A method for isolating and culturing duck microglial cells in vitro

Abstract

The application discloses a method for separating and culturing duck microglial cells in vitro, which comprises the following steps: aseptically collecting cortex tissue of duckling brain, removing the membrane, cutting, enzyme digestion, filtering, centrifugal washing, inoculating into a cell culture dish, and carrying out primary cell culture in a mixed culture system of astrocyte-microglial cells, separating microglial cells by oscillating the cell culture dish after a period of time, continuously inoculating and culturing, and identifying by Iba1 immunofluorescence. The application first establishes a high-purity model of duck microglial cells in vitro, and the procedure is simple and low in cost, and the high-purity microglial cells can be effectively separated, so that the duck microglial cells cultured in vitro can avoid the influence of the complex blood-brain barrier environment in vivo and individual immune difference, and provide a key cell model for the related researches on the immune regulation of the nervous system of ducks, the pathogenic mechanism of avian neurotropic virus and the disease-resistant breeding of waterfowls.

Description

Technical Field

[0001] This invention belongs to the field of cell isolation and culture technology, specifically relating to a method for isolating and culturing duck microglia in vitro. Background Technology

[0002] With the rapid development of waterfowl farming towards intensification and large-scale operations, disease prevention and control, as well as disease-resistant breeding, have become key bottlenecks restricting the industry's profitability, particularly for ducks, an important meat and egg-producing waterfowl. Frequent outbreaks of neurotropic infectious diseases such as duck plague, duck viral hepatitis, and duck Tembusu virus disease not only cause severe economic losses but also pose a potential threat to public health. The nervous system, as a major target organ for many avian pathogens, directly determines the occurrence, development, and outcome of diseases through the homeostasis of its immune microenvironment. Therefore, in-depth analysis of the immune regulatory mechanisms of the duck central nervous system is of significant scientific and economic value for elucidating the pathogenic mechanisms of neurotropic viruses and guiding disease-resistant breeding practices.

[0003] Microglia are innate immune cells residing in the central nervous system, accounting for approximately 5%–12% of the total brain cells. They perform core functions such as immune surveillance, synaptic pruning, and tissue repair. Under physiological conditions, microglia exhibit a highly branched, resting morphology, continuously monitoring changes in the microenvironment. When encountering pathological stimuli such as pathogen invasion, trauma, or ischemia, they rapidly activate into an amoeboid morphology, releasing inflammatory factors and mediating antigen presentation. Recent studies have shown that abnormal activation of microglia is closely related to neurodegenerative diseases, viral encephalitis, and autoimmune neurological damage. However, current understanding of microglia function is mainly based on mammalian models such as humans and mice; the biological characteristics, activation regulatory networks, and interaction mechanisms with neurotropic viruses of avian microglia remain largely unexplored.

[0004] As a typical precocious avian species, ducks exhibit significant differences in nervous system development and immune response patterns compared to mammals. The morphological characteristics, surface marker expression profiles, and immune response thresholds of duck brain microglia are species-specific. Unlike mammalian brain tissue, duck brain tissue has a dense matrix and is rich in vascular connective tissue, resulting in an extremely narrow digestion window. Simply applying isolation and purification protocols from mammalian sources is insufficient to obtain high-purity, high-viability duck primary microglia. For example, the traditional mild trypsin digestion method used for rodents is inefficient due to the high density and rich vascularity of duck brain tissue; furthermore, the lack of validated commercially available monoclonal antibodies on the surface of duck microglia hinders conventional magnetic bead sorting or flow cytometry sorting strategies.

[0005] Currently, there are sporadic reports on the preliminary culture of microglia derived from chicken embryos, but a systematic and stable in vitro isolation and culture system for microglia, an important waterfowl breed, has not yet been established. Unlike the thin and transparent skulls of rodents, the skull structure of ducks is hard and thick, with the meninges (especially the pia mater) tightly attached to the cerebral cortex and exhibiting fibrous chimeric structures at vascular passages. Directly applying the dissection method used in mice would inevitably lead to tearing of the brain parenchyma and rupture of microvessels, resulting in massive infiltration of erythrocytes and contamination by neuronal debris. In addition, microglia are highly sensitive to changes in the in vitro environment and are prone to spontaneously shifting towards a pro-inflammatory phenotype during culture. Therefore, developing a dedicated culture medium and standardized isolation and purification process for duck microglia has become an urgent problem to be solved in the fields of waterfowl neuroimmunology and disease resistance breeding research. It has important theoretical value and application prospects for advancing the understanding of the pathogenic mechanism of duck neurotropic viruses, evaluating vaccine immunogenicity, and screening for resistance genetic markers. Summary of the Invention

[0006] The purpose of this invention is to address the lack of a systematic duck microglia isolation and culture system in the existing technology. Furthermore, the small size of the duck brain, the hardness of the skull, and the tight attachment of the pia mater make anatomical sampling and tissue processing difficult, resulting in the inapplicability of existing culture methods for this species and the inability to effectively obtain high-purity duck microglia to support microglia research. This invention provides a method for isolating and culturing duck microglia in vitro.

[0007] To achieve the above objectives, the present invention provides a method for isolating and culturing duck microglia in vitro, comprising the following steps: S1. Pre-coat cell culture dishes with poly-D-lysine solution and place them in an incubator overnight for coating. S2. Aseptic procedure is used to collect the cerebral cortex of ducklings, remove the meninges and microvascular structures from the brain tissue and clean it with a cleaning solution. S3. After cleaning the tissue in S2, cut it into small pieces, transfer it into a centrifuge tube, add papain and digest it under suitable conditions. S4. Add an equal volume of stop culture medium to stop digestion, filter and centrifuge, discard the supernatant, resuspend in microglial cell culture medium, seed in pre-coated cell culture dishes, and place in an incubator for primary cell culture. S5. After the mixed glial cells have completely adhered to the culture medium, replace it with fresh microglial cell culture medium and place it in an incubator to continue culturing. S6. When the astrocyte layer is completely fused and the upper surface of the astrocyte layer is covered with a layer of highly refractive round cells, i.e., microglia, separate the microglia by shaking the cell culture dish. After centrifugation, discard the supernatant and resuspend the cells. After cell counting, seed them into pre-coated multi-well culture plates at an appropriate cell density and place them in an incubator for culture.

[0008] This invention involves aseptically collecting cerebral cortex tissue from ducklings. After demembranes, mincing, enzymatic digestion, filtration, centrifugation, and washing, the tissue is inoculated into cell culture dishes using DMEM complete medium and cultured as primary cells in a mixed astrocyte-microglia culture system. After a period of time, the cell culture dishes are shaken to isolate microglia, and the cells are inoculated and cultured again. Iba1 immunofluorescence is used for identification. This invention establishes a high-purity in vitro model of duck microglia for the first time. The procedure is simple and inexpensive, and it can effectively isolate microglia with high purity. In vitro culture of duck microglia can avoid the complex blood-brain barrier environment and individual immune differences in vivo, providing a key cell model for research on immune regulation of the duck nervous system and the pathogenic mechanism of avian neurotropic viruses in waterfowl disease resistance breeding.

[0009] The further optimized technical solution of this invention is as follows: In S1, the cell culture dish is a 60 mm cell culture dish, and the working concentration of the poly-D-lysine solution is 10 μg / mL.

[0010] In S2, the ducklings are 1 to 4 days old, and the washing solution includes 95% PBS and 5% penicillin-streptomycin solution.

[0011] In S3, the digestion conditions are: digestion in a 37°C water bath shaker for 30 minutes.

[0012] In S4 and S5, the termination culture medium and the microglia culture medium are both DMEM high glucose medium with 10% FBS and 5% penicillin-streptomycin solution added, and the final concentration of L-glutamine in the termination culture medium and the final concentration of sodium pyruvate in the microglia culture medium is 4mM and 1mM.

[0013] This invention reveals that duck microglia consume glutamine at a significantly higher rate than mouse cells, and rapidly enter a stress-induced apoptosis program without sodium pyruvate as an additional carbon source. Therefore, the culture medium of this invention, which specifies the synergistic use of high concentrations of glutamine and sodium pyruvate, constitutes a key nutrient microenvironment for maintaining the long-term inactivation and branching resting morphology of duck microglia in vitro. Conventional DMEM culture medium lacking this specific combination will lead to irreversible spontaneous activation or detachment and death of duck microglia within 24 hours.

[0014] In S4 and S6, the centrifugation conditions were both 1000 rpm for 10 min.

[0015] In step 5, the time for complete adhesion is 12 hours after inoculation; in step S6, the time for complete fusion of the astrocyte layer is day 4 of cell culture; the cell density after inoculation is 1.0 × 10⁻⁶. 6The culture plate is a 24-well plate with cells / mL.

[0016] In S6, the specific steps of oscillation are as follows: manually draw a cross-shaped trajectory and rotate the culture dish, with an oscillation frequency of 1–2 times / second, rotating 3–5 times in each direction, so that the culture medium generates multi-directional fluid shear force in the culture dish; During the oscillation process, intermittent microscopic observation was performed until most of the round, highly refractive microglia above the astrocyte layer detached. Collect the supernatant and centrifuge to obtain microglia.

[0017] In the above method, the culture dish is moved by hand at a frequency of 1 to 2 times per second, while simultaneously rotating the culture dish clockwise or counterclockwise. This is repeated 3 to 5 times in each direction. Through this vigorous and multi-directional shaking, the culture medium generates a strong multi-directional scouring force, thereby washing specific cells from the bottom of the culture dish.

[0018] The present invention also provides isolated duck primary microglia, which are obtained by the above-described method and have the following characteristics: (a) Expression of the microglia-specific marker Iba1; (b) Maintaining typical bipolar or unipolar resting morphology under unstimulated in vitro culture conditions; (c) Under in vitro culture conditions stimulated by LPS, it exhibits an amoeba-like activated state.

[0019] The present invention further provides the application of the above-described method for isolating and culturing duck microglia in vitro in establishing an in vitro model of duck microglia.

[0020] The beneficial effects of this invention are as follows: (1) The first in vitro culture model of duck microglia was established, filling the gap in the existing technology of lacking a systematic duck microglia isolation and culture system, and solving the problem of duck neuroimmunology research in the field of duck nervous system. (2) Papain digestion was used, with 30 minutes being the optimal digestion time to maximize the cell yield. Microglia and astrocytes were separated by mechanical shaking to obtain high-purity microglia and improve the purity of microglia separation. (3) The operation steps are clear, no complicated equipment is required, the components of the culture medium are easy to obtain, the separation and culture cost is reduced, and it is easy to promote and replicate. (4) In vitro models can avoid multiple interferences from the complex blood-brain barrier and neuro-immune-endocrine network in vivo, and are not affected by individual age, immune status and genetic background differences. They can accurately study the effects of specific conditions (such as neurotropic virus infection, inflammatory factor stimulation, candidate antiviral drugs) on the activation phenotype, phagocytic function and secretion of inflammatory factors of duck microglia, and provide key experimental materials for the immune regulation mechanism of duck nervous system, the pathogenic mechanism of avian neurotropic viruses and related research on disease-resistant breeding of waterfowl. Attached Figure Description

[0021] The technical solutions of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0022] Figure 1 The images show the primary duck microglia mixed culture system, resting state and activated state under an inverted microscope (scale bar 200µm) in Example 1 of this invention.

[0023] Figure 2 The image shows the immunofluorescence identification results of duck microglia in Example 2 of this invention (scale bar 100µm). Detailed Implementation

[0024] The technical solution of the present invention will be further described in detail below with reference to the embodiments: This embodiment is implemented under the premise of the technical solution of the present invention, and provides detailed implementation methods and specific operation processes, but the protection scope of the present invention is not limited to the following embodiments.

[0025] The instruments, equipment, reagents, and materials used in the examples were all obtained commercially. Example 1

[0026] A method for isolating and culturing duck microglia in vitro, specifically comprising the following steps: S1. Coat a 60 mm cell culture dish with a working concentration of 10 μg / mL poly-D-lysine solution (Sigma, P3513) and incubate overnight.

[0027] S2. Aseptically collect the cerebral cortex of 1-4 day old ducklings, remove the meninges and microvascular structures from the brain tissue, and wash with a cleaning solution containing 95% PBS (Jiangsu Kaiji Biotechnology Co., Ltd., KGL2206-500) and 5% penicillin-streptomycin double antibiotic solution (Suzhou Xinsaimei Biotechnology Co., Ltd., C100C5).

[0028] S3. Cut the tissue into small pieces, transfer it into a 15mL centrifuge tube using a pipette, add papain (sigma, 76216) diluted in DMEM medium, mix well, and then place it in a 37℃ water bath shaker for 30min for digestion.

[0029] S4. Add an equal volume of stop culture medium to terminate digestion, filter through a 70 μm cell filter, centrifuge, discard the supernatant, resuspend in microglial cell culture medium, and seed into pre-coated 60 mm cell culture dishes and place in an incubator for primary cell culture.

[0030] Both the termination medium and the microglia culture medium were prepared by adding 10% FBS and 5% penicillin-streptomycin solution to DMEM high glucose medium. The final concentrations of L-glutamine and sodium pyruvate in the termination medium and the microglia culture medium were 4 mM and 1 mM, respectively.

[0031] After 5 and 12 hours, once the mixed glial cells have completely adhered to the culture medium, replace it with fresh microglia culture medium and continue culturing in a 37°C, 5% CO2 incubator.

[0032] S6. On day 4 of cell culture, the astrocyte layer was completely confluent, and the upper surface of the astrocyte layer was covered with a layer of highly refractive round cells, i.e., microglia. Microglia were separated by shaking a 60 mm cell culture dish, the supernatant was collected, centrifuged, discarded, and resuspended. Cells were counted at 1.0 × 10⁻⁶. 6 Cells were seeded at a density of 10 cells / mL in pre-coated 24-well plates and incubated at 37°C in a 5% CO2 incubator.

[0033] The specific steps of the oscillation are as follows: manually draw a cross pattern and rotate the culture dish, with an oscillation frequency of 1–2 times / second, rotating 3–5 times in each direction, so that the culture medium generates multi-directional fluid shear force in the culture dish; During the oscillation process, intermittent microscopic observation was performed until most of the round, highly refractive microglia above the astrocyte layer detached. Collect the supernatant and centrifuge to obtain microglia.

[0034] S7. After the microglia adhered and grew for 2 days, an appropriate amount of LPS solution (final concentration: 10 μg / mL) was added to each well of the experimental group, while no LPS solution was added to the control group. The cells were placed in a 37℃, 5% CO2 incubator for 4 hours to stimulate the cells. The changes in cell morphology were observed under a phase contrast microscope.

[0035] like Figure 1As shown, duck microglia were observed under an inverted microscope two days after isolation. Figure A shows the mixed culture system of duck microglia and astrocytes under a phase contrast microscope; B shows purified resting microglia, which exhibit a typical "bipolar or unipolar" morphology with slender protrusions; C shows activated microglia after LPS stimulation, with an overall enlarged cell body, a large number of follicles produced in the cytoplasm, and thickened protrusions, exhibiting a typical "amoebic" appearance. Example 2

[0036] Fluorescent identification of microglia was performed using the microglia marker ion-calcium binding adapter molecule 1 (Iba1). The specific steps are as follows: (1) Preparation of cell slides: Take sterile slides coated with poly-L-lysine and inoculate them with cells.

[0037] (2) Observe the cell morphology, state and density under a microscope. When the cells are fully attached and growing well, discard the original culture medium, wash with PBS 3 times, 5 min each time, and discard the PBS.

[0038] (3) Add 500µL of cell fixation medium (i.e. PBS containing 4% paraformaldehyde) to each well, fix for 30 min and then discard the cell fixation medium.

[0039] (4) Add 500µL of PBS to each well, let stand at room temperature for 3 washes, 5 min each time, and discard the PBS.

[0040] (5) Add 500µL of 0.1% Triton-X-100 to each well, permeate the cell membrane at room temperature for 10 min, and then discard the 0.1% Triton-X-100.

[0041] (6) Add 500µL of PBS to each well, let stand at room temperature for 3 washes, 5 min each time, and discard the PBS.

[0042] (7) Add 300µL of blocking solution (i.e. 1% BSA) to each well, seal at room temperature for 1h, and then discard the blocking solution.

[0043] (8) Add 300µL of diluted Iba1 antibody (rabbit source, Hangzhou Huaan Biotechnology Co., Ltd., ET1705-78, and the Iba1 antibody is diluted at a ratio of 1:200) to each well and incubate overnight at 4°C.

[0044] (9) Discard the primary antibody, add 500µL of PBS to each well, and wash 3 times at room temperature for 5 min each time. Discard the PBS.

[0045] (10) Add 300µL of diluted FITC secondary antibody (goat anti-rabbit, Wuhan Aibote Biotechnology Co., Ltd., AS001, and the FITC antibody is diluted at a ratio of 1:100) to each well and incubate at 37°C for 1 hour under dark conditions.

[0046] (11) Discard the secondary antibody, add 500µL of PBS to each well, and wash 3 times at room temperature for 5 min each time. Discard the PBS.

[0047] (12) Add 1µg / mL DAPI, incubate for 30min, 200µL / well, wash three times with PBS for 5min each time, and discard the PBS.

[0048] (13) Under light-protected conditions, add 300µL of PBS to each well and use an inverted fluorescence microscope to image and photograph the cells to observe the specific expression of Iba1.

[0049] Immunofluorescence identification results of duck microglia are as follows Figure 2 As shown in the figure, FITC represents green fluorescence; DAPI stains the cell nucleus, producing blue fluorescence. Under a fluorescence microscope (10× / 20× objective), the isolated and cultured cells appear round and grow in clusters, consistent with the morphological characteristics of microglia. Under the fluorescence microscope, a distinct green fluorescent signal (FITC-labeled secondary antibody) is observed in the cytoplasm of the cells. The fluorescence is evenly distributed, clearly outlining the cell contours, and granular structures with fluorescent aggregation are visible in the cytoplasm, suggesting specific expression of Iba1 in the cytoplasm of microglia. The nuclei of all cells are stained blue by DAPI, with the fluorescence signal concentrated in the center of the cell, and the morphology is round or oval.

[0050] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any transformations or substitutions that can be conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for isolating and culturing duck microglia in vitro, characterized in that, Includes the following steps: S1. Pre-coat cell culture dishes with poly-D-lysine solution and place them in an incubator overnight for coating. S2. Aseptic procedure is used to collect the cerebral cortex of ducklings, remove the meninges and microvascular structures from the brain tissue and clean it with a cleaning solution. S3. After cleaning the tissue in S2, cut it into small pieces, transfer it into a centrifuge tube, add papain and digest it under suitable conditions. S4. Add an equal volume of stop culture medium to stop digestion, filter and centrifuge, discard the supernatant, resuspend in microglial cell culture medium, seed in pre-coated cell culture dishes, and place in an incubator for primary cell culture. S5. After the mixed glial cells have completely adhered to the culture medium, replace it with fresh microglial cell culture medium and place it in an incubator to continue culturing. S6. When the astrocyte layer is completely fused and the upper surface of the astrocyte layer is covered with a layer of microglia with strong refractive properties, the microglia are separated by shaking the cell culture dish. After centrifugation, the supernatant is discarded and the cells are resuspended. After cell counting, the cells are seeded into pre-coated multi-well culture plates at an appropriate cell density and placed in an incubator for culture.

2. The method for isolating and culturing duck microglia according to claim 1, characterized in that: In S1, the cell culture dish is a 60 mm cell culture dish, and the working concentration of the poly-D-lysine solution is 10 μg / mL.

3. The method for isolating and culturing duck microglia according to claim 1, characterized in that: In S2, the ducklings are 1 to 4 days old, and the washing solution includes 95% PBS and 5% penicillin-streptomycin solution.

4. The method for isolating and culturing duck microglia according to claim 1, characterized in that: In S3, the digestion conditions are: digestion in a 37°C water bath shaker for 30 minutes.

5. The method for isolating and culturing duck microglia according to claim 1, characterized in that: In S4 and S5, the termination culture medium and the microglia culture medium are both DMEM high glucose medium with 10% FBS and 5% penicillin-streptomycin solution added, and the final concentration of L-glutamine in the termination culture medium and the final concentration of sodium pyruvate in the microglia culture medium is 4mM and 1mM respectively.

6. The method for isolating and culturing duck microglia according to claim 1, characterized in that: In S4 and S6, the centrifugation conditions were both 1000 rpm for 10 min.

7. The method for isolating and culturing duck microglia according to claim 1, characterized in that: In step 5, the time for complete adhesion is 12 hours after inoculation; in step S6, the time for complete fusion of the astrocyte layer is day 4 of cell culture; the cell density after inoculation is 1.0 × 10⁻⁶. 6 The culture plate is a 24-well plate with cells / mL.

8. The method for isolating and culturing duck microglia according to claim 1, characterized in that: In S6, the specific steps of oscillation are as follows: manually draw a cross-shaped trajectory and rotate the culture dish, with an oscillation frequency of 1–2 times / second, rotating 3–5 times in each direction, so that the culture medium generates multi-directional fluid shear force in the culture dish; During the oscillation process, intermittent microscopic observation was performed until most of the round, highly refractive microglia above the astrocyte layer detached. Collect the supernatant and centrifuge to obtain microglia.

9. An isolated primary duck microglia, characterized in that, The cells are obtained by isolation and culture using the method described in any one of claims 1 to 8, and the cells have the following characteristics: (a) Expression of the microglia-specific marker Iba1; (b) Maintaining typical bipolar or unipolar resting morphology under unstimulated in vitro culture conditions; (c) Under in vitro culture conditions stimulated by LPS, it exhibits an amoeba-like activated state.

10. The application of the method as described in any one of claims 1-8 in establishing an in vitro model of duck microglia.

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