Isolation method of japanese quail primary embryonic neuronal cells and application thereof

Abstract

The application discloses a method for separating Japanese quail primary embryonic neuron cells and application thereof. The method comprises the following steps: selecting fertilized Japanese quail eggs, breaking the shells when the Japanese quail hatches for 16-17 days, selecting the head of the Japanese quail hatched for 15 days, and taking the embryonic brain tissue; the brain tissue is washed with pre-cooled PBS, cut into pieces, digested, and then complete culture medium is added to terminate the digestion; the cell suspension after digestion is subjected to two-stage gradient filtration to remove tissue fragments and cell clumps; the cell suspension after filtration is centrifuged and collected, resuspended with complete culture medium, inoculated into a culture vessel, and adherent cultured for 4-6 h; after adhesion, the complete culture medium is discarded, replaced with serum-free culture medium special for neurons, and cultured, the liquid is replaced every two days, and the culture is continued for 3-15 days to obtain Japanese quail primary embryonic neuron cells. The cell model can be applied to high-throughput developmental neurotoxicity screening of new nicotine insecticides and other chemicals and cross-species harmful outcome path analysis.

Description

Technical Field

[0001] This invention relates to the interdisciplinary field of ecotoxicology and in vitro alternative toxicology, specifically to a method for isolating neurons from primary embryonic Japanese quail and its application. Background Technology

[0002] Global bird populations are facing a rapidly declining biodiversity crisis, with the widespread use of neurotoxins such as neonicotinoids (NEOs) during agricultural intensification considered a key driving factor. Existing avian neurotoxicity assessment systems suffer from the following fundamental limitations: 1. Lack of species specificity in high-throughput screening tools: Current high-throughput screening (HTS) in ecotoxicology mainly relies on invertebrates (such as zebrafish embryos and nematodes) or mammalian cell lines (such as human SH-SY5Y). Birds, as a highly sensitive group among non-target vertebrates, still lack dedicated primary neuronal models to support high-throughput screening. Existing avian toxicology data mostly come from acute lethality tests (LD50) or behavioral observations, failing to capture developmental neurotoxicity at sublethal doses, severely limiting the need for rapid risk assessment of thousands of chemicals.

[0003] 2. Uncertainties in cross-species extrapolation: Within the current adverse outcome pathway (AOP) framework, bird-specific toxicological data are extremely scarce. Of the approximately 380 cases in the global AOP-Wiki database, the core data focuses on humans, rats, and zebrafish, with virtually no data on avian neurodevelopmental toxicity AOPs. Extrapolation from mammalian models to birds is highly unreliable due to species differences in nAChR subunit structure, metabolic enzyme (such as cytochrome P450) activity, and blood-brain barrier permeability.

[0004] 3. Low throughput bottleneck of traditional morphological assessment: The assessment of avian embryonic developmental toxicity has long relied on manual dissection, microscopic observation and manual measurement, which has defects such as strong subjectivity, low throughput and poor repeatability, and cannot meet the requirements of modern chemical regulation for standardized, automated and big data-driven morphological endpoint assessment.

[0005] 4. Fragmentation of existing bird neuron models: Although chicken embryo neuron culture technology is relatively mature, the following problems exist: (1) The chicken embryo incubation cycle is long (21 days), which is not conducive to rapid experimental turnover; (2) There is a lack of standardized cryopreservation and thawing system, making it impossible to establish a reusable cell bank; (3) The purity of neurons is low (75-80%), and glial cell contamination is serious (15-25%); (4) The synaptic network matures slowly (10-14 days), making it difficult to support chronic exposure experiments of more than 7 days; (5) There is a lack of compatibility verification with high-throughput detection platforms (such as 384-well plate transcriptomics, calcium imaging, and deep learning image analysis). Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a method for isolating neurons from primary Japanese quail embryos and its application. Using the isolation method of this invention to obtain primary Japanese quail embryo neurons (Quail), this invention constructs a complete technical system encompassing cell model construction, high-throughput toxicity screening, and cross-species AOP network analysis.

[0007] To achieve the above objectives, the technical solution designed by the present invention is as follows: This invention provides a method for isolating neurons from primary embryonic Japanese quail, comprising the following steps: (1) Select fertilized Japanese quail eggs, incubate them at 37~39°C for 13~16 days, and take the embryonic brain tissue; (2) After washing the brain tissue with pre-cooled PBS and cutting it into small pieces, digest it with trypsin solution containing EDTA at 35-39°C, and then add complete culture medium to terminate the digestion. (3) The digested cell suspension was filtered through a two-stage gradient filter, passing through a large-pore cell sieve and a small-pore cell sieve, to remove tissue fragments and cell clumps. (4) Collect the filtered cell suspension by centrifugation, resuspend it in complete culture medium, and inoculate it into a culture vessel for adherent culture for 4-6 h. (5) After adhesion, discard the complete culture medium and replace it with serum-free culture medium for neurons. Change the medium every two days and culture for 3 to 15 days to obtain primary embryonic neurons of Japanese quail.

[0008] Furthermore, in step (1), the incubation temperature is 38°C and the incubation time is 15 days; In step (2), the EDTA-containing pancreatic enzyme solution is specifically a 0.25% pancreatic enzyme solution containing 0.02% EDTA, with a digestion temperature of 37°C and a digestion time of 20~25 min; In step (3), the large-pore cell sieve is a 70-mesh cell sieve; the small-pore cell sieve is a 40-mesh cell sieve.

[0009] Furthermore, in step (5), the formulation of the serum-free culture medium specifically for neurons is: Neurobasal TM -A neuron basal culture medium, 1-3% B27 serum-free additive, 0.5-1% penicillin and streptomycin and 0.3-0.8 mM glutamine; In step (5), the culture medium is replaced with a serum-free medium specifically for neurons and cultured for 7 days.

[0010] Furthermore, the formulation of the serum-free culture medium specifically for neurons is: Neurobasal TM -A neuron basal culture medium, 2% B27 serum-free additive, 1% penicillin and streptomycin and 0.5 mM glutamine.

[0011] The present invention also provides a method for separating primary embryonic neurons of Japanese quail obtained by the aforementioned separation method.

[0012] The present invention also provides a method for cryopreservation and thawing of the aforementioned primary embryonic neuronal cells of Japanese quail, comprising the following steps: S1. The primary embryonic neuronal cells of Japanese quail were digested with trypsin, collected by centrifugation, resuspended in cryopreservation solution, transferred to cryopreservation tubes and then cooled to -80°C for 12-14 hours, and then transferred to liquid nitrogen for long-term storage. S2. Remove the cryovial from liquid nitrogen, thaw rapidly in a water bath at 35-39°C, collect the cells by centrifugation, resuspend them in serum-free medium specifically for neurons, and then seed them into culture dishes. After confirming uniform distribution under a microscope, culture the cells to complete the cell revival.

[0013] Furthermore, the cryopreservation solution is composed of fetal bovine serum and dimethyl sulfoxide in a volume ratio of 9-10:1; The formulation of the serum-free culture medium specifically for neurons is: Neurobasal TM -A neuron basal culture medium, 1-3% B27 serum-free additive, 0.5-1% penicillin and streptomycin and 0.3-0.8 mM glutamine.

[0014] The present invention also provides an application of the aforementioned Japanese quail primary embryonic neuronal cells in high-throughput transcriptome toxicity screening of insecticides.

[0015] This invention also provides a method for screening high-throughput transcriptomic toxicity of insecticides using the aforementioned primary embryonic neurons of Japanese quail, comprising the following steps: ① Dissolve various insecticides separately in serum-free culture medium specifically for neurons to obtain various culture media containing insecticides; ② Expose the primary embryonic neurons of Japanese quail to the culture medium in step ① and culture them. ③ After cultivation, high-throughput transcriptome sequencing was performed, and the sequencing results of multiple insecticides were combined to construct an Illumina sequencing library; ④ Based on the transcript information corresponding to each pesticide treatment, the Well Barcode was split into segments. Differentially expressed genes were calculated using the edgeR software package. Combined with BMD modeling, dose-response key events were screened, an AOP network was constructed, and the toxicity of various pesticides was analyzed.

[0016] Furthermore, the formulation of the serum-free culture medium specifically for neurons is: Neurobasal TM -A neuron basal culture medium, 1-3% B27 serum-free additive, 0.5-1% penicillin and streptomycin and 0.3-0.8 mM glutamine; The insecticide is any one of imidacloprid, acetamiprid, nitenpyram, thiamethoxam, thiamethoxam, dinotefuran, chlorpyrifos, piperazine, flonicamid, and flupyrflufenoxam. The concentration of the insecticide in the serum-free culture medium specifically for neurons is 0.01~10 μM.

[0017] The beneficial effects of this invention are: Compared with the prior art, the present invention has the following beneficial effects: (1) A primary avian neuron model suitable for high-throughput screening was established for the first time. The Quail model of this invention achieved a cell yield of 4.0 × 10⁻⁶ cells per embryo. 6 ~4.5×10 6 The number of neurons cultured is 68% higher than that of existing chicken embryo neuron methods; the purity of neurons (the proportion of MAP2 positive cells) reaches 92-96% on day 7, which is 18-26% higher than that of existing chicken embryo neuron methods (75-80%); the glial cell contamination rate is less than 5%, which is 3-5 times lower than existing methods; the neural network maturation time is shortened to 7 days, which is 30-50% shorter than existing methods; and the in vitro stable maintenance time reaches more than 15 days, which is extended by 50-100%.

[0018] (2) A stable and reproducible cryopreservation and thawing system was established. The cryopreservation and thawing system established in this invention achieved a cell viability rate of 87-92% (trypan blue rejection rate) 24 hours after thawing, maintained MAP2-positive cell purity at 88-94%, and fully recovered nAChR functional response. The neural network formed after thawing showed no significant difference from that of unfrozen cells, supporting the establishment of a reusable cell bank. The cryopreservation and thawing survival rate is approximately 19-28% higher than the 70-75% achieved by existing chicken embryo neuron methods.

[0019] (3) It realizes the integration of multi-scale high-throughput data from molecular initiation events to individual phenotypes. Based on Quail, this invention can complete the parallel screening of hundreds of chemicals in a single experiment. At the molecular level, transcriptome response detection of more than 400 samples can be achieved in a single experiment through 384-well plate transcriptomics. At the cellular level, functional detection can be carried out simultaneously through 96-well plate calcium imaging, synaptic growth, and cytotoxicity. At the individual level, embryo images can be processed in batches through EcoMorph morphological analysis, which improves efficiency by 2 to 3 orders of magnitude compared with traditional bird individual experiments.

[0020] (4) Provide data support for chemical substitution assessment and regulatory decision-making. This method has been successfully applied to the developmental neurotoxicity assessment of 10 typical neonicotinoid insecticides, such as imidacloprid, thiamethoxam, and acetamiprid. It was found that there are significant differences in toxic effects among different compounds, suggesting that the traditional practice of judging safety uniformly by chemical category may underestimate the risks of specific substitutes, thus providing a data basis for precise regulation. Attached Figure Description

[0021] Figure 1 Figure 1 shows the results of a cross-species developmental neurotoxicity comparison analysis of neonicotinoid insecticides (NEOs) on Quail and SH-SY5Y cell models; In the figure, a is the cross-species network overlap diagram after ACE treatment, with the independent networks of SH-SY5Y and Quail shown on the left and the merged view on the right; b is the cross-species similarity matrix heatmap; c is the AOP pathway importance comparison diagram; d is the detailed cascade network diagram of AOP 471. Figure 2 A comparison of the top 10 AOP pathways in both Quail and SH-SY5Y cell models. Detailed Implementation

[0022] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can understand it.

[0023] Experimental Materials Description 1. Neurobasal culture medium formula containing 2% B27: Neurobasal TM -A neuron basal culture medium, 2% B27 serum-free additive, 1% penicillin and streptomycin, 0.5 mM glutamine.

[0024] Example 1 Conditional screening of methods for isolating neurons from primary embryonic Japanese quail I. Selection of Development Time 1. Preparation before cultivation All culture dishes were coated with 0.1 mg / mL poly-L-lysine for 30 min before inoculation, rinsed with PBS, and then air-dried for later use.

[0025] 2. Cell isolation The incubation period for Japanese quail eggs is 16-17 days. Fertilized Japanese quail eggs were selected and incubated at 38.2℃ for 13, 14, 15, and 16 days. Embryos were harvested, and brain tissue was extracted by decapitation. The brain tissue was washed with pre-cooled PBS, minced, and then digested with 0.25% trypsin solution (containing 0.02% EDTA) at 37℃ for 20 min. Digestion was terminated with complete culture medium. After filtering through 70-mesh and 40-mesh cell sieves to remove debris, the tissue was centrifuged at 1500 r / min for 5 min, the supernatant was discarded, and the embryos were resuspended in complete culture medium and inoculated into 6-well plates for 4-6 h.

[0026] 3. Cell Culture After cell adhesion, discard the complete culture medium and replace it with neuronal basal medium containing 2% B27, changing the medium every two days. When the cells are growing well and need to be passaged (7 days of culture), trypsin digest for 1-2 min, stop digestion with complete culture medium, centrifuge at 1500 r / min for 5 min, discard the supernatant, resuspend in complete culture medium, and seed into 6-well plates for 4-6 h to obtain primary embryonic neurons from Japanese quail.

[0027] The developmental status and cell separation effect of the embryonic brain on days 13, 14, 15, and 16 were compared. The results are shown in Table 1. On day 15, the structures of the five cerebral lobes (teloencephalium, diencephalon, midbrain, cerebellum, and medulla oblongata) were fully developed, and neurons were in the late stage of highly active differentiation and migration. Cells separated at this time had the highest activity (94.2% trypan blue rejection rate) and a moderate proportion of neural progenitor cells (28.5%), which ensured the differentiation potential of cells while avoiding excessive undifferentiated state. At the same time, the embryos on day 15 had not yet hatched, and the amnion was intact, which could effectively avoid microbial contamination. The operation window was sufficient, and day 15 was determined to be the optimal time window for sampling.

[0028] Table 1. Screening and Results of Developmental Time II. Screening of Digestion Conditions 1. Preparation before cultivation: Same as step one (screening of development time).

[0029] 2. Cell isolation Fertilized Japanese quail eggs were selected and incubated at 38.2℃ for 15 days. Embryos were harvested, and brain tissue was extracted by decapitation. The brain tissue was washed with pre-cooled PBS, minced, and digested under different conditions (digestion temperature fixed at 37℃) (digestion conditions are shown in Table 2). Digestion was terminated with complete culture medium. After filtering through 70-mesh and 40-mesh cell sieves to remove debris, the tissue was centrifuged at 1500 r / min for 5 min, the supernatant was discarded, and the tissue was resuspended in complete culture medium and inoculated into 6-well plates for 4–6 h.

[0030] 3. Cell culture: Same as step one (screening for development time).

[0031] The developmental status and cell separation effect of the embryonic brain were compared under different digestion conditions. The results are shown in Table 2. Precise digestion with 0.25% trypsin containing 0.02% EDTA at 37°C for 20 min can achieve the following: (1) Maximize cell yield: (4.2±0.5)×10 cells per embryo 6 The single-cell yield is 40% higher than conventional methods; (2) Optimal single-cell ratio: 89.3% dissociation, which significantly reduces the interference of cell clumps on subsequent purification; (3) Survival rate guarantee: The trypan blue rejection rate of 94.7% proves that the digestion conditions were mild and did not cause significant membrane damage; (4) Morphological integrity: The intact neuronal processes are conducive to subsequent adhesion and network formation; (5) The synergistic mechanism of EDTA: EDTA works by chelating Ca 2+ Mg 2+ It disrupts calcium-dependent junctions between cells, assisting pancreatic enzymes in hydrolyzing extracellular matrix proteins more efficiently, while avoiding shear damage caused by excessive mechanical blowing.

[0032] Table 2. Screening and Results of Digestion Conditions III. Screening of Cell Sieve Filtration Mesh 1. Preparation before cultivation: Same as step one (screening of development time).

[0033] 2. Cell isolation Fertilized Japanese quail eggs were selected and incubated at 38.2℃ for 15 days. Embryos were harvested, and brain tissue was extracted by decapitation. The brain tissue was washed with pre-cooled PBS, minced, and digested with 0.25% trypsin solution (containing 0.02% EDTA) at 37℃ for 20 min. Digestion was terminated with complete culture medium. After filtering to remove debris using cell sieves of different mesh sizes (cell sieve mesh sizes are shown in Table 3), the cells were centrifuged at 1500 r / min for 5 min, the supernatant was discarded, and the cells were resuspended in complete culture medium and inoculated into 6-well plates for 4–6 h.

[0034] 3. Cell culture: Same as step one (screening for development time).

[0035] The developmental status of the embryonic brain and the cell separation effect were compared under different cell sieve mesh sizes. The results are shown in Table 3. A 70-mesh sieve (pore size 212 μm) primarily removed the meninges, larger blood vessels, and incompletely dissociated tissue fragments, preventing them from clogging the downstream filter membrane and releasing intracellular enzymes that could damage neurons. A 40-mesh sieve (pore size 38 μm) further retained small cell clusters, blood cells, and glial cell aggregates. Neuronal cell bodies have a diameter of 10–20 μm, and the length of their processes is shortened in the single-cell suspension due to shear force, allowing them to pass through the filter smoothly; however, glial cell clusters and erythrocyte aggregates (>40 μm) were effectively retained. Purity verification: MAP2 in the final single-cell suspension... + The proportion of neurons reached 91.4±2.5%, which was significantly higher than that of the unfiltered group (34.2%) and the single-sieve filtering group (70 mesh single filter: 56.7%), demonstrating the synergistic effect of the two-level gradient strategy.

[0036] Table 3. Screening and Results of Cell Screening Mesh Size IV. Screening of Special Culture Medium Formulations 1. Preparation before cultivation: Same as step one (screening of development time).

[0037] 2. Cell isolation Fertilized Japanese quail eggs were selected and incubated at 38.2℃ for 15 days. Embryos were harvested, and brain tissue was extracted by decapitation. The brain tissue was washed with pre-cooled PBS, minced, and digested with 0.25% trypsin solution (containing 0.02% EDTA) at 37℃ for 20 min. Digestion was terminated with complete culture medium. The tissue was filtered through 70-mesh and 40-mesh cell sieves to remove debris, centrifuged at 1500 r / min for 5 min, the supernatant was discarded, and the tissue was resuspended in complete culture medium and inoculated into 6-well plates for 4–6 h.

[0038] 3. Cell Culture After cell adhesion, discard the complete culture medium and culture in different media (as shown in Table 4), changing the medium every two days. When the cells are growing well and need to be passaged (after 7 days of culture), trypsin digest for 1-2 minutes, stop digestion with complete culture medium, centrifuge at 1500 r / min for 5 minutes, discard the supernatant, resuspend in complete culture medium, and seed into 6-well plates for 4-6 hours to obtain primary embryonic neurons from Japanese quail.

[0039] The developmental status and cell isolation effects of embryonic brains were compared under different culture media conditions. The results are shown in Table 4. Neurobasal™-A: a basal culture medium specifically designed for neurons, lacks growth factors (such as EGF and FGF) required for glial cell proliferation, thus inhibiting the proliferation of non-neuronal cells from the source. B27 additive (2%): provides essential factors for neuronal survival and synaptic growth. L-glutamine (0.5 mM): as a major energy metabolism substrate for neurons, it replaces glucose in the tricarboxylic acid cycle, supporting high-energy-demand synaptic transmission activities. Although the purity of the glutamine-free group was acceptable, the network density and maintenance time were significantly reduced, demonstrating its necessity for long-term culture.

[0040] After adhesion (4-6 hours), the complete culture medium should be replaced with Neurobasal™-A neuronal basal medium + 2% B27 serum-free additive + 1% penicillin and streptomycin + 0.5 mM glutamine, which is the optimal culture medium formulation.

[0041] Table 4 Screening and Results of Special Culture Medium Formulations V. Screening of Cell Maturity Time 1. Preparation before cultivation: Same as step one (screening of development time).

[0042] 2. Cell isolation: Same as step four (screening of special culture medium formula).

[0043] 3. Cell Culture After adhesion, the complete culture medium was discarded and replaced with neuronal basal medium containing 2% B27, with the medium changed every two days. After culturing for 1, 3, 5, 7, 10, and 15 days, trypsin digestion was performed for 1-2 minutes, digestion was stopped with complete culture medium, centrifuged at 1500 r / min for 5 minutes, the supernatant was discarded, and the cells were resuspended in complete culture medium and seeded into 6-well plates for 4-6 hours to obtain primary embryonic neurons from Japanese quail.

[0044] The developmental status and cell separation effect of embryonic brains under different culture times were compared. The results are shown in Table 5. Day 3: The cell body is round with short neuronal processes extending out, indicating an early differentiation stage. Day 5: The processes extend and branch rapidly, and preliminary connections are established between cells, but they are not fully mature. Day 7: The optimal maturation period. Axons extend fully in all directions and intertwine with each other, forming a highly complex, dense, and structurally mature neural network. Synaptophysin is distributed in a dotted pattern at the process contact sites, indicating the formation of functional synapses. Days 10-15: The network is generally maintained, but no further significant expansion is observed. Slight aggregation and senescence and shedding occur during long-term culture.

[0045] Day 7 is the optimal functional window for in vitro culture of neurons from primary Japanese quail embryos. At this time, the cell purity reaches 94.5%, the synaptic network is most dense, and it can be used for various subsequent neurobiological studies.

[0046] Table 5. Screening and Results of Cell Maturity Time Example 2 Isolation, cryopreservation, and revival of neuronal cells from primary embryonic Japanese quail 1. Preparation before cultivation All culture dishes were coated with 0.1 mg / mL poly-L-lysine for 30 min before inoculation, rinsed with PBS, and then air-dried for later use.

[0047] 2. Cell isolation Fertilized Japanese quail eggs were selected and incubated at 38.2℃ for 15 days. Embryos were harvested, and brain tissue was extracted by decapitation. The brain tissue was washed with pre-cooled PBS, minced, and digested with 0.25% trypsin solution (containing 0.02% EDTA) at 37℃ for 20 min. Digestion was terminated with complete culture medium. The tissue was filtered through 70-mesh and 40-mesh cell sieves to remove debris, centrifuged at 1500 r / min for 5 min, the supernatant was discarded, and the tissue was resuspended in complete culture medium and inoculated into 6-well plates for 4–6 h.

[0048] 3. Cell Culture After adhesion, the complete culture medium was discarded and replaced with neuronal basal culture medium containing 2% B27, with the medium changed every two days. After 7 days of culture, trypsin digestion was performed for 1-2 minutes, digestion was stopped with complete culture medium, centrifuged at 1500 r / min for 5 minutes, the supernatant was discarded, and the cells were resuspended in complete culture medium and seeded into 6-well plates for 4-6 hours to obtain primary embryonic neurons (Quail) from Japanese quail.

[0049] 4. Cell cryopreservation Discard the complete culture medium, wash the primary Japanese quail embryonic neuronal cells with PBS, add 2 mL of trypsin for 1 min, and terminate the digestion with 2 mL of complete culture medium. Centrifuge at 1000 r / min for 3 min, discard the supernatant, and resuspend the primary Japanese quail embryonic neuronal cells in 1 mL of pre-freezing buffer (FBS:DMSO = 9:1 mixture) and transfer to cryovials. Place in a programmed cooling box at -80℃ overnight (12~14 h), with a cooling rate of -1℃ / min. The next day, transfer to liquid nitrogen for long-term storage to obtain cryovials of primary Japanese quail embryonic neuronal cells.

[0050] 5. Cell resuscitation Prepare 5 mL of complete culture medium in advance. Remove the cryopreserved tube of primary Japanese quail embryonic neurons from liquid nitrogen and thaw it rapidly in a 37°C water bath (remove it when it is almost completely thawed and gently rub it by hand). Transfer the primary Japanese quail embryonic neurons into a centrifuge tube and rinse the cryopreserved tube with complete culture medium. Centrifuge at 1000 r / min for 3 min, discard the supernatant, and resuspend the cells in neuronal basal culture medium containing 2% B27. Inoculate the cells into a culture dish, mix well, and check under a microscope to confirm uniform distribution. Then place the dish in an incubator (to prevent localized high density from causing nutrient competition) to complete cell thawing.

[0051] Example 3 Stability verification of cell isolation, cryopreservation, and thawing methods The survival rates of the primary Japanese quail embryonic neurons isolated in step 3 of Example 2, the primary Japanese quail embryonic neurons 24 h after resuscitation in step 5, and the primary Japanese quail embryonic neurons 72 h after resuscitation were detected.

[0052] The results are shown in Table 6. The cryopreservation solution was formulated with FBS:DMSO = 9:1. The high proportion of FBS (90%) provided protein protection and reduced mechanical damage to the membrane caused by ice crystal formation; DMSO (10%) was a permeable cryoprotectant that lowered the intracellular freezing point and prevented intracellular ice crystal formation. The cell viability reached 89.5% 24 hours after cryopreservation and 88.7% after 72 hours; the MAP2 value was [missing data - likely related to cell viability and adhesion]. + The purity was 91.2% and 93.6% after 72 hours; the nAChR functional response (calcium signal) was fully restored, and the neural network formed 72 hours after thawing was not significantly different from that of the unfrozen cells.

[0053] Table 6. Survival rate of neurons in primary embryonic Japanese quail Example 4 High-throughput transcriptome toxicity screening based on Quail primary embryonic neurons. In this embodiment, Quail, a primary embryonic neuronal cell line from Japanese quail obtained in Example 2, was used to establish a high-throughput transcriptome toxicity screening process to evaluate the developmental neurotoxicity of 10 typical neonicotinoid insecticides.

[0054] I. Cell Seeding and Drug Exposure 1. Divide the Quail into 1×10 holes per hole. 5 One cell was seeded in a clear 96-well plate and cultured for 24 hours to allow it to fully adhere to the plate. SH-SY5Y cells were used as control cells.

[0055] 2. Preparation of neuronal culture media containing different concentration gradients of neonicotinoid insecticides: The basal neuronal culture medium containing 2% B27 was used as the base medium. The concentration gradients of neonicotinoid insecticides were 10 nM, 50 nM, 100 nM, 500 nM, 1 μM, 5 μM, and 10 μM. DMSO was used as a control group instead of neonicotinoid insecticides, with a final concentration of 0.1%. The neonicotinoid insecticides were selected from any one of the following 10 representative compounds: imidacloprid, acetamiprid, nitenpyram, thiamethoxam, thiamethoxam, dinotefuran, chlorpyrifos, piperazine, flonicamid, and flupyrflufenoxam.

[0056] 3. After cell adhesion in step 1, expose the cells to the culture medium prepared in step 2 for 7 days. Set up 5 biological replicates for each group.

[0057] II. High-throughput transcriptome sequencing Following exposure, high-throughput transcriptome sequencing was performed using the AccuraCode® HTP One Step RNAseq Kit (Syngene Biotech, Ltd.). 1. Discard the culture medium, wash Quail twice with PBS, and add 30 μL of AccuraCode Lysis Mix to each well for lysis at room temperature for 6 min.

[0058] 2. Transfer 2 μL of lysis product to a 384-well PCR plate. Each well should be pre-filled with a One Step reaction system (Singergen Biotech Co., Ltd.) containing 18 μL One Step Buffer, 0.3 μL RNase Inhibitor, 0.48 μL Amplification Enzyme, and 0.6 μL Reverse Transcriptase.

[0059] 3. The PCR reaction program is as follows: 42℃ for 1 h, 95℃ for 3 min, 4 cycles (98℃ for 20 s, 65℃ for 45 s, 72℃ for 3 min) + 10 cycles (98℃ for 20 s, 67℃ for 20 s, 72℃ for 3 min), 72℃ for 5 min.

[0060] 4. After purifying the cDNA mixture, construct an Illumina sequencing library. Sequencing is performed, and the transcripts are separated according to the Well Barcode to the corresponding neonicotinoid insecticide treatments. Differentially expressed genes are calculated using the edgeR software package. Combined with BMD modeling, dose-response key events are screened, and an AOP network is constructed. The specific process is as follows: (1) Data processing: ① Differential analysis: edgeR was used to calculate differentially expressed genes (P<<0.05); ②BMD modeling: Benchmark Dose software (such as PROAST or BMDS) was used to fit dose-response models to differentially expressed genes. The screening criteria included: (a) model fit goodness of fit p>0.1; (b) BMD / BMDL<5; (c) expression level showed a monotonically increasing or decreasing trend with dose. Genes that passed quality control were retained, and their BMD values ​​were calculated.

[0061] ③ Weighting: For genes that pass BMD quality control, ECDF global normalization is used, W = 1 - Percentile (BMD). Enrichment analysis is performed on AOP-Wiki pathways to screen for pathways with at least 5% differentially expressed genes that have significant dose-response relationships (passing BMD quality control). The BMD value of the pathway is calculated (median), and these pathways are included in the subsequent AOP network construction.

[0062] (2) AOP network construction: ① Network mapping: Map weights to the AOP-Wiki network to filter invalid paths; ② Signal propagation: Weight of missing node = mean of predecessor and successor × 0.8, iterated 50 times; ③ Path evaluation: The PageRank algorithm calculates the node centrality and accumulates it to obtain the path activation index; (3) Cross-species verification: ① Matrix alignment: Construct dimension-consistent adjacency matrices between Quail and SH-SY5Y; ② Similarity calculation: Cosine similarity is used to evaluate the consistency between the two model networks; (4) Visualization: ①Sugiyama layout: AOP cascade diagram; ② Heatmap: Cross-species / cross-drug similarity matrix; The results are as follows Figure 1 As shown in figure a, dark blue nodes represent events shared by both species, and solid lines represent shared pathways. The results show that there are obvious shared event modules between the Quail and SH-SY5Y models.

[0063] The results are as follows Figure 1 As shown in b, the blue area in the lower left corner shows the similarity between different drugs within the same model, and the white circle in the upper right corner represents the cross-species similarity of the same drug (the larger the circle, the more similar). The diagonal line shows that IMI (0.65) and DIN (0.53) have high cross-species consistency, while SUL and PAI have low consistency.

[0064] The results are as follows Figure 1As shown in Figure c, each row represents an AOP pathway, with the left and right sides displaying the frequency of activation by different NEOs (in color) in the SH-SY5Y and Quail models, respectively. AOP 471 was the most frequently activated by drugs in both models, making it the most conserved core toxicity pathway.

[0065] The results are as follows Figure 1 As shown in d, the red nodes on the left represent the SH-SY5Y model, and the blue nodes on the right represent the Quail model. The node size indicates the weight. Key events include ACHE inhibition (12), ACh accumulation (10), neuroinflammation (188), and neurodegenerative disease (352), demonstrating a complete toxicity cascade from cholinergic disorder to neurological damage.

[0066] The results are as follows Figure 2 As shown, AOP 471 is the most conserved core pathway across species. In the SH-SY5Y model, AOP 471 is significantly activated by various NEOs such as ACE, IMI, and SUL (large dark dots); in the Quail model, AOP 471 is uniformly activated by all 10 NEOs, making it the most frequently occurring pathway. Significant species differences exist: in the SH-SY5Y model, pathways such as AOP 540, Aop.48, and Aop.464 are selectively activated by specific compounds, with significant differences in dot size and color intensity; in the Quail model, the activation patterns of each AOP are more dispersed and uniform, suggesting that birds have a broader molecular response to NEOs. Compound specificity is also evident: IMI and SUL activate multiple highly important AOPs (large dark dots) in the SH-SY5Y model; ACE, CLO, and DIN show moderate activation in both models; TMX and PAI show relatively low overall activation levels. Conclusion: AOP 471 is the most conserved cross-species core mechanism of NEO neurotoxicity, but the AOP activation profiles differ significantly among different compounds and species.

[0067] In summary: (1) Exposure to 10 neonicotinoid insecticides (NEOs) caused significant transcriptome perturbations in Quail, involving the inhibition of multiple biological processes such as synaptic transmission, neurogenesis, and ion channel activation. (2) BMD modeling results show that molecular changes related to neurodevelopment occur much earlier than traditional toxic endpoints (such as cell death), and early risk identification can be achieved at sublethal doses; (3) Frequency analysis identified AOP 471 as the most commonly activated conserved pathway, which was triggered by 9 out of 10 NEOs in the Quail model and was also clearly enriched in the human SH-SY5Y cell model. (4) The toxicological cascade of events of AOP 471 includes: acetylcholinesterase inhibition (Event 12) → acetylcholine accumulation (Event 10) → synaptic dysfunction (Event 1944) → oxidative stress (Event 1392) → neuroinflammation (Event 188) → neurodegenerative disease (Event 352); (5) Cross-species similarity analysis showed that Quail and SH-SY5Y cells had significant conservation of AOP network activation patterns under NEOs exposure, with the highest interspecies similarity between dinotefuran (DIN) and thiamethoxam (CLO). (6) This AOP network provides a quantitative validation framework for extrapolating from mammalian data to birds, filling the gap in avian neurodevelopmental toxicity data in the global AOP database.

[0068] (7) Based on comprehensive multi-indicator analysis, the following compounds require special attention: High priority: ACE (acetamiprid). Despite significant toxicity across multiple indicators, it has been widely used under the misconception of being a "low-risk" alternative, posing a risk of cognitive bias.

[0069] High priority: SUL (fluoxetine). Most prominent abnormal head expansion, with severe pathological damage to brain tissue.

[0070] High priority: CLO (thiamethoxam). Highly conserved across species; low doses cause abnormal brain-to-body ratio.

[0071] Medium priority: NIT (acetamiprid). It has strong penetrability in brain tissue and significantly increases acetylcholine levels.

[0072] Medium priority: DIN (fipronil). Highest cross-species similarity, detects brain residues at multiple doses.

[0073] Attention should be paid to IMI (imidacloprid) and TMX (thiamethoxam). These are the most widely used, and although brain residues have not been detected, transcriptomic perturbations are significant, and the risks of long-term low-dose use cannot be ignored.

[0074] All other parts not described in detail are existing technologies. Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for isolating neurons from primary embryonic Japanese quail, characterized in that: Includes the following steps: (1) Select fertilized Japanese quail eggs, incubate them at 37~39°C for 13~16 days, and take the embryonic brain tissue; (2) After washing the brain tissue with pre-cooled PBS and cutting it into small pieces, digest it with trypsin solution containing EDTA at 35-39°C, and then add complete culture medium to terminate the digestion. (3) The digested cell suspension was filtered through a two-stage gradient filter, passing through a large-pore cell sieve and a small-pore cell sieve, to remove tissue fragments and cell clumps. (4) Collect the filtered cell suspension by centrifugation, resuspend it in complete culture medium, and inoculate it into a culture vessel for adherent culture for 4-6 h. (5) After adhesion, discard the complete culture medium and replace it with serum-free culture medium for neurons. Change the medium every two days and culture for 3 to 15 days to obtain primary embryonic neurons of Japanese quail.

2. The separation method according to claim 1, characterized in that: In step (1), the incubation temperature is 38°C and the incubation time is 15 days; In step (2), the EDTA-containing pancreatic enzyme solution is specifically a 0.25% pancreatic enzyme solution containing 0.02% EDTA, with a digestion temperature of 37°C and a digestion time of 20~25 min; In step (3), the large-pore cell sieve is a 70-mesh cell sieve; the small-pore cell sieve is a 40-mesh cell sieve.

3. The separation method according to claim 1, characterized in that: In step (5), the formulation of the serum-free culture medium for neurons is: Neurobasal TM -A neuron basal culture medium, 1-3% B27 serum-free additive, 0.5-1% penicillin and streptomycin and 0.3-0.8 mM glutamine; In step (5), the culture medium is replaced with a serum-free medium specifically for neurons and cultured for 7 days.

4. The separation method according to claim 3, characterized in that: The formulation of the serum-free culture medium specifically for neurons is: Neurobasal TM -A neuron basal culture medium, 2% B27 serum-free additive, 1% penicillin and streptomycin and 0.5 mM glutamine.

5. A primary embryonic neuronal cell of Japanese quail obtained by the separation method according to any one of claims 1 to 4.

6. A method for cryopreservation and resuscitation of neuronal cells from primary Japanese quail embryos as described in claim 5, characterized in that: Includes the following steps: S1. The primary embryonic neuronal cells of Japanese quail were digested with trypsin, collected by centrifugation, resuspended in cryopreservation solution, transferred to cryopreservation tubes and then cooled to -80°C for 12-14 hours, and then transferred to liquid nitrogen for long-term storage. S2. Remove the cryovial from liquid nitrogen, thaw rapidly in a water bath at 35-39°C, collect the cells by centrifugation, resuspend them in serum-free medium specifically for neurons, and then seed them into culture dishes. After confirming uniform distribution under a microscope, culture the cells to complete the cell revival.

7. The cryopreservation and thawing method according to claim 6, characterized in that: The cryopreservation solution is composed of fetal bovine serum and dimethyl sulfoxide in a volume ratio of 9-10:1; The formulation of the serum-free culture medium specifically for neurons is: Neurobasal TM -A neuron basal culture medium, 1-3% B27 serum-free additive, 0.5-1% penicillin and streptomycin and 0.3-0.8 mM glutamine.

8. The application of the Japanese quail primary embryonic neuronal cells as described in claim 5 in high-throughput transcriptome toxicity screening of insecticides.

9. A method for screening high-throughput transcriptomic toxicity of insecticides using primary embryonic neurons of Japanese quail as described in claim 5, characterized in that: Includes the following steps: ① Dissolve various insecticides separately in serum-free culture medium specifically for neurons to obtain various culture media containing insecticides; ② Expose the primary embryonic neurons of Japanese quail to the culture medium in step ① and culture them. ③ After cultivation, high-throughput transcriptome sequencing was performed, and the sequencing results of multiple insecticides were combined to construct an Illumina sequencing library; ④ Based on the transcript information corresponding to each pesticide treatment, the Well Barcode was split into segments. Differentially expressed genes were calculated using the edgeR software package. Combined with BMD modeling, dose-response key events were screened, an AOP network was constructed, and the toxicity of various pesticides was analyzed.

10. The method according to claim 9, characterized in that: The formulation of the serum-free culture medium specifically for neurons is: Neurobasal TM -A neuron basal culture medium, 1-3% B27 serum-free additive, 0.5-1% penicillin and streptomycin and 0.3-0.8 mM glutamine; The insecticide is any one of imidacloprid, acetamiprid, nitenpyram, thiamethoxam, thiamethoxam, dinotefuran, chlorpyrifos, piperazine, flonicamid, and flupyrflufenoxam. The concentration of the insecticide in the serum-free culture medium specifically for neurons is 0.01~10 μM.

Images