Microglial cell and neurosphere co-culture system as well as construction method and application thereof

Abstract

The invention discloses a microglial cell and neurosphere co-culture system as well as a construction method and application thereof, and belongs to the technical field of cell biology and developmental biology. According to the invention, after the microglial cells and the neurospheres are mixed, three-dimensional space mutual embedding of the microglial cells and the neurospheres is realized through a gravity settling method, and stable co-culture of the microglial cells and the neurospheres is realized, so that the physiological environment in the brain and the natural interaction of neurons-immune cells in the brain are simulated more truly. The construction method of the microglia and neurosphere co-culture system is simple to operate, does not need to depend on a complex three-dimensional culture system or a specific scaffold material, and realizes construction of the stable and extremely-strong-repeatability co-culture system of the microglia and the neurosphere; the method is of great significance to deep study of physiological and pathological mechanisms of the nervous system.

Description

Technical Field

[0001] This invention relates to a microglia and neurosphere co-culture system, its construction method and application, belonging to the fields of cell biology and developmental biology. Background Technology

[0002] Neurospheres are cell aggregates composed of neural stem cells, neurons, and astrocytes. They were first isolated and cultured in the mouse striatum by B.A. Reynolds and S. Weiss in 1992. Neurospheres are widely used to assess the toxic effects of environmental toxins on neural progenitor cells. Neurosphere culture methods are extensively applied in the distribution of stem cells during neural development, assessing the neurotoxicity of environmental toxins, and studying the mechanisms of action of neurotrophic factors. They also provide a source of transplanted cells for neurological diseases such as Parkinson's disease and spinal cord injury. However, traditional neurosphere culture methods often lack the participation of microglia, which limits their application in simulating the physiological environment of the brain and studying neuroinflammation.

[0003] Microglia, as immune cells in the central nervous system, play a crucial role in maintaining neuronal homeostasis and regulating neuroinflammatory responses. Studies have shown that microglia not only participate in the pathological processes of neurodegenerative diseases (such as Alzheimer's and Parkinson's diseases) but also play an important role in neural development and synaptic plasticity. However, existing neurosphere culture systems typically cannot realistically simulate the cellular composition and interactions within the brain, limiting their application in researching neuroinflammation, neurodegenerative diseases, and drug screening.

[0004] In their paper "Establishment of Neuron-Astrocyte-Microglia Co-culture System and Study on its Protective Effect on Neural Stem Cells," Xie Wenyi et al. constructed a neuron-astroglia-microglia co-culture system using Transwell. Neurons were seeded in the lower chamber, while astrocytes and microglia were seeded in the upper chamber. This multi-cell co-culture system simulated the dynamic microenvironment of the central nervous system to the greatest extent possible. However, this system, in which the three cell types were not in contact during culture, could not effectively simulate the nervous system in vivo and was not conducive to studying the interaction between neurons and glial cell synapses.

[0005] While 3D culture of microglia and neurospheres can solve the problem of non-contact co-culture systems, existing methods for co-culturing microglia and neurospheres are limited. Current protocols rely on complex three-dimensional biological scaffolds (such as hydrogels and matrix gels) or customized microfluidic devices, which are not only cumbersome and costly, but also suffer from poor reproducibility due to batch-to-batch variations in materials or fluctuations in equipment parameters. Furthermore, existing co-culture systems struggle to maintain cell viability long-term—neurospheres are prone to hypoxic necrosis, and microglia's migration ability is limited in a 3D environment, often leading to overactivation, overproliferation, or neurosphere structural disintegration, failing to stably simulate the physiological or pathological states of the neural microenvironment. Therefore, developing a simple and effective method for co-culturing microglia and neurospheres is of great significance for in-depth research into the physiological and pathological mechanisms of the nervous system. Summary of the Invention

[0006] The first objective of this invention is to provide a method for constructing a microglia and neurosphere co-culture system, in order to solve the problems of complex processes and poor stability and reproducibility in the co-culture of microglia and neurospheres in the prior art, which rely on complex three-dimensional systems or specific scaffold materials.

[0007] The second objective of this invention is to provide a microglia and neurosphere co-culture system, which provides a microglia and neurosphere co-culture system with high stability and good reproducibility.

[0008] The third objective of this invention is to provide an application of a microglia and neurosphere co-culture system in the study of neuroinflammatory responses, thereby providing an effective microglia and neurosphere co-culture system for neuroinflammatory response research.

[0009] To achieve the above objectives, the technical solution adopted in the method for constructing a microglia and neurosphere co-culture system of the present invention is as follows:

[0010] A method for constructing a microglia and neurosphere co-culture system includes the following steps: fusing microglia and neurospheres using natural gravity sedimentation, then seeding them into a co-culture medium, and culturing in suspension to obtain the microglia and neurosphere co-culture system; wherein the co-culture medium is a neurosphere medium supplemented with microglia growth factor.

[0011] The beneficial effects of the above technical solution are as follows: The method for constructing a microglia and neurosphere co-culture system of the present invention is a pioneering invention. This invention mixes microglia and neurospheres and achieves three-dimensional spatial intercalation of microglia and neurospheres through gravity sedimentation, realizing stable co-culture of microglia and neurospheres. This more realistically simulates the physiological environment within the brain and the natural interaction between neurons and immune cells in the brain. The method for constructing the microglia and neurosphere co-culture system of the present invention is simple to operate, does not rely on complex three-dimensional culture systems or specific scaffold materials, and achieves the construction of a stable and highly reproducible co-culture system of microglia and neurospheres, which is of great significance for in-depth research on the physiological and pathological mechanisms of the nervous system.

[0012] As a further improvement, the quantity is 1×10 5 The microglia mentioned above correspond to 100-110 neurospheres.

[0013] As a further improvement, the fusion time is 30-40 minutes.

[0014] As a further improvement, the suspension culture time is 1-2 weeks.

[0015] As a further improvement, the microglia growth factors are 50 ng / mL TGF-b1, 100 ng / mL IL-34, and 50 ng / mL M-CSF.

[0016] As a further improvement, the neurosphere culture medium is NeuralBasal 1×, GlutaMax 100×, B2725×, 50 ng / mL brain-derived neurotrophic factor (BDNF) and 20 ng / mL glial cell-derived neurotrophic factor (GDNF); the × indicates the final dilution factor.

[0017] As a further improvement, after inducing human induced pluripotent stem cells to differentiate into myeloid stem cells, the myeloid stem cells are then induced to differentiate into microglia; the source of the neurosphere is as follows: human induced pluripotent stem cells are induced to differentiate into neural stem cells, and then the neural stem cells are induced to differentiate into neurospheres.

[0018] To achieve the above objectives, the technical solution adopted in the microglia and neurosphere co-culture system of this invention is as follows:

[0019] A microglia and neurosphere co-culture system, wherein the co-culture system is constructed by the aforementioned construction method.

[0020] The beneficial effects of the above technical solution are as follows: As can be seen from the test, in the microglia and neurosphere co-culture system constructed by the present invention, microglia and neurospheres achieve perfect three-dimensional spatial interpenetration, which significantly improves the physiological relevance of the model and provides a reliable tool for neuroscience research and drug development.

[0021] To achieve the above objectives, the technical solution adopted in this invention for the application of a microglia and neurosphere co-culture system in the study of neuroinflammatory responses is as follows:

[0022] Application of a microglia and neurosphere co-culture system in the study of neuroinflammatory responses.

[0023] The beneficial effects of the above technical solution are as follows: the inflammatory response experiment of the microglia and neurosphere co-culture system constructed by the present invention fully demonstrates that the co-culture system significantly increases the relevant molecules under the stimulation of inflammatory factors, and can serve as a reliable model for studying neuroinflammation.

[0024] As a further improvement, the inflammation is caused by stimulation with lipopolysaccharide or interferon-γ. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the microglia differentiation process and morphological diagrams at each stage in Embodiment 1 of the present invention;

[0026] Figure 2 This is a schematic diagram of the neurosphere formation process and morphological diagrams at each stage in Embodiment 1 of the present invention;

[0027] Figure 3 This is a schematic diagram of the co-cultivation system in Embodiment 1 of the present invention;

[0028] Figure 4 This is a schematic diagram of the morphology of microglia and neurospheres after co-culture in Example 1 of the present invention (wherein, the scale bars are the same as those of the previous ones). Figure 3 (Scale of the central nerve sphere)

[0029] Figure 5 This is a schematic diagram of the immunofluorescence verification results in Example 1 of the present invention (wherein, red represents IBA1 positive microglia, green fluorescence represents MAP positive neurons, and blue represents DAPI-labeled cell nuclei). Detailed Implementation

[0030] Existing technologies for the co-culture of microglia and neurospheres are limited, and most rely on complex three-dimensional culture systems or specific scaffold materials, making it difficult to achieve a stable and reproducible co-culture environment. To overcome these shortcomings, this invention provides a method for constructing a microglia and neurosphere co-culture system.

[0031] Through extensive research and experimentation, this invention discovered that when microglia and neurospheres are mixed, the microglia can rapidly bind to the neurospheres through natural gravitational sedimentation. When seeded in a co-culture medium, these microglia quickly and effectively form a three-dimensional interlocking structure. Furthermore, the co-culture system constructed in this invention significantly improves the physiological relevance of the model, providing a reliable tool for neuroscience research and drug development.

[0032] The present invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the equipment and reagents used in the embodiments, experimental examples and comparative examples are all commercially available.

[0033] Unless otherwise specified, the following examples were conducted under conventional experimental conditions, such as those described in Sambrook et al.'s Molecular Cloning Laboratory Manual (Sambrook J & Russell DW, Molecular cloning: alaboratory manual, 2001), or as recommended by the manufacturer's instructions.

[0034] Main experimental reagents and materials:

[0035] The STEMdiff™ Hematopoietic Kit for myeloid stem cell differentiation was purchased from Stem Cell Technology; nutritional factors such as BDNF, GDNF, TGF-β1, and M-CSF were purchased from PeproTech; PSC neural induction medium, Neurobasal medium, DMEM / F12 medium, GlutaMax (35050061), B27 (0080085SA), and N2 (17502-048) were purchased from Gibco; NEAA (M7145) was purchased from Sigma; Insulin was purchased from Sigma MAP2; and IBA1 antibody was purchased from Abcam.

[0036] Biomaterials:

[0037] The human induced pluripotent stem cells (hiPSCs) used in the following embodiments of the present invention are the commercially available cell line NIBSC8.

[0038] I. Specific embodiments of the microglia and neurosphere co-culture system and its construction method of the present invention:

[0039] Example 1

[0040] In this embodiment, hiPSCs were cultured and differentiated into microglia and neurospheres, respectively. Then, a co-culture system of microglia and neurospheres was constructed using natural gravity sedimentation. The specific implementation steps are as follows:

[0041] 1. Differentiation of microglia from human induced pluripotent stem cells (hiPSCs)

[0042] The process involves inducing hiPSCs to differentiate into myeloid stem cells, and then further inducing these myeloid stem cells to differentiate into microglia. The specific procedure and typical cell morphology diagrams at each stage are shown below. Figure 1 As shown, the specific process is as follows:

[0043] Dilute Matrigel® 1:100 in pre-chilled DMEM / F12 medium to a final concentration of approximately 0.3–0.5 mg / mL. Then, transfer 500 μL / well to each well of a 12-well plate and incubate overnight at 4°C. Culture hiPSCs as dense cell aggregates with well-defined, smooth-edged clones (100–200 μm in diameter) in mTeSR™ Plus medium, seeding 16–40 clones per well in Matrigel®-coated 12-well plates. Initially, culture in medium A (from the STEMdiff™ Hematopoietic Kit) for 3 days; then switch to medium B, changing the medium every other day, until day 12 when myeloid stem cells can be harvested.

[0044] Add 500 μL of 0.1 mg / mL poly-L-lysine solution to each well of a 12-well plate and incubate overnight (16–18 hours) at 4°C or for 1 hour at 37°C. Discard the solution and rinse three times with sterile deionized water. Add 500 μL of 5 μg / mL laminin solution to each well and incubate at 37°C for 2 hours. The obtained myeloid stem cells are then cultured at 4 × 10⁻⁶ cells / well. 4 / wells were seeded in 12-well plates coated with poly-Lysine-lamin, and specific differentiation-inducing factors such as TGF-β, IL-34 and M-CSF were added. The specific culture medium formulation is shown in Table 1. The medium was changed every other day and cultured for 3 weeks to further induce myeloid stem cells to differentiate into microglia.

[0045] Table 1

[0046] 2. Differentiation of neurospheres from human induced pluripotent stem cells (hiPSCs)

[0047] The process involves inducing hiPSCs to differentiate into neural stem cells, and then further inducing these neural stem cells to differentiate into neurospheres. The specific procedure and typical cell morphology diagrams at each stage are shown below. Figure 2 As shown, the specific process is as follows:

[0048] hiPSCs were cultured in clonal form (100-200 μm in diameter) in mTeSR™ Plus medium and seeded in 6-well plates coated with Matrigel®. Neural stem cells were obtained by changing the medium continuously for 7 days using the differentiation medium from the Gibco PSC kit.

[0049] Neural stem cells were digested with 0.25% trypsin at a concentration of 2 × 10⁻⁶ per well. 6 One cell was seeded into a 6-well plate, which was then placed in a cell culture incubator on a shaker at 88 rpm to induce neural stem cells to form neurospheres using a suspension culture method. Initially, the culture medium used was the neural stem cell medium from the Gibco PSC kit; after two days, it was replaced with neurosphere medium (formulation shown in Table 2). The medium was changed every two days, and cultured for 7-8 weeks until neurospheres formed.

[0050] Table 2

[0051] 3. Construction of a co-culture system for microglia and neurospheres

[0052] The microglia obtained in step 1 and the neurospheres obtained in step 2 were uniformly mixed using natural gravity sedimentation, and then seeded into a co-culture medium to construct a microglia and neurosphere co-culture system. The specific process and typical cell morphology images at each stage are shown below. Figure 3 As shown, the specific process is as follows:

[0053] The cultured microglia were counted, and then 1×10⁶ cells were pipetted together. 5 Microglia were placed into a 2 mL EP tube. Using a pipette with a wide 1 mL tip, approximately 100 neurospheres from one well of a 6-well plate were gently added to the 2 mL EP tube. Neurosphere culture medium (as shown in Table 2) was added to bring the volume to 1.5 mL. The tube was then inverted to ensure thorough mixing of the two cell types. The EP tube was placed vertically in a cell culture incubator, allowing gravity to allow the microglia and neurospheres to fully fuse.

[0054] After standing for half an hour, transfer the cells from the EP tube back to one well of a 6-well plate and replace with fresh co-culture medium (formulation shown in Table 3). Continue culturing on a shaker at 37°C (88 rpm) for 1-2 weeks to obtain a co-culture system of microglia and neurospheres.

[0055] Table 3

[0056] 4. Validation of the co-cultivation system

[0057] The coexistence status of microglia and neurons in a co-culture system was verified using immunofluorescence technology. The specific process is as follows:

[0058] The morphology of microglia and neurons in the co-culture system is as follows: Figure 4 As shown in the figure, the morphology of the co-culture system was not significantly different from that before the addition of microglia to neurospheres. The cultures from the co-culture system were collected, washed with PBS, and fixed with 4% paraformaldehyde at room temperature for 30 min. Routine immunofluorescence experiments were performed using fluorescently labeled antibodies targeting specific markers for microglia (IBA1) and neurons (MAP2), respectively. The coexistence status of cells in the co-culture system was observed using a fluorescence microscope (e.g., ...). Figure 5 (As shown).

[0059] As shown in the figure, IBA1-positive microglia (red) and MAP-positive neurons (green) coexist in the co-culture system. The red channel, IBA1, marks microglia, showing punctate or short rod-shaped cell bodies with slender processes, indicating a predominantly resting distribution. The green channel, MAP2, outlines neuronal dendrites, showing a continuous network extension and intact dendritic structure. After Merge staining with blue DAPI nuclear staining, microglia are scattered among the neuronal dendritic network.

[0060] II. Specific embodiments of the application of the microglia and neurosphere co-culture system of the present invention in the study of neuroinflammatory responses:

[0061] Example 2: LPS stimulation to validate the inflammatory response of the model

[0062] This embodiment uses the microglia and neurosphere co-culture system constructed in Example 1 as a model. Lipopolysaccharide (LPS) is used to stimulate the co-culture system to verify its ability to detect inflammatory molecules. The specific implementation steps are as follows:

[0063] LPS (purchased from Sigma-Aldrich) was added to the co-culture system to a final concentration of 100 ng / mL. A co-culture system without LPS was set up as a control group, with three replicates in each group. After 24 hours of culture, the culture supernatant was collected, and total cellular protein was extracted. The levels of inflammatory factors interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) in the culture supernatant were detected using an ELISA kit (purchased from eBioscience), strictly following the kit instructions. The results are as follows:

[0064] Control group: IL-1β level was 15.2 pg / mL, TNF-α level was 22.4 pg / mL, and IL-6 level was 18.6 pg / mL.

[0065] LPS stimulation group: IL-1β level was 89.7 pg / mL, TNF-α level was 105.3 pg / mL, and IL-6 level was 76.5 pg / mL.

[0066] Compared with the control group, the levels of IL-1β, TNF-α, and IL-6 in the LPS-stimulated group increased by 5.9-fold, 4.7-fold, and 4.1-fold, respectively, and the differences were statistically significant (P<0.01). The experimental results showed that the levels of IL-1β, TNF-α, and IL-6 in the culture supernatant of the LPS-stimulated co-culture system were significantly higher than those in the control group, indicating that this co-culture system can effectively detect a significant increase in inflammatory molecules under LPS stimulation and can serve as a reliable model for studying neuroinflammation.

[0067] Example 3: IFN-γ stimulation validation of the inflammatory response model

[0068] This embodiment uses the microglia and neurosphere co-culture system constructed in Example 1 as a model. Interferon-γ (IFN-γ) stimulation of the co-culture system is used to verify its ability to detect inflammatory molecules. The specific implementation is as follows:

[0069] IFN-γ (purchased from PeproTech) was added to the co-culture system to a final concentration of 50 ng / mL, while a co-culture system without IFN-γ was set up as a control group. Three replicates were prepared for each group. After 24 hours of culture, the culture supernatant was collected, and total cellular protein was extracted. The levels of inflammatory factors interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) in the culture supernatant were detected using an ELISA kit (purchased from eBioscience), strictly following the kit instructions. The results are as follows:

[0070] Control group: IL-1β level was 14.8 pg / mL, TNF-α level was 20.5 pg / mL, and IL-6 level was 16.3 pg / mL.

[0071] In the IFN-γ stimulation group, the levels of IL-1β were 78.3 pg / mL, TNF-α were 92.6 pg / mL, and IL-6 were 65.2 pg / mL.

[0072] Compared with the control group, the levels of IL-1β, TNF-α, and IL-6 in the IFN-γ stimulation group increased by 5.3-fold, 4.5-fold, and 3.9-fold, respectively, and the differences were statistically significant (P<0.01). The experimental results showed that the levels of IL-1β, TNF-α, and IL-6 in the culture supernatant of the co-culture system after IFN-γ stimulation were significantly higher than those in the control group, indicating that this co-culture system can effectively detect a significant increase in inflammatory molecules under IFN-γ stimulation and can serve as a reliable model for studying neuroinflammation.

[0073] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for constructing a microglia and neurosphere co-culture system, characterized in that: The process includes the following steps: fusing microglia and neurospheres using natural gravity sedimentation, then seeding them into a co-culture medium and culturing them in suspension to obtain a microglia and neurosphere co-culture system; the co-culture medium is a neurosphere medium supplemented with microglia growth factor.

2. The method for constructing the microglia and neurosphere co-culture system according to claim 1, characterized in that: The quantity is 1×10 5 The microglia mentioned above correspond to 100-110 neurospheres.

3. The method for constructing the microglia and neurosphere co-culture system according to claim 1, characterized in that: The fusion time is 30-40 minutes.

4. The method for constructing the microglia and neurosphere co-culture system according to claim 1, characterized in that: The suspension culture time is 1-2 weeks.

5. The method for constructing the microglia and neurosphere co-culture system according to claim 1, characterized in that: The microglia growth factors are 50 ng / mL TGF-b1, 100 ng / mL IL-34, and 50 ng / mL M-CSF.

6. The method for constructing the microglia and neurosphere co-culture system according to claim 1, characterized in that: The neurosphere culture medium consisted of NeuralBasal 1×, GlutaMax 100×, B27 25×, 50 ng / mL brain-derived neurotrophic factor (BDNF), and 20 ng / mL glial cell-derived neurotrophic factor (GDNF); the × symbol indicates the final dilution factor.

7. The method for constructing the microglia and neurosphere co-culture system according to any one of claims 1 to 6, characterized in that: The microglia are obtained from the following sources: human induced pluripotent stem cells are induced to differentiate into myeloid stem cells, and then myeloid stem cells are induced to differentiate into microglia. The neurospheres are obtained from the following sources: human induced pluripotent stem cells are induced to differentiate into neural stem cells, and then neural stem cells are induced to differentiate into neurospheres.

8. A microglia and neurosphere co-culture system, characterized in that: The co-culture system is constructed using the construction method described in any one of claims 1 to 7.

9. The application of the microglia and neurosphere co-culture system as described in claim 8 in the study of neuroinflammatory responses.

10. The application of the microglia and neurosphere co-culture system according to claim 9 in the study of neuroinflammatory responses, characterized in that: The inflammation is caused by lipopolysaccharide or interferon-γ stimulation.

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