration of the test chemical or environmental water sample accordingly.
7. The method according to claim 6, characterized in that, In step (B), the nutrient salt stock solution includes: (1) stock solution A containing sodium, potassium and magnesium salts; (2) stock solution B containing calcium salts; (3) stock solution C containing phosphates; (4) stock solution D containing galactose; (5) stock solution E containing sodium pyruvate; In step (B), the L-15 / ex medium includes sodium, potassium, magnesium, calcium, phosphate, galactose and sodium pyruvate, and the solvent is water; In step (C), the mixed working solution A and working solution B include: 10% resazurin solution, 0.1% CFDA-AM and the remainder DPBS buffer; The working solution C includes: 1-2% neutral red solution and 98-99% DPBS buffer.
8. The method according to claim 7, characterized in that, The nutrient salt stock solution is prepared as follows: Stock solution A consists of 80 g NaCl, 4 g KCl, 0.98 g MgSO4, and 0.94 g MgCl2, diluted to 300 mL with deionized water and autoclaved; Stock solution B consists of 4.2 g CaCl2, diluted to 100 mL with deionized water and autoclaved; Stock solution C consists of 1.9 g Na2HPO4 and 0.6 g KH2PO4, diluted to 100 mL with deionized water and autoclaved; Stock solution D consists of 27 g galactose, diluted to 100 mL with deionized water and filtered and sterilized; Stock solution E consists of 16.5 g sodium pyruvate, diluted to 100 mL with deionized water and filtered and sterilized. The L-15 / ex culture medium is prepared in 500 mL batches as follows: 30 mL of salt solution A, 5 mL of salt solution B, 15 mL of salt solution C, and 5 mL of galactose solution. 5 mL of sodium pyruvate solution and 1 mL of sodium pyruvate solution were mixed evenly and then diluted to 500 mL with deionized water. Salt solution A consisted of 80 g NaCl, 4 g KCl, 0.98 g MgSO4, and 0.94 g MgCl2 dissolved in deionized water and diluted to 600 mL, then autoclaved. Salt solution B consisted of 1.4 g CaCl2 dissolved in deionized water and diluted to 100 mL, then autoclaved. Salt solution C consisted of 1.9 g Na2HPO4 and 0.6 g KH2PO4 dissolved in deionized water and diluted to 300 mL, then autoclaved. Galactose solution consisted of 9 g galactose dissolved in deionized water and diluted to 100 mL, then filtered and sterilized. Sodium pyruvate solution consisted of 5.5 g sodium pyruvate dissolved in deionized water and diluted to 100 mL, then filtered and sterilized.
9. The method according to any one of claims 5-8, characterized in that, Specifically, the steps include the following: (A) Take rare gudgeon eye cells (Gbr-eye) with a confluence of 90-100%, wash with HBSS buffer, add 1-2 mL of 0.25% trypsin for digestion, and after the cells become rounded, add passaged cell culture medium to adjust the cell density to 4-6 × 10⁶ cells / year. 5 The cell suspension to be inoculated was obtained by adding 0.5-1 mL of HBSS buffer to rinse all wells of the 24-well cell culture plate. After discarding the solution, the cell suspension to be inoculated was transferred to the 24-well cell culture plate at an inoculation rate of 500 μL / well. The plate was then incubated at 28°C for 24-48 h until the bottom of each cell culture plate was filled with rare gudgeon eye cells. (B) Dissolve the test chemical in DMSO to obtain a concentrated solution or dilute the environmental water sample obtained by adding nutrient salt stock solution in L-15 / ex medium according to the ratio to obtain an exposure solution; take the cell culture plate with rare gudgeon eye cells at the bottom from step (A), gently add 0.5-1 mL of HBSS buffer to each well, shake the plate slightly and discard it, add 2 mL of exposure solution to each well, and then place it in an environment of 28℃ for continuous exposure for 24 h to obtain a cell culture plate that has completed the exposure test; (C) Take the cell culture plate from step (B) after the exposure test, discard the exposure solution, add 0.5-1 mL of HBSS buffer to each well, gently shake the plate and discard it. Under sterile and light-protected conditions, add 400 μL of mixed working solution A and working solution B to each well, incubate in the dark for 30 min, and measure the fluorescence intensity of working solution A using an excitation wavelength of 530 nm and an emission wavelength of 590 nm, and measure the fluorescence intensity of working solution B using an excitation wavelength of 493 nm and an emission wavelength of 541 nm. Discard the mixed working solutions A and B. In a dark environment, add 400 μL of working solution C to each well and incubate in the dark for 60 min. Discard working solution C and add 400 μL of fixative to each well for 1-3 min. After discarding the fixative, add 400 μL of extraction buffer to each well. Gently shake the plate for 10 min in a microplate reader, then measure the fluorescence intensity using an excitation wavelength of 530 nm and an emission wavelength of 645 nm. Preferably, the fixative is prepared as follows: dissolve 2.5 g of CaCl2 in 200 mL of ultrapure water, add 3.375 mL of 37% formaldehyde solution, and then bring the volume to 500 mL with ultrapure water. The extraction buffer is prepared as follows: dissolve 5 mL of acetic acid in 100 mL of ultrapure water, add 250 mL of anhydrous ethanol, and then bring the volume to 500 mL with ultrapure water. (D) The value J is obtained by subtracting the cell-free blank wells from the wells containing cells and the wells containing exposed solutions, and the value P is obtained by subtracting the cell-free blank wells from the control group. Cell viability at each exposure concentration is then calculated using the formula: Based on the set concentration of the test chemical or the environmental water sample and the corresponding cell viability, the concentration-effect curve of the test chemical or the environmental water sample on the rare gudgeon eye cell Gbr-eye was plotted. The half-maximal effective concentration of the test chemical or the environmental water sample on the rare gudgeon eye cell Gbr-eye was calculated using the sigmoid nonlinear fitting curve.
10. Use of the rare gudgeon eye cells Gbr-eye as described in claim 1 in the acute toxicity assessment of chemicals and environmental water samples.